Note: Descriptions are shown in the official language in which they were submitted.
~ 2~ ~
N~/ A~OYINCA~iINO~ DE~IYA'~IVES
Thi~ invention relate~ to new apovincaminol derivati-
vee, a prOCe~8 for the preparation thereof and pharmaceutical
compo~ition~ cont~i n ~ ng the ~ame.
Accordlng to an a~pect of the present invention there
ar~ provlded new apo~incaminol derivative~ of the general For-
mula /I/, ~1
~ N
~l.... J /I/
c~o~&
~3~
o~3
namely the apovincaminol-3',4',5'-trimethoxy-benzoate o~ the
Formula /Ia/
C~
O~ -2~5
r~
tH~O ~ /la/
ct~
the 9-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate of the
~ormula /Ib/
A 2~87-67/Fe
~i~
~f~
N02
~c~l~
o C21ls
3 ~ ICO ' /Ib/
C1130 ~
cH3b
and the ll-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate of the
Formula /Ic/
0 N
o ~CH2 ~ 2H5
C / /Ic/
3 ~ J
C~130
CH.,b
and pharmaceutically acceptable acid addition salts thereof.
In the general Formula /I/ either both Rl and R stand for
hydrogen, or one of the symbols Rl and R2 represents hydrogen and the other
nitro.
The acid addition salts of the compounds of the general
Formula /I/ may be formed with inorganic or organic acids. From the salts
formed with inorganic acids the hydrochlorides, sulfates and phosphates
hile from the salts formed with organic acids the hydrogen tartarates,
succinates, citrates and ascorbates are particularly useful.
It is known that the apovincaminol and its acetate exhibit an
effect on the coronary artery /French Pa-tent Specification No. 2 035 784/.
It is also kno~m th~t the apovincamino] benzoate possesses general
asodilatory properties /llungarian Patent Specification No. 166 476;
CA 82, 129 279 v /1975// and the esters of apovincaminol formed with
alkane carboxylic a~id
~ ~3~
-- 3 --
having 3-12 carbon atoms exhibit cerebral vasodilatory effect /Hungarian
Patent Specification No. 171 662, the corresponding US Patent Specification
No. 4 108 996 and the BR German Federal Republic Patent Specification No.
26 32 118/.
Thus all the known esters of apovincaminol exhlbit vasotropic
effects. On the other hand the new compounds of the present invention
inhibit the enzyme activity of phosphodiesterase and can be used first of
all in the treatment of skin diseases attached to the pathological cell pro-
liferation and in the prophylaxis of the recurrence of such diseases.
Diseases attached to the pathological proliferation of the
epidermis are relatively frequent and involve a few per cents of the popula-
tion. These diseases can be of both benign and malignant character such as
psoriasis atopias dermatitis, primary contact dermatitis, allergical contact
dermatitis, baso- and spinocellular carcinoma, inchthyosis, premalignant
hyperceratosis, light induced ceratosis, acne and seborrhoeas dermatitis.
Some diseases occur only on humans while others both on humans and animals.
Since some of the skin diseases attached ~o pathological cell pro-
liferation /e.g. psoriasis/ do not occur on animals, the activity of the com--
pounds against psoriasis can be made probable in animal experiments only
indirectly.
Voorhees et al. /Arch. Derm. 104, 359-365, /1971// have found that
the pathological cell proliferation is accompanied by the decrease of the
level of cyclic adenozine monophosphate /c-AMP/. It is known that c-AMP is
formed under the effect of adenyl-cyclase and decomposed by phosphodiester-
ase. Voorhees succeeded in influencing the psoriasis by agents which stimu-
late the function of adenyl cyclase /such as nor-epinephrine/ or inhibit the
function of phosphodiesterase /e.g. papaverine/.
By planning our model experiment test we have started from the pre-
sumption that the statement of Voorhees is relevant to the contrary as well.
Thus if it can be proven that a certain compound inhibits the function of
-- 4 --
phosphodiesterase this makes it probable in an indirecc way that the said
compound is suitable for the treatment of skin diseases attached to the
pathological cell proliferation.
This presumption turned out to be true: compounds showing phos-
phodiesterase inhibiting activity in in vitro tests proved to be active in
the treatment of psoriasis in clinical experiments as well.
Our model tests are carried out with the aid of phosphodiesterase
isolated from animal body tissues /rat brain, bovine brain, bovine heart/.
The enzyme is isolated according to the method of J. Schr-oder and H. V.
Richenberg /Biochem. Biophys. Acta 302, 50 /1973//, the isolated phos-
phodiesterase is purified by the method of J. G. Hardman and E. W.
Sutherland /J. Biol. Chem. 240, 3704 /1965// and finally the activity of the
purified enzyme is measured according to the radioisotope method of G. Poch
in the presence of an excess of tritiated c-AMP /10.1 millimoles of c-AMP
substrate, from which the 3H-c-AMP is 2.59 K Bq/ in an incubation system at
first without an inhibitor and thereafter in the presence of apovincAm;nnl-
3',4',5'-trimethoxy-benzoate-derivative as inhibitor after an incubation
period of 20 minutes /N. S. Arch. Pharmacol. 268, 272 /1979//. From the
test compound a 1 millimolar stock solution is prepared and to the incubated
enzyme preparation various amounts are added with the aid of the said stock
solution so that the concentration of the test compound in the incubated
sample should correspond to 5 x 10 7, 1 x 10 6, 5 x 106, 1 x 10 5, 5 x 10 5
and 10 mole/litre, respectively. The aqueous solution of the compound
used for comparison /papaverine/ is added to the enzyme prepared in a
similar manner.
The actlvity of the control /enzyme solution containing no
inhibitor/ is taken as 100 %, while the activity of the solutions containing
the compounds of the general Formula /I/ as papaverine is expressed as the
percentage of the control. The results measured on the enzyme isolated from
rat brain are summarized in the following Table.
Test compound Concentration of the test compound,
/enzyme inhibitor/ mole/litre
5 x 10 6 1 x 10 5 5 x 10
/Effect on the enzyme activity, % of
the control/
Apovincaminol-3',4',5'-
-trimethoxy-benzoate . HCl 64.5 49.2 47.2
/-/-ll-nitro-apovin-
caminol-3',4',5'-tri-
methoxy-benzoate . HCl 53.6 44.4 37.3
/-/-9-nitro-apovincami-
nol-3',4',5'-trimethoxy-
-benzoate . HCl 62.9 61.6 37.7
Papaverine 91.2 89.7 60.5
The results on enzyme isolated from bovine brain and brovine heart
are measured in a similar manner. By using the results obtained, the enzyme
activity is plotted against the :Logarithm of the enzyme inhibitor concentra-
tion /expressed in ~moles/. The concentration of the enzyme inhibitor which
decreases the enzyme activity by 50 % /I50/ is read off the curve. The re-
sults obtained are summarized in the following Table.
Test compound I50 values, in ~moles on phosphodiesterase
enzyme isolated from
bovine heart rat brain
Apovin~minnl-3',4',5'-
-trimethoxy-benzoate . HCl 1.5 10
/-/-ll-nitro-apovin-
caminol-3',4',5'-tri-
methoxy-benzoate . HCl 1 8
/-/-9-nitro-apovincami-
nol-3',4',5'-trimethoxy-
-benzoate . HCl 1 15
Papaverine . HCl 50 70
It appears from the above Table that on the enzyme isolated from
bol~ine heart and rat brain the new compounds of the present invention are
33-50 and 4.5-9 times, respective]a~ more active than the papaverine used as
reference compound.
The first clinical tests were carried out with topical composi-
tions containing the active ingredient /ointment, cream, solution, trincture,
paste, aerosol/. Creams containing 2 %, 1 %, 0.5 %, 0.25 % and 0.1 % of
~ 9-nitro- or /-/-11-nitro-apovincaminol-3',4',5'-trimethoxy-ben~oate,
respectively, were used.
Patients suffering from psoriasis were treated. A further funda-
mental point of view of the selection was that the patients did not receive
simultaneously a systemic treatment of their basic disease /e.g. an immuno
suppressive, cytostatical or glucocorticoidal treatment/.
Groups consisting of five patients each were examined by the so
called plaque method. One side of the symmetrical skin lesions was treated
with the cream containing the active ingredient while the other side was
treated with a placebo. The other effected skin surfaces of the patient
were treated by other topical methods - among others with an ointment gener-
ally used for the treatment of psoriasis, containing flumethasone pivalate
and salicylic acid, said ointment being used as reference substance.
The test had been started with creams having high active ingredi-
ent content and further patients were treated with a cream having the lowestactive ingredient content but being still active. The treatment was carried
on for two weeks, twice a day, in open dressing.
The effect was evaluated by observing three different symptoms -
inflammation, infiltration and desquamation (peeling). The intensity of the
symptoms ~,ras expressed by the following scale between O and 3
O = no symptoms 2 = strong symptom
1 = moderate symptom 3 = very strong symptom
The symptoms were evaluated before treatment /I/, after a treat-
ment of seven days /II/ and after a treatment of fourteen days /III/. In
the following Table the average number of points /total number of points
~1.,'2(:~2V~3
divided by the number of patients/ is disclosed. A cream containing 2 %
active ingredient was used.
Test compound Average number of points
Infiltration Inflummation Desquamation
I II III I II III I II III
Compound of the
Formula /Ia/ 1.8 0.6 0.5 1.8 1.3 0.75 1.2 0.4 0
Compound of the
Formula /Ib/ 2.2 1 0.7 2.5 2.5 1.3 1.5 0.5 0
Compound of the
Formula /Ic/ 2.4 1.8 1.3 2.2 1.3 1.3 1.6 1.2 0.8
According to a further aspect of the present invention there is
provided a process for the preparation of new compounds of the general
Formula /I/ /wherein either R and R2 both stand
--8--
for hydrogen or one of the symbols Rl and R2 is hydrogen and thc other is
nitro/ and pharmaceutically acceptable acid addition salts thereof which
comprises reacting apovincaminol or an acid addition salt ther~of with, as
an acylating agent, 3,4,5-trimethoxy-benzoic acid or a reactive derivative
thereof capable of acylation and isolating the compound of the Formula /la/
or converting the same into a pharmaceutically acceptable acid addition
salt thereof, or if desired, nitrating the compound of the Formula /Ia/ or
an acid addition salt thereof separating the mixture of the compounds of
the Formulae /Ib/ and /Ic/ thus obtained into the two components, and if
desired, converting a compound of the general Formula /Ib/ or /Ic/ thus
obtained into a pharmaceutically acceptable acid addition salt thereof.
The process of the invention is carried out preferably in the
presence of the organic solvent, particularly a chlorinated hydrocarbon or
an aliphatic ketone or pyridine, particularly in methylene chloride,
chloroform or acetone. If a 3,4,5-trimethoxy-benzoyl halide is used as
acylating agent the reaction is carried out in the presence of an equimolar
amount or a small excess of an acid binding agent. For this purpose, e.g.
an alkali carbonate, alkali hydrogen carbonate or organic amine may be
used. If 3,4,5-trimethoxy-benzoic acid is used as acalating agent the
reaction is carried out in the presence of a catalytic amoun-t of an acid
/preferably hydrochloric acid or sulfuric acid/ or an activator of the
carboxylic group and/or a dehydrating agent. The carboxylic group may be
activated by a halogenated phenol, preferably pentachloro phenol. As
dehydrating agen~ e.g. N,N'-dicyclohexyl carbodiimid may be used. The
acylation may be carried out at a temperature between -20 C and the
boilin~ point of the reaction mixture, preferably at 20-60C.
The compound of the Formula /Ia/ may be isolated from the
reaction mixture by extraction and/or evaporation.
~,.
~I'Z~2~
The product thus obtained may be converted into a pharmaceutically
acceptable acid addition salt. Salt formation may be carried out by using
inorganic or organlc acid /e.g. hydrochloric acid, sulfuric acid or phos-
phoric acid or tartaric acid, succinic acid, citric acid or ascorbic acid/.
The salt formation is carried out by methods known per se. One may proceed
preferably by adding a solution of the acid in ethyl ether or acetone to
the solution of the base. Salt formation is accomplished at a pH value of
The compound of the Formula /Ia/ thus obtained may be nitrated if
desired. Nitration may be preferably carried out with the aid of concen-
trated nitric acid. The reaction is preferably accomplished in glacial
acetic as medium. Nitriation is preferably carried out under cooling at a
temperature of about 0 C. The reaction having been terminated the excess
of the acid is neutralised and the mixture of the compounds of the Formulae
/Ib/ and /Ic/ thus obtained is isolated from the reaction mixture by extrac-
tion and/or evaporation.
The isomer mixture thus obtained may be separated into the two com-
ponents. The separation of the isomers may be preferably carried out by
chromatographical methods.
The compounds of the Formulae /Ib/ and /Ic/ may be converted into
their pharmaceutically acceptable acid addition salts, if desired, by the
methods described in connection with the preparation of the acid addition
salts of the compound of the Formula /Ia/.
According to another feature of the present invention there are
provided pharmaceutical compositions having phosphodiesterase inhibitory
effect and being mainly useful in the treatment of skin diseases attached to
pathological cell proliferation and the prophylaxis of the recurrence of
such diseases, the said compositions comprising as active ingredient a com-
pound of the general Formula /I/ or a pharmaceutically acceptable acid addi-
tio}1s salt thereof and optionally further therapeutically active compounds
in admi~,ture with usua] pharmaceutical carriers and/or diluents.
-- 10 --
The active ingredient content of the pharmaceutical compositions
of the present invention is preferably 0.1-8.0 %, particularly 0.2-2.0 %.
rhe compositions may optionally contain further pharmaceutically therapeu-
tically active compounds, such as antibiotics, cytostatical agents, pro-
staglandines, ditranol, salicylic acid, tar, antiinflammatory agents, immuno-
supressants, glucocorticoid and - in the case if compositions suitable for
parenteral administration - local anaesthetical agents. As glucocorticoid
preferably triaminolon-acetonide may be used. The active ingredient may be
finished preferably in the form of compositions for topical use, such as
creams, ointments, solutions, gelées, aerosols, aerosol foams, adhesive
plasters, etc.
The active ingredient may be preferably used in the form of the
base but acid addition salts may be applied as well.
It is preferred to incorporate the active ingredient into a cream,
which can be washed off.
The creams may be prepared by dissolving the active ingredient in
a solvent of the alcoholic type, preferably in propylene glycole or ethylene
glycole or a mixture thereof formed with a small amount of water, and admix-
ing the solution thus obtained with a readily spreadable fatty phase being
skin compatible.
The said fatty phase may comprise cetyl alcohol, stearyl alcohol,
cetostearyl alcohol, paraffin oil, glycerine monostearate or etc.
The cream may also contain an emulsifying agent - preferably poly-
oxyethylene sorbitan monooleate or monostearate - and a preservative such as
benzoic acid derivatives, preferably methyl-p-hydroxy benzoate.
rhe creams may contain preferably 0.25-2.0 % of the active ingredi-
ent, 45-50 % of glycole, 23-27 % of paraffin oil, 11-15 % of stearyl alcohol
and optionally up to 100 % other auxiliary agents.
rrle active ingredient can also be formulated in the form of an
ointmen~ which cannot be ~lashed off with water by incorporating the active
ingredient directly in the fatty phase.
3~
~e active ingredient can also be formulated in the form of a 501u-
tion or tincture which may contain e.g. 20-40 % of propylene glycole or
dipropylene glycole, 40-55 % of 96 % ethanol and up to 100 % distilled water.
The aerosol formations may be prepared by adding to the solution
of the active ingredient in propylene glycole a fatty substance - e.g. iso-
propyl myristate - and a propellant /e.g. freon/.
Injectable solutions suitable for parenteral administrations, pre-
ferably applicable in a subcutanous or intracutaneous route may be prepared
by dissolving a salt of the active ingredient in a 0.72 % aqueous sodium
chloride solution and adjusting the pH of the solution to 5.
The pharmaceutical compositions of the present invention can be
prepared by methods known in the pharmaceutical industry. One may proceed
by admixing the active ingredient and optionally further therapeutically
active compounds with suitable inert non-toxical, known pharmaceutical car-
riers and/or additives and finishing a mixture thus obtained in a form suit-
able for medical use.
Further details of the present invention can be found in the fol--
lowing Examples without limiting the scope of protection to the said
Examples.
Example 1
Preparation of /-/-apovincaminol-3',4'5'-trimethoxy-benzoate
3.10 g /10.1 millimoles/ of /-/-apovineAm;T-~l are dissolved in
60 ml of anhydrous dichloromethane, whereupon 3.10 g of anhydrous sodium
carbonate and 2.50 g of /lO.9 millimoles/ of 3,4,5-trimethoxy-benzoyl
chloride are added and the reaction mixture is stirred at room temperature
for 24 hours. The reaction mixture is diluted with 100 ml of water, the
organic phase is separated and the aqueous layer is extracted twice with
20 ml of dichloromethane each. The united dichloromethane phases are dried
over rnagnesium suliate, filtered and the filtrate is e~aporated in vacuo.
Thus 4.50 g of the title compound are obtained, yield 89.1 %.
~ ~13~8
- 12 -
Brutto formula C30H34~l2o5. Molecular weight 502.61.
IR spectrum /film/: v 1725 cm /-C=O/; 1620 cm /=C=C=/
~MR spectrum /deuterochloroform/: ~: 1.01 /t, 3H, CH3CH2-/; ~: 3.72 /s, 6H,
2 x -OCH3/; ~: 3.85 /s, 3H, -OCH3/; ~: 4.25 /s, lH, anellation/; ~: 5.29
/s, lH, -CH=/; ~: 5.4 /m, 2H, -OCH2/; ~: 7.0-7.8 /m, 6H, aromatic/.
MS /m/e/: 502/53/, 432/100/, 290/17/, 261/41/, 220/l9/, 216/23/, 212/18/,
195/35/ -
/~/D5 = -22.0 /c = 0.7; dichloro methan/.
Example 2
Preparation of /-/-apovincaminol-3',4',5'-trimethoxy-benzoate hydrogen
tartarate
The compound prepared according to Example 1 is dissolved in
diethyl ether. To the solution a saturated solution of D-tartaric acid in
diethyl ether is added until the precipitation of the hydrogen tartarate
salt becomes complete. The salt is filtered off and dried. Mpl.: 120-121 C.
IR spectrum /KBr/: vmax 1730 cm /-C=O/; 1640-1665 cm /=C=C=/.
/~/D = -8.5 /c = 1; pyridine/. Molecular weight 652.7.
Example 3
Preparation of /-/-9-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate and
/-/-ll-nitro-apov; nr~m; nol-3 ~ ~ 4',5'-trimethoxy-benzoate
5 g of /-/-apovi nr~m; nnl-3 ~, 4',5'-trimethoxy-benzoate are dis-
solved in 50 ml of glacial acetic acid. To the solution obtained a mixtureof 20 ml glacial acetic acid and 10 ml of concentrated mitric acid /d = 1.52/
is added at O C under stirring. The reaction mixture is poured into 350 mg
of icecold water and the pH is adjusted to 9 by adding a 25 % aqueous
ammonium hydroxyde solution. The alkaline solution is extracted at first
with 250 ml and thereafter twice with 200 ml of dichloro methane each. The
united organic solution are dried over anhydrous sodium sulfate, filtered
and the filtrate is evaporated to dryness in vacuo.
The isomer Mixture thus obtained is separated into the two compon-
;,
ents by chromatographical methods by using ~ieselgel 60 as adsorbent and a10:2 mixture of benzene and acetone as developing agent. Under these condi-
tions the Rf value of the 9-nitro-compound is greater than that of the
ll-nitro-derivative. Thus 1.7 g of the 9-nitro-compound and 1.8 g of the
ll-nitro-derivative are obtained.
The physical constants of the /-/-9-nitro-apovincaminol-3',4',5'-
trimethoxy-benzoate are as follows:
Mp.: 77-78 C. /~/D = -78.5 /c = 1; chloroform/.
H-N~IR /CDC13/: ~: 3.8 /s, 6H, 2 x CH30-/; ~: 3.91 /s, 3H, 1 x CH30-/;
' ' 12/; ~: 7-96 /d, lH, Hll/; ~: 8.1 /d, lH, H
The physical constants of the /-/-ll-nitro-apovincaminol-3',4',5'-
trimethoxy-b~nzoate are as follows:
Mp.: 72-73 C. /~/D = -134.3 /c = 1; chloroform/.
H-NMR /CDC13/: ~: 3.8 /s, 6H, 2 x CH30-/; ~: 3.9 /s, 3H, 1 x CH30-/;
~: 7-56 /d, lH, Hg/; ~: 8.1 /d, lH, Hlo/; ~: 8.9 /d, lH, H12/.
Example 4
Preparation of 9-nitro-apovincaminol-3',4',5'-trimethoxy-benzoate-
hydrochloride and ll-nitro-apovincaminol-3',4',5'-trimethoxy~benzoate-
hydrochloride
The 9-nitro- or 11-nitro-apovincaminol-3',4',5'-trimethoxy-
benzoate prepared according to Example 3 is dissolved in methanol and the pH
of the solution is adjusted by adding a solution of hydrogen chloride and
methanol. The hydrochloride salt formed is precipitated by adding diethyl
ether. The salt is rriltered off, washed and dried. The hydrochloride of
the 9-nitro-derivative melts at 144-150 C, while the melting point of the
hydrochloride of the ll-nitro-derivative amounts to 141-144 C.
Example 5
A cream having the following composition is prepared:
- 14 -
Cornponent: Amount /g/:
Apovincaminol-3',4',5'-trimethoxy-benzoate 2
Propylene glycole 50
Paraffin oil 26
Polyethylene glycole 5
Stearyl alcohol 15
Glycerol monostearate 2
The active ingredient is dissolved in propylene glycole on a water
bath /bath temperature not exceeding 50 C/. The other components are
heated until they melt and thereafter cooled to 40-45 C under constant stir-
ring. To the melt the solution of the active ingredient is added under stir-
ring and the cream thus obtained is cooled under stirring.
In an analogeous manner creams containing 0.25 %, 0.5 %, 1.0 % and
1.5 % of the active ingredient, respectively, are prepared.
The active ingredient 9-nitro- or 11-nitro-apovincaminol-3',4',5'-
trimethoxy-benzoate may be used as well.
Example 6
A cream having the following composition is prepared:
Component: Amount /g/:
9-nitro-apov;nc~minol-3',4',5'-trimethoxy-
benzoate 2
Triamcinolon acetonide O.l
Glycerol monostearate 3.0
Polyethylene glycole 400 5.0
Stearyl alcohol 13.0
Paraffin oil 24.9
Propylene glycole 53.0
One proceeds in an analogeous manner to Example 5 except that two
active ingredients are dissolved in propylene glycole. As active ingredient
the ll-nitro-derivative or apovincaminol-3',4',5'-trimethoxy-benzoate may be
used as ~"ell.
. ,/
;7~ 8
- 15 -
Example 7
A tincture solution having the following composition is prepared:
Component: Amount /g/:
9-nitro-apov;n~m;n~1-3',4',5l-trimethoxy-benzoate
Propylene glycole 30
9G % methanol 69
The above process may also be carried out by using the ll-nitro-
derivatives as active ingredient.
Example 8
A tincture solution having the following composition is prepared:
Component: Amount /%/:
Apovincaminol-3',4',5'-trimethoxy-benzoate hydro~
gen tartarate
Propylene glycole 30
96 % ethanol 47
Distilled water 22
Example 9
An aerosol having the following composition is prepared:
Component: Amount /%/:
Apov;nc~m;n~1-3',4',5'-trimethoxy-benzoate hydro-
gen tartarate 0 5
Propylene glycole 30
Isoprop~yl myristate ~.5
~reon 65
As active ingredient the 9-nitro- or ll-nitro-derivative may be
used as ~,Jell.
Example 10
An aerosol foam having the following composition is prepared:
, "
2~
- 16 -
Component: Amount
~povincaminol-3',4',5'-trimethoxy-benzoate hydro-
gen tartarate 2
Cetostearyl alcohol
Benzyl alcohol 2
Polyoxyethylene-sorbitan-monostearate15
96 ~ ethanol 30
Distilled water 30
Freon 20
~'
