KR870001020B1 - Process for preparing silibinin derivatives - Google Patents

Abstract

Silibinin derivs. of formula (I) are prepd. by reacting II with III. In the formula n and m are 2; Alk, Alk1 and Alk2 are C1-4 alkylene or C2-4 alkenylen; M1 and M2 are H or alkali metal atom. (I) are useful for treatment of burns, liver cirrhosis, toxic-metabolic lower disease, and poisoning by fungi(parti cularly Amanita phalloides) or ovganic solvents, compared with silibinin itself. (I) have better solubility in water.

Description

Method of Preparation Silybinin Derivative

FIG. 1 shows the distribution of fatty acids on lipid change between micro-mallets due to thermal skin damage.

Figure 2 shows the effect of disodium of silybinin-C-2 ', 3-dihydrogensuccinate on embryonic development of lymphocytes.

nster는 실리빈이라는 성분을 분리한바 있다(Dissertation R. M

nster, M

nchen, 1966 참조). 이 화합물의 화학구조는 A. Pelter와 R. M

nsen에 의해 밝혀졌다(Tetrahedron Letters, London, 25, 2911-2916/1968 참조). 또한 실리마린(Ⅰ)으로 불리우던 실리빈은 간장 치료 물질로 유효하다는 것이 밝혀졌다(독일특허출원 제1767666호 참조). 실리빈의 제조방법이 독일특허출원 제1923082에서 공지되었다.The present invention relates to a novel silybinin derivative, a preparation method thereof and a pharmaceutical composition thereof. Lady's thistle (Silybum marianum) (L.) Gaertn. (Carduus marianus L.) is a medicinal plant that has been known for a long time. From Flaborgignan found in fruit of this plant R. M

nster has isolated a component called silybin (Dissertation R. M

nster, M

nchen, 1966). The chemical structure of this compound is A. Pelter and R. M

Revised by nsen (see Tetrahedron Letters, London, 25, 2911-2916 / 1968). Silybin, also called silymarin (I), has been found to be effective as a hepatic therapeutic substance (see German Patent Application No. 1767666). A method for the preparation of silibine is known from German patent application No. 1923082.

Around 1974, H. Wogner, P. Diesel and M. Seitz (Arzneimittelforschung, 24 (4), (466-471)) assumed two positional isomers for silybin, namely, silybin and isosilbin. This assumption was traced and experimentally demonstrated by A. Armone, L. Merlin and A. Zanarotti (J. Soc. Chem. Chem. Comm., 16, 496-697 / 1079). Accordingly, the known silicides consist of two different compounds, ie, compounds of the formulas (A) and (B).

From the above structural formulas, it can be proved that these compounds are positional isomers. The compound of formula (A) was recently known as 'silbinin' in the INN name. In the present invention, this name is used for the compound of formula (A).

Therapeutic use of silybin has the problem that the silicide-containing injectables or preparations cannot be produced if the specific solubility is required because the silin is insoluble in water.

German patent application 1963318 describes silinic derivatives having certain water solubility but they are very complex mixtures of semiesters of succinic acid. In this mixture, five esterifiable hydroxyl groups are present in the silybins, the silybins also have two such positional isomers and the succinic acid used in the esterification is capable of producing not only one monoester but also a diester. Because it is very complicated.

For pharmaceutical purposes, it is not possible to use preparations made of unexpectedly many different and unclear compounds.

Therefore, it is an object of the present invention to provide a water soluble silvin derivative which is also suitable for pharmaceutical purposes and is clearly characterized as an individual compound.

The inventors have found that the silicide derivatives of certain alkanes and alkylenedicarboxylic acids meet this requirement.

Thus, according to the present invention there is provided a silybinin derivative of the general formula (I):

Where n and m are each 2 and Alk ₁ and Alk ₂ are the same as each other and have an alkylene radical having 4 or less carbon atoms or an alkenylene radical having 2 to 4 carbon atoms and M ₁ and M ₂ are independently hydrogen or alkali Metal atom)

Preferred compounds of formula (I) are those in which Alk ₁ and Alk ₂ are each methylene radicals and M ₁ and M ₂ are the same alkali metal atoms.

Particularly preferred is the disodium salt of silybinin-C-2 ', 3-dihydrogen succinate.

In the case of the compounds according to the invention, the hydroxyl groups which are not bound to the benzene nucleus of the silybinin are partially or fully esterified with, for example, oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid or fumaric acid. It is preferred that the two non-aromatically bound hydroxyl groups of the silybinin are simply esterified by one of the carboxylic acids described above.

The present invention also dissolves about 1 part by weight of the silybinin of the general formula (A) in 1 to 2 parts by weight of pyridine and dicarboxylic acid anhydride (General Formula: O = C (Alk) _n -C = O during stirring. , Alk is _one of the Alk ₁ and Alk ₁ radicals above), and ethanol is added until the homogeneous mixture is obtained, and water is continuously mixed with gentle stirring. Upon completion, the reaction mixture was diluted with ethyl acetate and washed with acidic water saturated with ethyl acetate, the ethyl acetate phase was evaporated and the residue was taken up in ethanol to give the alcoholic solution of alkali metal hydroxide in the glass non- Provided are methods for the preparation of novel compounds of general formula (I) which are converted to salts of esterified carboxylic acid residues. The reaction with the dicarboxylic acid anhydride is preferably carried out at 40 to 50 ° C. It is advantageous for the pH of ethyl acetate-saturated acidic water to be maintained at 1.5 to 2.4.

These compounds, in particular the somewhatdadium salts of silybinin-C-2 ', 3-dihydrogen succinate, surprisingly show excellent pharmaceutical action in the treatment of burns. In addition, despite these changes, they retain the full pharmaceutical efficacy of the known silicides for the treatment of livers. They are especially suitable for the treatment of liver cirrhosis and toxic-metabolic damage.

Surprisingly the compounds according to the invention are also very dangerous infections by fungal infections, especially fungi known as green death cap or deadly ageric (Amanita phallo-ides). Proved to be particularly effective in treatment. Poisoning with halogenated organic solvents such as carbon tetrachloride, trichloroethylene, chloroform and the like can also be treated very well thereby. In case of disease prophylactic use the compounds according to the invention prevent the above-mentioned damage.

Therefore, the present invention also provides a weak composition containing at least one of the novel compounds together with a pharmaceutically acceptable solid or liquid diluent or carrier. They are generally used for systemic phases in the form of pills, capsules, solutions and the like, usually in the usual carriers, with the usual medium as possible.

The daily dose of an adult is about 50 to 500 mg depending on the condition of the patient and the extent of the disease symptoms.

Experiment of Disodium (Sili-suc-na) of Silybinin-C-2,3-dihydrogensuccinate

Symptoms of burns are caused, in particular, by poisoning by the products of thermal tissue necrosis. The process of self-intoxication after severe skin burns has been demonstrated in many ways. In particular, cross-transplantation of burns and non-burned skin to healthy and burned animals has been shown to kill non-burn receptors on burn skin while burn receptors on burns do not exhibit any detrimental action. (See K.H. Schmidt, New aspects of autointoxication after severe burnings: The burning disease; F.W. Ahnefeld et al., Eds.pub.Springer Verlag, Berlin, pp. 42-52).

In the case of skin burns, free or new production of many chemical components occurs. Despite its many structures, it is possible for the structure of some of these compounds to be identified. Among others, the compounds resulting in skin burns have similarities to those occurring in the case of lipid peroxidation. There is also similarity in the toxic effects of these substances.

Particularly impressive are Benedetti et al., Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochem.Biochem.Biophys. KH Schmidt et al., Studies on the structure and biological effects of pyrotoxins purified from burned skin, World J. Surg., 3,361-365 / 1979) .Saturated and unsaturated aldehydes act as toxic to varying chain length. Is the generation of. Therefore, it is assumed that the burn causes oxidative damage of the cell structure.

Therefore, autooxidative changes in membrane lipids have been investigated as a result of autotoxicity after severe burns. In particular, changes in the fatty acid composition of membrane lipids have been investigated. It was also tested to what extent the amount of the silybinin derivative of the present invention would affect the change in fatty acid composition.

Changes in Fatty Acid Composition of Membrane Lipids after Severe Burns

Female wistar rats weighing an average of 360 grams are divided into three groups with free contact with water and dry feed. Until the start of the experiment, room temperature is 22 ° C. and after the experiment is maintained at 30 ° C.

The skin burns using a copper step of 20 cm 2 at a constant pressure and 250 ° C. temperature. The skin is exposed to an air-cooled hollow spatula to prevent thermal damage to deep organs. Highly accurate burn trauma can be made with animal models that provide a constant survival rate.

Prior to the start of the experiment, the animals are anesthetized with 50 mg / kg of nembutal. After the burn, 20 ml of Ringer lactate is injected orally to prevent shock.

Five experimental groups are used:

a) normal group: completely intact animals

b) Control I: 65.5 days of treatment with silybinin with disodium salt of 75.5 mg of silybinin-C-2 ', 3-dihydrogen succinate.

c) control group II: gastric manipulated animals

d) Burn animal: 25%, 250 ℃, 20 sec, 0.5 atm

e) Experimental group: Animals orally administered 75.5 mg of the disodium salt of silybinin-C-2 ', 3-dihydrogensuccinate for 6 days starting from day 1 before burn.

After the end of the experiment, the animals are bleeded under anesthesia for microsomal isolation. Subsequently, livers were removed and weighed immediately and transferred to an ice cold separation medium (0.25M Sacrose, 1mMEDTA, 10mMol Tris. Hydrochloric acid, pH7.2). Liver is ground and homogenized in the medium. Microsome fractions are compressed and crystallized by differential centrifugation. Centrifuge again after resuspending the microsomes. Subsequently, 1 ml of suspension corresponding to 1 g of liver tissue is prepared.

The lipids were analyzed by J. Folch's method (the Simple method for the isolation and purification of total lipids from animal tissues, J. biol. Chem., 226, 497-508 / 1957) and by Bligh and Dyer (A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37, 911-917 / 1959).

The extracted microsomal lipids are saponified with aqueous sodium hydroxide solution. Free fatty acids are esterified by adding BF ₃ -methanol. The fatty acid esters are quantitatively determined after evaporation of methanol and removal of hydrophilic by-products.

In the case of non-burn groups, no noticeable change in fatty acids was identified. Thus, anesthesia and a second operational intervention do not result in a change of microsomal lipids. For this reason, the normal group and the control group are combined into one control group for another comparison.

Comparison of unburned and burned animals with respect to their microsomal fatty acids indicates that a series of unsaturated fatty acids have been changed to saturated fatty acids. The accompanying FIG. 1 shows the distribution of fatty acids in microsomal lipids and changes due to thermal skin damage. The proportion of palmitic acid (C 16) increases from 25.1 to 34.4% of total fatty acids after burns.

In the case of stearic acid (C 18), in the case of burn animals, the proportion is 46.3%, which is 13.2% higher than that obtained with the control animal. In the case of oleic acid (C 18: 1), a slight decrease was detected. Linoleic acid (C 18: 2) was reduced to about one third of the initial value after burn. Finally, in the case of arachidonic acid (C 20: 4), only 31% of the initial content after burn was detected.

The table below shows the effect of disodium salt of silybinin-C-2 ', 3-dihydrogensuccinate on fatty acid ratios in burned and non-burned animals.

Treatment with the silibinine derivatives according to the present invention indicates that in the case of unburned control animals no significant changes were made as compared to untreated animals. In the case of burn animals, the treatment results in the complete elimination of unsaturated fat loss.

Briefly, it can be explained as follows. The fatty acid pattern of the microsomal lipids is changed by the burn. This is assumed to be due to oxidative damage of the membrane. This is especially demonstrated by the marked reduction in polyunsaturated fatty acids. Therefore, the silibinine derivative according to the present invention can suppress oxidative cell damage. Therefore, they are particularly effective in preventing oxidative damage miracles after severe burns.

As already mentioned, the autotoxic reaction after severe burns in particular leads to oxidative cell damage. Therefore, the inventors studied how standard thermal trauma affects PHA-induced T-lymphocyte embryonic development from the spleen and peripheral blood of rats. The inventors also studied how the silivinin derivatives according to the present invention affect such lymphocyte dysfunction after severe burns.

Effect of Standardized Thermal Trauma on the PHA-Induced Embryogenesis of T-lymphocytes from Spleen and Peripheral Blood of Rats (Let)

The back skin of a Wister rat is burned with copper staff according to the method described above. As a control group, a group of stomach burns in which all manipulation exaggerations were performed without a burn is used. After 2,4,7,9 days blood and spleen are removed to the burn group and control group under ether anesthesia.

For isolation of lymphocytes, heparinized blood is coated on Ficoll Hypaque solution (concentration 1.077). Subsequently, this is centrifuged and the resulting lymphocytes examined with trypan blue for their vitality. For isolation of splenic lymphocytes, the trachea is pulverized, passed through a sieve, and red blood cells are removed with a lysis solution of Gay. Subsequently, in order to reduce (5%) the proportion of mono-nucleocytes in the suspension by sticking at the container wall, the cell mixture was placed in the container in the presence of 5% heat-inactivated fetal calf serum for 30 minutes. Preserve For incubation, the cells are placed on a flat bottom microtiter plate. 20% fetal calf serum is added. In this way, spontaneous embryonic development is examined by measuring the binding of 3H-thymidine- (2Ci / mM) to the DNA of the cell.

In this experiment, it was evident that optimal mitogen stimulation occurred in the case of 5 μg / ml PHA (mitogen agglutinin). In the case of these experiments for the optimization of cell test systems, it was also confirmed that after 72 hours a new stimulation of DNA synthesis occurred. In addition, the optimal concentration of fetal calf serum to obtain its best stimulus was also found to be 20%.

As mentioned above, spontaneous embryonic development was measured by measurement of binding of 3H-thymidine to DNA of cells. The cells were harvested 18 hours after adding 3H-thymidine so that the zero point for 18 hours coincides with the time point of maximum stimulation.

For the study investigation of the action of the silivinin derivatives according to the present invention, the Lett group is treated with the silivinin derivative. For this purpose, 75.5 mg of the disodium salt of silybinin-C-2 ', 3-dihydrogen succinate is orally administered once daily. This treatment is performed from the day of the burn to the day the organ is removed (up to 9 days).

Stimulus indices are calculated to evaluate the results obtained in the control group and the results obtained with disodium salt treated group of the silybinin-C-2 'and 3-dihydrogen succinate. This figure represents the quotient of the mean of the control sample and that of the stimulated sample. Therefore, the average stimulation index for each animal group is calculated from the stimulation index obtained in each experimental group. The calculated result is represented by an index.

The accompanying Figure 2 shows the effect of the disodium salt of sillyvinyl-C-2 ', 3-dihydrogen succinate used in the embryonic development of lymphocytes. In the case of burn animals, the reduced stimulatory capacity of the cells was significantly increased by the silybinin derivative.

Already on the second day, in the case of the disodium salt treatment group of silybinin-C-2,3-dihydrogen succinate, about 10 times higher reactivity of blood lymphocytes to plant coagulants was found. On the fourth day of the burn, the stimulation index value was 8 for the blood) lymphocytes for the treated group and 1.5 for the untreated group.

In the case of splenocytes, the stimulation index of the untreated group of images was all significantly less than one. Significant improvements developed on all irradiation days with the administration of the silibinine derivative, with its maximum found on day 7.

Comparative experiments are also conducted, in healthy animals, with disodium salt of silybinin-C-2 ', 3-dihydrogen succinate alone in the stimulatory capacity of T-lymphocytes to stimulate plant agglutinogenesis in the spleen and peripheral blood. It was shown that no significant change was caused.

Thus, the silivinin derivatives according to the present invention significantly promote the embryonic development of lymphocytes in burned animals.

In addition, in the case of animals treated with the silibinin derivative of the present invention, the general catabolism is lowered because the animal gains weight quickly again after a burn.

Fungal infections

The most serious in medicine is due to Deadly Azeri (Amanita phalloides). Although only 10-30% of all fungal infections are caused by Amanita phalloides, the infection of the fungus is always of great medical interest because of its risk. In older documents, the lethality is described as 30-50%.

Thanks to modern gradual medicine, according to a group study of 205 patients by Floersheim et al., This value was reduced to an average of 22.4%.

Amanitin, a toxic from Amanitapalloides, can be fatal to adults, even at 7 mg, which is present in about 50 g of fresh fungi.

After a series of animal experiments, the disodium salt of the active substance silibinin-C-2 ', 3-dihydrogensuccinate was used for the treatment of infection by aminitapalloides.

Aminitapalloides infected patient 28 was treated with disodium salt of silybinin-C-2 ', 3-dihydrogen succinate in addition to conventional treatment. Of these 28 patients, only one died, who had been severely infected with a large amount of fungi in suicide attempts. This result represents an enormous therapeutic advance in the field.

Preparation of Silybinin Free of Isosilbinin

500 g of product according to German patent application No. 1923082 (see column 8, lines 14-19) in 2 kg of methanol (about 2.53 L) and about 70% content of silytin and about 3: 1: 1 silybin / silidiinine / Suspension of the silicaxritine (the silicin containing 1/3 isosilbin) is heated and boiled for 15 minutes while stirring. Subsequently some silicebinin may already precipitate in the resulting solution. Successively, 0.75 to 1.25 kg (about 0.96-1.581) of methanol are removed under vacuum and the residue is left at ambient temperature for 10 to 28 days. Precipitated silybinin is filtered off and washed twice with 50 ml of cold methanol. After drying in vacuo at 40 [deg.] C., the crude crude silibinine is purified again as follows.

60 g of crude silybinin is dissolved in industrial grade ethyl acetate 31 while heating. Subsequently, 20 g of activated charcoal is added thereto and the mixture is heated under reflux with stirring for another 2 hours. Then it is purified by filtration and the solution is evaporated to 250 ml at 50 ° C. under reduced pressure.

The concentrate is stirred for 15 minutes using an Ultra-Turax instrument and mixed with 25 ml of methanol while stirring. Subsequently, the mixture is allowed to stand overnight at ambient temperature. Prior to filtration of the precipitated silybin by suction, agitation is again performed with a UItra Turrax instrument for 5 minutes.

The suction-filtered precipitate is washed twice with 50 ml of ethyl acetate and dried overnight at 40 ° C. in a vacuum drying cabinet. The resulting product was triturated and dried again under the same conditions for 48 hours.

The following examples are intended to illustrate the invention.

Example 1

Preparation of Silybinin-C-2 ', 3-Dihydrogen Succinate

50 g of silybinin is dissolved in 70 ml pyridine at 45 ° C. and anhydride is added thereto, and the mixture is stirred at 45 ° C. for 8 hours, 30 ml of ethanol is added thereto and the mixture is stirred again to obtain a homogeneous mixture. . Subsequently, 60 ml of water is added within 30 minutes with vigorous stirring to saponify the phenyl ester. After stirring at 30 ° C. for about 1 hour, the phenyl ester is quantitatively hydrolyzed. Completion of the hydrolysis is examined by HPLC. Hydrolysis is stopped by rapidly adding 1.71 ethyl acetate to the reaction mixture.

The reaction solution diluted with ethyl acetate is extracted twice with backing 5 L of water for separation of excess succinic acid and pyridine, saturated with ethyl acetate, and a pH of 1.85 (adjusted with dilute aqueous hydrochloric acid). Ethyl acetate-saturated, acidified wash water is pumped into the reaction solution diluted with a cycle counter and diluted hydrochloric acid is added to maintain the pH at 1.85 until this pH value is constant after passing through ethyl acetate.

Subsequently, for excess hydrochloric acid wash, the ethyl acetate phase is extracted twice with 3.4 L of water countercurrent and saturated with ethyl acetate. As soon as the pH value of the wash water is above 4.5, the organic phase is quantitatively separated and evaporated in vacuo at 40-50 ° C. to 1/12 of the initial volume (about 0.2 L) and diluted in 125 ml ethanol.

The title compound is obtained by reprecipitation with ethanol / water and dried at 50 ° C. in vacuo for 15 hours.

For the preparation of analytical specimens, the title compound is reprecipitated three times from ethanol / water and subsequently dried under vacuum at 50 ° C. for 15 hours in succession.

The molecular peak in the FD mass spectrum is at the expected molecular weight of 682.

The IR spectrum shows two overlapping bands in the region of the valence frequency of CO, one of which is also the same as in the case of silybinin and appears to be related to the carbonyl action of pyroning at a wavelength of 1635 cm ⁻¹ . The second band is 1730 cm ⁻¹ and originates from two ester carbonyl actions.

The 1 H-NMR spectrum demonstrates that double esterification occurred. Thus, the ratio of aromatic protons to methylene protons of succinic acid residues measured by integration amounts to 8: 8 (ppm range 5.9-7.1), and the ratio of these methylene protons to methyl protons of methoxy radicals (ppm3.8) is Reaches 8: 3 and thus is united by it.

In the case of 13C-irradiation the chemical substitution also indicates that the esterification of the two alcoholic hydroxyl groups takes place, since the chemical substitution changes most strongly at C ₁₁ and at adjacent carbon atoms C ₁₂ -C ₁₄ and C ₂ -C ₄ .

Elemental analysis

C ₃₃ H ₃₀ O ₁₆ (Molecular Weight 682.60)

Calculated Value: C 58.07%: H 4.4%; O 37.50%

Found: 58.05%; 4.57%; 37.31%

Example 2

Disodium Salt Preparation of Silybinin-C-2 ', 3-Dihydrogen Succinate

To the ethanolic solution of Example 1, 6% ethanolic sodium hydroxide solution relative to the solids content of the ethanolic solution was added while stirring and cooling at -5 to 9 ° C. The suspension is stirred again at ambient temperature for 1 hour and the beige precipitated solid is filtered off with suction, suspended twice using Turrax in 150 ml ethanol twice for 5-10 minutes each and filtered again by suction.

To remove residual ethyl acetate, the product is suspended in 280 ml of ethanol continuously for 14 hours at ambient temperature and filtered again by suction, washed with 70 ml of ethanol and dried in a 40-45 ° C. vacuum drying cabinet for 15 minutes. . The dried product is continuously milled, sifted to a particle size of 0.2 mm or less and again dried in vacuo at 40 to 45 ° C. for 48 hours. This yields 52 g (69% of theory) of the title compound.

The title compound does not have an accurate melting point. At about 80 ° C. the compound begins to sinter and melts, forming bubbles at about 100 ° C.

The UV spectrum in methanol is as follows:

λ = _max = 288 nm, ε = 1.73.10 ₄ .

The molecular weight of the title compound is 726.56. This compound is light beige, microcrystalline powder, with no particular smell and salty taste. The compound is easily soluble in water, insufficiently soluble in ethanol and practically insoluble in acetone, diethyl ether and chloroform.

Example of use

Preparation of Lyophilizer for Clear Dose

Disodium salt of silybinin-C-2 ', 3-dihydrogen succinate 75.0 mg

Mannitol 10.0mg

A sufficient amount of 4 total 1.5 ml of distilled water for injection

1.5 ml of the solution is placed in a 5 ml pupil of a sharp ampoule and freeze-dried by a known method. For storage purposes, the ampoule containing the final freeze drying agent is sealed by a known method. In use, the lyophilizer is dissolved in 5 ml of sterile physiological saline solution to obtain a clear solution.

Claims (3)

1 part of the siliebinin of the following general formula (II) is dissolved in 1 to 2 parts of pyridine and reacted with 1 to 3 parts of the dicarboxylic anhydride of the following general formula (III) while stirring and ethanol until a uniform mixture is formed. Hydrolysis of the ester of the hydroxyl group bound to aromatics by successive addition of water, and upon completion of this hydrolysis, the mixture was diluted with methyl acetate, washed with acidified water saturated with ethyl acetate and then ethyl acetate A process for producing a silybinin derivative of the general formula (I) below, wherein the residue obtained by evaporating the phase is taken up in ethanol and converted into a free carboxylic acid residue salt with an alcoholic alkali hydroxide metal solution.

Wherein n and m are each 2, Alk, Alk ₁ and Alk ₂ are the same as each other, an alkylene radical having 4 or less C atoms, or an alkenylene radical having 2 to 4C atoms, and M ₁ and M ₂ is a hydrogen atom or an alkali metal atom irrespective of each other). The method of claim 1 wherein the reaction with the dicarboxylic acid anhydride is carried out at a temperature of about 40-50 ° C. The process of claim 1 or 2, wherein the ethyl acetate-saturated, acidified number is maintained at a pH of about 1.5 to 2.4.

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