NL192387C - Pure Silibinine Derivatives, Process for Their Preparation and Pharmaceutical Preparation Containing Such Derivative. - Google Patents

Description

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Pure Silibinine Derivatives, Process for Their Preparation and Pharmaceutical Preparation Containing Such Derivative

The present invention relates to silibinine derivatives represented by the formula 1 of the formula sheet, wherein Alk1 and Al1, which may be the same or different, represent an alkylene group of 1-4 carbon atoms or an alkenylene group of 2-4 carbon atoms, M, and M2, which may be the same or different, represent a hydrogen atom or an alkali metal atom, and n and m, which may be the same or different, are 0 or 1.

It is known (Wagner, et al., Arzneimittel Forschung, 24, 466-471 (1974) and Amone, et al., J. Chem. Soc. 10 Chem. Comm. 16, 696-697 (1979)) that silibinin (Formula 3) one site isomer is of silybin, further comprising isosilybin (formula 4). Silybin is a valuable liver therapeutic. Due to the poor water solubility, silybin is derivatized such that the water solubility increases sharply and the compound becomes suitable for, in particular parenteral, administration.

Derivatives mentioned in the opening paragraph have been described by H.P. Koch et al. In Archiv der Pharmazie 316, 15 pp. 385-394 (1983). That article describes the hydrolysis kinetics of silybin diemisuccinate, as an example of a half ester of silybin with a dicarboxylic acid, at different pH values. As the structural formula for silybin, the formula as formula 1 of the present formula sheet is given, with the proviso that the present groups -O-CO-fAlk, ^), -COOM1 / 2 stand there for a bamstone group or an H-atom (in the latter case, the compound is unsubstituted silybin). Although in that article the structural formula of silibinine, derivatized or not, is shown, this does not mean that the tested compounds are actually the pure compounds. This is evident from the comments at the top of page 386, where it is stated that it is assumed that the compounds tested satisfy the structural formula shown.

However, the compounds investigated by Koch et al. Are mixtures of derivatives of silibinin and isosylibine. This is clear from the fact that the compounds examined by them were manufactured on 12 December 1975 (see batch number 75 1212 / Fy mid-page 393 of that article). At that time it was not yet possible to manufacture pure silibinine derivatives.

It has been found that the compound silibinine and its derivatives, defined above, in pure form provide improved pharmaceutical performance of those compound-containing preparations. A preparation of pure silibinine and a pharmaceutical preparation with isosylibine-free silibinine together with a pharmaceutically acceptable carrier and / or excipient is the subject of Dutch patent application 8503136.

The present invention now provides silibinine derivatives as mentioned in the preamble, characterized in that the silibinine derivatives are free from isosilybin or derivatives thereof and are in pure form.

Preference is given to such a pure silibinine derivative characterized in that Alk, and Ali <2 represent an alkylene group with 2 carbon atoms, M, and M2, which may be the same or different, represent an alkali metal atom, and n and m, which are equal or can be different, be 0 or 1.

Further preferred is such a pure silibinine derivative, characterized in that Alk, and Alkg, M, and M2, and n and m in formula 1 have the same meanings.

Finally, the pure disodium salt of silibinin C-2 ', 3-dihydrogen succinate of the formula 6 is preferred. This compound shows good pharmacological action in the treatment of burns.

The action of the pure silibinine derivatives is better than that of the silibin derivatives which also contain isosilybin derivatives. They are particularly suitable for the treatment of hepatic osrosis and toxic-metabolic liver lesions.

The invention further relates to a process for preparing pure silibinine derivatives according to any one of claims 1 to 4, wherein 1 part by weight of silibinine according to formula 3 is dissolved in pyridine; while stirring is reacted with 1-3 parts by weight of dicaibonic anhydride of formula 2, wherein Alk has the meaning indicated in claim 1 for Alk, or Alks; ethanol is added until a homogeneous mixture has formed; the product obtained is washed with 50 water saturated with ethyl acetate and reacting acid; the washed product which is in the ethyl acetate phase is taken up in ethanol and reacted with an alcoholic alkali metal hydroxide solution until the salt of the free carboxylic acid is characterized in that water is slowly added to the homogeneous mixture with intensive stirring to hydrolyze aromatic ester groups, after completion of the hydrolysis is diluted with ethyl acetate, and before the washed product is taken up in ethanol, the ethyl acetate phase is concentrated.

Preferably, the reaction with the dicarboxylic acid is carried out at a temperature of 40-50 ° C.

Furthermore, it is preferred that the acid-reacting and wash water saturated with ethyl acetate is kept at a pH of 1.5-2.4.

Finally, the invention relates to a pharmaceutical composition, which composition contains at least one compound according to any one of claims 1 to 4, together with a pharmaceutically acceptable carrier or excipient.

In addition to liver diseases, the pure compounds according to the invention have a good effect in the treatment of mushroom poisoning, in particular the very dangerous poisoning by amanita (Amanita phalloides). Furthermore, it was not anticipated that poisoning by halogenated organic solvents such as carbon tetrachloride, trichlorethylene, chloroform, etc. can be treated surprisingly well with the compounds of the invention. In addition to being curative, the compounds of the invention are also prophylactic against the aforementioned disorders.

The preparations according to the invention are usually used systemically, for example as pills, capsules and solutions in conventional carriers and optionally together with conventional auxiliary substances. For adults, the dose is 50-500 mg per day depending on the patient's condition and the severity of the symptoms.

15

Pharmacological studies with the disodium salt of silibinin-C-Z, 3-dihydrogen succinate. Burning symptoms are caused in particular by intoxication by products of thermal tissue necrosis. Evidence that autointoxication after severe skin burns is responsible for this has been provided in several ways. Particularly convincing are the results of 20 cross-grafts of burnt or unburned skin on healthy and burnt host animals, respectively, where it has been shown that the unburnt recipient of burnt skin perishes while the burnt recipient of healthy skin does not experience any harmful effects (see KH Schmidt et al., Neuere Aspekte zur Autointoxikation nach schweren Verbrennungen; Die Verbrennungskrankheid (published by FW Ahnefeld et al., L), Springer, Berlin 1982, pp. 45-52.

In skin fat burning, the formation of a large number of different compounds occurs or is released. Despite their large number, some of them have been cleared up.

For example, it could be shown that skin burns produce similar compounds as lipid peroxidation. There are also analogies between the toxic effects. Particularly striking is the formation of toxic saturated and unsaturated aldehydes of different chain length due to lipid peroxidation (Benedetti et al., Identification of 4-hydroxynoneal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochim. Biophys. Acta 620, 281 296 (1980)) and due to thermal tissue damage (KH Schmidt al., Studies on the structure and biological effects of pyrotoxins purified from burned skin, Worid J. Surg. 3, 361-365 (1979)). It is therefore believed that combustion leads to oxidative damage to cell structures.

Based on the foregoing, with the disodium salt of silibinin C-2 ', 3-dihydrogen succinate of the invention, (Sili-sucna), auto-oxidative changes in membrane lipids due to autointoxication after heavy combustion were investigated. In particular, changes in the fatty acid composition of membrane lipids were examined. The extent to which the silibin derivative influences changes in the fatty acid composition of membrane lipids was also investigated.

40

Changes in the fatty acid composition of membrane lipids after heavy combustion. Males Wistar rats with an average weight of 360g, divided into three groups, were made available water and dry food ad libitum. The ambient temperature was 22 ° C up to the start of the study and 30 ° C after the start.

45 Skin burns were induced with a copper stamp with a surface of 20 cm 2 at a temperature of 250 ° C and under constant pressure. To prevent thermal damage to deeper organs, the skin was pulled over an air-cooled hollow spatula. Proceeding in this way, very accurate burns could be caused without the rats dying.

Before the start of the study, the animals were anesthetized with 50 mg / kg nembutal. After combustion, 20 ml of Ringer's lactate solution were injected intraperitoneally for shock prophylaxis.

Five test groups were composed: a) normal group: completely unharmed animals b) control group I: only silibinine treatment for 6 days with 75.5 mg Sili-suc-na c) control group II: sham-operated animals 55 d) group with burnt animals: 25% , 250 ° C 20 sec, 0.5 at e) test group: animals of which 75.5 mg Sili-suc-na ip for 6 days, starting one day before combustion.

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To isolate the microsomes, the animals were bled under anesthesia at the end of the study. The liver was then removed, weighed and immediately placed in ice-cold isolation medium (0.25 mM sucrose, 1 mM EDTA, 10 mM Tris.HCl, pH 7.2). The liver was thinly sliced and homogenized in the medium. The microsome fractions were pelleted by differential centrifugation. The microsomes were resuspended and centrifuged again. A suspension was then prepared, 1 ml of which corresponded to 1 g of liver tissue.

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)), modified according to Bligh and Dyer (A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol. 37, 10 911-917 (1959)).

The extracted microsome lipids were saponified with sodium hydroxide. The free fatty acids were esterified with BF-1 / methanol. After evaporation of the methanol and removal of hydrophilic by-products, the fatty acid esters were quantitated.

No significant change in the fatty acid samples could be observed in the group of non-fibrous animals. This means that anesthesia and minor surgery do not alter the microsomal lipids.

On this basis, the normal group and the control group were combined into a single control group for further comparison.

Comparison of the unburned animals with the burned animals with respect to their microsomal male fatty acid samples showed that a serious shift from unsaturated to saturated fatty acids had occurred.

For explanation, reference is made to Figure 1. This shows the fatty acid distribution in the microsomal liver lipids and the changes caused by thermal skin damage. The palmitic acid (C16) content increases from 25.1 to 34.4% of all fatty acids together after combustion. For stearic acid (C18), the content in the burnt animals is 46.3% as against 13.2% in the control group. For oleic acid (C18: 1), a slight, insignificant decrease is evident. The linoleic acid (C18: 2) content fell to 1/3 of baseline after combustion. Finally, for arachidonic acid (C20: 4), the content after combustion was only 31% of the starting value.

Table A below shows the influence of Sili-suc-na on the quota of fatty acids in burnt and unburnt animals.

TABLE A

Fatty acid samples of microsomal lipids from rat liver after Silica sucna therapy in burnt and unburnt animals 35 - C16 C18 C18: 1 C18: 2 C20: 4

Unburned 29.8% 37.2% 8.9% 9.6% 16.2% (control group!) ± 6.2 ± 12.3 ± 1.1 ± 3.3 ± 4.9 40 Burned 25 .4% 37.5% 7.8% 11.4% 18.0% ± 6.0 ± 8.6 ± 1.0 ± 5.3 ± 9.1

Table A shows that treatment with a silibinine derivative according to the invention does not cause any substantial changes with respect to the untreated animals in the unburned control animals. In the burned animals, the loss of unsaturated fatty acids is completely eliminated by the therapy.

In summary, it can therefore be stated:

Burns lead to changes in fatty acid samples from microsomal lipids. This is believed to be traceable to oxidative damage to the membranes. This is particularly evident from the strong decrease in the polyunsaturated fatty acids.

Thus, the silibin derivatives of the invention have the ability to inhibit oxidative cell damage. Therefore, they lend themselves particularly to interrupting oxidative damage mechanisms after heavy burns.

As already mentioned, it is believed that autotoxic reactions after heavy burns, in particular, lead to oxidative cell damage. On this basis, it was investigated what the effect of a standardized burn wound is on the PHA-induced blastogenesis of T lymphocytes from the spleen and peripheral blood of rats. Furthermore, it was investigated how the silibinine derivatives according to the invention influence such lymphocytic function disorders after heavy fat burns.

Effect of a standardized thermal wound on the PHA-induced blastogenesis of spleen and peripheral blood T-lymphocytes from rats. As described above, the back skin of Wistar rats was burned with a copper stamp. Sham-burned animals in which all surgical procedures were performed without burning were used as the control group. After 2, 4, 7 and 9 days, blood was drawn from the burnt and control animals under ethemarcose and the spleen dilated.

To isolate the lymphocytes, Ficoil-Hypaque solution (density 1.077) was coated with heparinized blood. Then centrifugation was performed and the resulting lymphocytes were tested for vitality with trypan blue. To isolate the spleen lymphocytes, the organ was reduced, forced through a sieve, and freed from accompanying erythrocytes by Gay lysis dissolution.

Subsequently, the cell mixture was incubated in a container for 30 minutes in the presence of 5% 1g heat-inactivated calf serum to reduce the number of mononudal cells in the suspension by adhesion to the wall of the container (5%). First, the cells were placed in the wells of a flat bottom microtiter plate. 20% pure calf serum was then added. Spontaneous blastogenesis was then determined by measuring 3 H-thymidine (2C i / mM) built into the DNA of the cells.

It had already become clear from previous studies that optimal mitogen stimulation takes place at a PHA concentration (mitogen-phythaemagglutin) of 5 µg / ml. In these tests to optimize the cellular test system, it was further found that the synthesis of new DNA is maximally stimulated after 72 hours. In addition, it was found that the optimal fetal calf serum concentration should be 20% to achieve maximum stimulation.

As described above, spontaneous blastogenesis was determined by measuring 3 H-thymidine incorporated into the DNA of the cells. The cells were harvested 18 hours after addition of 3 H-thymidine with the zero point coinciding with the time of maximum stimulation for those 18 hours.

In order to investigate the action of the silibinine derivatives according to the invention, a group of rats was treated with such a derivative. To this end, 75.5 mg of Silica suc was injected intraperitoneally once a day. This treatment is sustained from the burn day to the day the organs are removed (with the ninth day as the ultimate).

The stimulation index was calculated from the results obtained with the control animals, non-burnt animals and animals treated with Silcuccine. This is the quotient of the mean value from the results with the stimulated rats and the mean value from the results with the control rats. An average stimulation index per animal group was calculated from the stimulation index for each animal. This 35 average stimulation index is indicated by SI.

For explanation, reference is made to Figure 2. This shows the influence of Sili-sucna on the blastogenesis of lymphocytes. In the burned animals, the reduced stimulation of the cells by silibinin was clearly increased.

As early as the second day, the response of the blood 40 lymphocytes to PHA was approximately 10-fold higher in the animals treated with Sili-sucna. On the fourth day after burn, the blood lymphocyte stimulation index for treated animals was 8 vs. 1.5 for untreated animals.

In the spleen cells, the stimulation indices of the burnt, untreated animals are all clearly below 1. Administration of silibinin leads to a significant improvement on all study days, which is maximum on the seventh day after the burn.

In addition, comparative tests were also conducted which showed that Sili-sucna alone does not produce significant changes in the stimulatability in PHA-induced blastogenesis of spleen and peripheral blood T lymphocytes in healthy animals only.

Silibinin thus significantly stimulates the blastogenesis of lymphocytes in burned animals.

It was further found that the overall catabola of the animals treated with the silibin derivatives was 50 less because the weight of the animals increased rapidly after combustion.

Mushroom poisoning

Of all the poisons that the physician has to deal with, it is the worst by the amanita. Although poisoning by the green amanita only affects 10-30% of all mushroom poisons, it is very popular in 55 medical circles because it is so dangerous.

According to older publications, the lethality is 30-50%. Thanks to modern medical science, after a group study by Floersheim et al. In 205 patients, lethality has decreased by an average of 22.4%.

The venom from the amanita, amanitine, can be lethal at a dose of 7 mg for adults. This amount of poison can be obtained from about 50 g of a fresh amanita.

After a number of successful animal experiments, Sili-suc-na has been tested for its resistance to amanita poisoning in humans.

28 Patients with amanita poisoning were treated with Sili-sucna in addition to the usual therapies. Daaivan died only one, but that was because this patient had taken a lot of amanita poison for the purpose of car authanasia. This result represents a great therapeutic advance in this area.

10

Preparation of isosilybin-free silibinine

A suspension of 500 g of product according to DE-AS 1,923,082 column 8, lines 14-19 with a silymarin content of about 70% at an isomer ratio of silybin / silidianine / silicristine of about 3: 1: 1, the silybin being about 1 / 3 contains isosilybin, and 2 kg of methanol (2.53 liters) is boiled under stirring for 15 minutes. A little silibinin can then precipitate from the solution formed. Subsequently, 0.75-1.25 kg (0.96-1.58 liters) of methanol is suctioned off under vacuum and the residue is left to stand at ambient temperature for 10-28 days. The precipitated silibinin is filtered and washed twice with 50 ml of cold methanol each time. After drying at 40 ° C in vacuo, the isolated crude silibinine is further purified as follows: 60g crude silibinine is dissolved in 3 liters of technical ethyl acetate under heating. 20 g of activated charcoal are then added, followed by refluxing for an additional 2 hours. Afterwards, it is clarified by filtration and the solution is concentrated to about 250 ml at 50 ° C under reduced pressure. The concentrate is stirred with an Ultra-Turrax apparatus for 15 minutes while adding 25 ml of methanol. The mixture is then left to stand overnight at ambient temperature. Before filtering the precipitated silybin under suction, stir again with an Ultra-Turrax device for another 5 minutes. The suction precipitate is rinsed twice with 50 ml of ethyl acetate and dried in a vacuum drying cabinet at 40 ° C overnight. The product is then ground and dried under the same conditions for 48 hours.

30 Example 1

Preparation of silibinin-C-2, 3-dihydrogen succinate (formula 5) 50g of Silibinin are dissolved in 70 ml of pyridine at 45 ° C. 50 g of succinic anhydride are added to the solution and the mixture is stirred at 45 ° C for about 8 hours. Then 30 ml of ethanol is added and stirring is continued until a homogeneous mixture has formed. Then, with vigorous stirring, 60 ml of water are added over 30 minutes to saponify phenyl esters. After stirring for 1 hour at 30 ° C, the phenyl esters are quantitatively hydrolyzed according to high performance liquid chromatography (HPLC). Hydrolysis is terminated by quickly adding 1.7 liters of ethyl acetate to the reaction mixture.

To separate excess tartaric acid and pyridine, the reaction mixture diluted with ethyl acetate is extracted twice countercurrently with 5 liters each of water saturated with ethyl acetate 40 and having a pH of 1.85 (adjusted with dilute hydrochloric acid). Thereby, the acidified saturated wash water saturated with ethyl acetate is circulated in a countercurrent pump with the dilute reaction solution and the pH is then kept at 1.85 with dilute hydrochloric acid until this pH remains constant after the ethyl acetate passage.

Then, to wash out excess hydrochloric acid, the ethyl acetate phase is extracted twice in countercurrent 45 with 3.4 liters of water each saturated with ethyl acetate. Once the pH of the wash water has risen above 4.5, the organic phase is separated quantitatively, concentrated in vacuo at 40-50 ° C to 1/12 of the initial volume (2 liters) and diluted with 125 ml of ethanol.

The title compound is obtained by redissolving in and precipitating from ethanol / water and drying at 50 ° C in vacuo for 15 hours.

50 To obtain a sample for elemental analysis, the title compound is dissolved in and precipitated three times in ethanol / water and then dried in vacuo at 50 ° C for 15 hours.

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

The IR spectrum shows two overlapping bands in the region of the CO valentine frequency, one of which, as with silibinin, can be attributed to the carbonyl function in the pyron ring at a wavelength of 1635 cm -1. The other band is at a wavelength of 1730 cm -1 and arises from both ester carbonyl functions.

That a two-fold adjustment has taken place is confirmed by the 1 H-NMR spectrum. So

Claims (8)

Example: 58.05 4.57 37.31 Example 2 Preparation of the disodium salt of silibinin C-2 ', 3-dihydrogen uccinate (Formula 6) The ethanolic solution formed in Example 1 is stirred and external cooling at -5 to 15 ° C a 6% ethanolic sodium hydroxide solution is added dropwise in an amount sufficient to suspend the determined solid content in the ethanolic solution. The suspension is stirred for an additional 1 hour at ambient temperature after which the precipitated beige solid is aspirated. This is suspended twice in 5 ml minutes with a Turrax in 150 ml ethanol and suctioned again. To remove residual ethyl acetate, the product is then stirred in 280 ml of ethanol at ambient temperature for 14 hours, after which the suspension is again aspirated, the residue is washed with 70 ml of ethanol and dried in a vacuum drying cabinet at 40-45 ° C for 15 hours. The pre-dried product is then comminuted, sieved to a particle size of less than 0.2 mm and dried for another 48 hours at 40-45 ° C in vacuo. Thus, 52 g of the title compound (69% yield) are obtained. The title compound has no sharp melting point and starts sintering at about 80 ° C to melt at about 100 ° C with vesicle formation. UV spectrum in methanol: = 288 nm, E = 1.73.104. The molecular weight of the title compound is 726.56. The compound is a light beige, micro-crystalline powder with no specific odor and tastes salty. The title compound is readily soluble in water, sparingly soluble in ethanol and practically insoluble in acetone, ether and chloroform. Example 3 Preparation of lyophylisates for i.v. administration Disodium salt of silibinin-C-2 ', 3-dihydrogen succinate 75.0 mg 35 Mannite 10.0 mg Water for injections ad 1.5 ml 5 ml pointed ampoules, filled with 1.5 ml solution and then known freeze-dried. In order to store the ampoules with the finished lyophylisate, they are closed in the usual manner. Before use, the lyophylisate is dissolved in 5 ml of sterile, physiological sodium chloride solution until clear. 45 1. Silibinine derivatives, represented by formula 1 of the formula sheet, wherein Alk., And All ^, which may be the same or different, represent an alkylene group of 1-4 carbon atoms or an alkenylene group of 2-4 carbon atoms, M1 and M2, which are the same or may be different, represent a hydrogen atom or an alkali metal atom, and n and m, which may be the same or different, are 0 or 1, characterized in that the silibin derivatives are free of isosilybin or derivatives thereof and are in pure form . Silibinin derivatives according to claim 1, characterized in that Alk 1 and Al 4 represent an alkylene group with 2 carbon atoms, M1 and M2, which may be the same or different, represent an alkali metal atom, and n and m, which may be the same or different be 0 or 1. Silibinin derivatives according to claim 1, characterized in that Alk1 and Al1, M, and M2, and n and m in formula 55 have the same meanings. 4. Disodium salt of isosilybine free silibinin C-2 ', 3-dihydrogen succinate of the formula 6 of the formula sheet. A process for preparing pure silibinine derivatives according to any one of claims 1 to 4, wherein 1 part by weight of silibinine according to formula 3 of the formula sheet is dissolved in pyridine; while stirring is reacted with 1-3 parts by weight of dicarboxylic anhydride of formula 2 of the formula sheet, wherein Alk has the meaning indicated in claim 1 for Al 2 or Alk 2; ethanol is added until a homogeneous mixture has formed; the livestock product is washed with water saturated with ethyl acetate and reacting acid; the washed product which is in the ethyl acetate phase is taken up in ethanol and is reacted with an alcoholic alkali metal hydroxide solution until the salt of the free carboxylic acid, characterized in that water is added slowly to the homogeneous mixture with intensive stirring to give aromatic ester groups hydrolysis, after completion of the hydrolysis, dilute with ethyl acetate, and before the washed product is taken up in ethanol, the ethyl acetate phase is concentrated. Process according to claim 5, characterized in that the reaction with the dicaibonic anhydride is carried out at a temperature of 40-50 ° C. 7. Process according to claim 5 or 6, characterized in that the acid-reacting wash water saturated with ethyl acetate is kept at a pH of about 1.5-2.4. Pharmaceutical composition suitable as a liver therapeutic, characterized in that the composition contains at least one vein according to any one of claims 1 to 4, together with a pharmaceutically acceptable carrier or excipient. Hereby 3 sheets drawing