JPH0338252B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a cobaltichlorophyllin complex compound 1
The present invention relates to an anti-ulcer agent containing a novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated. Conventionally, thiamine chlorophyllin complex compounds in which two molecules of thiazole-type thiamine or two molecules of thiol-type thiamine are coordinated to one molecule of a cobaltichlorophyllin complex compound are disclosed in Japanese Patent Publications Nos. 50-2005 and 51-
They are described in Publication No. 28687, and are said to be useful drugs because they can have medical effects in the field of collagen diseases, and, for example, in steroid therapy, when used in combination with steroids, steroids can be used sparingly. However, the thiamine-cobaltichlorophyllin complex compound of the present invention is a substance in which one molecule of thiol-type thiamine is coordinated to one molecule of the cobaltichlorophyllin complex compound, and is different from the above-mentioned known thiamine-cobaltichlorophyllin complex compound. Shows remarkable medicinal effects on gastrointestinal ulcers. The thiamine cobaltichlorophyllin complex compound of the present invention is constructed 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 process, the high-purity cobalt chlorophyllin complex compound obtained in the first step is dissolved in methanol, and thiamine, which has been made into an alkaline methanol solution, is mixed and reacted to form a thiamine chlorophyllin complex compound, and then reacted. Remove as much methanol as possible from the mixture. Finally, as a purification step, the crude thiamine chlorophyllin complex compound obtained as described above is subjected to liquid-liquid extraction using a benzene alcohol/water system (removal of unreacted thiamine and decomposed products derived therefrom).
The mixture is then subjected to silica gel chromatography and gel perchromatography (removal of chlorophyllin and by-products generated during the reaction). Another method is to dissolve the crude thiamine cobaltichlorophyllin complex compound in, for example, aqueous alcohol and subject it to hydrophobic porous polymer column chromatography or gel permeation chromatography. The method for producing the thiamine cobaltichlorophyllin complex, which is the active ingredient, will be explained in more detail below. The chlorophyllins used as raw materials are derived from plant chloroplasts, such as higher plant leaves such as mulberry leaves, alpha alpha leaves, timothy grass leaves, algae such as Chlorella, Scenedesmus, or even highly enriched plant chloroplasts. It can be obtained from grains such as silkworm feces through solvent extraction, saponification, acidification, and purification operations such as solvent fractional extraction, fractional crystallization, and chromatographic separation. This product may be partially mixed with chlorin p ₆ mainly consisting of chlorin e ₆ , purpurin 7, and the like. 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 10
~30%. As the divalent cobalt salt, cobalt chloride, cobalt sulfate, cobalt acetate, etc. can be used, but cobalt acetate is preferable. This divalent cobalt salt is subjected to the reaction, for example, as a methanol solution. The reaction temperature ranges from room temperature to 50-60°C, preferably 40-50°C. At this time, it is important to avoid the presence of water as much as possible in terms of the quality and yield of the purified product. The above reaction is carried out in the presence of simplex. 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 completion of the reaction, a highly purified cobaltichlorophyllin complex compound was obtained by removing the solvent by vacuum concentration or by adding a solvent that insolubilizes the reaction product to the reaction solution and separating the formed precipitate. Examples of the solvent in which the reaction product is insolubilized include water, ether, isopropyl ether, and chloroform. Another method is to add an alkali to adjust the pH and cause precipitation and crystallization, but this is not so preferred as it causes not only industrial problems but also affects quality. According to the above findings of the present inventors, the produced cobaltichlorophyllin complex compound is stable even when heated to a certain extent in an alcohol-acetic acid mixed medium, and the high purity cobaltichlorophyllin complex compound can be directly concentrated under reduced pressure from the reaction solution. It was surprising that it could be recovered. The cobaltichlorophyllin complex compound thus obtained is of high purity and can be provided as it is to the next step. A methanol solution of a cobaltichlorophyllin complex compound and a hydrochloride, nitrate, or free base of thiamine (vitamin B ₁ ) is adjusted to pH 7 to 11, and the raw material thiamine is mixed and reacted in the thiol form. 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 if necessary. The reaction is desirably carried out at room temperature to 50° C. while blocking oxygen, preferably under light-shielding conditions. 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 to perform chromatographic fractionation. At this time, it is not necessary to concentrate the benzyl alcohol. methanol,
Although a single solvent or a mixed solvent such as 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 means of absorption ratios in the visible 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. Another purification method is to dissolve the crude thiamine-cobaltichlorophyllin complex compound in 30-70% aqueous alcohol, adjust the pH to 3-7, preferably 4-6, and perform hydrophobic porous column chromatography fractionation. You can also do that. The larger the unit surface area of the resin is, the more suitable the porous polymer is, but it is usually 100 m ² /g or more, preferably 400 m ² /g or more. The pore diameter of the porous synthetic resin is usually preferably 5.0 Å or more, and the larger the average pore diameter, the better the result. Examples of these porous polymers include styrene-divinylbenzene copolymer [Amberlite XAD-2 (ROHM & Co., Ltd.)]
(manufactured by Haas Corporation) and Diaion HP (manufactured by Mitsubishi Chemical Industries, Ltd.). By passing the purified thiamine cobaltichlorophyllin complex compound fraction through a gel permeation chromatography column, a thiamine cobaltichlorophyllin complex compound of constant quality is obtained. Examples of gel perchromatographic carriers include Sephadex.
Dextran gels such as LH-20 or synthetic gelling agents such as Toyopearl HW-40 are preferred. Suitable developing solvents include methanol, ethanol, acetone, and the like, with methanol being preferred. 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 fine black-green glossy powder is obtained by concentration to dryness, spray drying, or the like. This product has extremely high purity and can be provided as a pharmaceutical raw material as is. 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 physical properties. (1) The thiamine cobaltichlorophyllin complex compound, which is the active ingredient 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 approximately 100 mg of this thiamine cobaltichlorophyllin complex compound, and dissolve in methanol to make exactly 50 ml. Take 1 ml of the liquid and add methanol to make exactly 50 ml. Furthermore, take 2 ml of this solution and add 3 ml of methanol to use it as a sample solution. When the visible absorption spectrum is measured using the Japanese Pharmacopoeia General Test Method "Absorbance Measurement Method" using methanol as a reference liquid for the sample solution, the maximum absorption is at wavelengths 428 to 432 nm and 640 to 667 nm, and the absorption strength is 428 to 432 nm. ＞640～
The order is 667nm. (3) Analysis by infrared absorption spectra Infrared absorption spectra (KBr tablet method) were conducted for the active ingredients of the present invention, such as thiamin cobaltichlorophyllin complex compound, cobaltichlorophyllin (cobaltichlorophyllin e ₆ ), and chlorophyllin (chlorin e ₆ ). It was measured. Its infrared spectra were as shown in Figures 1, 2 and 3, respectively. The absorption of 3400 cm ^-1 derived from NH and water of the raw material chlorophyllin (Fig. 3) shifts to 3430 cm -1 in the cobaltichlorophyllin complex compound, and at the same time, the absorption of 3430 cm ^-1 derived from NH and water of the cobaltichlorophyllin complex compound (Fig. 2) and In the thiamin-cobaltichlorophyllin complex compound, it is derived from the cobaltichlorophyllin complex.
Absorption of 1080cm ^-1 (νCo-N) occurs (first
figure). From this, it is inferred that the cobalt metal of thiamine cobaltichlorophyllin and cobaltichlorophyllin complex compound is complex bonded to the four N atoms of the pyrrole nucleus of the raw material chlorophyllin. In addition, 1625 cm ^-1 of the complex compound of the present invention
(NH) and 1040 cm ^-1 (C-O) absorption thought to be derived from thiamine (vitamin B ₁ ) (the former is derived from the primary amine of thiamine, and the latter is derived from -C ₂ H ₄ OH of thiamine). , cobaltichlorophyllin and chlorophyllin did not exist. From the above results, it was estimated that cobalt in the complex compound was coordinately bonded to four N atoms of the pyrrole nucleus of chlorophyllin, and that thiamine was bound in this complex compound. (4) Analysis by visible region absorption spectrum The three components of the active ingredient of the present invention, the thiamine-cobaltichlorophyllin complex compound, its precursor, the cobaltichlorophyllin complex compound, and the raw material, chlorophyllin, were analyzed using a methanol solvent and a layer. Visible absorbance measurements were taken from 340 to 700 nm with a length of 10 mm. The absorption spectrum in the visible region 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 active ingredient of the present invention and the cobaltichlorophyllin complex compound, the characteristic 500 to 530n of chlorophyllin is observed.
The m absorption band almost disappears, the red band shifts to a slightly shorter wavelength region, and the Soret's band (Soret's
band) moves to the longer wavelength region, and the ε value decreases. 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 such thiaminecobaltichlorophyllin compounds 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, oral administration was performed 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 experimental generation of ulcers by the restraint cortisone method was published in ``Journal of the Japanese Society of Fire and Extinguishing Diseases,'' No. 62.
(12), No. 1533 (1965). The results are shown in the table below.

【table】

[Table] Next, the clinical test results are shown. Test method Thiamine cobaltichlorophyllin complex compound
A stomach-soluble coated oral preparation containing mg was administered 4 times a day (1 hour before each meal and before bedtime) for 6 weeks at 1 to 2 tablets each time. The test results are shown in the table below.

[Table] As is clear from the above test results, the active ingredient compound of the present invention can be administered by intravenous injection, subcutaneous injection, intramuscular injection, orally, and oral administration and intramuscular injection are particularly preferred. When used in the treatment of adults, the dosage range is preferably 10 to 120 mg, particularly 20 to 40 mg per day. When the active ingredient of the present invention is orally administered, it may be administered in the form of tablets, granules, or powders, and in particular, granules and powders can be made into unit dosage forms in the form of capsules, if necessary. These oral solid preparations contain commonly used excipients, such as silicic anhydride, magnesium aluminate metasilicate, synthetic aluminum silicate, lactose, sugar, corn starch, microcrystalline cellulose, hydroxypropyl starch, or glycine. , binders such as gum arabic, gelatin, tragacanth, hydroxypropylcellulose, or polyvinylpyrrolidone, lubricants such as magnesium stearate, talc or silica, disintegrants such as potato starch, calcium carboxymethyl cellulose or wetting agents such as polyethylene glycol, sorbitan monooleate, It may also contain polyoxyethylene hydrogenated castor oil, sodium lauryl sulfate, etc. The tablets may be coated according to conventional methods. Oral liquid preparations are aqueous or oily emulsion solutions,
It may be made into a syrup or the like, or it may be a dry product which can be redissolved in a suitable vehicle before use. Such liquid preparations contain commonly used additives such as emulsifying aids such as sorbitol syrup, methylcellulose, gelatin, hydroxyethylcellulose, etc., and emulsifying agents such as lecithin, sorbitan monooleate, polyoxyethylene hydrogenated castor oil, and Aqueous vehicles such as fractionated coconut oil, almond oil, peanut oil, preservatives such as methyl p-hydroxybenzoate, p-
Propyl hydroxybenzoate or sorbic acid may be added. Furthermore, these preparations for oral administration may contain preservatives, stabilizers, etc., if necessary. When this compound is used as an injection, an oil solution,
It may be in the form of an emulsion or an aqueous solution, and these solvents may contain commonly used emulsifiers, stabilizers, and the like. These compositions can contain the compound in an amount of 1% or more, preferably 3% to 6%, depending on the administration method. Next, synthesis examples and specific formulation examples of the active ingredient of the present invention will be shown as examples. Synthesis 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 substance 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 with 50 g of Sephadex 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%. Synthesis 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.
Add 30% hydrogen peroxide to the 50ml solution.
Add 0.75 ml and drop the resulting solution to the previously prepared chlorophyllin solution at 50°C while stirring. After further stirring at the same temperature for 1 hour, the mixture was cooled to room temperature while continuing to stir. After separating the insoluble matter from the reaction solution, the solvent was distilled off under reduced pressure, and the resulting dry solid was approx.
Suspend in 100ml of water, filter through a glass filter, and wash by pouring the same amount of water over the glass filter. The residue was then dried to obtain 4.26 g (yield 73.7%) of cobaltichlorophyllin. This cobaltichlorophyllin is dissolved in 300 ml of methanol. Separately, 3.20 g of thiamine hydrochloride was dissolved in 190 ml of methanol, and the pH was further adjusted to 10.5 with a methanol solution containing about 5% sodium hydroxide. This solution is added dropwise to the above methanol solution of cobaltichlorophyllin at 40° C. while stirring. Stirring is continued for an additional 3 hours after the addition. Insoluble materials generated during the reaction were separated, and the liquid was concentrated to dryness under reduced pressure to obtain crude thiamin cobaltichlorophyllin.
5.15g (yield 63.3%) was obtained. Next, add Diaion HP to 40% methanol in advance.
20 60ml is suspended and packed into a column to prepare a hydrophobic porous polymer column. 40% of the crude thiamine cobaltichlorophyllin
After adjusting the pH to 5.0 with a methanol solution containing about 5% sodium hydroxide, the resulting precipitate is removed by filtration.
A column (diameter 2.5 cm, polymer part length 15 cm) filled with this liquid was filled with the hydrophobic porous polymer Diaion HP-20 (manufactured by Mitsubishi Chemical Industries, Ltd.).
50ml/hour. Then 40% of about 300ml
Methanol is passed through at 50 ml/h. Further about
200 ml of pure methanol is passed through the column at 100 ml/hour, and a green eluate fraction (approximately 100 ml) is collected. Methanol was distilled off from the fractionated eluate under reduced pressure, and the dark green color was extracted from thiamine cobaltichlorophyllin.
Obtain 942 mg (yield 18.3%). Next, a chromatography column is prepared by suspending 50 g of Cephadex LH-20 in methanol and filling a column with a diameter of 3 cm and a length of 30 cm. 42 mg of the previously obtained thiamine cobaltichlorophyllin was dissolved in about 10 ml of methanol and placed on this column, and then developed and eluted with methanol. Approximately 100 ml of thiamine cobaltichlorophyllin fraction was collected by thin layer chromatography and visible absorption spectrum absorption ratio of the eluate, and methanol was distilled off under reduced pressure to obtain 460 mg of dark green thiamine cobaltichlorophyllin powder ( Yield: 48.8%). When this thiamine cobaltichlorophyllin powder was dissolved in methanol and used as a sample solution to perform visible absorption spectrum measurement, maximum absorption was found at wavelengths of 430 nm and 646 nm. The total yield from chlorophyllin (chlorin e ₆ ) is 5.65%. Example 1 Hard capsule for oral use 3.7 g of thiamincobaltichlorophyllin and polyoxyethylene castor oil (Nitsukor)
Dissolve 7.5 g of HCO60) in acetone and then mix with 25 g of silicic anhydride. After evaporating the acetone, further carboxymethyl cellulose calcium 5
g, 5 g of corn starch, 7.5 g of hydroxypropyl cellulose (HPC-L), and 20 g of microcrystalline cellulose were mixed, and 30 ml of water was added, kneaded, and granulated. This is then carried out using a granulator (excpelletter) equipped with a No. 24 mesh (BS) screen.
It was granulated using Fuji Paudal KK (manufactured by). The granules were dried to a moisture content of 5% or less and sifted through a No. 16 mesh (BS) sieve. Next, 190 mg of these particles were filled into one capsule using a capsule filling machine. Example 2 Oral Soft Capsules 14 g of thiamine cobaltichlorophyllin and 130 g of fractionated coconut oil (miglyol 812) are mixed to form a homogeneous solution. Separately, a gelatin solution consisting of 93 g of gelatin, 19 g of glycerin, 10 g of D-sorbitol, 0.4 g of ethyl paraoxybenzoate, 0.2 g of propyl paraoxybenzoate, and 0.4 g of titanium oxide was prepared and used as a capsule coating agent by manual plate punching. Soft capsules containing 180 mg of content were manufactured by the following method. Example 3 Injection 1 g of thiamincobaltichlorophyllin, an appropriate amount of peanut oil and 1 g of benzyl alcohol are mixed, and the total amount is made up to 100 c.c. using peanut oil. Dispense 1 c.c. of this solution into ampoules using aseptic technique and seal.

[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 as the active ingredient of the present invention, cobaltichlorophyllin as the intermediate, and chlorophyllin as the raw material, respectively. , and FIG. 4 are curves showing the visible absorption of the three above.

Claims (1)

[Claims] 1 formula An anti-ulcer agent containing a thiamine cobaltichlorophyllin complex compound represented by: