JPS63264420A - Antiulcer agent containing thiamine cobaltichlorophylin complex compound - Google Patents

Abstract

NEW MATERIAL:The compound having estimated structural formula of I or II and produced by coordinating 1mol. of thiol-type thiamine to 1mol. of a cobaltichlorophyllin complex compound containing Co forming coordinate bonds to 4 N atoms of the pyrrole nuclei of chlorophyllin. USE:An antiulcer agent. PREPARATION:A thiamine cobaltichlorophyllin complex compound which is an active component of the objective agent can be produced by (1) dissolving chlorophyllin in anhydrous acetic acid, preferably alcoholic (e.g. methanolic) acetic acid, (2) reacting the dissolved chlorophyllin with a bivalent cobalt salt such as cobalt acetate in the presence of oxygen to form a cobaltichlorophyllin complex compound, (3) removing the solvent from the reaction mixture in vacuum, (4) dissolving resultant crystalline compound in methanol and reacting with an alkali methanol solution of thiamine to obtain a crude compound and (5) subjecting the product to liquid-liquid extraction with a benzyl alcohol/water system, removal of by-products, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antiulcer agent containing a novel thiamine cobaltichlorophyllin complex compound in which one molecule of thiol type thiamine is coordinated to one molecule of the cobaltichlorophyllin complex compound.

Conventionally, 2 molecules of thiazole-type thiamine and 2 molecules of thiol-type thiamine are cobaltichlorophyllin complex compound 1
Thiamine chlorophyllin-based complex compounds coordinated with molecules are described in Japanese Patent Publications No. 50-2005 and No. 51-28887, and they can have medical effects in the field of collagen diseases, as well as in steroid therapy, for example. It is said to be a useful drug because it reduces the need for steroids when used in combination with steroids.

However, the thiamine-cobaltichlorophyllin complex compound of the present invention is a substance in which one molecule of the cobaltichlorophyllin complex compound is coordinated with one molecule of thiol-type thiamine, and is a different substance from the above-mentioned known thiamine-cobaltichlorophyllin complex compound. Shows significant medicinal effect in ulcers.

The thiamine cobaltichlorophyllin complex compound of the present invention is constructed by the following steps. In the first step, cobaltichlorophyllin is complexed by reacting a divalent cobalt salt such as cobalt acetate in the presence of oxygen with chlorophyllin in a water-free acetic acid solution, preferably an alcoholic, for example, methanolic acetic acid solution. A complex compound is prepared, and the solvent is removed from this compound under vacuum and reduced pressure to obtain a highly pure crystalline cobaltichlorophyllin complex compound. In the second step, the high-purity cobaltichlorophyllin complex obtained in the first step is dissolved in methanol, and thiamin prepared in an alkaline methanol solution is reacted with the mixture to form a thiamine cobaltichlorophyllin complex. Remove as much methanol as possible. Finally, as a purification step, the crude thiamine cobaltichlorophyllin complex compound obtained as described above is extracted with liquid-liquid extraction using a benzyl alcohol/water system (
The mixture is subjected to silica gel chromatography and gel filtration chromatography (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 and gel filtration chromatography.

The method for producing the thiamine cobaltichlorophyllin complex compound, which is the active ingredient, will be explained in more detail below. The chlorophyllin used as a raw material is derived from plant chloroplasts, for example leaves of higher plants such as mulberry leaves, alfalfa and timothy grass leaves, algae such as chlorella and scenedesmus, or also highly concentrated grains of plant chloroplasts. It can be obtained from certain silkworm dung by solvent extraction, saponification and acidification, and purification operations such as solvent fractional extraction, fractional crystallization, and chromatographic separation. This material may be partially mixed with chlorin p6, which mainly consists of chlorin e6, bar blinf, 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 preferable.

In this case the acetic acid content is between 5 and 80%, preferably between 1O and 309
It is 6. 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. Reaction temperature ranges from room temperature to 50~
The temperature range is up to 60°C, preferably 40 to 50°C. It is important to avoid the presence of this water removal as much as possible in terms of product quality and yield.

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 causes instability and minimizes the effect on the lolin ring, resulting in a highly purified product. 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, the solvent was removed by vacuum concentration, or a solvent in which the reaction product was insolubilized was added to the reaction solution, and the resulting precipitate was separated in a furnace to obtain a highly purified cobaltichlorophyllin complex compound. Examples of the solvent in which the reaction product is insolubilized include water, ether,
Examples include 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 some extent in an alcohol-acetic acid mixed solvent, and the highly purified cobaltichlorophyllin complex compound can be directly recovered from the reaction solution by vacuum concentration. That was unexpected.

The cobaltichlorophyllin complex compound thus obtained is of high purity and can be provided to the next step as it is. Cobaltichlorophyllin complex and thiamine (vitamin B,
) A methanol solution of hydrochloride, nitrate or free base 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 p11 if necessary. The reaction is desirably carried out at a temperature of 50° C. 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.

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. At this time, 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 operation, gel filtration 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 column chromatographic fractionation of the hydrophobic porous polymer. You can also do that. As for the porous polymer, the larger the unit surface area of the resin, the more suitable it is, but it is usually 100 go/g or more, preferably 400 go/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 copolymers [Amberlite XAD-2 (manufactured by Rohm and Haas), Diaion HP (manufactured by Mitsubishi Chemical Corporation)].

The thiamine cobaltichlorophyllin complex compound fraction purified as described above is passed through a gel filtration chromatography column to obtain a thiamine cobaltichlorophyllin complex compound of constant quality. As the gel filtration chromatographic carrier, for example, dextran gel such as Sephadex LH-20 or synthetic gel filtration agent such as Toyopearl HW-40 is desirable.

Suitable developing solvents include methanol, ethanol, acetone, and the like, with methanol being preferred. Confirmation of the thiamine cobaltichlorophyllin complex compound fractionation 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 a pharmaceutical raw material as is.

The thiamine cobaltichlorophyllin complex compound thus obtained has the following estimated structural formula.

(Left below) or C48H55CON s Oto S (molecular weight 971
．． The elemental analysis results as 08) are as follows,
This measured value agrees well with the theoretical value, which assumes that one cobalt atom and one vitamin Bl (thiol-type thiamine) molecule are bound to one molecule of chlorophyllin (chlorin e6).

C (%) H (%) N (%) 0 (%) S (%) Co (%)
Theoretical value 758.895.72 11.541B, 483
．． 30 8.07 Actual nj value: 57.835.33 1
1.7817.073.23 5.99 (hereinafter blank) The thiamine cobaltichlorophyllin complex compound obtained in 22 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) This thiamine cobaltichlorophyllin complex compound about 1
Weigh 00mg accurately, add methanol to dissolve it,
It should be exactly 50m1. Take 1 ml of the liquid and add methanol to make exactly 50 ml. Furthermore, 2 m of this liquid
1 and add 3 ml of methanol to make a sample solution. When the visible absorption spectrum is measured using the Japanese Pharmacopoeia General Test Method "Absorbance Measurement Method" using methanol as the control solution for the sample solution, the wavelength is 428-432n* and 840-667ni.
It has a maximum absorption at 428-432 nm.
> 840 to 887 nm.

3) Analysis by infrared absorption spectrum Thiamine cobaltichlorophyllin compound, cobaltichlorophyllin (cobaltichlorin e6), which is the active ingredient of the present invention
), and chlorophyllin (chlorin ee) were measured for their infrared absorption spectra (KBr tablet method). Its infrared spectra were as shown in Figures 1, 2 and 3, respectively. The absorption of 3400c+n-' derived from NH and water of raw material chlorophyllin (Figure 3) shifts to 3430(to)-1 in the cobaltichlorophyllin complex compound,
At the same time, in the cobaltichlorophyllin complex compound (Fig. 2) and the thiamine cobaltichlorophyllin, an absorption of 1080 cm-1 (νCo -N) resulting from the cobalticomplexation occurs (Fig. 1). From this, it is inferred that the cobalt metal in thiamin cobaltichlorophyllin and cobaltichlorophyllin complex compound has a complex bond with four N atoms of the pyrrole nucleus of the raw material chlorophyllin. In addition, absorption considered to be derived from thiamine (vitamin B1) at 1625 cm-' (NH) and 104104 O' (C-0) of the complex compound of the present invention (the former is derived from the primary amine of thiamine, and the latter is due to the absorption of thiamine). -C2H4OH
) was not present in cobaltichlorophyllin or chlorophyllin.

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 absorption spectrum About the thiamine cobaltichlorophyllin compound which is the active ingredient of the present invention, its precursor cobaltichlorophyllin complex compound, and the king of chlorophyllin which is the raw material,
Visible absorption measurements were performed in the range of 340 to 700 nm using a methanol solvent and a layer length of Lhm. The visible absorption spectrum is shown in Figure 4, and the results are summarized in the table below.

(Left below) Maximum absorption part in visible absorption spectrum In the visible part absorption spectrum of the complex compound of the active ingredient of the present invention and the cobaltichlorophyllin complex compound, the characteristic absorption band of 500 to 530 nm of chlorophyllin almost disappears,
The red band shifts to a slightly shorter wavelength region, and the ray 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 metal cobalt in the complex compound and cobaltichlorophyllin complex compound of the present invention.

The toxicity of such thiaminecobaltichlorophyllin compounds is as follows.

(Left below) 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 Wistar male rat (body weight 200-3
00g) and 10 animals in each group. All drugs to be administered were dissolved in physiological saline. Dosage is 100mg/day
kg body weight, and 1 ml of physiological saline for saline as a control.
1 was administered. In an acute ulcer prevention test using stress method, the drug was continuously administered orally for 7 days before stress loading.

In addition, in a treatment test for chronic ulcers using the restraint cortisone method, the drug was continuously administered orally for 7 days after the restraints were removed. Experimental ulcer generation by stress method is described in Rchem, Pharm, B.
Ull, J Vol. 12, p. 485 (19B4), and the generation of experimental ulcers by the restrained cortiscine method is described in "Journal of the Japanese Society of Gastroenterology," Vol. 82 (12), p. 1533 (19B4).
65). The results are shown in the table below.

stress method Restraint cochcin method Next, we will show the clinical test results.

Test method A stomach-soluble coated oral preparation containing 5 mg of thiamine cobaltichlorophyllin complex compound was administered 4 times a day (1 hour before each meal and before bedtime) for 6 weeks at 1 to 2 tablets. The test results are shown in the table below.

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 for the treatment of adults, the preferred dosage is 10 to 120 mg per day, 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 solid preparations for oral administration include 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 carboxymethylcellulose or wetting agents such as polyethylene glycol, sorbitan monooleate, polyoxyethylene hydrogenated castor oil. ,
It may also contain sodium lauryl sulfate and the like. Tablets may be coated according to conventional methods.

Oral liquid preparations may be aqueous or oily emulsion solutions, syrups, etc., or they 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, methyl cellulose, gelatin 5, hydroxyethyl cellulose, etc., and emulsifying agents such as lecithin sorbitan monooleate, polyoxyethylene hydrogenated castor oil, non-aqueous vehicles such as fractionating Coconut oil, almond oil, peanut oil, preservatives such as p
- Methyl hydroxybenzoate, propyl p-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 for injections, oil solutions, emulsions,
It may be in the form of an aqueous solution, and these solvents may contain commonly used emulsifiers, stabilizers, etc.

These compositions contain 1% or more of the compound depending on the administration method.
Preferably, it can be contained in an amount of 3% to 6%.

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 ee) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, add 0.75 30% hydrogen peroxide solution to a solution of cobalt acetate tetrahydrate lσ, Og in 50 ml of methanol.
m1 is added, and the resulting solution is added dropwise to the previously prepared chlorophyllin solution at 50° C. with stirring.

The mixture was further stirred at 50° C. for 1 hour to complete the reaction.

After eliminating insoluble matter from the reaction mixture, it was concentrated to dryness under reduced pressure, and the residue was washed with water and dried to obtain cobaltichlorophyllin 4.
．． 12 g are obtained (yield 71.4%).

4.0 g of cobaltichlorophyllin are dissolved in 280 ml of methanol. Separately, 3.0 g of thiamine hydrochloride was dissolved in 180 ml of methanol, and a methanol solution of caustic soda was added to pH 0. After adjusting the temperature to O, the resulting solution was added dropwise to cobaltichlorophyllin methanol solution 1 while stirring in a nitrogen stream at 40°C. In addition, 40
Stir at ℃ for 3 hours. After removing insoluble matter from the reaction mixture, it was concentrated under reduced pressure to obtain 6.18 g of crude thiamine cobaltichlorophyllin (yield 109.2%).

5.0 g of crude thiamine cobaltichlorophyllin was dissolved in 50 ml of benzyl alcohol and washed twice with 50 ml of water each time, then this benzyl alcohol solution was loaded into a silica gel column and acetone/methanol (3:2) was added.
After washing with 500 ml to remove impurities, the target fraction is eluted with 400 ml of methanol. The eluate was concentrated under reduced pressure to obtain purified thiamin cobaltichlorophyllin 645m.
g (yield 12.9%).

3 with Sephadex LH-2050g and methanol
(2) A 30-an X column is prepared, a methanol solution of 600 mg of purified thiamincobaltichlorophyllin is loaded thereon, and the column is developed and eluted with methanol and fractionated. The eluate obtained by removing the impurity fraction was concentrated under reduced pressure to obtain 380 mg of thiamine cobaltichlorophyllin (yield: 63.
3%). The total yield from chlorophyllin (chlorin ee) is 6.37%.

Synthesis Example 2 5.0 g of chlorophyllin (chlorin ea) is dissolved in a mixed solvent of 500 ml of methanol and 100 ml of glacial acetic acid. Separately, cobalt acetate tetrahydrate 10. Add 30% hydrogen peroxide to a solution of Og dissolved in 50ml of methanol.
．． 75 ml was added, and the resulting solution was added dropwise 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 with continued stirring. Insoluble matter from the reaction solution? After separating each house, the solvent was distilled off under reduced pressure, and the resulting dry solid was suspended in about 100 ml of water and filtered through a glass filter. Filter and wash by running an equal amount of water over a glass filter. The residue was then dried to give cobaltichlorophyllin 4.26+r (yield 7
3.7%).

This cobaltichlorophyllin is dissolved in 300 ml of methanol. Separately, 3.20K of thiamine hydrochloride was dissolved in 190 ml of methanol, and the solution was further adjusted to a pillo of 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.

The insoluble matter generated during the reaction was separated in a furnace, and the liquid was concentrated to dryness under reduced pressure to obtain crude thiamin cobaltichlorophyllin 5.1
5g (yield 63.3%) was obtained.

Next, add Diaion HP-206 to 40% methanol in advance.
A column of hydrophobic porous polymer is prepared by suspending 0 ml and filling the column.

The crude thiamine cobaltichlorophyllin was dissolved in about 100 ml of 40% methanol, and the pH was adjusted to 5.0 with a methanol solution containing about 5% sodium hydroxide, and the resulting precipitate was removed by filtration.

This furnace solution was poured into a column packed with the above-mentioned hydrophobic porous polymer Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation).
50m in diameter 2.5cm, polymer part length 15cm)
Pass at 1/hour. Approximately 300 ml of 40% methanol are then passed through at 50 ml/h. Approximately 200 more
ml of pure methanol is passed through the column at a rate of 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 1 g of dark green thiamin cobaltichlorophyllin 942+I was obtained.
(yield 18.3%).

Next, a chromatography column is prepared by filling a column with a diameter of 3 (1) and a length of 30 cm with 50 g of Sephadex LH-20 suspended in methanol.

On this column, 42 mg of thiamincobaltichlorophyllin obtained earlier was dissolved in about 10 ml of methanol and loaded.
Then, it is developed and eluted with methanol. Approximately 100 ml of thiamine cobaltichlorophyllin 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 μl of dark green thiamine cobaltichlorophyllin powder (yield 48.8 %)
I got it.

When this thiamin cobaltichlorophyllin powder was dissolved in methanol and a visible absorption spectrum was measured as a sample solution, maximum absorption existed at wavelengths of 430n11 and 646111. The total yield from chlorophyllin (chlorin ee) is 5.65%.

Example 1 Oral hard capsule 3.7 g of thiamincobaltichlorophyllin and tunyl HCO60 [F])7 in polyoxyethylene castor oil
．． Dissolve 5 g in acetone and then mix with 25 g of silicic anhydride. After evaporating the acetone, add 5 g of carboxymethylcellulose calcium and 5 g of corn starch.
s Hydroxypropyl cellulose (RPC-Lo) 7
．． 5g and microcrystalline cell, loin 20g: mix 30
Add ml of water, knead, and granulate. This is N(L2
Granulator with 4-mesh (B, S,) screen (
It was granulated using Eckberetter (manufactured by Fuji Paudal KK). The granules were dried to a moisture content of 5% or less and sieved through a No. 16 mesh (B, S, ) sieve. Next, 190 mg of these particles were filled into one capsule using a capsule filling machine.

Example 2 Soft capsules for daily use 14 g of thiamine cobaltichlorophyllin and 130 f of fractionated coconut oil (Miglyol 812'9) were mixed to form a homogeneous solution. Separately, 93 g gelatin 19 g glycerin 5 D-sorbitol 1 (Ig, ethyl paraoxybenzoate 0.4 g, propyl paraoxybenzoate 0
．． A gelatin solution containing 2 g of titanium oxide and 0.4 g of titanium oxide was prepared, and this gelatin solution was used as a capsule coating agent to produce soft capsules containing 180 mg of content by manual plate punching.

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 cc using peanut oil. Pour 100 portions of this solution into ampoules using aseptic technique and seal.

[Brief explanation of drawings]

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; The figure shows a curve showing the visible absorption of the champion. Patent applicant: Midori Juji Co., Ltd. and 2 people other than Nisshin Seifun Co., Ltd.

Claims (1)

[Claims] An anti-ulcer product containing a thiamine-cobaltichlorophyllin complex compound in which one molecule of thiol-type thiamine is coordinated to one molecule of a cobaltichlorophyllin complex compound in which four N atoms of the pyrrole nucleus of chlorophyllin and cobalt are coordinated. agent.