Note: Descriptions are shown in the official language in which they were submitted.
~4~ 7
- 1 - o.Z~ Oo~o/033453
Pharmaceutical agent containing
all-E- or 13-Z-7,8-dehydroretinic acid,
processes for its preparation, and its use
The present invention relates to a pharmaceutical
agent which contains all-E- or 13-Z-7,8-dehydroretinic
acid as the active compound in addition to conventional
pharmaceutical carriers and diluents, to the preparation
of these agents and to their use as drugs
It is known that vitamin A compounds, for example
esters of retinol or of retinic acid, control the normal
cell di~ferentiation of epithelial tissue. The
effectiveness of retinol and retinic acid in the preven-
tion of lung, -skin, bladder and
mammary cancer in animal experiments has also been des-
cribed by several groups of workers (~. Bollag, Int.Z.
Vitaminforsch; 40 (1970), 299 et seq.; M.B. Sporn et al.,
Fed.Proc Fed.Am.Socs~Exp. Biol. 35 (1976), 1332 - 1338)
However, natural vitamin A compounds exhibit, alongside
their cancerostatic activity in animal experiments and
in man, the disadvantage of a relatively high toxicity
resulting from an un~avorable distribution in the
organism (disadvantageous pharmacokinetics). For
example, retinol is in the main stored as retinyl
palmitate in the liver, where, in higher concentration,
it can cause serious functional disorders. Higher
doses of retinic acid lead to signs of severe systemic
toxicity which can be characterized by destabilization
of membranes, rupture of lysosomes and the like, even
.~ .
~` -
~3~S7
xanging up to bone fractures.
We have found that these disadvantages can be avoided
by a pharmaceutical agent which contains all-E- or 13-Z-7, 8-
dehydroretinic acid as the active compound, in addition to
conventional pharmaceutical carriers or diluents, with or with-
out conve~tional pharmaceutical auxiliaries.
In their cancer-prophylactic action, all-E- and
13-Z-7,8-dehydro-retinlc acid resemble retinic acid and are,
for example, still active in as little as 10 9 molar concen-
10 tration in hamster trachea tissue keratinized by vitamin A
hypovitaminosis. The methods employed are described in G.H.
Clamon et al., Nature 250 (1974), 64-66 or M.B. Sporn et al.,
Nature 253 (1975), 47-50. The keratinization is viewed as a
pre-cancerous process. Compared to retinic acid, the compounds
used according to the invention surprisingly exhibit a lower
toxicity and more advantageous pharmacokinetics. The actlve
compound concentration required for therapy or prophylaxis of
pre-cancerous conditions or carcinomas can therefore be achieved
in the organs in question without undesired side-efifects.
The compounds according to the invention-have signi-
ficant tumor-inhibiting action. Growth control may be obser-
ved n cells cultivated in vitro and may be detected by the
method described by R. Lotan et al in Journal of the National
Cancer Institute 60 (1978), 1035-1041. The compounds also
inhibit the proliferation of spontaneously chemically or virally
transformed cells in tissue culture, the preferred culture
being S 91 melanoma cells. ` ~
.... _ _ _ . ... .
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- 3 ~ . 00~0/03~4~0
Accordingly, the drug according to the invention
can be used for the topical and systemic therapy and
prophylaxis of pre-cancerous conditions and carcinomas
of the skin, mucous membranes and internal organs and
for the topical and systemic therapy o~ acne, psoriasis
and other dermatological disorders accompanied by
pathologically altered kera-tinization.
A preferred indlcatlon is the prophy- -
lactic and therapeutic treatment of pre-cancerous con-
ditions and tumors of the bladder, the mammary gland,
the skin and the mucous membranes.
13-Z-7,8-Dehydroretinic acid (I) can be prepared
in a simple manner, starting from 7,8-dehydro-~-ionylidene-
acetaldehyde III via 7,8-dehydro-~-ionylidene-ethyl-
triphenyl-phosphonium bromide IV, by a ~,~ittig
olefinization reaction with the lactol of Z-3-formyl-2-
butenoic acid V.
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EI
,~ dioxane, butyl-
lithium
' . 0~0~0 V
3 steps . ~ ~ P~(P~)3 Br
III
~ NaOC2H5
O ~,' O VIII
OH
.' ~ ~0
. ~ ~ II
: ~. . 6 steps
J.( 'hem. Soc.1952, 1094
, ~ . ~,..0~'-
VI VIT
5~
_ 7 _ 3.z. 0050/033450
A synthesis of all-E-7,~-dehydroretinic acid (II)
ln six steps from 2-ethynyl-1,3,3-trimethylcyclohex-l-
ene V~ and. hepta-3,5~dien-2-one VII is described by J.
Attenburrow et al , J. Chem~ Soc. (London) ~ , 1094 et
seq.
Compound II is more easily accessible, starting
from compound IV, by Wlttig olefiniza-tion witn E-3-
formyl-2-butenoic acid ethyl ester VIII, and hydrolysis
of the ester group.
It should be emphasized that all-E- and 13-Z-
7,8-dehydroretinic acid are also distinguished by sub-
stantially greater stability than that of retinol and
retinic acid~
The present invention also embraces the prepara~
tion of therapeutic agents or pharmaceu-tical formulations
which are obtained in the conventional manner, in parti-
cular by mixing a dose o~ the active compound approp-
riate for the particular application wi-th conventional
carriers or diluents, with or without conventional
pharmace~tical excipients , in accorda~ce with the
desired route of administration~
For local application the -therapeutic agents
contain the compounds, to be used according to the in~ren-
tion, at a concentration of from 0.001 to 1.0%, prefer-
ably from 0.01 to 0.1%, whilst for systemic adminis-tra-
tion a single dose is preferably from 0.1 to 5 mg.
Su.itable daily dosages are from 50 to lO0 mg,
and can vary in accordance with the nature and severity
of the disorder, the formulation used, and the route of
~a ~43657
_ ~ _ o z. 0050/033450
administration~
The conventional galenical formulations are
employed, for example, for oral administration, tablets,
film tablets, dragees, capsules, pills, powders, gran-
ules, solutions or suspensions. For external use,
suitable forms are in particular pastes, ointments,je~l;es,
creams, lotions, powders, solutionsor emulsions and sprays.
The drugs according to the invention may be employed
either internally or externally. Preferably, they
o are administered orally or applied topically
Examples of conventional pharmaceutical excipi-
ents for local application are alcohols, eg. isopropanol,
oxyethylated castor oil or oxyethylated hydrogenated
castor oil, polyacrylic acid, glycerol monostearate,
paraffin oil, vaseline, wool grease, polyethylene glycol
400, polyethylene glycol 400 stearate and oxyethylated
fatty alcohols, whilst examples for systemic administra-
tion are lactose, sucrose, propylene glycol, ethanol,
starch, talc and polyvinylpyrrolidone.
Examples of further conventional additives are
preservatives, antioxidants, flavorings, stabili~ers,
emulsifiers, lubricants, wetting agents and the like.
It is a precondition that all materials used in the
preparation of pharmaceutical formulations should be
non-toxic and compatib~e with the active compounds
-
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employed (cf. L.G~ Goodman and A. Gilman, The Pharma-
cological Basis of Therap~utics).
~YAMPLE 1
7,~-Dehydro-~-ionylidene-ethyl-triphenylphos-
phonium bromide:
11 6 parts by weight of sodium borohydride are
introduced in portions into a solution of 66.4 parts by
weight of 7,8-dehydro-~-ionylidene-acetaldehyde in 400
parts by volume of methanol, whilst cooling by means of
an ice bath, stirring is continued for 2 hours and the
mixture is then left to stand ove~night at about 20C.
1 t parts by volume of n-hexane are added, the mix-ture
is washed with 1,000 parts by volume of 5 per cent
strength sodium hydrogen tartrate solution, and the
phases are separated The aqueous phase is re-
- extracted with three times 150 parts by volume of n-
hexane. After conventional wor~ing up, 63.~ parts by
weight of 7,8-dehydro-~-i.onylidene-ethanol are obtained
as a colorless oil.
The 7,8-dehydro-~-ionylidene-ethanol (63.5 parts
by weight) and 6 5 parts by weight of pyridine are dis-
solved in 150 parts by volume of ether and a solution of
31.1 parts by weight of phosphorus tribromide in 80 parts
by volume of ether is added dropwise at -20C Stirr-
ing is continued for 2 hours at about 22C, 300 parts by
weight of ice are added and the mi~ture is partitioned
between ether and water in the conventional manner.
After concentrating the ether solution on a rotary
evaporator, 75.2 parts by weight of 7,8-dehydro-~-
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- 8 - O.Z. 0050/0334~0
ionylidene-ethyl bromide are obtained as a pale yellow
oil.
A solution of 70.1 parts by weight of triphenyl
phosphine in 150 parts by volume of me-th~lene chloride
is added dropwise at about 25C to a solution of 75 parts
by weight of 7,3-dehydro-~-ionylidene-ethyl bromide in
150 parts by volume of methylene chloride, stirring is
continued for 2 hours and the mixture is then concen-
trated at 50C on a rotary evaporator. The residue
is taken up in 120 parts by volume of methylene chloride,
ether is added until clouding occurs, and the mixture is
then seeded and kept at -8C for 16 hours. After
filtering off the crystals and drying them in a stream
of nitrogen, 67 parts by weight of 7,8-dehydro-~-
ionylidene-ethyl-triphenylphosphonium bromide are
obtained. A further 25 parts by weight of crystals
can be lsolated from the mother liquor after adding more
ether; melting point 166.5C (with decomposition).
EXAMPLE 2
all-E-7,8-Dehydroretinic acid:
38 1 parts by weight of 7,8-dehydro-~-ionylidene-
ethyl-triphenylphosphonium bromide and 11.7 parts by
weight of E-~-formyl-2-butenoic acidethylester are dis-
solved in 140 parts by volume of ethanol and a solution
of 2 56 parts by weight of sodium in 75 parts by volume
of ethanol is added dropwise whilst cooling the reaction
mixture in an ice bath. The mixture is stirred for 2
hours at about 12C and is then concentrated to about
25% of its volume in a rotary evaporator, 250 parts by
3~S7
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volume of n-hexane are added, and after se-ttling out the
clear solution is decanted; this operation is repeated.
The hexane solution is washed with three times 70 parts
by volume of 60 per cent strength aqueous methanol and
is concentrated. The yield is 17.8 parts by weight
of all-E-7,8-dehydroretini¢ acid ethyl ester in the form
of a red oil.
In order to dissolve 17.5 parts by weight of all-
E-7,8-dehydroretinic acid ethyl ester in 150 parts by
volume of e~hanol, a solution of 7.1 parts by weight of
85 per cent strength potassium hyJroxide in 50 parts by
volume of ethanol is added and the mixture is refluxed
for 2 hours. The solution is cooled, 100 parts by
volume of water are added and the pH is brought to 2 with
about 10 parts by volume of concentrated hydrochloric
acid. The aqueous-ethanolic phase is extracted with
one portion of 100 parts by volume and three portions
each of 50 parts by volume of chloroform, and the chloro-
form phase is washed with three times 50 parts by volume
of water and concentrated. The yield is 18.8 parts
by weight of crude all-E-7,8-dehydroretinic acid.
The crude product is recrystallized from diisopropyl
ether at -20C. 6 6 parts by weight of product, of
melting point 106.6C, are obtained. The mother
liquor is concentrated, the residue is taken up in a
small amount of n-hexane and seeding crystals are added
to the solution After about 26 hours at 0C, a
further 4.6 parts by weight of crystalline product have
been obtained; melting point 158.8C. Analysis by
3~S7
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high pressure liquid chromatography shows that the pro-
duct is free from cis-isomer,
EXAMPLE 3
13-Z-7,8-Dehydroretinic acid:
241 parts by weight of n-butyl-lithium, dis-
solved in 1,370 parts by volume of n-hexane, are run at
10C into a suspension of 652 parts by weight of 7,8-
dehydro-~-ionylidene-ethyl-triphenylphosphonium bromide
in 3,000 parts by volume of dry dioxane and stirring is
continued for 30 minutes, A solution of 150 parts by
weight of 2-hydroxy-3-methyl-dihydrofuran-2-one is then
added at 10C and stirring is continued for 16 hours,
To work up the mixture, 2,000 parts by volume of
water and 1,000 parts by volume of n-hexane are added
cautiously, the phases are separated and the aqueous
phase is again e~tracted with a further 2,000 parts by
volume of n-hexane. Thereafter the aqueous phaseisacidi-
fied with 2N sulfuric acid to pH 2 and is extracted
with three times 2,000 parts by volume of n-hexane, and
the hexane solution is washed with twice 2,000 parts by
volume of 60 per cent strength aqueous methanol and is
concentrated to 1,500 parts by volume at 50C. The
product which crystallizes out in the course of 2 hours
at 22C is filtered off, washed wlth 200 parts by volume
of n-hexane at -30C and dried in a stream of nitrogen.
127.5 parts by weight of 13-Z-7,8-dehydroretinic acid
are obtai~ed; melting point 168-170C, El (iso-propanol)
1537 and 1545, The structure was confirmed by H-NMR
spectroscopy, According to analysis by high pressure
S7
liquid chromatography, the product contains 97~ of the
desired isomer.
The mother liquor, after being kept for 16 hours
at 20C, gives a further 22 parts by weight o~ crystal-
line 13-Z-7,8-dehydroretinic acid, melting point 167C.
Examples of pharmaceutical formulations or
active compound/carrier mixtures for external application
are given below:
EXAMPLE 1
Solution
all-E-7,8-dehydroretinic acid I 0.25 g
oxyethylated hydrogenated castor oil35.0 g
(Cremophor*RH 40, from BASF AG,
Ludwigshafen)
Polyethylene glycol 400 35.0 g
oxyethylated castor oil (Softigen 767,10.0 g
from Chemische Werke Witten
deionized water to give 100.0 g
The Cremophor RH 40 and Softigen 767 are mixed
and the mixture is heated to 70C. Thé active com-
pound is dissolved therein whilst stirring and the poly-
ethylene glycol 400 is added. The solution is thencooled to 40C and water at 40C is added slowly, whilst
stirring The finished solution is ~iltered and
packaged in, for example, 100 ml flasks.
EXAMPLE 2
Cream
13-Z-7,8-dehydroretinic acid 0.1 g
butylhydroxytoluene 0.1 g
glycerol monostearate ll.O g
polyethylene glycol 400 steara-te 6.o g
*
Trademark.
. .. , .. ,, . . _ .. , . . = _ __ . _ . . .. .... . _ . , .
~3~7
-12
oxyethylat2d fatt~J alcohol 4.0 g
paraffin oil lO.0 g
p-hydroxybenzoic acld ester (Nipasteril,
from Nipalaboratorium Hamburg) 0.2 g
perfume oll O.l g
deionized water to giye lO0.0 g
The fats are melted and the very finely pulverized
active compound and butylhydroxytoluene are dispersed
therein whils-t stirring at 65C (solution I). The
water is boiled up with the Nipaester
and then cooled to 65C (solution II). Solution II
is then emulsified, a little at a time, in solution I,
with thorough stirring, After the mixture has cooled
to 45C, the perfume oil is added and the emulsion is
cooled to room temperature, whilst stirring. The
finished cream is packaged in tubes carrying an internal
protective coating.
EXAMPLE 3
Jelly
all-E-7,8-dehydrore-tinic acid O.Ol g
butylhydroxytoluene O.l g
oxyethylated castor oil (Cremophor EL,
from BASF AG, Ludwigshafen) 35,0 g
isopropanol 20.0 g
polyacrylic acid (Carbopol, from
Goodrich Hamburg) l.5 g
triethanolamine 0.002 g
p-hydroxybenzoic acid ester (Nipasteril,
from Nipalaboratorium Hamburg) 0.2 g
deionized water to give lO0.0 g
The Cremophor EL is heated to 60C, the active
Trademark.
~1~3~57
compound and the butylhydroxytoluene are dissolved therein,
whilst stirriny, and the isoporpanol, in which the Nipaesters*
have been dissolved are admixed (solution I). The Carbopol*
is dispersed in the water, with vigorous stirring (solution
II). Solution II is added, a little at a time, to solution
I, wi-th thorough stirring. The pll oE the mixture is brought
to 4.5 with triethanolamine. The finished jelly is packaged
in tubes carrying an internal protective coating.
Examples oE formulations or active compound/carrier
10 mixtures particularly suitable for systemic use are the
following:
EXAMæLE 4
Drops
all-E-7,8-dehydroretinic acid0.1 g
propylene glycol 25.0 g
ethyl alcohol to give 50.0 g
The ethyl alcohol and propylene glycol are mixed and
the active compound is dissolved in the mixture by heating
at 35C and stirring. After filtration, the solution is
20 packaged in dark-colored drop bottles.
EX~MPLE 5
Hard gelatin capsules
13-Z-7,8-dehydroretinic acid1 ~mg
lactose to give 0.25 g
The constituents are sieved, mixed and used to fill
hard gelatin capsules of size 2 on a suitable capsule fil-
ling and sealing machine.
* Trademark.
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