Note: Descriptions are shown in the official language in which they were submitted.
-
-1- 1337124
Flavolignan derivatives
The present invention is concerned with new
flavolignan derivatives, with processes for the
preparation thereof and with pharmaceutical composit-
ions containing these new compounds.
Lady's thistle (Silybum marianum (L.) Gaertn.
(Carduus marianus L.) is a medicinal plant which has
long been known. From the flavolignans occurring in
the fruits of this plant, R. Munster isolated a compon-
ent called silybin (cf. Dissertation R. Munster,
Munchen, 1966). The chemical structure of this com-
pound was elucidated by A. Pelter and R. Hansel (cf.
Tetrahedron Letters, London, 25, 2911-2916/1968).
It is known that silybin, previously also called
silymarin I, is a valuable liver therapeutic substance
(cf. Federal Republic of Germany Patent Specification
No. 17 67 666). A technical process for the prepar-
ation of silybin (silymarin I) is described, for
- 1337124
--2--
example, in Federal Republic of Germany Patent Spec-
ification No. 19 23 0~2.
As long ago as 1974, H. Wagner, P. Diesel and
M. Seitz (Arzneimittelforschung, 24 (4), (466-471)
assumed, with regard to silybin, two positional isomers,
namely silybin and isosilybin. This conjecture was
investigated and confirmed experimentally by A. Arnone,
L. Merlini and A. Zanarotti (J. Chem. Soc. Chem. Comm.,
16, 696-697/1079). According to this, the known silybin
consists of two different compounds, namely, the com-
pounds of the following structural formulae A and B:
~, o CH20H
HO ~ ~ OCH3
OH OH
OH O
(A) Silibinin
OH
HO ~ ~ ~ OCH3
OH
OH o
(B) Isosilybin
From these structural formulae, it can be seen
that these compounds are positional isomers. The
compound of formula A has recently been given the INN
- - 1 337 1 24
designation silibinin. This designation will now be
used herein for the compound of formula A.
The therapeutic use of silybin gives rise to the
difficulty that silybin is practically insoluble in
water.so that silybin-containing injection solutions
or preparations, in the case of which a certain water-
solubility is necessary, could not be produced.
Federal Republic of Germany Patent Specification No.
19 63 318 admittedly describes silybin derivatives
which possess a certain water-solubility but these are
very complex mixtures of semiesters of succinic acid.
This mixture is so complex because there are five
esterifiable hydroxyl groups in silybin, the silybin
also contains the two above-described positional isomers
and the succinic acid used for the esterification is a
dicarboxylic acid which can form not only monoesters
but also diesters. For ph~rm-ceutical purposes, a
product which consists of an unforeseeable number of
greatly differing and non-elucidated compounds cannot
be used.
It is, therefore, an object of the present
invention to provide water-soluble silibinin derivatives
which are suitable for phAnm~ceutical purposes and which
are precisely characterised as chemically individual
compounds.
We have found that silibinin derivatives of
certain alkane and alkylenedicarboxylic acids fulfil
1 337 1 24
--4--
these requirement~.
Thus, according to the present invention, there
are provided silibinin derivatives of the general
formula:-
~ o CH2-0-CO- ( Alkl ) n-COOMl
5 HO ~ / O ~ OHH3 (I)
OH 0 ( 2)m OOM2
wherein n and m, independently of one another, stand
for 0 or 1, Alkl and Alk2, independently of one another,
are alkylene radicals containing up to 4 carbon atoms
or alkenylene radicals containing 2 to 4 carbon atoms
and Ml and M2, independently of one another, are
hydrogen atoms or alkali metal atoms.
Preferred compounds of general formula (I) are
those in which m and n, independently of one another,
stand for 0 or 1, Alkl and Alk2 each stand for an
alkylene radical containing 2 carbon atoms and Ml and
M2, independently of one another, stand for alkali
metal atoms, m and n, Alkl and Alk2 and Ml and M2
preferably have, in each case, the same meanings.
The disodium salt of silibinin-C-2'!3-dihydrogen
succinate is especially preferred.
.
1 337 1 24
-- 5
In the case of the compounds according to the
present invention, the hydroxyl groups which are not
attached to a benzene nucleus of the silibinin are
partly or completely esterified, for exa~lple, by oxalic
acid, malonic acid, succinic acid, adipic acid, maleic
acid or fumaric acid. Preferably, the two non-
aromatically bound hydroxyl gro~ps of the silibinin
are simply esterified by one of the mentioned carboxylic
âcids.
In accordance with another aspect of the
invention there is provided a process for preparing a
silibinin derivative of formula (I) which comprises
esterifying the non-aromatic hydroxyl groups in
pure isosilybin-free silibinin of formula (11):
HO ~ ~ ~ ~O ~ 3
OH O
- with at least one dicarboxylic acid anhydride of
formula (III):
O=C-Alk-C=O
\ / (III)
lD
1 3371 2~
_ - 5a -
in which Alk is at least one of Alkl and Alk2 as
defined above. When the alkali metal salt is desired
the free carboxylic acid residues derived from the
anhydride are converted in conventional manner, for
example, by reaction with the alkali metal hydroxide,
carbonate or bicarbonate.
It will be understood that a mixture of
anhydrides (III) may be employed to produce derivatives
(I) in which Alkl and Alk2 are different. In such
case one of the non-aromatic hydroxyl groups in
- 1337124
, - 6 -
silibinin may be temporarily protected while the other
is esterified with an anhydride (III) in which Alk is
Alk~, whereafter the protective group may be removed
and the hydroxyl group esterified with an anhydride
(III) in which Alk is Alk2, Alk1 and Alk2 being
different.
Suitable protective groups and procedures
for temporarily protecting such hydroxyl groups and
subsequent removal of the protecting groups are well
1 0 k nown.
Preferably Alk~ and Alk2 are the same so that
a single anhydride (III) is employed.
Since the aromatically bound hydroxyl
groups in silibinin may also be esterified by the
anhydride (III), it will unusually be necessary to
saponify or hydrolyse the esterification product to
free the aromatically bound hydroxyl groups.
In a particular embodiment of the process
of the invention about 1 part by weight of silibinin
of the above-given formula (A) is dissolved in 1 to
2 parts by weight of pyridine and reacted, while
stirring, with 1 to 3 parts by weight of a di-
carboxylic acid anhydride of the general formula -
O=C-Alk-C=O
o
-~_ ~ 7 ~ 1 337 1 24
in which Alk stands for one of the above-defined Alk1
and Alk2 radicals, ethanol is then added until a homo-
geneous mixture is obtained, subsequently water is
admixed therewith, with intensive-stirring, esters
present in the aromatically bound hydroxyl groups
thereby being hydrolysed, as soon as this hydrolysis is
complete, the reaction mixture is diluted with ethyl
acetate, washed with acidified water which is saturated
with ethyl acetate, the ethyl acetate phase is evaported
~ - 8 - 1 3371 2~
and the residue is taken up in ethanol and converted
with an alcoholic solution of an alkali metal
hydroxide into the salt of the free, non-esterified
carboxylic acid residue.
The reaction with the dicarboxylic acid anhydride
is preferably carried out at 40 to 50C. The pH of the
ethyl acetate-saturated acidified water is advantage-
ously kept at about 1.5 to 2.4.
These compounds, especially the disodium salt of
silibinin-C-2',3-dihydrogen succinate, show, surprisingly,
an outstanding pharmacological action in the case of the
treatment of burn injuries. Furthermore, in spite of
the described derivatisation, they retain the complete
p~rm~cological effectiveness of the known silybin as
a liver therapeutic. They are especially suitable for
the treatment of liver cirrhosis and of toxic-metabolic
liver damage.
Surprisingly, the compounds according to the
present invention also prove to be extraordinarily
effective for the treatment of fungal poisonings, espec-
ially the very dangerous poisoning caused by the fungus
known as green death cap or deadly ageric (Amanita
phalloides). Poisonings by halogenated organic solvents,
such as carbon tetrachloride, trichloroethylene,
chloroform and the like, can also be surprisingly well
treated therewith. In the case of a prophylactic use,
the compounds according to the present invention
1 3371 24
prevent the above-described damage.
Therefore, the present invention also provides
pharmaceutical compositions containing at least one of
these new compounds, together with pharmaceutically
acceptable solid or liquid diluents or carriers. They
are usually employed systemically, for example, in the
form of pills, capsules, solutions and the like, in
conventional carriers and possibly together with
conventional adjuvants.
The daily dosage for an adult human amounts to
about 50 to 500 mg., depending upon the state of the
patient and the severity of the symptoms of the disease.
In a particular embodiment there is provided a
pharmaceutical composition comprising a silibinin
derivative (I) in association with a pharmaceutical
diluent or carrier, the compositions being sub-
stantially free of the corresponding isosilybin
derivative and substantially free of esters of the
silybinin derivative (I) and esters of the correspond-
ing isosilybin derivative.
The invention is illustrated by reference tothe accompanying drawings in which:
Figure 1 shows the fatty acid distribution in
microsomal liver lipids and the changes caused by
thermal skin damage, and
-- 1 o 1 3 3 / ~ 2 ~
Figure 2 shows the influence of a derivative
(I) of the invention on theblastogenesis of lymphocytes.
Experiments with the disodium salt of silibinin-
C-2',3-dihydrogen succinate (sili-suc-na).
The symptoms which arise in the case of burns
are especially brought about by intoxication by the
products of thermal tissue necrosis. Evidence that
autointoxication processes after severe skin burnings
are responsible therefor have been carried out in a
large variety of ways. Especially convincing are
cross-transplantations of burnt and non-burnt skin to
healthy and burnt recipient animals, it thereby being
demonstrated that unburnt recipients of burnt skin die,
whereaS burnt recipients of unburnt skin do not show
any harmful actions (see K. H. Schmidt, New Aspects
of Autointoxication after Severe Burnings: The
Burning Disease; F. W. Ahnefeld et al, eds. pub.
Springer Verlag, Berlin, pp. 45-5Z).
-- 11 1337124
In the case of skin burnings, the liberation or
new formation of a number of chemical compounds occurs.
In spite of the plurality thereof, it has been possible
to elucidate the structure of some of these compounds.
5 It could be shown, inter alia, that the con~pounds
resulting in the case of skin burnings possess a
similarity with those compounds which arise in the case
of lipid peroxidation. There are also analogies with
regard to the toxic actions of these substances.
10 Especially in~ressive is the formation of toxically-
acting saturated and unsaturated aldehydes of varying
chain length a~ a result of lipid peroxidation
(Benedetti et al., Identification of 4-hydroxynonenal
as a cytotoxic product originating from the peroxidation
15 of liver microsomal lipids, Bio~~h;~. 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
20 burnings lead to an oxidative ~n -g; ng of cell structures.
~ herefore, autooxidative changes of membrane
lipids were investigated as a result of an autointoxic-
ation after severe burnings. In particular, changes in
the fatty acid composition of the membrane lipids were
25 investigated. Furthermore, it was tested to what
extent the silibinin derivatives according to the
present invention influence the changes in the fatty
-12- 133712~
acid composition of the membrane lipids.
Chanqes in the fatty acid composition of membrane
lipids after severe burninqs.
Male Wistar rats with an average body weight of
360 g. were kept in groups of three with free access
to water and dry feed. Up to the commencement of the
experiment, the room temperature was 22C. and after
the commencement of the experiment the An;mAls were
kept at 30C.
The skin burnings were made with a copper stamp
with a surface area of 20 cm2 with constant pressure
and a temperature of 250C. In order to prevent a
thermal damage to deeper lying organs, the skin was
drawn over an air-cooled hollow spatula. Very exact
burning traumata can be made with this An;~-l model
which provide constant survival rates.
Before romm~ncement of the experiment, the
~n;mAl8 were narcotised with 50 mg./kg. of nembutal.
After the burning, 20 ml. Ringer lactate were injected
i.p. for shock prophylaxis.
Five experimental groups were used:
a) normal group: completely intact An;m-18
b) control group I: only silibinin treatment for 6
days with 75.5 mg. sili-suc-na
c) control group II: pseudo-operated ~n;~ -1 S
d) group with burned An;m~ls: 25%, 250C., 20 sec.,
0.5 at.
-13 - ~ 3371 24
e) test group: ~n;m~l s to which had been administered
75.5 mg. sili-suc-na i.p. for 6 days,
starting 1 day before burning.
For the isolation of microsomes, after the end
of the experimental period, the An;m~ls were bled under
narcosis. Subsequently, the liver was removed, weighed
and immediately transferred to an ice-cold isolation
medium (0.25 M saccharose, 1 mM EDTA, 10 mMol Triq.HCl,
pH 7.2). The liver was cut up and homogenised in the
medium. The microsome fraction was pelletised by
differential centA fuging. The microsomes were re-
suspended and again centrifuged. Subsequently, a sus-
pension was prepared in the case of which 1 ml. of
suspension corresron~d to 1 g. of liver tissue.
The lipids were determined by the method of
J. Folch (A simple method for the isolation and purific-
ation of total lipids from ~n;m-l tissues, J. biol. Chem.,
226, 497-508/1957, modification of Bligh and Dyer (A
rapid method of total lipid extraction and purification,
Can. J. Biorh~m. Physiol., 37, 911-917/1959).
The extracted microsome lipids were saponified
with an aqueous sodium hydroxide solution. The free
fatty acids were esterified by the addition of BF3-
methanol. After evaporation of the methanol and Lel,oval
of hydrophilic by-products, the fatty acid esters were
determined quantitatively.
~ - 14 - 1 337 1 2~
In the case of the unburned group of Ani~Als, no
noteworthy change of the fatty acid pattern could be
ascertained. Thus, the narcosis and minor operational
intervention did not result in a change of the micro-
somal lipids. For this reason, for further comparison,the normal group and the control group were combined
to one control group.
A comparison of the unburned and of the burned
An;~ls with regard to their micrsomal fatty acid
pattern showed a serious displacement of unsaturated
to saturated fatty acids.
Fig. 1 of the accompanying drawings shows the
fatty acid distribution in the microsomal liver lipids
and the changes caused by the thermal skin damage. The
proportion of palmitic acid (C16) increased after
burning from 25.1 to 34.4% of the total fatty acids.
In the case of stearic acid (C18), the proportion in
the case of the burned A~ lS was, with 46.3~, 13.20~
above the value obtained with the control An;~-ls, In
the case of oleic acid (C18:1), a slight, insignificant
decrease is detectable. The proportion of linoleic
acid (C18:2) was decreased after burning to about one
third of the initial value. Finally, in the case of
arachidonic acid (C20:4), after burning there was found
only 31% of the initial content.
The following Table shows the influence of sili-
suc-na on the proportions of fatty acids in the burned
_ - 15 - 1337124
and in the unburned An;m~lS
Table
Fatty acid pattern of the microsomal lipids of the rat
liver after sili-suc-na therapy in the case of burned
- 5and unburned animals
C16 C18 C18:1 C18:2 C20:4
unburned 29. 8~o 37.~/O 8. 9yo 9`. 6% 16. 2%
(control
group I) + 6.2 +12.3 +1.1 +3.3 +4.9
25.4% 37.5% 7.8% 11. 4% 18 . 0~
burned
+6.0 +8.6 +1.0 +5.3 +9.1
It can be seen that treatment with a silibinin
derivative according to the present invention does not
give rise to any important changes in the case of the
unburned control ~n;m~l s in ~omr~rison with the
untreated An;m~l s. In the case of the burned An;m-ls,
the therapy resulted in a complete removal of the loss
of unsaturated fatty acids.
To summarise, the following can be said: Burnings
result in changes of the fatty acid pattern of micro-
somal lipids. It is to be assumed that this is to beattributed to oxidative damage of the membranes. This
is shown especially by the marked decrease of the poly-
unsaturated fatty acids.
"-
.
~ - 16 _ 1 337 1 2~
The silibinin derivatives according to the
present invention are thus able to inhibit oxidative
cell damage. Therefore, they are especially useful
for interrupting oxidative damage mechaniqms after
severe burnings.
As already mentioned, it is assumed that auto-
toxic reactions after severe burnings lead especially
to oxidative cell damage. We have, therefore, investi-
gated what effect a standardised thermal trauma has on
the PHA-induced blastogenesis of T-lymphocytes from the
spleen and the peripheral blood of rats. Furthermore,
we have investigated how the silibinin derivatives
according to the present invention influence such
lymphocytaeric functional disturbances after severe
burnings.
Action of a standardised thermal trauma on the PHA-
induced blastoqenesis of T-lymphocytes from the spleen
and the peripheral blood of rats.
The back skin of Wistar rats was ~urned with a
copper stamp in the above-described manner. As control
group, there were used pseudo-burned ~n;mAls on which
were carried out all the operative manipulations but
without burning. After 2, 4, 7 and 9 days, blood and
spleen were removed from the burned and from the control
~n;m~l S under ether narcosis.
For the isolation of the lymphocytes, heparinised
blood was coated on to FICOLL-HYPAQUE* solution (density
* Trademark
^-`~'`"
A-`
~ - 17 - 1 337124
1.077). Subsequently, it was centrifuged and the
lymphocytes obtained tested with tryptane blue for
their vitality. For the isolation of the spleen lymæho-
cytes, the organ was comminuted, passed through a sieve
and freed from accompanying erythrocytes by means of
Gay' 8 lysis solution.
Subsequently, the cell mixture was incubated in
a vessel in the presence of 5% heat-inactivated foetal
calf serum for 30 minutes in order to reduce the pro-
portion of mono-nuclear cells in the suspension by
adhesion to the vessel wall (5%). For culturing, the
cells were introduced into flat-bottom microtitre plates.
20~ foetal calf serum was then introduced. In this way,
the spontaneous blastogenesis was determined by measure-
ment of the incorporation of 3H-thymidine-(2Ci/mM) into
the DNA of the cells.
In previous experiments, it was elucidated that
an optimum mitogen stimulation takes place in the case
of a PHA concentration (mitogen phyt~e~gglutinin) of
5 ~mg./ml. In the case of these experiments for the
optimisation of the cellular test system, it was also
ascertained that the m~i ml~m stimulation of the new
synthesis of DNA took place after 72 hours. Further-
more, it was ascertained that the optimum concentration
of foetal calf serum is 2~ in order to achieve the
highest stimulation.
- 18 ~ 1 337 t 2~
As described above, the spontaneous blastogenesis
was determined by measurement of the incorporation of
3H-thymidine into the DNA of the cells. The cells were
collected 18 hours after the addition of 3H-thymidine,
the zero point for the 18 hours thereby coinciding with
the time point of mAX; mllm stimulation.
For the investigation of the action of the
silibinin derivatives according to the present invention,
a group of rats were treated with a silibinin derivative.
For this purpose, 75.5 mg. of sili-suc-na were injected
i.p. once per day. This therapy was carried out from
the day of burning up to the day on which the organs
were lel.io~ed (m~; ml-m Up to the 9th day).
For the evaluation of the results obtained with
the control Anim-ls and with the sili-suc-na-treated
An;mAlS, the stimulation index was calculated. This
numerical value represents the quotients of the average
value of the stimulated sample and the average value of
the control sample. From the stimulation index thus
obtained in the case of each experimental An;mAl, there
was calculated an average qtimulation index per An;~-
group. The results obtained are expressed by the
index SI.
Fig. 2 of the accompanying drawings shows the
influence of the sili-suc-na used on the blastogenesis
of lymphocytes. In the case of the burned An;mAls,
the reduced stimulation ability of the cells was
~ - 19 -
- 1 337 1 24
markedly increased by the silibinin derivative.
Already on the second day, in the case of the
sili-suc-na-treated ~n;m~ls~ there was found an about
10 times higher responsivity of the blood lymphocytes
S towards PHA. On the fourth post-traumatic day, the
value for the stimulation index in the case of blood
lymphocytes for treated An;m~ls was 8, whereas the
corresponding value in the case of the untreated An;mAls
was 1.5.
In the case of spleen cells, the stimulation
indices of the burned, untreated An;m~ls were all
markedly under 1. The administration of the silibinin
derivative results in a significant imp-o~e~-Rnt on all
investigated days, a maximum being found on the 7th
post-traumatic day.
Comparative experiments were also carried out
which showed that sili-suc-na alone in the case of
healthy An;m-ls did not result in any significant
changes in the stimulation ability of the PHA-induced
blastogenesis of T-lymphocytes from the spleen and from
the peripheral blood.
Thus, the silibinin derivatives according to the
present invention stimulate the blastogenesis of
lymphocytes of burned Ani~ls in a significant manner.
It was also ascertained that, in the case of
An;mAlS treated with the silibinin derivatives according
to the present invention, the general catabolism was
~ - 20 - 1337124
lower since, after the thermal trauma, the An;m~ls
again rapidly increased in weight.
Funqal poisoninqs.
Poisonings due to deadly ageric (Amanita
phalloides) belong to the most serious ones found in
medicine. Although only 10 to 30~ of all fungal
poisonings are caused by the Amanita phalloides,
poi onings with this fungus have always attracted great
medical interest because of their danger. In older
publications, the lethality was said to be 30 to 50%.
Thanks to modern intensive medicine, according to a
collective study by Floersheim et al. on 205 patients,
this value has been reduced, on average, to 22.4%.
The poison from Amanita phalloides, amanitin,
can be fatal for adult humans even in an amount of 7 mg.,
this amount of poison being present in about 50 g. of
fresh fungus.
After a series of ~n;mAl e~periments which
promised success, the active material sili-suc-na was
used in the therapy of poisoning by Amanita phalloides.
28 patients with Amanita phalloides poisoning
were treated with sili-suc-na, in addition to conventional
therapeutic measures. Of these 28 patients, only one
died who had taken comparatively large amounts of the
fungus with suicidal intent. This result ~emon~trated
an enormous therapeutic advance in this field.
- 21 - 1 337 1 24
Preparation of isosilybin-free silibinin
A suspension of 500 g. of the product according
to Federal Republic of Germany Patent Specification
~o. 19 23 082 (see column 8, lines 14-19), with a
silymarin content of about 7~/O and an isomer ratio of
silybin/silidianin/silicristine of about 3:1:1, the
silybin cont~; ni ng about one third of isosilybin, in
2 kg. methanol (about 2.53 litres) is heated to the
boil for 15 minutes, while stirring. After thi~ time,
some silibinin can already precipitate out of the sol-
ution thus obtained. Subsequently, 0.75 to 1.25 kg.
(about 0.96 - 1.58 litres) of methAnol are ~ .o~ed in
a vacuum and the residue is left to stand for 10 to 28
days at ambient temperature. The precipitated silibinin
is filtered off and washed twice with 50 ml. amounts of
cold methanol. After drying in a vacuum at 40C., the
isolated crude silibinin is further purified in the
following manner:
60 g. of crude silibinin are dissolved, with
heating, in 3 litres of technical grade ethyl acetate.
Subsequently, 20 g. active charcoal are added thereto
and the mixture is heated under reflux for a further
2 hours, while stirring. Thereafter, it is clarified
by filtration and the solution is evaporated at 50&.
under reduced pressure to about 250 ml. The concentrate
is stirred for 15 minutes with the use of an Ultra-
Turrax apparatus and, while stirring, mixed with 25 ml.
- 22 - 1 337 1 24
of methanol. Subsequently, the mixture is left to
stand overnight at ambient temperature. Before filter-
ing off with suction the thereby precipitated silybin,
stirring is again carried out with an Ultra-Turrax
apparatus for 5 minutes. The suction-filtered precip-
itate is washed twice with 50 ml. amounts of ethyl
acetate and dried overnight at 40&. in a vacuum drying
cabinet. The product obtained is subsequently ground
and further dried for 48 hours under the same conditions.
The following Examples are given for the purpose
of illustrating the present invention:
Example 1.
Preparation of silibinin-C-2',3-dihydroqensuccinate.
I ~ o ~ ~ CH2--0-CO--CH2--CH2--COOH
HO ~ ~ O~3
OH 0-CO-CH2-CH2-COOH
50 g. of silibinin are dissolved at 45C. in
70 ml. pyridine, 50 g. succinic anhydride are added
thereto, the mixture is stirred for about 8 hours at
45C., 30 ml. eth~nol are added thereto and the mixture
is further stirred until a homogeneous mixture has
formed. Subsequently, 60 ml. of water are added
thereto, with intensive stirring, for the saponific-
- 23 _ 1 337 1 2~
ation of the phenyl esters, wnthin the course of about
30 minutes. After stirring for about 1 hour at 30C.,
the phenyl esters are quantitatively hydrolysed. The
completeness of the hydrolysis is tested by means of
HPLC. The hydrolysis is stopped by rapidly adding
1.7 litres of ethyl acetate to the reaction mixture
thus obtained.
For the separation of excess succinic acid and
of pyridine, the reaction solution diluted with ethyl
acetate is extracted twice in countercurrent with 5
litre amounts of water which has been saturated with
ethyl acetate and has a pH of 1.85 (adjusted with
- dilute aqueous hydrochloric acid). The ethyl acetate-
saturated, acidified wash water is thereby pumped in
a cycle counter to the diluted reaction solution and
subsequently the pH is maintained at 1.85 by the
addition of dilute hydrochloric acid until this pH
value remains constant after the ethyl acetate passage.
Subsequently, for washing out excess hydrochloric
acid, the ethyl acetate phase is extracted twice in
countercurrent with 3.4 litre amounts of water which
has been saturated with ethyl acetate. As soon as the
pH value of the wash water is greater than 4.5, the
organic phase is separated off quantitatively, evapor-
ated in a vacuum at 40 to 50C. to one twelfth of theinitial volume (about 0.2 litres) and then diluted with
125 ml. ethanol.
- 24 _ 1337124
The title compound is obtained by reprecipitation
from ethanol/water and drying in a vacuum at 50C. for
15 hours.
For the preparation of an analytical sample, the
title compound is reprecipitated three times from
ethanol/water and subsequently dried in a vacuum for
15 hours at 50C.
In the FD mass spectrum a molecule peak appears
at the expected molecular weight of 682.
The IR spectrum shows, in the region of the C0
valency frequency, two overlapping bands, whereby one,
as is also the case for silibinin, is to be associated
with the carbonyl function of the pyrone ring at a
wavelength of 1635 cm 1, The second band is at 1730 cm 1
and originates from the two ester carbonyl functions.
The H-NMR spectrum confirms that a twofold
esterification has taken place. Thus, the ratio,
determined by integration, of aromatic protons to
methylene protons of the succinic acid residue, amounts
to 8:8 (ppm range 5.9 - 7.1). The ratio of these
methylene protons (ppm 2.6) to the methyl protons of
the methoxy radical (ppm 3.8) amounts to 8.3 and is
thus in agreement therewith.
The chemical displacements in the ca~e of the
3C-investigations also show that the esterification
of the two alcoholic hydroxyl groups has taken place
since the chemical displacements change the most
-25_ l 337 1 24
strongly at Cll and the adjacent carbon atomq C12-C14,
as well as at C2-C4.
Elementary analysis:
33H30016 (M.W, 682,60)
calc.: C 58.07%, H 4.4%0 37.50~
found: 58.05%, 4.57%,37.31%
Example 2.
Preparation of the disodium salt of silibinine-C-
2',3-dihydroqen succinate.
To the ethanolic solution obtained according to
Example 1, there is added, while stirring and cooling
externally at -5 to 9C., 6% ethanolic sodium hydroxide
solution in an amount based upon the determination of
the solids content of this ethanolic solution. The
suspension is further stirred for 1 hour at ambient
temperature, the beige-coloured precipitated solid is
filtered off with suction, suspended twice, in each
case for 5 to 10 minutes, with the he~p of a Turrax,
in 150 ml. ethanol and again filtered off with suction.
For the ell~o~al of residual ethyl acetate, the product
is subsequently suspended for 14 hours at ambient temp-
erature in 280 ml. ethanol, again filtered off with
suction, then washed with 70 ml. ethanol and dried for
15 hours in a vacuum drying cabinet at 40 to 45&.
The previously dried product is subsequently ground,
sieved to a particle size of less than 0.2 mm. and
again dried in a vacuum for 48 hours at 40 to 45&.
` - - 26 - 1 337 1 24
There are thus obtained 52 g. (6~ of theory) of the
title compound.
The title compound does not possess a sharp
melting point. At about 80C., it begins to sinter
and melts, with bubble formation, at about looc,
The W spectrum in methanol shows: ~max = 288 nm,
= 1 73,104.
The molecular weight of the title compound is
726.56. The compound is a light beige-coloured,
microcrystalline powder without a specific odour and
with a salty taste. It is readily soluble in water,
sparingly soluble in ethanol and practically insoluble
in acetone, diethyl ether and chloroform.
Example of use.
Preparation of a lyophilisate for intravenous
administration.
disodium salt of silibinin-C-
2',3-dihydrogen succinate75.0 mg.
mannitol 10.0 mg.
water for injection purposesad 1.5 ml.
1.5 ml. of solution is placed in a pointed
ampoule of 5 ml. capacity and then freeze-dried in known
manner. For storage purposes, the ampoule containing
the final lyophilisate is closed in the usual manner.
For use, the lyophilisate is dissolved in 5 ml.
of sterile, physiological saline solution to give a
clear solution.
