Note: Descriptions are shown in the official language in which they were submitted.
77~2~
This invention relates to novel non-pyrogenic steroidal erythro-
poietic agents and methods for their preparation. More particularly, the
invention concerns the use of 3-ësters and 3-ethers of 3-hydro~y-5~-oestran-
17-one for the stimulation of erythropoieses.
Erythropoiesis is the process of formation of red blood cells.
The term anemia implies an abnormally low number of circulating red ~ -
cells or a decreased concentration of hemoglobin in the blood. The appearance
of anemia reflects either marrow failure or excessive red cell loss, or both.
Marrow failure, i.e., reduced erythropoiesis, may occur as a result of a nu-
tritional deficiency, toxic exposure, tumor invasion, or other and sometimes
unknown causes.
For the treatment of anemias of bone marrow failure ~hypoplastic
and aplastic anemias), it has been proposed to use substances which might
stimulate the marrow, such as androgens or corticosteroids. United States
Patent 3,383,282 discloses various 3,5-androstadiene-3,17-diol derivatives as
possessing erythropoietic activity. United States Patents 3,519,659 and
3,519,660 disclose various prednisolone derivates having antileukemia
x activity.
It is known that erythropoietic activity is exhibited by metabolites
of certain androgenic, anabolic, or progestational steroids. Thus, Levere et
' al. Proceedings of a Symposium held in conjunction with the American Society
of Hematology, December 4, 1971, Chapter III, discloses that etiocholanolone,
a human metabolite of testosterone, possesses erythropoietic activity. Jepson,
ibid., Chapter II, discloses that nandrolone (l9-nortestosterone; 17~-hydroxy-
l9-nor-4-androsten-3-one), an anabolic steroid, possesses erythropoietic
activity similar to testosterone. This substance, however, has the drawback
~,~ of exhibiting androgenic side-effects. Etiocholanolone possesses the sub-
stantial drawback of being a pyrogen in man.
In accordance with the present invention, it has been found that
~i,
certain 3-hydroxy-oestrane derivatives which contain the 5~-H-configuration
exhibit erythropioetic activity, while at the same time they are nonpyrogenic
and exhibit little or no hormonal side effects.
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The compounds now found to be nonpyrogenic and active in stimula-
ting erythropoieses are the 3-esters and 3-ethers, of 3~-hydroxy-5~-estrane-
17-one as well as the 3-esters and 3-ethers of the corresponding 3a-epimer
i.e. 3C-L-hydroxy-5~-oestran-l7-one.
l9-Noretiocholanolone ~3~-hydroxy-5~-oestran-17-one) is a known
compound and is disclosed by Engel et al., J. Biol. Chem. 231, 1, 159 ~1958).
The preparation of this compound and the 3~-epimer thereof are also disclosed
in an article by Counsell in Tetrahedron, Vol. 15, 202-211 ~1961), by reduc-
tion of 5~-estrane-3,17-dione with Raney nickel in ethanol. l9-Noretiochola-
nolone may also be synthesized by hydrogenating nandrolone 17-acetate to the
corresponding 5~-3-keto-17~-acetate by the method described in J. Org. Chem.
31, 2394 (1966), then reducing the 3-keto group to form the ~-hydroxy group
using lithium-aluminium tri-tert.-butoxyhydride, protecting the 3a-hydroxy
group, hydrolyzing the 17~-acetate, oxidizing the 17~-hydroxy group to 17-
keto with CrO3-pyridine, and finally removing the 3C~-protecting group.
The 3~-epimer can be obtained by epimerisation of l9-noretiochola-
nolone, for example by conversion of the 3-hydroxy group in its 3-tosylate
and saponification of the tosylate with potassium acetate, or even better
according to the method described in Tetrahedron Letters, Vol. 18, 1619 ~1973), -
using triphenylphosphine, diethylazodicarboxylate and formic acid.
The 3-esters and 3-ethers of both 3a-hydroxy-5~-estran-17-one and
!~, 3~-hydroxy-5~-estrane-17-one which exhibit erythropoietic activity are novel
compounds and this invention thus in one embodiment relates to these novel
steroids having erythropoietic activib.
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It also relates to a process for the preparation of a 3-ester or a
3-ether of 3-hydrox~-5~-estran-17-one which comprises esterifying or etheri-
fying 3-hydroxy-5~-estran-17-one.
The 3-esters include those with pharmaceutically acceptable acids
which may be elther inorganic or organic. Examples of inorganic acids include
hydrochloric, sulfurlc and phosphoric acid, the steroid esters of which are
usually applied in the alkali metal salt form, such as the sodium salt. As
organic esters, saturated and unsaturated organic carboxylic acids having 1
to 18 carbon atoms, preferably 3 to 12 carbon atoms, may be employed. The
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preparation of these esters can be carried out in conventional manner by
reacting the 3-hydroxy steroid with the acid, or with its corresponding an-
hydride or acyl halide.
As examples of organic carboxylic acids, mention is made of the
following: formic, acetic, propionic, butyric, valeric, isocapronic, de-
canoic, undecylic, lauric ~ridecylic, myristic, oleic, palmitic~ stearic,
trimethylacetic, diethylacetic~ undecylenic, malonic, succinic, glutaric,
pimelic and tartaric acids. There may also be employed cycloaliphatic car-
boxylic acids, such as cyclohexanecarboxylic, cyclopentylpropionic, cyclo-
hexylpropionic and cyclohexylbutyric acids, and also araliphatic carboxylicacids, such as phenylacetic, phenylpropionic, and phenyl butyric acids, and
also aromatic acids, such as benzoic acid.
For obtaining the 3-ethers, the corresponding 3-hydroxy steroid
is etherified with a group derived from an aliphatic, cycloaliphatic, aro-
matic, araliphatic, or heterocyclic hydrocarbon. Examples of suitable ether
groups include methoxy, ethoxy, propoxy, cyclopentyloxy, benzyloxy, phenyl-
ethoxy and tetrahydropyranyloxy. The etherification can be carried out ac-
cording to methods known in the art, for example by conversion of the steroid
alcohol to its sodium alcoholate and reac~ion of the sodium alcoholate with
a hydrocarbon halogenide, in a suitable solvent such as tetrahydrofuran or
; dimethylsulphoxide. The tetrahydropyranylether can be obtained by reacting
the steroid alcohol with dihydropyran.
The 3-esters have the advantage of exhibiting a protracted action.
The 3-ethers exhibit oral activity.
The foregoing compounds are adapted for the administration thereof
- to humans and other warm-blooded animals in amounts effective to stimulate
erythropoiesis, such amounts being generally in the range from about 5 to
about 500 mg per unit dosage. The usual method of administration of the
' 3-esters of this invention is parenterally, for which purpose the compound
may be prepared in a form suitable for injection as a solution or suspen-
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~7792'~
sion in 1 ml ampoules. The compounds may also be administered enterally,
inclucling oromucosally, in the form of tablets, pills, capsules, suppositories
and the like.
The pharmaceutical compositions according to the invention may be
prepared according to methods known in the art. The unit dosage form may con-
tain in addition to the active ingredient one or more of the usual excipients,
such as vegetable oils, benzyl alcohol, propylene glycol, glycerin, lactose,
starch, magnesium stearate, waxes. Other agents, such as preservatives, emul-
sifying agents, stabilizing agents, flavours, dyes, binding agents and/or coat-
ing agents may optionally be present. The capsules may be soft or hard gela-
tine capsules.
The following experiments illustrate the erythropoietic activity of
r~ 1
es~hY t-n~ compounds according to the invention as well as providing
additional information for reference or comparison purposes.
a) Erythropoietin Bioassay
l9-Noretiocholanolone was administered to mice as a single subcuta-
neous injection of 2.5 mg in a 2-propanediol vehicle, at various intervals
-- following induced hypoxia. The first injection was made on the third day post-
hypoxia; on the fifth post-hypoxic day, 0.5 ~ Ci 59FeC13 was injected intra-
venously; the percent 59Fe-incorporation into the red cells was determined on
; day 7 post-hypoxia. The l9-noretiocholanolone stimulated iron incorporation
significantly, the figure for ~ RBC - Fe incorporation being 5.82 + 1.21
p < 0.05)-
b) Rat Marrow Bioassay
l9-Noretiocholanolone was added in 1 ~1 of 2-propanediol to rat bone
marrow, ~S-D~, ~ 100 - 150 g~ at the initiation of the cultures; about 72
hours later 0.5 ~ Ci 59Fe, bound to transferrin, was added to the cultures;
radioheme was extracted 6 hours later and quantitated. The data suggest that
l9-noretiocholanolone is active in this system:
` 30 Concentration tM) 3x10 7 3x10 8 3x10 9 3x10 10
54 46 172 180+47.9
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c) 14C-Labeled Hemoglobin
Human marrow cultures were treated with l9-noretiocholanolone for
three days; 3 ~ Ci of 14C-valine was added for the last 24 hours of culture.
Hemoglobin was isolated simultaneously from cells cultured with either 2-
propanediol or the steroid (3 x 10 lOM~. The specific activity (14C-cpm/A540)
of each was calculated and the ratio determined. The data show that the ste-
roid was stimulatory:
C-hemoglobin
l9-noretiocholanolone 1.34
Ratio of the specific activity of a steroid-treated culture to a 2-propane-
diol-treated culture.
d) Human Marrow Cultures
Radioironincorporation in~o heme was determined in the same manner
as in the rat marrow cultures of Experiment b). The l9-noretiocholanolone was
evaluated at a concentration of 3xlO ~ except in the marrow obtained from a
patient with no demonstrable disease where a concentration of 5xlO lOM was
used. The test data are as follows:
% 59Fe-Heme Incorporation (vehicle is considered as 100%)
No Systemic
Demonstrable Lupus Mycosis Hemolytic Rhabdomyo-
Disease Erythrematosus Fungoides Anemia sarcoma
90 144 148 93 105
The foregoing data indicate that l9-noretiocholanolone stimulates
erythropoiesis both in vivo and in vitro. Repeat of the test with 3~-hydroxy-
5~-oestran-17-one gave similar results.
e) Primary avian liver cell cultures
The induction of porphyrin synthesis and heme formation in cells was
investigated in the primary avian liver cell culture system described by
Granick and Kappas, J. Biol. Chem. 242, 4587-93 ~1967~. In accordance with
this technique, livers of 16 to 17 day old chick embryos are minced, and the
cells separated by trypsin. Suspensions containing 3 to 5 x 10 cells are inocu-
lated into vials which contain a cover slip and 1.0 ml of Eagle's basal medium
supplemented with glutamine, fetal bovine serum, and antibiotics. The vials
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are incubated at 37C in 5% C02 and air for 20 hours. The growth medium is
then replaced with fresh medium, and the addition of the steroid is made as
required. Following reincubation for an additional 20 hours, the cover slips,
now overgrown with a monolayer of hepatic parenchymal cells, are examined under
phase and fluorescence optics. Semi-quantitative estimates of cellular por-
phyrins are made on the basis that values of fluorescence intensity ranging
from +1.0 to ~4.0 are equivalent to approximately 5 to 50 x 10-11 moles of
protoporphyrin per mg of protein on the cover slip. The amounts of protopor-
phyrin formed reflect the levels of ~-aminolevulinate synthetase which is the
rate-limiting enzyme in heme formation.
The compounds tested were 19-noretiocholanolone (A) and its 3-deca-
noate (B), and 3~-hydroxy-5~-estrane-17-one (C) and its decanoate (D). All
compounds were found to be non-pyrogenic.
The results are shown in the following table:
Table I
Induction of Porphyrin Synthesis by Steroids in Cultured
Chick Embryo Liver Cells
Treatment No. of Samples ~ Dose Protoporphyrin found ~pmol/
mg protein, 20 hr)
_ _ .
Steroid A 4 - 2 ~g/ml 736.0 + 27.6
Steroid A 4 - 10 ~g/ml~ 814.8 + 40.7
Steroid B 4 - 2 ~g/ml 341.1 + 32.0
Steroid B 4 - 10 ~g/ml 434.5 + 45.9
Steroid C 4 - 2 ~g/ml 647.6 + 87.1
Steroid C 4 - 10 ~g/ml 751.4 + 103.4
Steroid D 4 - 2 ~g/ml Approx. 1/2 the
Steroid D 4 - 10 ~g/ml values for C
The following Examples, some of which are included for reference pur-
poses, illustrate the invention, but are not to be regarded as limiting.
Example 1
Ampoule Dosage Form
The dosage form is prepared by admixing 500 g of l9-noretiocholano-
- lone into 2 liters of sterile sesame oil containing about 500 ml of ben7yl
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alcohol, and heating the resulting mixture to about 80C to obtain a solution.
The solution is allowed to return to room temperature and the volume is in-
creased to 10 liters by addition of sesame oil. The solution is filtered
through a bacteriological membrane filter and is packaged into dosage forms,
e.g., vials of 2 or 5 ml or ampoules of 1 ml. The strength of the steroid
solution is about 50 mg per ml.
Similarly, the following compounds were made up to an oilysolution
for packaging into 1 ml ampoules:
l9-noretiocholanolone 3-propionate
l9-noretiocholanolone 3-isocapronate
l9-noretiocholanolone 3-decanoate
3~-hydroxy-5~-oestran-17-one
3~-hydroxy-5~-oestran-17-one 3-undecanoate
3~-hydroxy-5~-oestran-17-one 3-phenylpropionate
Example 2
Tablets
l9-noretiocholanolone 3-cyclopentylether 25 mg
potato starch 25 mg
polyvinylpyrrolidone 10 mg
magnesiumstearate 2 mg
tocopherol 0.1 mg
lactose up to 200 mg
3~-hydroxy-5~-oestran-17-one 3-tetrahydropyranylether 10 mg
potato starch 10 mg
polyvinylpyrrolidone 5 mg
magnesiumstearate 1 mg
tocopherol 0.05 mg
lactose up to 100 mg
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19-noretiocholanolone 3-(2'-me~hyl)decanoate 10 mg
capric acid 20 mg
tocopherol 0.1 mg
potato starch 80 mg
lactose up to 250 mg
Example 3
Soft-shell gelatine capsules
A sterile solution of l9-noretiocholanolone 3-~2'-methyl-3'-
cyclohexyl)propionate in arachis oil, containing 83.33 g per liter, was en-
capsulated in soft-shell gelatine capsules, having a content of 0.12 ml, so
that the amount of active substance per capsule was 10 mg.
The combination of a branched chain ester and an oilyvehicle in-
creases the oral acitivity.
Example 4
Supppositories
Suppositories are prepared in the usual manner on the basis of
theobroma (cocoa butter). l9-noretiocholanolone in finely divided form is
, dispersed in the base, the mixture is melted, poured into moulds and cooled;
1 g suppositories are obtained containing 50 mg l9-noretiocholanolone.
Example 5
To a suspension of 10 g of 3~-hydroxy-5~-oestran-17-one in 100 ml
pentane and 10 ml pyridine 10 ml decanoyl chloride is added at 15C in 1 hour.
After stirring for 2.5 hours water is added to the reaction mixture to decom-
pose the excess acid chloride. The reaction mixture is extracted with pentane.
The pentane extract is dried on sodiu~ sulphate and evaporated to dryness.
The residue is crystallized from pentane. Yield 14.1 g 3~-hydroxy-5~-oestran-
17-one 3a-decanoate, m.p. 39-43C; [a]20 = ~90 (in CHC13).
In a similar manner the following esters are prepared from the cor-
responding steroid and the corresponding acid chloride:
3~-hydroxy-5~-oestran-17-one 3-propionate
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3a-hydroxy-5~-oestran-17-one 3-phenylpropionate
3a-hydroxy-5~-oestran-17-one 3-caprylate
3a-hydroxy-5~-oestran-17-one 3-(2'-methyl-3-cyclohexyl)propionate
3~-hydroxy-5~-oestran-17-one 3-oenanthate
3~-hydroxy-5~-oestran-17-one 3-decanoate
3~-hydroxy-5~-oestran-17-one 3-~2'-methyl)decanoate
Example 6
To a solution of 2.3 g 3a-hydroxy-5~-oestran-17-one in 20 ml of
2,3-dihydropyran, 0.15 ml POC13 was added, after which the mixture was stirred
at room temperature for 3 hours. The mixture was poured into water and ex-
tracted with methylene chloride. The extract was washed with water to neutral
and dried on sodium sulphate. Evaporation of the solvent in vacuo and crystal-
lization of the residue gave 2.4 g 3-hydroxy-5~-oestran-17-one 3-tetrahydro-
pyran-2'-yl ether.
In a similar manner 3~-hydroxy-5~-oestran-17-one3-tetrahydropyran-
2'-yl ether was prepared.
Example 7
A mixture of 0.96 g NaH ~50% suspension in oil) in 18 ml of dimethyl-
sulphoxide was stirred in a nitrogen atmosphere for 1.5 hours at a temperature
of 75C. After cooling down to room temperature a solution of 3.2 g 3a-hydroxy-
. 5~-oestran-17-one in 40 ml of dimethylsulphoxide was added and the mixture was
stirred for 1 hour.
After the addition of 3.7 ml methyliodide the reaction mixture was
stirred for 3 hours at room temperature. Usual processing of the reaction mix-
ture yielded 3.1 g 3a-methoxy-5~-oestran-17-one.
In a similar manner the following ethers were prepared:
3a-hydroxy-5~-oestran-17-one 3-cyclopentyl ether
3~-hydroxy-5~-oestran-17-one 3-ethyl ether
3~-hydroxy-5~-oestran-17-one 3-cyclopentyl ether.
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Example 8
3~-Hydroxy~5~-oestran-17-one (25 g) was suspended in 250 ml pentane
and 25 ml pyridine. Decanoyl chloride (25 ml) was added to the suspension at
15C in 1 hour, while stirring. After further stirring for 2.5 hours water
was added to the reaction mixture to decompose the excess acid chloride. The
reaction mixture was extracted with pentane, the extract dried on sodium sul-
phate and evaporated to dryness. Crystallisation of the residue from pentane
yielded 28.3 g 3~-hydroxy-5~-estran-17-one 3~-decanoate, m.p. 50-51.5C;
[~]D0 = +71 (in CHC13).
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