FI84064B - FOERFARANDE FOER FRAMSTAELLNING AV FARMAKOLOGISKT VAERDEFULLA SILIBININDERIVAT. - Google Patents

Description

84064

The process for the preparation of pharmacologically valuable silibinin derivatives - Förfarande för framställning av Pharmacakologiskisk värdefulla si 1ibininderivat.

The invention relates to a process for the preparation of pharmacologically valuable siibinin derivatives of the formula I: / v / CH2_0-C0_CH2 '~ CH2 ~ C00M1

Wr ^ r - OH O CO-CH2-CH2-COOM2 wherein 20 Mj and M2 are independently a hydrogen atom or an alkali metal atom.

Marianohdake - Silybum marianum (L.) Gaertn. (Carduus marianus L.) - has been a well-known 25 medicinal plants since ancient times. R.Munster isolated one component, silybin, from f 1avolignans present in the fruit of this plant, cf. dissertation R.Munster, Munich 1966. A.Pelter and R.Hänsel have investigated the chemical structure of this compound, cf. Tetrahedron Letters. London, Volume 25, Pages 3Ό 2911-2916 (1968).

Silybin, formerly also known as sily-marine I, is known to be a valuable liver drug, cf.

DE-AS 17 67 666. For example, 35 DE-AS 19 23 082 describes a technical process for the preparation of silybin (silymarin I).

As early as 1974, H. Wagner, P. Diesel, and M. Seitz, Arzneimittelforschung, Volume 24 (4), pages 466-471, assumed two structural isomers for silybin 40, namely silybin and isosilybin-2 84064 ni. A. Arnone, L. Merlini, and A. Zanarotti, Journal Chemical Society Chem.comm., 1979, Volume 16, pages 696/97 refined and experimentally confirmed this assumption. The known silybin thus consists of two different compounds, namely compounds of the following structural formulas A and B: ch9oh

15 OH O

(A) Silibinin 20 ffV ^ YX ^ OCH3. '25 Nr '' · ch2oh

.;: 'OH O

: 30 (B) Isosilybin It can be seen from these structural formulas that these compounds are positional isomers. The compound of formula (A) is currently silibinin in the INN nomenclature. This term is used for a compound of formula (A) hereinafter in the present application.

A difficulty with the therapeutic use of silybin is that sily 40 bin is virtually insoluble in water, so 84064 3 cannot be prepared into silybin-containing injectable solutions or formulations that require a certain amount of water solubility.

DE-PS 19 63 318 describes siybin derivatives with a certain solubility in water, but it is also a question of a very complex mixture of succinic acid half-esters. This mixture is complicated because silybin has five esterifiable hydroxyl groups and, in addition, silybin 11 has both of the aforementioned positional isomers, and the succinic acid used for esterification is a dicarboxylic acid that can form both mono- and diesters. For pharmaceutical purposes, it is not possible to use a product consisting of an undetectable amount of a wide variety of unresolved compounds.

DE-PS 19 63 318 describes highly complex mixtures of succinic acid half-esters, e.g. mixtures of sodium salts of various siibine dihemisuccinates. The explanation for this is as follows: The libinin backbone contains three phenolic and two alcoholic hydroxyl groups (previously called silybin). To prepare the disodium salt of pure siibinin C-2 ', - 3-dihydrogen succinate (which mono-compound is shown for the first time and the right substitution as a result of the present invention), all hydroxyl groups of silibinin are first esterified according to the invention. The esters of the aromatic-linked hydroxyl groups (phenolic hydroxyl groups) are then hydrolyzed. Thus, two things are achieved: a) Complete esterification with the dicarboxylic acid compound and under the reaction conditions according to the invention, monosubstitution or monoesterification of the dicarboxylic acid compound is always achieved.

b) Specific hydrolysis of esters of aromatically linked hydroxyl groups by a simple and inventive method leads to the formation of a pure mono-compound, in particular for the two alcoholic hydroxyl groups of silibinin, to a diester.

The mono-compounds described in the application (siibinin diesters in which the two alcoholic hydroxyl groups 440 of silibinin are monosubstituted with a dicarboxylic acid compound as an ester) 4,84064 are first described in our present application. At the same time, the advantageous pharmacological properties of these new compounds have also been reported.

It is therefore an object of the invention to provide water-soluble sibinin derivatives suitable for pharmaceutical purposes, which have been precisely characterized as individual chemical compounds.

The invention is characterized in that 1 part by weight of the silibinin of the formula (A)

n CH OH

·

I 11 OH

OH O

(A) Silibinin is dissolved in 1-2 parts by weight of pyridine and reacted while mixing with 1-3 parts by weight of dicarboxylic anhydride of the formula o = c-ch2-ch2-c = o \ /

O

then ethanol is added until a homogeneous mixture is formed, followed by slow addition of water with vigorous stirring to hydrolyze the esters of aromatically bound OH groups present, immediately after this hydrolysis complete addition of ethyl acetate, washing with ethyl acetate saturated acidic water, ethyl acetate, ethyl acetate is dissolved in ethanol and optionally converted into the free carboxylic acid ester salt by means of an alcoholic solution containing 5,84064 alcohol.

The reaction with the dicarboxylic anhydride takes place par-5 at 40-50 ° C. The pH of the acidic wash water saturated with ethyl acetate is preferably maintained at about 1.5 to 2.4.

Preferred compounds of formula (I) are those in which and M2 are in each case independently of one another a metal atom. The groups and M2 have the best meaning in each case.

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

15

In the compounds of the invention, the OH groups of silibinin which are not bound to the benzene nucleus are completely esterified with succinic acid.

These compounds, in particular siibinin C-2 ', 3-dihydrogen succinate disodium salt, surprisingly have a clear pharmacological effect in the treatment of burns. In addition, despite the derivatization described, they have the full pharmacological efficacy of the known silybin as a hepatic drug.

25 They are particularly well suited for the treatment of cirrhosis of the liver and toxic metabolic liver damage. Surprisingly, the compounds according to the invention have also proved to be very effective in the treatment of fungal poisonings, in particular very dangerous poisonings caused by fly agarics (Amanita phalloides). Surprisingly, they can also be used to treat poisonings caused by halogenated organic solvents such as carbon tetrachloride, trichlorethylene, chloroform, etc. In prophylactic use, the compounds of the invention prevent the damage described above.

35

Experiments with Si-libinin-C-2 ', 3-dihydrogen succinate disodium salt (as Si 1i-suc).

The symptoms of burns are due in particular to the intoxication caused by the products of tissue necrosis caused by heat .40. It has been shown in many different ways that this is due to autointoxic processes after severe skin burns. Particularly convincing are the transplants of burnt and non-burnt skin into healthy 5 or burnt host animals, where it has been shown that non-burnt recipients of burnt skin die, while no deleterious effects can be observed in burnt recipients of healthy skin, cf.

K. H. Shmidt et al., Neuere Aspekte zur Autointoxikation 10 nach schweren Verbrennungen; Die Verbrennungskrankheit (F.W. Ahnefeld et al., Ed. L), Springer, Berlin 1982, pp. 45-52.

Numerous chemical compounds are released or re-formed in skin burns. Despite their multiplicity, it has been possible to elucidate the structure of some of these compounds.

Among other things, it has been shown that compounds formed in skin burns have similarities to compounds formed by lipid peroxidation. There are also analogies between the toxic effects of these compounds. Particularly impressive is the formation of toxic and saturated unsaturated aldehydes of different chain lengths by peroxidation of lipids. '(Benedetti et al., Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal Lipids, Biochim. -296, 1980) and heat-induced tissue damage (KH Schmidt et al., Studies on the structure and Biological effects of • 30 pyrotoxins purified from burned skin, World J. Surg. 3, 361-365, 1979). Therefore, it is hypothesized that burns lead to oxidative damage to cellular structures.

Therefore, the auto-oxidative changes of membrane lipids due to autointoxification after severe burns were studied. In particular, changes in the fatty acid composition of membrane lipids were studied. It was further investigated to what extent the siibin derivatives 7,84064 according to the invention affect changes in the fatty acid composition of membrane lipids.

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

Wistar male rats weighing an average of 360 g were housed in groups of three by giving water and dry food freely. Until the beginning of the experiment, the room temperature was 22oC.

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

Skin burns were caused by a copper stamp with a surface area of 20 cm 2, a constant pressure and a temperature of 250 ° C. Damage to the deeper organs by heat was prevented by brushing the skin with an air-cooled hollow spatula. With this animal model, highly accurate burn seams could be induced, giving constant survival times.

Prior to the start of the experiment, the animals were anesthetized with 50 mg / kg nembutal. After burning, 20 ml of Ringer-1-lactate solution was injected i.p. for shock prophylaxis.

5 experimental groups were formed: a) Normal group: Completely intact animals b) Control group I: Treatment with siibinin alone for 6 days by administration of 75.5 mg of Sili-suc-na.

30 c) Control group II: Phase-treated animals d) Group with burnt animals: 25%, 250oC, 20 s, 0.5 at e) Test group: Animals given 75.5 mg

Sili-suc-na i.p. Within 6 days from 35 the day before incineration.

To isolate microsomes, blood was counted from anesthetized animals after the experimental period. The livers were then removed, weighed and immediately transferred to ice-cold 40 isolation solution (0.25 mm sucrose, 1 mM EDTA 10 mMol 8 84064

Three. HCl, pH 7.2). The livers were dissected and homogenized in medium. The microsome fraction was pelleted by differential centrifugation. The microsomes were resuspended and recentrifuged. A suspension was then prepared in which 1 ml of the suspension corresponded to 1 g of liver tissue.

Lipids were determined by 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)), 10 Bligh and Dyer variant ( 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 the addition of BF3-methanol.

After evaporation of the methanol and removal of the hydrophilic by-products, the fatty acid esters were quantified.

20 In the non-combustible groups of animals, no significant changes in the fatty acid pattern could be observed. Thus, anesthesia and minor surgical procedures do not cause a change in microsomal lipids. Therefore, in subsequent comparisons, the normal group and the control group were combined into one control group.

Comparison of unburned and burned animals with respect to their microsomal fatty acid patterns showed a clear migration of unsaturated fatty acids into saturated ones.

:: 30

Figure 1 shows the distribution of fatty acids in microsomal liver lipids and the changes caused by thermal damage to the skin. The proportion of palmitic acid (C16) in the total amount of fatty acids after combustion increases from 35.1% to 34.4%. The proportion of stearic acid (C18) in burned animals is 46.3% compared to 13.2% in control animals. In oleic acid (C18-: 1) a smaller, insignificant decrease can be shown. After combustion, linoleic acid (C18: 2) decreased by 9 84064 to about 1/3 of the initial value. After combustion, arachidonic acid (C20: 4) was only 31% of the initial concentration.

The following Table 1 shows the effect of Si 1i-suc-na on the amounts of fatty acids in burned and non-burned animals.

TABLE 1 10 Fatty acid pattern of rat liver microsomal lipids in burnt and non-combustible animals after Sili-suc-na treatment _C16_C18_C18: 1 C18: 2 C20: 4 15 NON-COMBUSTIBLE 29.8% 37.2% 8.9% 9.6% 16.2 % (control group I> _ ± 6.2_ ± 12.3_ ± 1.1_ ± 3.3_ ± 4.9 BURNED 25.4% 37.5% 7.8% 11.4% 18.0% 20 _ ± 8,6_ 6.0_ ± ± ± 1,0_ 5,3_ ± 9.1

Treatment with a siibinin derivative according to the invention does not show any significant changes in non-combustible control animals compared to untreated: '25 animals. In burned animals, treatment completely reverses the loss of unsaturated fatty acids.

In summary, burns cause changes in the fatty acid pattern of microsomal lipids. 20 This is thought to be due to oxidative damage to the membranes. This is particularly evident in the strong reduction in polyunsaturated fatty acids.

The siibinin derivatives used according to the invention are capable of inhibiting oxidative cell damage. They are therefore particularly suitable for inhibiting oxidative damage mechanisms after severe burns.

It has already been described above that the belief in autotoxic reactions; 40 severe burns, leading in particular to oxidative cell damage. Therefore, the effect of standardized heat-induced trauma on PHA-induced blastogenesis of T-1 lymphocytes from rat spleen and periteria blood was investigated. In addition, the effect of the silyl-5 bin derivatives used according to the invention on such lymphocytic dysfunction after severe burns was investigated.

Effect of standardized heat trauma on PHA-induced blastogenesis of T-1 vmf from spleen and inherited blood

The dorsal skin of Wistar rats was burned as described above with a copper stamp. As a control group, phase-burned animals were used, which underwent all seasonal treatments without burns. After 2, 4, 7 and 9 days, blood and spleen were removed from burned animals under ether anesthesia or control animals.

Heparinized blood was coated with Ficol1-Hypaque-1 solution (density 1.077) to isolate lymphocytes. The resulting lymphocytes were then centrifuged and tested for Trypan-Blau staining. To isolate spleen lymphocytes, the organ was minced, passed through a sieve, and entrained erythrocytes were removed according to Gay's Lyse-1 solution.

j The cell mixture was then incubated for 30 minutes in a vessel in the presence of 5% heat-inactivated fetal calf serum to reduce the number of mononuclear cells in the 3Ό suspension by fixing the vessel to the wall (5%). For culture, cells were plated on a "1-bottom" microtiter plate. 20% fetal calf serum was then added. In this way, spontaneous blastogenesis was determined by measuring thymidine (2Ci / mM) incorporation into cellular DNA.

35

Preliminary experiments showed that optimal mitogen stimulation occurs at a concentration of 5 pg / ml PHA (mitogen-phytemagglutinin). In these optimization experiments of the Sei 1 uiaar test system, it was further found that • 40 DNA resynthesis is activated after a maximum of 72 hours at 11,84064. It was further found that the optimal concentration of fetal calf serum is 20% to provide greater stimulation.

5 It has already been described above that spontaneous blastogenesis was determined by measuring the incorporation of 3 H-thymidine into cellular DNA. Nuclei were removed from the cells 18 hours after the addition of 3 H-thymidine, with the 18-hour zero point coinciding with the time of maximal stimulation.

10

To study the effect of the siibin derivatives useful in the invention, one group of rats was treated with the siibinin derivative. To this end, 75.5 mg of the Si-suc-na compound was injected intraperitoneally once a day. This treatment was performed from the day of burn until the day of organ removal (up to day 9).

To evaluate the results obtained in control and non-combustible animals and in animals treated with Sili-suc-na, a stimulation index was calculated. This number is the quotient of the mean of the stimulated sample and the mean of the control sample. From the stimulation index of each experimental animal thus obtained, the average stimulation index per group of animals was calculated. The results obtained are expressed by the SI:.

Figure 2 shows the effect of the Si 1i-suc-na compound used on lymphocyte blastogenesis. In burned animals, silibinin clearly increased the decreased cellularity.

Already on the second day of Si 1i-suc-na treatment, the reactivity of blood lymphocytes against PHA was found to be about 10-fold higher. On the fourth day of trauma, the stimulation index value was 8 for the blood lymphocytes of the treated animals, while the corresponding value for the untreated animals was 1.5.

Stimulation indices of burnt, untreated animals; -40 spleen cells are all well below 1. Administration of silibinin 12,84064 results in significant improvement on all days studied, with the maximum being reached on day 7 post-traumatic.

5 In addition, comparative experiments were performed which showed that Sili-suc alone did not cause any significant changes in the stimulation of PHA-induced blastogenesis of T lymphocytes from spleen and peripheral blood in healthy animals.

10

The silibinin used according to the invention thus significantly stimulates the blastogenesis of lymphocytes from burned animals.

It was further found that the animals treated with the siibinin derivative 11a had a lower overall catabolism because, after the heat trauma, the animals rapidly increased their growth again.

20 Fungal Poisonings: Poisoning caused by fly fungi is considered to be the most severe that occurs in medicine. Although only 10 to 30% of all fungal poisonings are caused by the green fly fungus, the poisoning of this fungus is medically the most interesting because of its danger. In older publications, the mortality rate is 30-50%. Thanks to modern intensive care, FLOERSHEIM el ai. based on a study collected by.

The fly agaric venom, amanitine, can be lethal to an adult as high as 7 mg. This amount of poison is about 50 g in a fresh sample.

35

Following a successful series of animal experiments, the active ingredient Sili-suc was used to treat fly agaric poisoning. 20: T; Patients with fly agaric poisoning were treated with Sili-suc -:.: 40 na in addition to standard treatment procedures. Of these 28 patients, only one died who had taken a larger number of poisonous fungi for suicidal purposes. The result of this is a testament to the tremendous therapeutic progress in this field.

5 Preparation of isosi 1ybin-free silibinin:

A suspension containing 500 g of a product prepared according to DE-AS 19 23 082 (vertical row 8, rows 14-19) with a siymarin content of about 70% and a 10: 1/1: 1 ratio of 1: 1: 1 silybin contains about 1/3 of isocybin, and 2 kg of methanol * 2.53 l, is heated at reflux for 15 minutes with stirring. After this time, some silibin may already precipitate out of the solution thus obtained. 0.75 to 1.25 (a 0.96 to 1.58 l) of methanol are then removed in vacuo and the residue is left to stand for 10 to 28 days at room temperature. The precipitated silibin is filtered off and washed twice with 50 ml of cold methanol. After drying at 40 ° C under vacuum, the isolated crude silibin 20 is purified as follows: 60 g of crude silibinin are dissolved by heating in 3 liters of technical acetic acid ester; then 20 g of activated carbon are added and the mixture is stirred for a further 2 hours at reflux. It is then filtered to clear and the solution is evaporated at 50 ° C under reduced pressure to about 250 ml. The concentrate is stirred for 15 minutes using an Ultra-Turrax-1 pad and 25 ml of methanol are added with stirring. The mixture is then allowed to stand overnight at room temperature. Before filtering off the precipitated silibin, the Ultra-Turrax-1a is also stirred for a further 5 minutes. The precipitate filtered off is washed twice with 50 ml of acetic acid ester and dried in a vacuum oven overnight at 40 ° C. The product 35 is then ground and dried under the same conditions for 48 hours.

Example 1

Preparation of silibinin C-21,3-dihydrogen succinate.40 14 84064

^ -0-. CH0-0-CO-CH, -CH, -COOH

V ^ UQC 7! ^ O ^ ner ”· I π ° v OH O \ ^ -711

CO-CH 2 CH 2 COOH

Dissolve 50 g of siibinin at 45 ° C in 70 ml of pyridine, add 50 g of succinic anhydride, stir for about 8 hours at 45 ° C, add 30 ml of ethanol and mix until a homogeneous mixture is formed. 60 ml of water are then added with vigorous stirring over a period of about 30 minutes to saponify the phenyl esters. After stirring for about 1 hour at 30 ° C, the phenyl esters have been quantitatively hydrolyzed. The completeness of the hydrolysis is examined by HPLC. The hydrolysis is stopped by rapidly adding 1.7 l of acetic acid ethyl ester to the reaction mixture obtained.

Excess succinic acid and pyridine are separated by extracting the reaction solution 25 diluted with acetic acid ethyl ester twice countercurrently with 5 liters of water saturated with acetic acid ethyl ester and having a pH of 1.85 (adjusted with dilute aqueous hydrochloric acid). In this case, the acetic acid ethyl ester is pumped by circulating the acidified wash water against the dilute reaction solution and then the dilute hydrochloric acid is maintained by adding the pH to 1.85 as long as this pH remains constant after the acetic acid ethyl ester cycle.

The excess hydrochloric acid is then washed off by extracting the acetic acid ethyl ester phase twice countercurrently with 3.4 liters of water saturated with acetic acid ethyl ester. As soon as the pH of the washings is greater than 4.5, the organic phase is separated quantitatively, evaporated at 40-50 ° C under vacuum to 1/12 part of the starting volume of '40 (about 0.2 l) and diluted with 125 ml of ethanol.

84064 15

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

To prepare the test sample, the title compound is reprecipitated three times from ethanol / water and then dried for 15 hours at 50 ° C under vacuum.

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

The IR spectrum has two overlapping peaks in the CO valence frequency range, the other being due to the carbonyl function of the pyrone ring at 1635 cm-1, as is the case of silin-binin. The second peak is at a wavelength of 15 1730 cm-1 and is derived from both ester carbonyl functions.

The 1 H-NMR spectrum confirms that double esterification has taken place. The ratio of aromatic protons 20 obtained by integration to the methyl protons of the succinic acid group is 8: 8 (ppm range 5.9 to 7.1). The ratio of these methylene protons (ppm 2.6) to the methoxy group methyl protons (ppm 3.8) is 8: 3 and is thus compatible with this.

: - '25 The chemical shifts in ^ C studies also show that esterification has taken place in both alcohol-OH groups, as the chemical shifts are most strongly altered in the carbon Cu and adjacent carbon atoms C ± 2 “c14 and C2 - C4.

; .3o

Ai ukaineanal yysi 11 e C33H30O15 (682.60)

C H H

Calculated: 58.07 4.43 37.50 35 Found: 58.05 4.57 37.31

Example 2

Preparation of silibinin C-21,3-dihydrogen succinate disodium salt •; 40 tus ie 84064 CH2-O-C0-CH2-CH2-CO3Na

OH

OH 0 CO-CH2_CH2_CONa 10

The ethanol solution obtained according to Example 1 is added dropwise with stirring and cooling externally at -5 to 9 ° C to 15% of 6% ethanolic sodium hydroxide obtained by determining the dry matter content of this solution, the suspension is stirred for a further hour at room temperature, the precipitated beige solid is filtered off twice. In 5-10 minutes, use a Tur-Rax mixer to 150 ml of ethanol and re-separate by suction. To remove the residual ethyl acetate, the product is then suspended for 14 hours at room temperature in 280 ml of ethanol, filtered off with suction, 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: vacuum. 52 g of the title compound are thus obtained (yield 69%).

The title compound 1 has no sharp melting point.

It begins to sinter at about 80oC and melts at about 100oC to form vesicles.

UV spectrum in methanol: max = 288 nm, = 1.73 10 ^.

35

The molecular weight of the title compound is 726.56. The compound is a slightly beige, microcrystalline powder with no particular odor and salty taste. It is readily soluble in water, sparingly soluble in ethanol and practically insoluble in acetone, ether and chloroform.

Claims (4)

A process for the preparation of pharmacologically valuable silibinin-5 derivatives of the formula I is Ns / CH2-O-CH2-CH2-CH2-C00M1 10 H0 \ OCH, (I) OH o Nco-ch2-ch2-coom2 c wherein 20 and M2 are independently a hydrogen atom or an alkali metal atom, characterized in that 1 part by weight of silibinin of the formula (A); 25 n CH OH OH O 35 (A) Silibinin is dissolved in 1-2 parts of piano pyridine and reacted while mixing with 1 to 3 parts by weight of dicarboxylic anhydride of formula:; .40 18 84064 O = C - ch2 - ch2 - C = O 5 / N o then ethanol is added until a homogeneous mixture is formed, 10 then water is slowly added with vigorous stirring, whereby the esters of aromatically bound OH groups present are hydrolysed, immediately after this hydrolysis is complete, ethyl acetate is added, washed with acidic water saturated with ethyl acetate, the ethyl acetate phase is evaporated and, if desired, dissolved in ethanol to prepare the alkali salt and converted into the free carboxylic acid ester with an alcoholic alkali hydroxide solution. 20 84064 19 For the preparation of pharmacologically active substances 5 and 1 of the formulation I v> CH2-0-C0-CH2-CH2-C00M1 10 HO lOl I 15 "\ OH O CO-CH2-CH2-COOM2 from 20 and M2 are listed of varandra en väteatom eller alka- limetal1 Atom, kännetecknat därav, att 1 vikt-del silibinin med formuleln (A) or CH-OH 30 OH OH O 35 (A) Silibinin in 1 to 2 parts of pyridine and cycles reacting under the same velocity with 1-3 parts of dicarboxylic cyanohydride in the form of: 40 20 84064 O = C - ch2 - CH2 - C = O 5 ^ / O varefter ethanol tillsätts, tills en homogeneous blandning uppstär, varefter vatten vängsamt tillsätts under velvetig 10 kraftig omröring, varvid de aromatic bundna OH-pernas närvarande estrar hydrolyseras, omedelbart efter det denna hydrolys fullbordats, The ethyl acetate phase is further converted to an alkali salt of 15 with ethanol and a mixture of alcohol and an alcoholic hydroxide solution to give the carboxylic acid ester.