Note: Descriptions are shown in the official language in which they were submitted.
S3;2
A 3,4-dihydroxy cinnamic aldehyde derivative, its
preparation and use.
The invention relates to a nove:L derivative of 3,4-
dihydroxy cinnamic aldehyde and its preparation, the invention
is also concerned with pharmaceutical compositions containing
the derivative~
The new derivative has a surprisingly satisfactory
anti-hepatotoxic effectproviding a welcome extension in the
effective spectrum of liver agents.
According to the invention there is provided a 3,4-
dihydroxy cinnamic aldehyde derivative of formula:
X~~ ~
~C/~O CH2H ~H2H
e ~I ~H
O = ~ H or , ~
H ~ OH
and the salts thereof, particularly the pharmaceutically
acceptable, pharmacologically compatible salts.
The new derivative has been named "Americanin".
In another aspect of the invention there is provided
a process for preparing the novel derivative comprising
a) extracting a Phytolacca fruit containing said derivative
with an organic solvent system, concentrating the extract to t
obtain a residue and fractionating the residue to~recover said
derivative, or b) oxidatively reacting 3,4-dihydroxy cinnamic
~ ~ 1 - ~ .
532
aldehyde with 3,4 dihydroxy cinnamic alcohol, or c) oxidatively
reacting 3,4-dihydroxy cinnam.ic aldehyde with allylpyrocatechin.
In an embodiment of the process of the invention the
novel derivative is converted into a corresponding pharma-
ceutically acceptable, physiologically compatible salt.
l'hus in one embodiment the new clerivative is ohtained
by extraction and fractionating of a fruit of the Phytolacca
type, more particularly Phytolacca americana L.. In a particular
embodiment the finely-ground dried fruit is extracted with
acetone after treatment with petroleum-ether 7 the residue from
the concentrated extract is washed with chloroform and dissolved
in n-butanol, the n-butanol solution is wash~d with water,
concentrated, and the residue obtained i~ dissolved
in acetone, and the acetone solùtion is chromatographed in a
column with for example, Sephadex L~I 20~ (a three-dimensional
network of polysaccharide with crossllnked dextran chains), using
acetone as the elution-agent; the pharmacologically active frac-
tions are washed, and the residue is recrystallized out of methanol.
The process may be carried out in accordance with the
following preferred procedure:
a) finely-ground dried fruit of PhYtolacca americana L. is
extracted three times, for 0.5 to 1.5 hour~, preferably about
one hour, with from 2.5 to 4.5, preferably about 3.5 times the
amount by weight of petroleum-ether having a boiling range of
60 - 70C, at a temperature of 45C to 55C, preferably about
50C, with vigorous stirring;
b) the thus treated fruit i5 extracted three times, for from
0.5 to 1.5 hours, preferably about one hour, with from 2.5 to
4.5, preferably about 3.5 times the amount by weight of acetone,
at from 45C to 55C preferably about 50C, w,ith vigorous
stirring,
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c) the acetone extract from b) is concentrated, at from 35C
to 45C, preferably about 40C, in a vacuum, at from 5 to 50,
preferably about 20 mbars;
d) the residue obtained in c) is stirred out twice with from
1/4 to 1/2, preferably about 1/3 of chloroform, by weight, based
on the weight of the original drug residue, and is filtered;
e) the solid from d) is dissolved in n-butanol in a weight
ratio of from 1:10 to 1:14, preferably 1:12;
f) the n-butanol solution is shaken out ~rom 3 to 5 times,
preferably about 4 times, with from 1/3 to 2/3, preferably about
1/2 the volume of water;
g) the n-butanol phase from f) is concentrated at from 35 to
45~, preferably about 40C, under vacuum, at from 5 to 50,
preferably about 20 mbars;
h) the residue in g) is dissolved in from 0.05 to 0~15,
preferably about 0.10 times the amount of acetone, by weight,
based on the weight of the original drug, and is filtered;
i) the acetone solution.is chromatographed in a column with
a Sephadex LH 20* using acetone as the elution-agent;
j) the pharmacologically active fractions thus obtained are
washed and are concentrated at from 35 to 45C, preferably
about 40C, under a vacuum, at from 5 to 50, preferably about
20 mbars;
~: k) the resulting residue is recrystallized out of methanol.
The product of the invention thus obtained is a
: light-yellow crystalline substance having the following pro-
perties: melting point 246-247C with decomposition, empirical
formula C18H1606: molecular mass 328; element analysis: C 64.98%~
H 4.98%~ O 28~/o~ Rf value 0.17 by thin-layer chromatography,
using silica-gel-60 finished plates by Merck and chloroform/-
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acetone/formic acid 9/2/1 as tne fluidizing system, practically
insoluble in water, chloroform, benzene and petroleum ether;
soluble in dimethyl sulphoxide, dimethyl formamide, tetrahydro-
furane, dioxane and acetone, optical rotatory power C~317 ~ 23.7
and IR absorption at 3200 cm (-OH), 1650 cm 1 (~,~ unsaturated
C = 0), 1610 cm 1 ( ~ C - C ~ ) and 1580, 1510 and 1450 cm 1
(aromat. system).
The new derivative may also be obtained by dimerizing
- 3,4-dihydroxy cinnamic aldehyde and 3,4 dihydroxy cinnamic
alcohol by oxidative addition.
The procedure is preferably as follows: equimolecular
amounts of 3,4-dihydroxy cinnamic aldehyde and 3,4-dihydroxy
; cinnamic alcohol are allowed to react in 5 times the amount of
the total reagents in a solvent, suitable solvents include
benzene, dioxane, tetrahydrofurane, and acetone, at from 15C
to 30C preferably about 23C, for from 1.5 to 3, preferably
about 2 hours, under the action of silver oxide, in an amount
of 0.5 to 0.6, preferably about 0.55 mole per mole of total
reactants; the filtered reaction solution is dried and the
residue is separated by column chromatography.
The product thus obtained synthetically has the same
characteristics as that obtained by isolating from fruit of the
Phytolacca type. ~-~
The new derivative may also be obtained by reacting -~
3,4-dihydroxy cinnamic aldehyde with allylpyrocatechin,
suitably in an organic solvent, by oxidative addition; whereafter
the methyl substituent in the benzodioxane group is oxidized
to an alcohol function.
It is appropriate to protect the hydroxyl groups
during the oxidation of the methyl group and any conventional
protecting groups for aromatic hydroxy groups may be employed,
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which protecting groups can be readily removed after the
oxidation, as by hydroly~is. Suitable protecting groups inelude
acyl groups, for example acetyl and benzoyl,. Thus in one
embodiment the intermediate product from the oxidativa addition
is acetylated and after the oxidation of the methyl, the aeetyl
protective groups are removed by weak alkali hydrolysis.
In a preferred embodiment the procedure is as follows:
equimoleeular amounts of 3,4-dihydroxy cinnamie aldehyde and
allylpyroeatechin are allowed to reac-t in 5 times the amount of
total reaetants in a solvent,suitable solvents inelude benzene,
-dioxane, tetrahydrofurane and aeetone, at a temperature of from
15C to 30C preferably about 25C, for from L.5 to 3, preferably
about 2 hours, under the action of s.ilver oxide, in an amount
of from 0.5 to 0.6, preferably about 0.55 mole per mole of
total reaetants; the product thus obtained is acetylated in
known fashion, and is isolated by column ehromatography, after
which the methyl substituent on the benzodioxane group is
oxidized in known fashion to an alcohol funetion, whereafter
the aeetyl groups are removed by hydrolysis.
The synthetic product thus obtained has the same
characteristics as that obtained by i~olation from fruit of
the Phytolacea type.
The reaction seheme is illustrated as follows:
- 5 ~
~Z~3~i3~
C ~ H ~ o 55 Mol/Ml 2 ~oQrs
/ H in benzene with : -
: H trace of CH30H
-: ,CH3
O ~ o ~ Oxidation
"CH2QH
~ ~/c~o~o~ ~
H OH ~:
;An initial structural clarification of the derivative
of the invention produced the following formula:
'' ~ .
OH
H ~ O ~ CH2~ ~ CH2H
C ~ \ H OH
H or
. .
. .
,, . ~ '
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This substance may be converted in the usual manner
into its pharmaceutically acceptable, physiologically compatible
salts. Weakly-basic agents suitable for salt formation
include, for example inorganic bases including alkali car-
bonates and bicarbonates, and organic amines for example tri-
ethanolamine, trimethylamine and amino-sugars, for example
methyl glucamine.
In this specificatibn it will be understood that the
qualification that the salts be "physiologically compatible"
is to be understood as extending to salts of non-toxic inorganic
or organic bases which have no adverse effects to the extent that
such salts would be unsuitable for administration to living bod-
ies. For incorporation into pharmaceutical compositions it
will be recognized that such salts should also be pharmaceuti-
cally acceptable in the sense that the salts should have the
necessary physical characteristics, for example, stability, to
render them suitable for formulation into pharmaceutical
compositions.
Salts which are not pharmaceutically acceptable and
pharmacologically compatible form a useful aspect of the in-
vention of the novel derivatives, inasmuch as they can be readily
converted, to different salts having the requixed physical and
chemical characteristics to make them suitable for administration
in pharmaceutical compositions to living bodies.
The novel derivative of the invention may be used
eit~er as a crude extract or as a crystalline product for
medicinal purposes, as a liver therapeutic. It may be used in
various pharmaceutical forms including tablets, capsules,
granular substances, dra~ees, suppositories and liquid pre-
parations, for oral, anal or parenteral administrationD Thedose for human beings is between 200 and 450 mg~day, depending
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upon the gravity of the case.
In the course of investigations involving experiments
with animals, the active substance Americanin, described above,
was found, surprisingly enough, to provide pronounced protection
against influences damaging to the liver. Americanin produces
a particularly protective and stabilizing effect upon cellular
and intracellular biomembranes, especially liver cells, and so
provides excellent protection for the liver and may therefore
be used in treating diseases of the liver. -
The anti-hepatotoxic effectiveness of Americanin
is demonstrated as follows: ~-
Female mice were injected with the liver toxin
phalloidin (3 mg/kg; i.p.). The test animals were injected
with Americanln one hour prior to the phalloidin injections,
the remainder represent control animals.
The Americanin was dissolved in (1:1) dimethyl~
sulphoxide (OMSo) - polyethylene ylycol and was administered
intravenously, in doses of 50 and 100 mg/kg one hour prior
to the phalloidin injections~
- 20 Results:
85% of the mice injected with phalloidin died, whereas
treatment with 50 and 100 mg/kg of Arnericanin reduced the death
rate to 60 and 3~/0 (period of observation: 7 days, 20 animals
in each group).
The i.v. toxicity of Americanin was checked in male
and female mice. The values obtained were 820 mg/kg for male
mice and 800 mg/kg for female mice.
- The invention is further illustrated by reference
to the accompanying drawings in which:
Figure 1 shows the I.R. spectrum of the nove:L deri-
vative, and
Figure 2 shows the mass spectrum of the novel
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derivative.
The inven~ion is still further illustrated by re-
ference to the accompanying examples which describe particular
and preferred emhodiments.
Example 1
100 Xg of finely-ground dried Phytolacca americana L.
fruit were extracted three times, with the aid of an Ultra-
Turrax unit, each time for a period of 1 hour, with 400 litres
of petroleum ether having a boiling range of 60 to 70C, at
50C. The drug thus pretreated was then subjected three times
to 1 hour Turrax extraction, each time with 400 litres of
acetone, at 50C. The combined acetone extracts were con-
centrated in vacuo at 40C. The residue obtained was stirred
out twice, each time with 30 litres of chloroorm. The mixture
was filtered and the remaining solid was dissolved in n-butanol
in a l:l~ ratio. The n-butanol solution was shaken out four
times with half its volume of waterO The butanol phase was
thereafter concentrated in vacuo at 40C, the residue was dis-
solved in 10 litres of acetone. After separation of undissolved
material, the solution was subjected to column-chromatography
with Sephadex LH 20*, using acetone as the elution~agent. The
fractions containing the active substance were washed and con-
centrated in vacuo at 40C. The residue was recrystallized
out of methanol.
The end product obtained had the following chemical
and physical parameters:
Appearnace: a light-yellow microcrysta~line solid
Solubility: soluble in dimethyl sulphoxide, dimethyl
~ - formamide, tetrahydrofurane, dioxane and
;~ 30 acetone; slightly soluble in methanol,
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ethanol and butanol; almost insoluble in
water, chloroforml benzene and petroleum
ether
Melting point: 246-247C ~decomposition)
Empirical
formula: C18H16~6 ~-~
Molecular mass: 328
Element
analysis C64.98 ~calculated: 65.85~
EI4.9~ ( " 4.91)
o28.3 ( " 2~.24)
Thin-layer chromatograp~y:
silica-gel finished plates (Merck)
Fluidizer: chloroform, acetone, formic acid 3/2/1 tV/V/V)
Development: under chamber saturation
Detection: spray reagent 2,4-dinitrophenylhydrazine/-
sulphuric acid (1 g of dinitrophenyl-
hydrazine suspended in 2 ml of conc.
sulphuric acid, made up to 100 ml with
methanol).
After the plate had been sprayed, heating
was carried out for 20 minutes at 120C.
The substance appeared as an intense
yellow zone on a light-yellow ground.
Rf value: 0.17.
The IR spectrum and mass spectra are reproduced in
Figs. 1 and 2.
Galenical_examples:
Production of tablets
70 Xg of the active substance of the invention was
mixed with the following accessory substances:
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10,000 kg of polyvinyl pyrrolidone
14,400 kg of microcrystalline cellulose
17,400 kg of amylum tritici
6,500 kg of aerosil
10,000 kg of stearic acid
- 371,700 kg of lactose DIN 80.
- 0.50 g tablets, containing 70 mg of active substance, were
moulded from the mixture.
Production of preparations for iniectinq ~ampoules),
For the production of 10,000 ampoules, 15~945 kg of
methyl-glucamine salt of the active substance of the invention
were dissolved in 48.6 litres of a physiological solution of
kitchen salt (sodium chloride) to which 4% of polyvinyl
pyrrolidone (M~G.^V 10,000) had been addedr The pH value
was maintained at at least 7.6. The solution was sterile-filter-
; ed and was filled into sterile, brown 5 ml ampoules. Each
ampoule thus contained 159.45 mg of the N-methyl-glucamine
salt~ corresponding to 100 mg of the active substance of the
invention,
Production of suppositories
318.9 g of the methyl-glucamine salt of the active
substance of the invention were rubbed with 500 g of melted
DAB 7 hard fat. 1,181.1 g of Deutsches Arzneibuc~ - German
Parmacopoeia 7 melted hard fat was stirred in and suppositories
were cast from the mixture. Each 2.0 g suppository contained
318.9 mg of the methyl-glucamine salt, corresponding to 200 mg
of the active substance of the inventionO
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