SE465676B - FLAVOL SIGNIFICANT DERIVATIVES, PROVIDED TO MANUFACTURE THIS AND A PHARMACEUTICAL AGENT - Google Patents

Description

465 676 2 10 15 20 25 30 35 40 ./j_\_/°H Ií Ü í/§./°\í/\/\OCHB . \ /o\\// \ f/\0/\cn on ' 2 ir/ \/ \OH OH Ä (B) Isosilybin It is clear from these structural formulas that these compounds are position isomers. The compound of formula A is currently designated silibinin in accordance with the INN nomenclature. This designation is used hereinafter in the present description for the compound of formula A.

The therapeutic use of silybin caused problems because silybin is practically insoluble in water, which is why silybin-containing injection solutions or preparations, for which a certain water solubility is required, could not be produced. Although DE-PS-1 963 318 describes silybin derivatives which exhibit a certain water solubility, this involves a very complex mixture of succinic acid half-esters. This mixture is precisely so complex because silybin contains four esterifiable hydroxyl groups, and because silybin also contains the two positional isomers mentioned above, and the succinic acid used for the esterification is a dicarboxylic acid which can form both monoesters and diesters. A product which consists of an incalculable number of the most diverse, undefined compounds is not suitable for pharmaceutical purposes.

The invention is therefore based on the task of providing water-soluble silibinin derivatives suitable for pharmaceutical purposes, which are accurately characterized as chemical individuals.

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

The invention therefore relates to silibinin derivatives of the general formula I 10 15 20 25 30 3 465 676 //CH2-O-CO-(Alk1)n-COOM1 /\/°\ OCH3 "°\./,_n\_/°\/\/\0/\/\/ l m» « _____/ .\ . . \\/ \o \oH ¿H 3 \\CO-(Alk2)m-COOM2 where n and m are each independently of each other 0 or 1, Alkí and Alkz each independently of each other denote an alkylene radical having 1-4 carbon atoms, or an alkenylene radical having 2-4 carbon atoms, and M1 and M2 each independently of each other denote a hydrogen atom or an alkali metal atom. _ Preferred compounds of the formula I are compounds in which n and m are each independently of each other 0 or 1, Alk1 and Alkz each denote an alkylene radical having 2 carbon atoms and M and M2 each independently of one another denote an alkali metal atom. Preferably, n and m, Alk1 and Alkz as well as M1 and M2 have the same meanings as one another.

Silibinin-C-2',3-dihydrogen succinate, disodium salt is particularly preferred.

In the case of the compounds according to the invention, the OH groups in the silibinin not bound to the a-benzene nucleus are partially or completely esterified, for example with oxalic acid, malonic acid, succinic acid, adipic acid, maleic acid or fumaric acid. Preferably, the two non-aromatically bound OH groups in the silibinin are simply esterified with one of the carboxylic acids mentioned above.

The process according to the invention for preparing the silibinin derivatives in question is characterized in that approximately 1 part by weight of silibinin of the formula A is dissolved in 4e5 676 4 10 15 20 25 30 35 40 _/ . O H OH /m 7-1,/ \\.l/c 2 1 ¿ .

In f/ Ho\ . o å_ _ pä ¿_ R ocn \/:g\\_// \~._,/ \0/ __ / 3 I- a f ;" 1 *_.._..... _. /f-« f. 5' à k.) L x \\t \\r//'_\oH àfi 3 (A) Silibinin in 1-2 parts by weight of pyridine and under stirring is reacted with 1-3 parts by weight of a dicarboxylic acid anhydride with the formula 0 = C - Alk - C = 0 O/ where "Alk" denotes one of the above-mentioned Alk1 and Alká residues, that ethanol is then added until a homogeneous mixture is formed, that water is then added slowly under intensive stirring, whereby the esters of the aromatically bound OH groups present are hydrolyzed, that as soon as this hydrolysis is complete, one dilutes with ethyl acetate, washes with ethyl acetate-saturated, acidic water, evaporates the ethyl acetate phase, a take-up in ethanol and converting the resulting compound with an alcoholic alkali hydroxide solution to the salt of the free, non-esterified carboxylic acid ester.

Preferably, the reaction with the dicarboxylic acid anhydride takes place at 40-50°C.

Advantageously, the pH of the ethyl acetate-saturated, acidic wash water is maintained at from about 1.5 to 2.4.

These compounds, especially the compound silibinin-C-2', 3- dihydrogen succinate, disodium salt, surprisingly exhibit a pronounced pharmacological effect in the treatment of burns. In addition, despite the described derivatization, they retain the full pharmacological effect of the known silybin 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 caused by the insidious fly agaric (Amanita phalloides). Even poisoning caused by halogenated organic solvents, such as chlorine tetrachloride, trichloroethylene, chloroform, etc., can surprisingly be treated with good results. When used prophylactically, the compounds according to the invention prevent the damage described above.

The invention thus also relates to pharmaceuticals containing these compounds. They are mostly used systemically, for example in the form of pills, capsules, solutions, in conventional carriers and possibly together with conventional excipients. The daily dose for an adult amounts to approximately 50-500 mg depending on the patient's condition and the severity of the disease symptoms.

Try silibinin-C-2', 3-dihydrogen succinate, disodium salt (sili-suc-na).

The symptoms that occur in burns are caused in particular by intoxication with products of thermal tissue necrosis. It can be proven in many ways that autointoxicating processes after severe burns of the skin are responsible for this.

Particularly convincing are cross-transplantations of burned and non-burned skin onto healthy recipient animals and burnt recipient animals, in which it is shown that the non-burned recipients of burnt skin perish while burnt recipients of healthy skin do not suffer any harmful effects; compare K H Schmidt et al, "Neuere Aspekte sur Autointoxikation nach schweren Verbrennungen"; "Die Verbrennungskrankheit" G'W2&meflfld et al, eds L), Springer, Berlin 1982, pp. 45-52.

When skin burns, a number of chemical compounds are released or newly formed. Despite their diversity, the structure of some of these compounds has been successfully explained.

It was shown, among other things, that the compounds formed in burns on the skin are similar to those formed in lipid peroxidation. There are also analogies regarding the toxic effects of these substances. Particularly striking is the formation of toxic saturated and unsaturated aldehydes of 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 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 assumed 465 676 , 6 10 15 20 25 30 35 40 therefore that burns lead to oxidative damage to cell structures.

Thus, autooxidative changes in membrane lipids as a result of autointoxication after severe burns were investigated. In particular, changes in the fatty acid composition of the membrane lipids were investigated. Furthermore, the extent to which the silibinin derivatives according to the invention affect the changes in the fatty acid composition of the membrane lipids was tested.

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

Male Wistar rats with an average weight of 360 g were housed in groups of 3 with free access to water and dry food. Until the beginning of the experiment, the room temperature was 22°C and after the beginning of the experiment the animals were kept at 30°C.

The burns on the skin were produced by means of a copper stamp with a surface area of 20 cm2 at constant pressure and a temperature of 250°C. To avoid thermal damage to deeper organs, the skin was moved over an air-cooled, perforated spatula. With this animal model, very precise burns are produced, which give constant survival values.

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

Five experimental groups were formed: a) Normal group, completely uninjured animals b) Control group I: only silibinin treatment for 6 days with 75.5 mg of sili-suc-na. c) Control group II: sham-operated animals d) Sham-operated animals e) Test group: 25%, 250°C, 20 sec, 0.5 at. animals, which received 75.5 mg of sili-suc-na intraperitoneally for 6 days starting one day before burning the skin.

For isolation of the microsomes, the animals were bled under anesthesia at the end of the experimental period. The liver was then removed, weighed and immediately transferred to ice-cold isolation medium (0.25 m sucrose, 1 mM EDTA, 10 mmol Tris-HCl, pH 7.2). The liver was cut into pieces and homogenized in the medium. The microsome fraction was pelleted by differential centrifugation. The microsomes were resuspended and centrifuged again. A suspension was then prepared, where 1 ml of suspension corresponded to 1 g of liver tissue.

The lipids were determined using the method according to J Folch ("A simple method for the isolation and purification of total lipids from animal tissues", J Biol Chem, 226, 497-508 (1957)), modified 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 were saponified with sodium hydroxide. The free fatty acids were esterified by addition of BF3-methanol. After evaporation of the methanol and removal of hydrophilic by-products, the fatty acid esters were determined quantitatively.

In the case of the animal groups without burns, no significant change in the fatty acid pattern could be determined. The anesthesia and the minor surgical intervention therefore do not lead to any change in the microsomal lipids. For this reason, for the further comparison, the normal group and the control group were combined into a single control group.

A comparison between the animals without burns and the animals with burns regarding their microsomal fatty acid patterns showed strong shifts from unsaturated to saturated fatty acids.

The accompanying drawing, Fig. 1, shows the fatty acid distribution in the microsomal liver lipids and the changes caused by thermal damage to the skin. The proportion of palmitic acid (C16) increases after the resulting burn from 25.1 to 34.4% of the total fatty acids.

In the case of stearic acid (C18), the proportion in the burned animals amounts to 46.3%, which is 13.2% higher than the value obtained in the case of the control animals. In the case of oleic acid (C18fU), a slight, non-significant decrease is detectable. The proportion of linoleic acid (C18:2) fell after the burns to about 1/3 of the initial value. In the case of arachidonic acid (C20:4), the content finally amounted to only 31% of the initial content after the burns.

The following table 1 shows the effect of sili-suc-na on the fatty acid proportions in the animals with and without burns. 465 676 8 10 15 20 25 30 35 40 TABLE 1 Fatty acid pattern of the microsomal lipids from rat liver after sili-suc-na therapy in cases of animals with burns and animals without burns.

C 16 C18 CT8:1 C18:2 C20:4 Without burns 29.8% 37.2% 8.9% 9.6% 16.2% (conformity group I) 16.2 112.3 11.1 23.3 14.9 With burns 25.4% 37.5% 7.8% 11.4% 18.0% t6.0 18.6 21.0 ïs.3 :9.1 It appears that the treatment according to the invention with a silibinin derivative in the case of the control animals without burns did not cause any significant changes in comparison with the untreated animals. In the case of the animals with burns, the therapy leads to a complete reversal of the loss of unsaturated fatty acids.

In summary, the following can be stated: burns lead to changes in the fatty acid pattern of microsomal lipids. It is assumed that this may be due to oxidative damage to the membranes. This is particularly evident in the sharp decrease in polyunsaturated fatty acids.

The silibinin derivatives used according to the invention now have the ability to inhibit oxidative cell damage. They are therefore particularly suitable for disrupting oxidative damage mechanisms after severe burns.

As already mentioned, it is assumed that autotoxic reactions after severe burns lead in particular to oxidative cell damage. The effect of a standardized thermal injury on the PHA-induced blastogenesis of T lymphocytes from the spleen and peripheral blood of rats was therefore investigated. Furthermore, it was investigated how the silibinin derivatives usable according to the invention affect such lymphocyte dysfunction after severe burns.

Effect of a standardized heat injury on the PHA-induced blastogenesis of T lymphocytes from the spleen and peripheral blood of rats.

In the manner described above, burns are produced on the dorsal part of Wistar rats by means of a copper stamp. The control group consisted of apparently burned animals on which all operative manipulations were carried out without causing burns.

After 2, 4, 7 and 9 days, blood and spleen were taken under ether anesthesia from the animals showing burn injuries and the control animals, respectively.

Heparinized blood was layered for isolation of lymphocytes on Ficoll-Hypague solution (density 1.077). Centrifugation was then carried out and the lymphocytes obtained were tested with trypan blue for their vitality. For isolation of spleen lymphocytes, the organ was disintegrated, passed through a sieve and freed from the accompanying erythrocytes by means of a lysis solution according to Gay.

The cell mixture was then incubated in a vessel in the presence of 5% heat-inactivated fetal calf serum for 30 min to reduce (5%) the proportion of mononuclear cells in the suspension by adhesion to the vessel wall. For the culture, the cells were introduced into "flat-bottomed" microtiter plates. Then 20% fetal calf serum was added. In this way, spontaneous blastogenesis was determined by measuring the incorporation of 3H-thymidine (2 Ci/mM) into the DNA of the cells. t After preliminary experiments, it became clear that optimal mitogen stimulation takes place at a PHA concentration (mitogen phyta agglutinin) of 5 pg/ml. In these experiments to optimize the cellular test system, it was further determined that the maximum stimulation of new DNA synthesis occurs after 72 hours. It was further determined that the optimal concentration of fetal calf serum is 20% to achieve the highest stimulation.

As described above, spontaneous blastogenesis was determined by measuring the incorporation of 3H-thymidine into the DNA of the cells.

The cells were harvested 18 hours after the addition of 3H-thymidine, the zero point for said 18 hours coinciding with the time of maximal stimulation.

To investigate the effect of the silibinin derivatives usable according to the invention, a group of rats was treated with a silibinin derivative. For this purpose, 75.5 mg of sili-suc-na was injected intraperitoneally once a day. This therapy was carried out from the day on which the burns were inflicted until the day on which the above-mentioned organs were removed (maximum up to the ninth day).

For the evaluation of the results obtained in the case of the control animals and the animals without burns and the animals treated with sili-suc, the stimulation index was determined. This number is the quotient of the mean value of the stimulated sample and the mean value of the control sample. Based on the stimulation index thus obtained for each experimental animal, an average stimulation index was calculated per animal group. The results obtained are expressed with this index SI.

The attached drawing shows the effect of the derivative sili-suc-na used on the blastogenesis of lymphocytes, Fig. 2. In the case of the burned animals, the reduced stimulability of the cells by silibinin was clearly increased.

Already on the second day, the animals treated with silibinin showed an approximately tenfold higher responsiveness of the blood lymphocytes to PHA. On the fourth day after the burns, the stimulation index value for the blood lymphocytes of the treated animals was 8, while the corresponding value for the untreated animals was 1.5. In the case of the spleen cells, the stimulation index for the burned, untreated animals was all clearly below 1. The administration of silibinin led to a marked improvement on all days examined, with a maximum value occurring on the seventh day after the burns.

Comparative experiments were also conducted, which showed that sili-suc-na alone in the case of healthy animals does not lead to any marked changes in the stimulating ability of PHA-induced blastogenesis of T lymphocytes from spleen and from peripheral blood.

Thus, the silibinin used according to the invention significantly stimulates the blastogenesis of lymphocytes in burn-damaged animals.

It was further determined that in the case of the animals treated with the silibinin derivatives, the general catabolism was less, since the animals quickly regained weight after the heat injury.

§Yê@Bɧš¶iÉÉQi§9êE= Poisonings caused by the insidious fly agaric are among the most serious that exist in medicine. Although only 10-30% of all mushroom poisonings are caused by the green, insidious fly agaric, poisoning with this mushroom has attracted the greatest medical interest since ancient times due to its dangerous nature.

Older literature reported a mortality rate of 30-50%. Thanks to modern intensive care, this figure has been reduced to an average of 22.4%, according to a comprehensive study by Floersheim et al. of 205 patients.

The poison in the insidious fly agaric, amanitin, can be fatal to an adult in a dose of 7 mg. This amount of poison 10 15 20 25 30 35 11 465 676 is contained in approximately 50 g of a fresh mushroom specimen.

After a series of promising animal experiments, the active compound sili-suc-na was included in the therapy for mushroom poisoning caused by the insidious fly agaric. 28 patients with poisoning caused by the insidious fly agaric were treated with sili-suc-na in addition to the usual therapeutic measures. Of these 28 patients, only one died, having ingested large quantities of the poisonous mushroom with the intention of killing himself. This result shows an enormous therapeutic advance in this field.

EšêI¿1§Eë2l.lf_1.i.f¿¶_êY iSOS-ilybiflfšltt Siäëllaäß A suspension of 500 g of the product according to DE-AS-1 923 082, column 8, lines 14-19, with a silymarin content of about 70% at an isomer ratio silybin/silydianin/silichristin of about 3:1:1, the silybin containing about 1/3 isosilybin, and 2 kg methanol ë"2.53 l are heated with stirring for 15 minutes to boiling. Some silibinin can then already precipitate from the solution thus obtained. Then 0.75-1.25 kg E 0.96 - 1.58 l methanol is evaporated in vacuo and the residue is left at room temperature for 10-28 days. The precipitated silibinin is filtered and washed twice times with 50 ml of cold methanol each time. After drying at 40°C in vacuum, the isolated crude silibinin is purified as follows: 60 g of crude silibinin is dissolved in 3 l of technical acetic acid ester under heating; then 20 g of activated carbon is added and stirring is carried out for a further 2 h under reflux conditions.

Then, clear filtration is carried out and the solution is evaporated at SOOC under reduced pressure to about 250 ml. The concentrate is stirred for 15 min using an ultra-Turrax apparatus and 25 ml of methanol are added while stirring. The mixture is then left to stand overnight at room temperature. Before filtering the silybin that has precipitated, it is stirred again for 5 min using an ultra-Turrax apparatus. The filtered precipitate is washed twice with 50 ml of acetic acid ester and dried in a vacuum drying cabinet overnight at 40°C. Finally, the product is ground and dried under the same conditions for 48 h.

EXAMPLE 1 Preparation of silibinin-C-2', Bëdihydrogensuccinate 10 15 20 25 30 35 40 0465 676 12 -o-co-cH -cH2-coon J/ 0\;/CH2 2 H g yr] /OCHB g g l 01.

W?\¶H \\C0-CH2-CH -COOH OH O 2 Dissolve 50 g of silibinin at 45°C in 70 ml of pyridine, add 50 g of succinic anhydride, stir the mixture for about 8 h at 45°C, add 30 ml of ethanol and stir until a homogeneous mixture is formed. Then, with vigorous stirring, 60 ml of water is added within about 30 min to saponify the phenyl esters.

After about 1 hour of stirring at 30°C, the phenyl esters are quantitatively hydrolyzed. The completeness of the hydrolysis is checked by means of HDLC. The hydrolysis is stopped by rapidly adding 1.7 l of ethyl acetate to the resulting reaction mixture.

To remove excess succinic acid and pyridine, the reaction solution diluted with ethyl acetate is extracted twice with 5 l of water each time, which is saturated with ethyl acetate and has a pH of 1.85 (adjusted with dilute aqueous hydrochloric acid), in countercurrent. The acidified wash water saturated with ethyl acetate is then pumped in a circuit against the diluted reaction solution and finally the pH is maintained at 1.85 by adding dilute hydrochloric acid until this pH value remains constant after the ethyl acetate has passed through.

The ethyl acetate phase is then extracted twice in countercurrent to wash out excess hydrochloric acid with 3.4 l of water saturated with ethyl acetate. As soon as the pH of the washing water is higher than 4.5, the organic phase is separated quantitatively, evaporated at 40-50°C in vacuo to 1/12 of the starting volume (approximately 0.2 l) and diluted with 1.25 ml of ethanol.

The compound specified in the title above is obtained by reprecipitation from ethanol/water and drying at 50°C in vacuum for 15 h.

To prepare an analytical sample, the compound specified in the title above is reprecipitated three times from ethanol/water and then dried for 15 h at 5000 in vacuum. 10 15 20 25 30 35 40 13 465 676 In the FD mass spectrum, the molecular peak appears at the expected molecular weight of 682.

The IR spectrum shows two overlapping bands in the CO valence frequency range, one of which, as is also the case for silibinin, is to be assigned to the carbonyl group in the pyrone ring at a wavelength of 1,635 cm-1. The other band is at 1,730 cm-1 and originates from the two ester carbonyl groups. The 1H-NMR spectrum confirms that a double esterification has taken place. Thus, the ratio obtained by integration between aromatic protons and methylene protons in the succinic acid residue is 8:8 (ppm range 5.9 - 7.1). The ratio between these methylene protons (ppm 2.6) and the methyl protons in the methoxy group (ppm 3.8) is 8:3 and thus agrees with it. The chemical shifts at 13 also show that the esterification at the two alcoholic OH groups has taken place, because the chemical shifts change most strongly at C11 and the nearest carbon atoms C12-C14 as well as at C2- C C investigations Elemental analysis for C33H30O16 (682.60): C H O Calculated: 58.07 4.43 37.50 Found: 58.05 4.57 37.31 EXAMPLE 2 Preparation of silibinin-C-2', 3-dihydrogen succinate, disodium salt.

CH -0-CO-CH -CH -CONa T/_'_\l/°\/2 2 2 /\___I u//O __/,/í~\\o/_a\\( /OCH3 I , '9 lm \ N/ \oH \co-cH2-cH2-coNa To the ethanol solution obtained according to example 1, add- A - -- -r dnxmannflnlflerfimfiàïng and cooling from the outside at from -SOC to 9°C a quantity of a 6% ethanolic sodium hydroxide solution calculated on the basis of a determination of the solids content in this solution, stir the suspension for a further hour at room temperature, separate the precipitated, beige solid while applying suction, suspend this twice for 5-10 min with the aid of a Turrax type stirrer in 150 ml ethanol and suction is applied again. To remove the remaining ethyl acetate, the product is then suspended for 14 hours at room temperature in 280 ml of ethanol, suction is applied again, washed with 70 ml of ethanol and dried for 15 hours at 40-45°C in a vacuum drying cabinet. The previously dried product is then ground, sieved to a particle size of less than 0.2 mm and dried for a further 48 hours at 40-45°C in vacuum. In this way, 52 g of the compound specified in the title above are obtained (69% yield).

The compound given in the title above does not have a sharp melting point. It begins to sinter at about 80°C and melts with the formation of bubbles at about 100°C. x=288 nm, e=1.73 x 104.

The molecular weight of the above compound is 726.56. The compound is a slightly beige-coloured, microcrystalline powder with no specific odour and with a saline taste. It is freely soluble in water, sparingly soluble in ethanol and practically insoluble in acetone, ether and chloroform. Preparation of lyophilisate for intravenous administration.

Silibinin-C-2', 3-dihydrogen succinate, disodium salt 75.0 mg Mannitol 10.0 mg Water for injection to 1.5 ml 1.5 ml of solution is filled into 5 ml tip ampoules and then freeze-dried according to known technology. The ampoules with the finished lyophilisate are closed for storage in the usual way.

For use, the lyophilisate is dissolved in 5 ml of sterile, physiological saline solution to form a clear solution.

Claims (8)

Ü 465 676 PATENTKRAV Silibinin derivatives of the general formula I / Ow / Hz -o-co- (A1k1) n-cooM1 1 (I) o \ co- (A11 <2) m-cooM2 where n and m are each independently 0 or 1, Alk 1 and Alk 2 each independently represent an alkylene radical having 1 to 4 carbon atoms or an alkenylene radical having 2 to 4 carbon atoms and M 1 and M 2 each independently represent a hydrogen atom or an alkali metal atom. Silibinin derivatives according to claim 1, characterized in that n and m each independently represent 0 or 1, Alk1 and Alkz each represent an alkylene radical having 2 carbon atoms and M1 and M2 each independently represent an alkali metal atom. . Silibin derivatives according to Claim 1, characterized in that n and m, Alkí and Ålkz and M1 and M2 have the same meanings. Silibinin derivative according to any one of claims 1-3, characterized in that it consists of silibinin-C-2 ', 3-dihydrogen succinate, disodium salt of the formula: HO íššâššï // Û \\ í // CH2- O * CO-CH2-CH2_COONä \ / \ lß \ 5G- JG: \\\ ¥ // // V if; \ co-cH2 -cnz -cooNa I OH 465 676 _ 16 5. A process for the preparation of silibinin derivatives according to claims 1 to 4, characterized in that a part by weight of silibinin of the formula A is dissolved: CH 2 OH / _ 2 / O 2 C) Û \ \. (H) Silibinin in 1-2 parts by weight of pyridine and reacting with stirring with 1-3 parts by weight of a dicarboxylic acid anhydride of the formula: o = c-A1k- = o O where "Alk" denotes an Alk1 or Alk2 residue according to claim 1, then add ethanol until a homogeneous mixture is formed, then slowly add water with vigorous stirring, the present esters of the aromatically bonded OH groups being hydrolyzed, so that as soon as this hydrolysis is complete dilute with ethyl acetate, wash with ethyl acetate saturated acidic water; evaporates the ethyl acetate phase, takes up ethanol and transfers with an alcoholic alkali hydroxide solution the product obtained to the salt of the free carboxylic acid ester. 6. A process according to claim 5, characterized in that the reaction with the dicarboxylic anhydride is carried out at about 40-50 ° C. A process according to claim 5, characterized in that the ethyl acetate saturated acidic wash water is maintained at a pH of about 1.5 - 2.4. Pharmaceutical composition containing as active ingredient at least one compound according to any one of claims 1-4, optionally in a pharmaceutical carrier or excipient.