NO160205B - PROCEDURE FOR THE PREPARATION OF SILIBINE INGREDIENTS. - Google Patents

Description

The present invention relates to a method for the production of silibinin derivatives with the general formula I

in which

n is 0-4, and

Mi means a hydrogen atom or an alkali metal atom.

The milk thistle - Silybum marianum (L.) Gaertn. (Carduus marianus L.) - has been known since ancient times as a healing plant. From the flavolignans occurring in the fruits of this plant, a component, silybin, was isolated by R. Munster, compare Dissertation R. Munster, Miinchen, 1966. The chemical structure of this compound was elucidated by A.Pelter and R.Hansel, cf Tetrahedron Letters, London, Vol. 2:5, pp. 2911-2916 (1968).

It is known that silybin, formerly also called silymarin I

is a valuable liver therapeutic, compare German explanatory document 17 67 666. A technical method for producing silybin (silymarin I) is e.g. described in German specification document 19 23 082.

Already in 1974, H. Wagner, P.Diesel and M. Seitz, Arzneimittelforschung, volume 24(4), pages 466-471, had assumed two positional isomers of silybin, namely silybin and isosilybin. This assumption was specified and experimentally confirmed by

A. Arnone, L. Merlini and A. Zanarotti, Journal Chemical Society Chem. comm., 1979, volume 16, page 696/97.

Consequently, the known silybin consists of two different compounds, namely the compound with the following structural formulas A and B:

From these structural formulas it can be seen that these compounds are positional isomers. Compounds of formula (A) more recently have the INN designation silibinin. This designation is used from now on in the present application for the compound with formula (A).

The therapeutic use of silybin led to difficulties because silybin is practically not soluble in water, so that silybin-containing injection solutions or preparations, in which a certain water solubility is necessary, could not be produced. German. patent 19 6 3 318 does indeed describe silibynderi-vats which have a certain water solubility, but this concerns a very complicated mixture of half-esters of succinic acid. This mixture is so complicated because there are five esterifiable hydroxyl groups in silybin, the silybin also contains the two above-mentioned positional isomers and the succinic acid used for the esterification is a dicarboxylic acid which can form both mono- and diesters. For pharmaceutical purposes, a product consisting of e-t non-overview number of different unresolved compounds is not usable.

The task of the invention therefore lies in providing water-soluble silybin derivatives which are suitable for pharmaceutical purposes, which are precisely characterized as chemical entities.

It has now been found that silibinin derivatives of certain alkane and alkylenedicarboxylic acids fulfill these requirements.

Preferred compounds of formula (I) are those where n means 0 or 1, and Mi an alkali metal atom.

Particularly preferred is silibinin-C-2', 3-dihydrogensuccinate, disodium salt.

The compounds produced according to the invention have the OH groups of the silibinin which are not bound to a benzene nucleus partially or completely esterified, e.g. with oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid or fumaric acid. Preferably, the two non-aromatically bonded OH groups of silibinin are esterified once with one of the aforementioned carboxylic acids.

The method according to the invention for the production of the silibinin derivatives is characterized by dissolving one part by weight of silibinin with formula (A): in 1 to 2 parts by weight of pyridine and, while stirring, reacting with 1 to 3 parts by weight of a dicarboxylic acid anhydride of formula

then ethanol is added until a homogeneous mixture is formed, then slowly mixed with water with vigorous stirring, during which the present esters on the aromatically bound OH groups are hydrolysed as soon as this hydrolysis is finished, dilute with ethyl acetate, wash with ethyl acetate-saturated acidic water and evaporate the ethyl acetate phase, take up the concentrate in ethanol, and either the free carboxylic acid is obtained from the ethanolic solution by precipitation from ethanol/water, or

the ethanolic solution is transferred into the salt of the free carboxylic acid ester with an alcoholic alkali hydroxide solution.

Preferably, the reaction with the dicarboxylic acid anhydride takes place at 40 to 50°C. Advantageously, the pH in the ethyl acetate-saturated acidic washing water is kept at approx. 1.5 to 2.4.

These compounds, especially the compound silibinin-C-2', 3-dihydrogensuccinate, disodium salt surprisingly show a distinct pharmacological effect in the treatment of burn injuries. Moreover, despite the above-described derivative formation, they retain their full pharmacological effect

they knew silibinin as a liver therapeutic. They are particularly suitable for the treatment of liver cirrhosis and toxic metabolic liver damage.

Surprisingly, the compounds according to the invention also prove to be extremely effective in the treatment of mushroom poisoning, especially the very dangerous poisoning with Amanita phalloides. Poisonings with halogen-containing organic solvents such as carbotetrachloride, trichloroethylene, chloroform etc. can also be treated surprisingly well with this. When used preventively, the manufactured compounds prevent the damage described above.

Medicines containing these compounds are preferably used systemically, e.g. in the form of pills, capsules, solutions,

in usual carriers and possibly together with usual excipients. The daily dose for an adult is approx. 50-500 mg depending on the patient's condition and the strength of the disease symptoms.

Experiment with silibinin-C-2',3-dihydrogensuccinate disodium salt (sili-suc-na).

The symptoms that occur with burns are especially caused by poisoning with products with thermal tissue necrosis. The demonstration that self-poisoning processes are responsible for this after severe skin burns has been carried out in many ways. Particularly convincing are cross-transplantations of burned and non-burned skin on healthy or burned recipient animals, whereby it turns out that the non-burned recipients of burned skin perish, while burned recipients of healthy skin suffer no harmful effects, see K.H.Schmidt et al., Recent aspects of self-poisoning after severe burns: Die Verbrennungskrankheit (F.W. Ahnefeld et al., eds.L), Springer, Berlin 1982, pp. 45-52.

Skin burns cause the release or new formation of a number of chemical compounds. Despite the large number, the structure of some of these compounds has been successfully elucidated.

Among other things, it could be shown that the compounds formed by skin burns are similar to such compounds formed by lipid peroxidation. There are also analogies with regard to the toxic effects of these substances. Particularly striking is the formation of toxic-acting saturated and unsaturated aldehydes with different chain lengths as a result of lipid peroxidation (Benedetti et al., Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochim.Biophys.Acta 620, 281-296, 1980) and the thermal tissue damage (K.H. Schmidt et al., Studies on the structure and biological effects of pyrotoxins purified from burned skin, World J. Surg. 3, 361-365, 1979). It is therefore assumed that the burns lead to oxidative damage to cell structures.

Therefore, self-poisoning changes in membrane lipids were investigated as a result of self-poisoning after severe burns. In particular, the change in the fatty acid composition of the membrane lipids was investigated. Furthermore, it was investigated whether the produced silibinin derivatives affected the changes in the fatty acid composition of the membrane lipids.

Changes in fatty acid levels. the composition of membrane lipids after severe burns.

Male Wistar rats with an average weight of 360 g were kept in groups of three with free access to water and dry food. Up until the beginning of the experiment, the room temperature was 22°C,

and after the start of the experiment, the animals were kept at 30°C.

The skin burns were set with a copper stamp of 20 cm<2 >surface with constant pressure and a temperature of 250°C. To rule out thermal damage to deeper-lying organs, the skin was pulled through an air-cooled perforated spatula. With this animal model, it was possible to set very precise burn injuries that gave constant survival rates.

Before the start of the experiment, the animals were anesthetized

50 mg/kg Nembutal. After the burn, 20 ml of Ringer's lactate solution was injected intraperitoneally for shock prophylaxis.

5 experimental groups were formed:

a) Normal group: completely undamaged animals

b) Control group I: only silibinin treatment for 6 days with 75.5 mg sili-suc-na.

c) Control group II: Skin-operated animals.

d) Group with burnt animals: 25%, 250°C, 20 sec., 0.5 at.

e) Experimental group: Animals that had been given 75.5 mg sili-suc-na i.p. through 6 days starting 1 day before

the combustion.

To isolate microsomes, blood was taken from the animals after the end of the experimental period under anesthesia. The liver was then removed, weighed and immediately transferred into ice-cold isolation medium (0.25 m sucrose, 1 mM EDTA 10 mMol tris-HCl, pH 7.2). The liver was cut open and homogenized in the medium. By differential centrifugation, the microsome fraction was pelleted. The microsomes were resuspended and centrifuged again. A suspension was then prepared where 1 ml of suspension corresponded to 1 g of liver weight.

The lipids were determined according to the method of J. Folch (A simple method for the isolation and purification of total lipids from animal tissues, J. biol.Chem. 226, 497-508 (1957)), modification of Bligh and Dyer (A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37, 911-917 (1959).

The extracted microsomal lipids were saponified with caustic soda. The free fatty acids were esterified by the addition of BF3-methanol. After evaporation of the methanol and removal of hydrophilic by-products, the fatty acid esters were determined quantitatively.

In the burned animal groups, no significant change in the fatty acid pattern could be determined. The anesthesia and the small operative intervention therefore do not lead to a change in microsomal lipids. For this reason, the normal group and the control group were combined into a control group for the further comparisons.

A comparison of the unburned and burned animals with regard to their microsomal fatty acid pattern showed dramatic shifts of unsaturated to saturated fatty acids.

Fig. 1 shows the fatty acid distribution in the microsomal liver lipids and changes caused by thermal skin damage. The proportion of palmitic acids (C16) rises after combustion from 25.1 to 34.4% of the total fatty acids. In the case of stearic acid (C18), the proportion of 46.3% in the burned animals is 13.2% above the value obtained with the control animals. In the case of oleic acid (C18:1), a slight but not significant reduction is demonstrable. The proportion of linoleic acid (C18:2) went back to approx. 1/3 of the initial value. In the case of arachidonic acid (C20:4) only 31% of the initial content was found after combustion.

The following table 1 shows the influence of sili-suc-na on the fatty acid proportions of burned and non-burned animals.

It turns out that the treatment with silibinin derivative in the non-burnt control animals does not cause significant changes compared to the untreated animals. In the burned animals, the therapy leads to a complete reversal of the loss of internalized fatty acids.

As a conclusion, one can state: Burns lead to changes in the fatty acid pattern of microsomal lipids. It is believed that this is due to an oxidative damage to the membranes. This is particularly evident in the strong decline in polyunsaturated fatty acids.

The silibinin derivatives used are now able to inhibit oxidative cell damage. It is therefore particularly suitable for breaking through oxidative damage mechanisms after severe burns.

As already described, it is assumed that self-poisoning reactions after severe burns lead in particular to oxidative cell damage. It was therefore investigated which effect a standardized thermal trauma has on the PHA-induced blastogenesis of T-lymphocytes from the spleen and the peripheral blood of rats. It was further investigated how the silibinin derivatives produced according to the invention affect such lymphocytic functional disorders after severe burns.

Effect of a standardized thermal trauma on the PHA-induced blastogenesis of T lymphocytes from the spleen and peripheral blood of rats

As previously described, the dorsal skin of Wistar rats was burned with a copper punch. skin-burned animals served as a control group, which had undergone all operative manipulations without burns. After 2, 4, 7 and 9 days, the incinerated, respectively the control animals were drained for blood and spleen in Eternar-koze.

Heparinized blood was layered for isolation of lymphocytes on Ficoll-Hypaque solution (density 1.077). They were then centrifuged and the lymphocytes obtained were examined for vitality with Trypan blue. For the isolation of splenic lymphocytes, the organs were finely divided, passed through a sieve and freed with lysis solution according to Gay for the included erythrocytes.

Then the cell mixture was incubated in a container in the presence of 5% heat-inactivated fetal calf serum for 30 minutes to reduce the proportion of mononuclear cells in the suspension by adhesion

to the container wall (5%). For cultivation, the cells were introduced into "flat-bottom" microtiter plates. Then 20% fetal calf serum was added. In this way, spontaneous blastogenesis was determined by measuring the incorporation of <3>H-thymidine-(2Ci/mM) into the cells'

DNA.

In preliminary experiments, it was clarified that optimal mitogen stimulation takes place at a PHA concentration (mitogen-phythaemagglutinin) of 5 ug/ml. In these experiments for the optimization of the cellular experimental system, it was further found that the maximum stimulation of new synthesis of DNA takes place after 72 hours. Furthermore, it was found that the optimal concentration of fetal calf serum is 20% to achieve the highest stimulation.

As described above, the spontaneous blastogenesis was determined by measuring the incorporation of "^H-thymidine into the cells' DNA. The cells were harvested 18 hours after the addition of <3>H-thymidine, whereby the zero point for the 18 hours coincides with the time point for the maximum stimulation.

In order to investigate the effect of the silibinin derivatives produced, a group of rats was treated with a silibinin derivative. In addition, 75.5 mg sili-suc-na was injected once per day intraperitoneally. This therapy was carried out from the day of burning until the day of organ removal (maximum up to the 9th day). The assessment of the results obtained with the control animals and the unburned animals and the animals treated with sili-suc-na was made based on the calculation of the stimulation index. This numerical value is the coefficient of the mean value of the stimulated probe and the mean value of the control probe. From the thus obtained stimulation index for each experimental animal, an average stimulation index was calculated per animal group. The results obtained are expressed in this index BT.

Fig. 2 shows the influence of the sili-suc-na used on the blastogenesis of lymphocytes. In the burned animals, the reduced stimulability of the cells with silibinin was clearly increased.

Already on the 2nd day, the animals that had been treated with silibinin showed an approx. 10 times higher reactivity of blood lymphocytes towards PHA. On the 4th post-traumatic day, the value for the stimulation index of blood lymphocytes for treated animals was 8, while the corresponding value for the untreated animals was 1.5.

In the spleen cells, the stimulation indices of burned, untreated animals are all clearly below 1. The administration of silibinin leads to a significant improvement on all days examined, during which a maximum occurs on the 7th post-traumatic day.

Comparison experiments were also carried out which show that sili-suc-na alone in healthy animals does not lead to any significant changes in the stimulability of PHA-induced blastogenesis of T-lymphocytes from the spleen and from the peripheral blood.

Thus, the produced silibinin significantly stimulates the blastogenesis of lymphocytes of burned animals.

It was further found that in the animals that had been treated with silibinin derivatives, the general catabolism was less,

when the animals quickly gained weight again after the thermal trauma.

Mushroom poisoning:

Poisonings with fly agaric are among the strongest available in medicine. Although only 10 to 30% of all mushroom poisonings are caused by green fly agaric, poisoning with this fungus has always had the greatest medical interest due to its danger.

In older publications, the mortality rate was stated at 30 to 50%. Thanks to modern intensive care medicine, according to

a combined study by FLOERSHEIM et al. in 20 5 patients redu-

cert this figure to an average of 22.4%.

The poison of the fly agaric, amanitrin, can be fatal already at a dose of 7 mg for an adult human. This amount of poison is available for approx. 50 g of a fresh specimen.

After a series of promising animal experiments, the active substance sili-suc-na was tried in the therapy of fly poisoning. 2 8 Patients with fly poisoning were additionally treated with sili-suc-na in addition to the usual therapy. Of these 28 patients, only one died, who had ingested large quantities of this poisonous mushroom with suicidal intent. This result shows an enormous therapeutic advance in this area.

Preparation of isosilybin-free silibinin:

A suspension of 500 g of the product according to German specification 19 23 082 column 8, line 14-19 with a silymarin content of approx. 70% at an isomer ratio of silybin/silydianin/silicristin of ~ 3:1:1-, under which the silybins contain approx. 1/3 isosilybin and 2 kg of methanol = 2.5 3 1 are heated while stirring for 15 minutes at the boiling point. From the solution thus obtained, some silibinin may already fall out after this time. Then 0.75 to 1.25 kg = 0.96-1.58 1 methanol is distilled off in a vacuum and the remainder is left to stand for 10-28 days at room temperature. The precipitated silibinin is filtered and washed twice with 50 ml of cold methanol each time. After drying at 40°C in a vacuum, the isolated crude silibinin is further purified as follows: 60 g of crude silibinin are dissolved in 3 1 technical acetic acid while heating, then mixed with 20 g of activated carbon and stirred further for 2

for hours under reflux conditions. It is then filtered clear and the solution is evaporated at 50°C under reduced pressure to approx.

250 ml. The concentrate is stirred for 15 minutes using an ultra-turrax apparatus and mixed while stirring with 25 ml of methanol. The mixture is then allowed to stand overnight at room temperature. Before filtering off, the precipitated silibinin is stirred again 5 times in the same way using an ultra-turrax apparatus. The filtered precipitate is washed twice with 50 ml of vinegar and dried in a vacuum oven overnight at 40°C. The product is then painted and dried for 4-8 hours under the same conditions.

Example 1

Preparation of silibinin-C-2', 3-dihydrogensuccinate

Dissolve 50 g of silibinin at 45°C in 70 ml of pyridine, add 50 g of succinic anhydride, stir the mixture for approx. 8 hours at 45°C, add 30 ml of ethanol and stir until a homogeneous mixture is formed. 60 ml of water is then added with vigorous stirring over the course of 30 minutes to saponify the phenyl esters. After approx. After 1 hour of stirring at 30°C, the phenyl esters are quantitatively hydrolysed. The completeness of the hydrolysis is checked using HPLC. The hydrolysis is stopped by quickly adding 1.7 1 acetic acid ethyl ester to the reaction mixture thus obtained.

To separate excess succinic acid and pyridine, the reaction solution, which is diluted with ethyl acetate, is extracted twice with 5 1 of water which is saturated with ethyl acetate and has a pH of 1.85 (adjusted with dilute aqueous hydrochloric acid) in a countercurrent flow. Underneath, the wash water which is saturated with acetic acid ethyl ester and acidified in circulation is pumped towards the diluted reaction solution and then, by adding diluted hydrochloric acid, the pH is kept at 1.75 so that this pH value remains constant after the acetic acid ethyl ester passage.

The acetic acid ethyl ester phase is then extracted to wash out excess hydrochloric acid twice in a countercurrent flow with 3.4 1 of water each time which is saturated with acetic acid ethyl ester. As soon as the pH value of the wash water is greater than 4.5, the organic phase is quantitatively separated, evaporated at 40-50°C in a vacuum to 1/12 of the initial volume (approx. 0.2 1) and diluted with 125 ml of ethanol.

The title compound is obtained by precipitation from ethanol/water

and drying at 50°C in vacuum for 15 hours.

To prepare an analytical sample, the title compound is precipitated three times from ethanol/water and then dried for 15 hours at 50°C in a vacuum.

In the FD mass spectrum, the molecular peak occurs at the expected molar mass of 682.

The IR spectrum shows two overlapping bands in the region of the C0 valence frequency, below which one, which is also the case with silibinin, must be assigned to the carbonyl function of a pyrone ring at the wave number 1635 cm^.

The second band lies at 1730 cm and originates from the two ester carbonyl functions.

The H-NMR spectrum confirms that a double esterification has taken place. Thus, the ratio measured by integration of aromatic protons to methyl protons in succinic acid ester is 8:8 (ppm range 5.9-7.1). The ratio of these methyl protons (ppm 2.6) to the methoxy group's methyl protons (ppm 3.8) is 8:3 and is thus in agreement.

The chemical shifts in ^ C studies also show that the esterification has taken place on the two alcoholic OH groups,

- for the chemical shifts change strongly at C-, -, and the adjacent carbon atoms C2.2-<"14 as well as <^ C2-C4"

Elemental analysis for C33<H>3o°i6 (682.60)

Example 2

Preparation of silibinin-C-2',3-dihydrogensuccinate, disodium salt

To the ethanol solution obtained according to example 1, while stirring and cooling from the outside at -5 to 9°C, a quantity measured on the basis of a determination of the solids content in this solution of 6% ethanolic caustic soda is used, the suspension is stirred for another hour at room temperature, filter off the precipitated beige solid, slurry it twice every 5 to 10 minutes using a turrax in 150 ml of ethanol and filter off again. To remove the rest of the acetic acid ethyl ester, the product is then suspended for 14 hours at room temperature in 2 80 ml of ethanol, filtered off again, washed with 70 ml of ethanol and dried for 15 hours at 40-45°C in a vacuum oven. The pre-dried product is then ground, sieved to a grain size of less than 0.2 mm and dried for a further 48 hours at 40-45°C in a vacuum. 52 g of the title compound are thus obtained (69% yield).

The title compound has no sharp melting point. It starts to sinter at approx. 80°C and melts under blister formation at approx. 100°C.

The UV spectrum in methanol shows: A ms. x..= 288 nm, e = 1.73 10 4.

The molecular weight of the title compound is 726.56. The compound is a light beige, microcrystalline powder without a specific odor and with a salty taste. It is easily soluble in water, sparingly soluble in ethanol and acetone, practically insoluble in ether and chloroform.

Claims (3)

1. Procedure for the production of silibinin derivatives with the general formula I in which n is 0-4, and Mi means a hydrogen atom or an alkali metal atom, characterized by dissolving 1 part by weight of silibinin with formula (A): in 2 parts by weight of pyridine and react with 1-3 parts by weight of a dicarboxylic acid anhydride with formula: with stirring, then add ethanol until a homogeneous mixture has formed, then mix with water slowly with vigorous stirring, during which the present esters on the aromatically bound OH groups are hydrolysed, as soon as this hydrolysis is finished, dilute with ethyl acetate, wash with ethyl acetate-saturated acid water and evaporates the ethyl acetate phase, absorbs the concentrate in ethanol, and either the free carboxylic acid is obtained from the ethanolic solution by reprecipitation from ethanol/water, or the ethanolic solution is transferred into the salt of the free carboxylic acid ester with an alcoholic alkali hydroxide solution. 2. Method according to claim 1, characterized by that the reaction with the dicarboxylic acid anhydride is carried out at approx. 40-50°C. 3. Method according to claims 1 and 2, characterized by that the ethyl acetate-saturated acidic washing water is kept at a pH of approx. 1.5 - 2.4.