JPH036152B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a cobaltichlorophyllin complex compound 1
This invention relates to a method for producing a novel thiamine cobaltichlorophyllin complex compound in which one thiol-type thiamine molecule is coordinated. The thiamine cobaltichlorophyllin complex compound obtained by the present invention is a substance useful as a medicine, and is expected to have many physiological effects in addition to having medical effects on gastrointestinal ulcers, for example. Conventional thiamin cobaltichlorophyllin complex compounds are disclosed in Japanese Patent Publication No. 1986-2005 and Japanese Patent Publication No. 1972-2005.
No. 28687 and the techniques described in each publication are known.
That is, in Japanese Patent Publication No. 50-2005, a cobalt salt or an iron salt is added to a salt-saturated acetic acid solution of chlorophyllin, and a chlorophyllin-cobalt complex compound is precipitated by adjusting the pH (PH 5 to 7), and then an alkaline solution of thiamine is added. and then set the pH to 4 again.
The product was obtained by adjusting the temperature to ~6. The method disclosed in Japanese Patent Publication No. 51-28687 is based on carrying out the reaction with the pH level at the time of thiamine addition being made more alkaline. However, in any case, 1 equivalent of thiamine was coordinated to the cobaltichlorophyllin complex compound, that is, 1
A thiamine cobaltichlorophyllin complex compound having a thiol-type thiamine molecule as one ligand has not been confirmed so far, and no method for selectively producing it is known. Therefore, according to the present invention, there is provided a method for obtaining a novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated to one molecule of the cobaltichlorophyllin complex compound.
The method of the present invention is constituted by the following steps. The first step is to complex chlorophyllin in a water-free acetic acid solution, preferably an alcoholic, for example, methanolic acetic acid solution, with a divalent cobalt salt such as cobalt acetate in the presence of oxygen. A chlorophyllin complex compound is obtained, and the solvent is removed from this product under vacuum and reduced pressure to obtain a highly pure crystalline cobalt chlorophyllin complex compound. In the second step, the high-purity cobaltichlorophyllin complex compound obtained in the first step is dissolved in methanol, and thiamine, which has been made into an alkaline methanol solution in advance, is mixed and reacted to form a thiamine cobaltichlorophyllin complex compound, and then the reaction takes place. Remove as much methanol as possible from the mixture. Finally, as a purification step, the crude thiamine cobaltichlorophyllin complex compound obtained as above was subjected to liquid-liquid extraction with benzyl alcohol/water system (removal of unreacted thiamine and decomposed products derived therefrom), silica gel chromatography, and Subjected to gel perchromatography (removal of chlorophyllin and by-products generated during the reaction). The present invention will be explained in more detail below. The chlorophyllin used as a raw material in the present invention is derived from plant chloroplasts, such as higher plant leaves such as mulberry leaves, alpha alpha leaves, timothy grass leaves, algae such as chlorella and scenedesmus, or plant chloroplasts. It can be obtained from highly concentrated grains such as silkworm feces through solvent extraction, saponification and acidification, and purification operations such as solvent fractional extraction, fractional crystallization and chromatographic separation. This product mainly consists of chlorin e ₆ , but a portion of chlorin p ₆ , purpurin 7, etc. may be mixed in without any problem. When converting the raw material chlorophyllin into an acetic acid solution, it is preferable to coexist with alcohol, and methanol, ethanol, isopropanol, etc. can be used, but methanol is preferred. In this case, the acetic acid content is between 5 and 80%, preferably between 10 and 80%.
It is 30%. As the divalent cobalt salt, cobalt acetate is preferable, of which cobalt chloride, cobalt sulfate, cobalt acetate, and the like can be used. This divalent cobalt salt is mixed and reacted, for example, in the form of a methanol solution, usually in a dropwise manner. Reaction temperature ranges from room temperature to 50-60℃
It is preferably 40 to 50°C. At this time, it is important to avoid the presence of water as much as possible in terms of product quality and yield. In the present invention, the above reaction is carried out in the presence of oxygen. As a result of various studies, the inventor discovered that the addition of hydrogen peroxide minimizes the effect on the unstable chlorin ring, and as a result, a highly pure product can be obtained. That is, by adding hydrogen peroxide in advance to the cobalt salt solution to be introduced into the reaction system, a reaction that normally requires several hours can be completed within one hour. It is also possible to carry out the reaction with air or oxygen blowing.
After the reaction was completed, the solvent was removed by vacuum concentration to obtain a highly pure cobaltichlorophyllin complex compound. In this case, it is possible to add an alkali to adjust the pH and cause precipitation and crystallization, but this is not so preferred since it not only causes industrial problems but also affects quality. According to the findings of the present inventors, the cobaltichlorophyllin complex compound produced is stable in an alcohol-acetic acid mixed solvent even when heated to a certain extent, and the high purity cobaltichlorophyllin complex compound is directly recovered from the reaction solution by vacuum concentration. What was possible was unexpected. The cobaltichlorophyllin complex compound thus obtained is of high purity and can be provided as it is to the next step. The cobaltichlorophyllin complex compound is dissolved in methanol, and a methanol solution of the hydrochloride, nitrate, or free base of thiamine (vitamin B ₁ ), which has been previously converted into thiol form with sodium hydroxide, is adjusted to pH 9 to 11 and mixed and reacted. Less than 2 moles of thiamine should be used per mole of cobaltichlorophyllin complex compound. Preferably, substantially equimolar amounts are used. At this time, an ammonia buffer can be used to adjust the pH.
The reaction is carried out under non-oxidizing conditions. The term "non-oxidizing conditions" as used herein means that conditions are not such as to actively bring the reaction system into contact with oxygen, such as by introducing air into the reaction system. Therefore, the reaction takes place at room temperature to 50°C.
Although it is preferable to carry out the process under light-shielding conditions while blocking oxygen, it is possible to carry out the process without taking any special measures such as blocking oxygen if care is taken such as gentle stirring. It is. When the reaction solution is concentrated under reduced pressure, a crude thiamine cobaltichlorophyllin complex compound is obtained. For purification, it is dissolved in benzyl alcohol and washed with water to transfer unreacted thiamine and decomposition products derived therefrom to the aqueous phase. In this process, the target thiamine cobaltichlorophyllin complex compound is not lost and remains stably in the benzyl alcohol phase. This benzyl alcohol phase is passed through a silica gel column chromatographic column to perform chromatographic fractionation. At this time, it is not necessary to concentrate the benzyl alcohol. Although a single solvent or a mixed solvent such as methanol, ethanol, chloroform, acetone, etc. can be used as a developing agent, methanol is preferably used industrially. After the benzyl alcohol is eluted, a predetermined thiamine cobaltichlorophyllin complex compound is eluted. In this case, impurities and decomposition products flowing before and after a given product can be excluded by checking the absorption ratio of the visible absorption spectrum and thin layer chromatography. This silica gel column chromatography also eliminates contamination of the gel carrier in the next final purification step, gel permeation column chromatography. A thiamine cobaltichlorophyllin complex compound of constant quality is obtained by passing the thiamine cobaltichlorophyllin complex compound fraction obtained by silica gel chromatography through a gel permeation chromatography column. Gel perchromatography carriers include Cephadex LH-20 and Toyopearl.
HW-40 is preferable. As the developing solvent, methanol, ethanol, acetone, etc. are suitable, but methanol is preferable. Confirmation of the fractionation of the thiamine cobaltichlorophyllin complex compound is carried out by visible absorption spectroscopy and thin layer chromatography. From the thus obtained thiamine cobaltichlorophyllin complex compound fraction, a thiamine cobaltichlorophyllin complex compound in the form of a black-green glossy fine powder is obtained by concentration to dryness, spray drying, or the like. This product has extremely high purity and can be provided as is as a raw material for pharmaceuticals. The thiamine cobaltichlorophyllin complex compound thus obtained has the following estimated structural formula. The elemental analysis results for C ₄₆ H ₅₅ CoN ₈ O ₁₀ S (molecular weight 971.08) are as follows, and this actual value is 1 molecule of chlorophyllin (chlorin e ₆ ), 1 atom of cobalt and 1 molecule of vitamin B ₁ (thiol). This agrees well with the theoretical value, which assumes that one molecule (type thiamine) is bound. C (%) H (%) N (%) Theoretical value: 56.89 5.72 11.54 Actual value: 57.63 5.33 11.76 O (%) S (%) Co (%) Theoretical value: 16.48 3.30 6.07 Actual value: 17.07 3.23 5.99 Here The obtained thiamine cobaltichlorophyllin complex compound further has the following physicochemical properties. 1. The thiamine cobaltichlorophyllin complex compound of the present invention is a dark green amorphous or shiny fine lump with a weak specific odor. This substance is extremely soluble in methanol and ethanol, slightly soluble in water, and almost insoluble in ether, chloroform, benzene, and ethyl acetate. 2. Accurately weigh about 100 mg of the thiamine cobaltichlorophyllin complex compound of the present invention, and dissolve it by adding methanol to make exactly 50 ml. 1ml of the liquid
and add methanol to make exactly 50ml. Furthermore, take 2 ml of this liquid and add 3 ml of methanol.
ml to make the sample solution. When the absorption spectrum in the visible region is measured using the Japanese Pharmacopoeia General Test Method "Absorbance Measurement Method" using methanol as the control solution for the sample solution, it has maximum absorption at wavelengths of 428 to 432 nm and 640 to 667 nm, and the absorption strength is at 428 to 432 nm. ＞640〜
The order is 667nm. 3 Analysis by infrared absorption spectrum Thiamine cobaltichlorophyllin compound according to the present invention, cobaltichlorophyllin (cobaltichlorophyllin e ₆ ), and chlorophyllin (chlorin e ₆ )
The infrared absorption spectrum (KBr tablet method) was measured. The infrared spectra are first and second, respectively.
It was as shown in Figures 2 and 3. The absorption of 3400 cm ^- ₁ derived from NH and water of the raw material chlorophyllin (Fig. 3) shifts to 3430 cm - ' in the cobaltichlorophyllin complex compound, and at the same time, the absorption of 3400 cm ^- 1 derived from NH and water of the cobaltichlorophyllin complex compound (Fig. 2) and thiamine In cobaltichlorophyllin, absorption at 1080 cm ^- ' (νCo-N) (Fig. 1) arises due to cobalticomplexation. From this, it is inferred that the cobalt metal in the thiamine-cobaltichlorophyllin and cobaltichlorophyllin complex compounds is complex-bonded with the four N atoms of the pyrrole nucleus of the raw material chlorophyllin. In addition, 1625 cm ^- ′ (NH) and
1040cm ^- '(C-O) absorption thought to be derived from thiamine (bimin B ₁ ) (the former is the primary thiamine
amine, the latter from -C ₂ H ₄ OH of thiamine), which was not present in cobaltichlorophyllin and chlorophyllin. From the above results, it is estimated that cobalt in the complex compound of the present invention is coordinately bonded to the four N atoms of the pyrrole nucleus of chlorophyllin, and that thiamine is bound in the complex compound of the present invention. Ta. 4 Analysis by visible region absorption spectrum The thiamin cobaltichlorophyllin compound of the present invention, its precursor cobaltichlorophyllin complex compound, and the raw material chlorophyllin were analyzed using a methanol solvent with a layer length of 10 mm at 340 ~
Visible absorption measurements at 700 nm were performed. The visible absorption spectrum is shown in Figure 4, and the results are summarized in the table below.

[Table] In the visible absorption spectra of the complex compound of the present invention and the cobaltichlorophyllin complex compound, the characteristic absorption band of 500 to 530 nm of chlorophyllin almost disappears, and the red band shifts to a slightly shorter wavelength region, and The Soret's band has moved to longer wavelengths, and the ε value has decreased. These changes are considered to indicate a complex bond between chlorophyllin and metallic cobalt in the complex compound and cobaltichlorophyllin complex compound of the present invention. The toxicity of the thiaminecobaltichlorophyllin compound obtained by the present invention is as follows.

[Table] The pharmacological effects of thiamine cobaltichlorophyllin on experimental gastric ulcers induced by the stress method and the restraint cortisone method are shown below. The animal is a male Wistar rat (body weight
200-300g) and 10 animals in each group. All drugs to be administered were dissolved in physiological saline. The dose was 100 mg/Kg body weight per day, and 1 ml of physiological saline was administered to the control saline group. In an acute ulcer prevention test using the stress method, the drug was orally administered continuously for 7 days before stress loading. In addition, in a treatment trial for chronic ulcers using the restraint cortisone method, 7 days after the restraint was removed.
It was orally administered continuously for several days. Experimental ulcer generation by stress method is described in "Chem.Pharm.Bull." Volume 12, No. 465
(1964), and the generation of experimental ulcers by the restraint cortisone method was published in ``Journal of the Japanese Society of Gastroenterology,'' No. 62.
(12), No. 1533 (1965). The results are shown in the table below.

[Table] Example 1 5.0 g of chlorophyllin (chlorin e ₆ ) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, add 10.0 g of cobalt acetate tetrahydrate to methanol.
0.75 ml of 30% hydrogen peroxide solution is added to the solution dissolved in 50 ml, and the resulting solution is added dropwise to the previously prepared chlorophyllin solution at 50° C. while stirring. moreover
Stir at 50°C for 1 hour to complete the reaction. After removing insoluble materials from the reaction mixture, the reaction mixture was concentrated to dryness under reduced pressure, and the residue was washed with water and dried to obtain 4.12 g of cobaltichlorophyllin (yield: 71.4%). Dissolve 4.0 g of cobaltichlorophyllin in 280 ml of methanol. Separately, dissolve 3.0 g of thiamine hydrochloride in 180 ml of methanol and adjust the pH to 10.0 with a methanol solution of caustic soda, then add the resulting solution to a methanol solution of cobaltichlorophyllin.
Add dropwise while stirring in a nitrogen stream at 40°C. moreover,
Stir for 3 hours at 40°C in a nitrogen stream. After removing insoluble matter from the reaction mixture, it was concentrated under reduced pressure to obtain 6.16 g of crude thiamine cobaltichlorophyllin (yield:
109.2%). 5.0 g of crude thiamine cobaltichlorophyllin was dissolved in 50 ml of benzyl alcohol, washed twice with 50 ml of water each time, and then this benzyl alcohol solution was loaded into a silica gel column and washed with 500 ml of acetone/methanol (3:2). After removing impurities, elute the target fraction with 400 ml of methanol.
The eluate was concentrated under reduced pressure to obtain 645 mg of purified thiamine cobaltichlorophyllin (yield 12.9%). A 3 cm x 30 cm column was prepared using 50 g of Cephadex LH-20 and methanol, and a methanol solution of 600 mg of purified thiamine cobaltichlorophyllin was loaded onto this column, developed and eluted with methanol, and fractionated. The eluate obtained by removing impurities was concentrated under reduced pressure to obtain thiamine cobaltichlorophyllin.
Obtain 380 mg (yield 63.3%). The total yield from chlorophyllin (chlorin e ₆ ) is 6.37%. Example 2 5.0 g of chlorophyllin (chlorin e ₆ ) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, add 10.0 g of cobalt acetate tetrahydrate to methanol.
The solution obtained by dissolving in 50 ml is added dropwise to the above chlorophyllin solution while stirring at 40°C while passing air. Further, the reaction mixture was stirred at 50° C. for 3 hours under air circulation to react. After removing the insoluble matter, the reaction solution was concentrated to dryness under reduced pressure, and the residue was washed with water and dried to obtain 3.88 g of cobaltichlorophyllin (yield: 67.2%). Dissolve 3.5 g of cobaltichlorophyllin in 250 ml of methanol. Separately, a solution of 2.63 g of thiamine hydrochloride dissolved in 160 ml of methanol and adjusted to pH 10.0 was added dropwise to the above methanol solution of cobaltichlorophyllin while stirring at 40°C. Stirring is continued for an additional 3 hours after the addition. After removing insoluble matter, the reaction solution is concentrated under reduced pressure. The residue was then dissolved in ethanol to remove insoluble materials, and the solution was concentrated under reduced pressure to obtain 4.62 g of crude thiamine cobaltichlorophyllin (yield 93.7%). 4.5 g of crude thiamine cobaltichlorophyllin was dissolved in 45 ml of benzyl alcohol, washed twice with 45 ml of water each time, and then this benzyl alcohol solution was loaded into a silica gel column and washed with 500 ml of acetone/methanol (3:2). After removing impurities, elute the target fraction with 400 ml of methanol.
The eluate was concentrated under reduced pressure to obtain 734 mg of purified thiamine cobaltichlorophyllin (yield 16.3%). 3cm with Toyopearl HW-40F and methanol
Prepare a 30 cm x 30 cm column, load a methanol solution of 700 mg of purified thiamincobaltichlorophyllin thereon, develop and elute with methanol, and fractionate. The fraction obtained by removing impurities was concentrated under reduced pressure to obtain 369 mg of thiamine cobaltichlorophyllin (yield
52.7%). The total yield from chlorophyllin (chlorin e ₆ ) is 5.41%. Example 3 5.0 g of chlorophyllin (chlorin e ₆ ) is dissolved in a mixed solvent of 500 ml of ethanol and 100 ml of glacial acetic acid. Separately, add 5.0 g of cobalt acetate tetrahydrate to ethanol.
The solution obtained by dissolving in 100 ml is added dropwise to the above chlorophyllin solution while stirring at 40°C while circulating air. The reaction was further completed by stirring at 50°C for 3 hours under air flow. After removing the insoluble materials, the reaction solution was concentrated to dryness under reduced pressure, and the residue was washed with water and dried to obtain 3.45 g of cobaltichlorophyllin (yield: 59.8%). Dissolve 3.0 g of cobaltichlorophyllin in 210 ml of ethanol. A solution of 2.25 g of thiamine hydrochloride dissolved in 270 ml of ethanol and adjusted to pH 10.0 was added to the above cobaltichlorophyllin ethanol solution at 50°C.
Add dropwise while stirring. It is then stirred for an additional 3 hours. After removing the insoluble matter, the reaction solution was concentrated under reduced pressure to obtain 4.41 g of crude thiamine cobaltichlorophyllin (yield 104.3%). 4.0 g of crude thiamine cobaltichlorophyllin was dissolved in 40 ml of benzyl alcohol, washed twice with 40 ml of water each time, and then this benzyl alcohol solution was loaded into a silica gel column and washed with 500 ml of acetone/methanol (3:2). After removing impurities, elute the target fraction with 400 ml of methanol.
The desired eluate is concentrated under reduced pressure to obtain 432 mg of purified thiamine cobaltichlorophyllin (yield 10.8%). A 3 cm x 30 cm column is prepared using 50 g of Sephadex LH-20 and methanol, and a methanol solution of 400 mg of purified thiamine cobaltichlorophyllin is placed on top of the column, and the column is developed and eluted with methanol for fractionation. The eluate obtained by removing the impurity fraction was concentrated under reduced pressure to obtain 326 mg of thiamine cobaltichlorophyllin.
(yield 81.5%). The yield from chlorophyllin (chlorin e ₆ ) is 5.49%. Example 4 5.0 g of chlorophyllin (chlorin e ₆ ) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, add 10.0 g of cobalt chloride hexahydrate to methanol.
The solution obtained by dissolving in 50 ml is added dropwise to the above chlorophyllin solution while stirring at 40°C while passing air. Further, the reaction was completed by stirring at 50° C. for 3 hours under air circulation. After removing the insoluble matter, the reaction solution was concentrated to dryness under reduced pressure, and the residue was washed with water and dried to obtain 4.45 g of cobaltichlorophyllin (yield 77.1%). 4.0 g of this cobaltichlorophyllin is dissolved in 280 ml of methanol. Dissolve 3.0 g of thiamine nitrate in 180 ml of methanol and adjust the pH to 10.0. Add the solution to the above cobaltichlorophyllin methanol solution.
Add dropwise while stirring at 40°C, and further stir at 40°C for 3 hours. After removing the insoluble matter, the reaction solution was concentrated under reduced pressure to obtain 6.48 g of crude thiamine cobaltichlorophyllin (yield 114.9%). 6.0 g of crude thiamine cobaltichlorophyllin was dissolved in 60 ml of benzyl alcohol, washed twice with 60 ml of water each time, and the benzyl alcohol solution was loaded into a silica gel column, followed by washing with 500 ml of acetone/methanol (3:2). to remove impurities. Elute the target fraction with 400ml of methanol,
The eluate was concentrated under reduced pressure to obtain 453 mg of purified thiamine cobaltichlorophyllin (yield 7.55%). Prepare a 3 cm x 30 cm column with 50 g of Cephadex LH-20 and methanol, and add purified thiamincobaltichlorophyllin 400 to the top of this column.
Load mg of methanol solution, develop and elute with methanol, and fractionate. The solution obtained by removing impurities was concentrated under reduced pressure to obtain thiamine cobaltichlorophyllin.
Obtain 380 mg (yield 50.3%). The yield from chlorophyllin (chlorin e ₆ ) is 3.36%.

[Brief explanation of the drawing]

In the accompanying drawings, FIGS. 1, 2, and 3 are infrared absorption spectrum curves of the thiamine cobaltichlorophyllin complex compound of the present invention, cobaltichlorophyllin as an intermediate, and chlorophyllin as a raw material, respectively; The figure shows curves showing the visible absorption of the three types.

Claims (1)

[Claims] 1. Reacting chlorophyllin (chlorin e ₆ ) with a dicobalt salt in the presence of oxygen in a water-free acetic acid solution to form a cobalt chlorophyllin complex, and then removing the solvent from the resulting reaction mixture, To a solution in which the cobaltichlorophyllin complex compound recovered as a residue is dissolved in alcohol, thiamine, which has been made into an alkaline alcohol solution in advance, is added in an amount less than 2 moles per 1 mole of the cobaltichlorophyllin complex compound, and the mixture is treated under non-oxidizing conditions. The reaction mixture is allowed to react, the alcohol is distilled off as much as possible from the reaction mixture, the nearly solid residue is dissolved in benzyl alcohol, the resulting solution is extracted with water to remove unreacted materials, and the remaining benzyl alcohol phase is removed. A novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated to one molecule of the cobaltichlorophyllin complex compound, which is characterized by being subjected to silica gel chromatography and gel overtreatment as it is. Manufacturing method.