5 -estrane compounds and pharmaceutical compositions containing same

The present invention relates to new steroids useful as pyrogen-free erythropoietic drugs.

More particularly, the invention relates to nor-19 etiocholanolone (hydroxy-3 "5ß-estranone-17), its epimer-3ß and corresponding derivatives substituted in position 5, which are useful for stimulating 1¹erythropoiesis.

Erythropoiesis is the process of red blood cell formation.

Anemia is an abnormal decrease in the number of red blood cells in circulation or a decrease in the blood hemoglobin concentration. The appearance of anemia is due either to insufficient. this spinal cord, either an excessive loss of red blood cells or both. Medullary insufficiency, that is to say a reduction in erythropoiesis, can be due to a nutritional deficiency, intoxication, a tumor invasion or other sometimes unknown causes.

To treat anemias due to bone marrow failure (hypoplastic and aplastic anemias), it has been proposed to use substances stimulating the marrow such as androgens or corticosteroids. EÜA Ho 3,383,282 indicates that various derivatives of androstadiene-5,5 diol-3,17 have erythropoietic activity. EÜA Eos patents 3,519,659 and 5,519,660 describe various prednisolone derivatives having antileukemic activity.

It is known that the metabolites of certain androgenic, anabolic or progestin steroids have erythropoietic activity. Levere et al., In the proceedings of a symposium held jointly with the American Society of Hematology, December 4, 1971, Chapter III, indicate that etiocholanolone, which is a human metabolite of testosterone, has erythropoietic activity. Jepson, ibid., Chapter II, indicates that nandrolone (nor-19 testosterone; hydroxy-17ß nor-19 androstene-4 · one-3) is an anabolic steroid that has erythropoietic activity like testosterone. However, this substance has the disadvantage of having androgenic side effects. Etiocholanolone has the significant disadvantage of being pyrogenic for humans.

, The invention is based on the discovery that certain derivatives of hydroxy-3 estrane, whose hydrogen atom in position 5 is in the β configuration, have erythropoietic activity ^ are non-pyrogenic and have little or no d hormonal side effects.

The pyrogenic compounds stimulating erythropoiesis have the formula:

<img file="LU75901A1_D0001.tif" />

<img file="LU75901A1_D0002.tif" />

where R represents nn free hydroxy radical, esterified or etherified if nor-19 etiocholanolone (hydroxy-3 "5β-estranone-17) is 'nn known compound described by Engel et al,' J. Biol. Chem. 231, 1,

159 (1958). This compound is also described in an article by Connsell in Tetrahedron, Vol. 15, 202-211 (1961). The nor-19 etiocholanolone can be synthesized by hydrogenating the nandrolone acetate-17 to the corresponding acetate-17β 5β-keto-3 according to the method described in J. Org. Chem. , 2i, 2394 (1966), then reducing the keto-3 radical to form an hydroxy radical ~ 3a using lithium aluminum tri10 tert-butoxyhydride, by protecting the 3-hydroxy radical, by hydrolyzing acetate-17ß, by oxidizing the hydroxy-17ß radical to keto-17 radical with a mixture of anhydride, chronic and pyridine, then finally eliminating the protective group of the hydroxy-5 radical ".

The 3-epimer can be obtained by epimerizing nor-19 etiocholanolone, for example by transforming the hydroxyl radical ~ 3a into to-fj sylate-3 then by saponifying the tosylate with potassium acetate or better according to the process described in Tetrahedron Betters,

Flight. 18, 1619 (1973), using triphenylphosphine, diethyl azodi20 carboxylate and formic acid.

the ester and ether derivatives in position 5 of the above-mentioned compounds, which exhibit erythropoietic activity, are new.

!

The invention therefore also relates to new steroids with erythropoietic activity of formula:

i;

J

<img file="LU75901A1_D0003.tif" />

oh h¹ represents an acyloxy radical or an ether function.

The esters in position 3 are derived from acids suitable for pharmacy, which can be mineral or organic. We can cite corn10

35'

<img file="LU75901A1_D0004.tif" />

·&gt; '· Ne examples of mineral acids sulfuric acid and phosphoric acid, the esters of which are generally used as steroids in the form of an alkali metal salt such as the sodium salt. esters of organic acids, saturated or unsaturated carboxylic acids having 1 to 18 carbon atoms and preferably 3 to 12 carbon atoms. These esters can be prepared in a conventional manner by reacting the 3-hydroxy steroid with the acid or with the corresponding acyl anhydride or halide. As examples of organic carboxylic acids, mention may be made of formic, acetic, propionic, butyric, valeric, isocaproic, decanoic, undecylic, lauric and tridecylic acids. myristic, oleic, palmitic, stearic, trimethylacetic, diethylacetic, cyclohexanecarboxylic, cyclopentylpropionic, cyclohexylbutyric, cyclohexylpropionic, undecylenic, benzoic, phenylacetic, phenylpropionic, phenylbutyric, malonic, succinic, succinic, succinic.

To obtain the 3-ethers, the hydroxy3 "or hydroxy-3β steroid is etherified with a radical derived from an aliphatic hydrocarbon. cycloaliphatic, aromatic, araliphatic or heterocyclic. Mention may be made, as examples of radicals suitable for the formation of an ether, of the methoxy, ethoxy, propoxy, cyolopentyloxy, benzyloxy, phenylethoxy and tetrahydropyrannyloxy radicals. Etherification can be carried out according to known methods, for example by

I converting the hydroxysteroid to the sodium alcoholate and reacting the sodium alcoholate with a halogenated hydrocarbon in an appropriate solvent such as tetrahydrofuran or dimethyl sulfoxide. Derivatives of the tetrahydropyrannyloxy type can be obtained by reacting the hydroxysteroid with the dihydropyran.

esters-3 have the advantage of having a prolonged action. 3-ethers are active orally.

The above compounds are used in pharmaceutical forms suitable for administration to humans and other warm-blooded animals in amounts effective to stimulate erythropoiesis, which generally corresponds to unit doses containing from about 5 to about 500 mg of active ingredient. The 3-hydroxy compounds and the corresponding 3-esters are generally administered f

parenteral, for example in the form of a solution or suspension for injection in 1 ml ampoules. The compounds can be administered digestively, for example, in the form of tablets, pills, capsules, suppositories and the like;

The pharmaceutical compositions containing the medicaments of the invention can be prepared according to known methods. Unit doses may contain, in addition to the active ingredient, one or more common excipients such as vegetable oils, benzyl al5 cool, propylene glycol, glycerin, lactose, starch, magnesium stearate and raincoats. Other agents, such as preservatives, emulsifiers, stabilizers, flavors, colors, binders and / or coating agents may also be present. The capsules may be soot of hard or soft gelatin.

The following experiments illustrate the erythropoietic activity of the compounds and compositions according to the invention.

a) Biological determination of erythropoietic activity

Nor-19 etiocholanolone is administered to mice as a single 2.5 mg subcutaneous injection into a vehicle consisting of 1,2-propanediol at various intervals after causing hypoxia. The first injection is carried out on the third day following hynoxia: on the fifth day following hypoxia, “IV injects 0.5 / ^ 01 of FeCl ~ intravenously; the percentage of incorporation of red cells into the red cells is determined on the seventh day following hypoxia. Nor-19 etiocholanolone stimulates iron incorporation significantly, the percentage of incorporation due to red cells being 5.82 + 1.21 (p &lt;0.05).

b) Biological test on rat bone marrow

Nor-19 etiocholanolone in l-l propanediol-1,2 is added to rat bone marrow (SD ^, a / 100-150 g) at the start of cultivation; about 72 hours later, transferrin labeled with 0.5 µl Fe is added to the cultures; the labeled heme is extracted 6 hours later and assayed, the results suggest that nor-19 etiocholanolone is active in this system:

Concentration (M) 5x10 *? ^ χΐο ~ θ 5x1

46 172 180+47,9

c) Hemoglobin labeled with 140 is

Human bone marrow cultures are treated with nor-19 etiocholanolone for 5 days; 3 / ^ 01 of C-valine is added during the last 24 hours of culture. Hemoglobin is simultaneously isolated from cultures made with propanediol-10

1,2 or with the steroid (5x10 ΙΊ). The specific activity détermine is determined.

fique (Ιλ-οριη / Δ ^ θ) of the hemoglobins extracted and we determine the! ' report. The results show that the steroid has a stimulating activity; the ratio of the specific activity of the culture treated with the steroid to the specific activity of the culture treated with propanediol-1,2 is 1.34.

d) Cultures of human bone marrow

The incorporation of the radioactive iron into the heme is determined in 5 operate as for the rat bone marrow cultures of experiment b). Nor-19 etiocholanolone is studied at the concentration -S of 3x10 ”ΙΊ, except in the case of the marrow coming from a patient apparently suffering from no affection where a concentration of 5x10 Ii is used. The results are as follows:

Percentage of¹ incorporation of Fe into the heme (the value of 100 / g is assigned to the vehicle)

No disease Lupus Mycosis Anemia Rhabdomyosarapparent erythematosus fungus hemolytic disseminated horn r

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<img file="LU75901A1_D0005.tif" />

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<img file="LU75901A1_D0006.tif" />

144 148 93 105

The above results show that nor-19 etiocholanolone stimulates erythropoiesis in vivo and in vitro. When the tests with hydroxy-3ß 5ß-oestranone-17 are repeated, similar results are obtained.

e) Primary avian henatin cell cultures

The induction of porphyrin synthesis and heme formation in cells is studied using primary avian liver cell cultures described by Granick and Kappas, J. Biol. Chem. 242, 4587-93 (1967). According to this technique, the livers of chick embryos from 16 to 17 days are minced and the cells are separated by trypsin. Inoculated with suspensions containing 3 to 5 × 10 6 cells, containers fitted with a culture plate and containing 1.0 ml of Eagle base medium supplemented with glutamine, fetal calf serum and antibiotics. The containers are incubated at 37 ° C in air supplemented with 5% carbon dioxide for 20 hours. The culture medium is replaced with fresh medium and the steroid is added, if necessary. It is again incubated for 20 hours and the culture plates covered with a single-cell layer of hepatic parenchyma cells are examined by phase contrast and fluorescence microscopy.

A semi-quantitative evaluation of the cellular porphyrins is carried out by considering that the fluorescence intensity values ​​of between +1.0 and +4.0 correspond approximately to 5 to 50 × 10 ~ mole of protoporphyrin per milligram of proteins on the culture plate. . The quantities of protoporphyrin formed cor * · sa ► · ;.

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The

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f £

B

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I c&gt;_s correspond to the contents of ζ-aminolevulinate synthetase which is the substance limiting the rate of heme formation.

the compounds studied are (Λ) nor-19 etiocholanolone, (B) its decanoyloxy-5 derivative, (0) hydroxy-5ß 5ß-oestranone-17 and (B) its deeanoyloxy derivative · -;?. All of these compounds are nonpyrogenic.

the results obtained are shown in the following table î f '

The

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there.

BOARD

Induction of porphyrin synthesis by steroids in

<td>cultures of</td><td>liver cells</td><td colspan="2">chicken embryos.</td>

<td>Treatment</td><td>number of samples and doses</td><td>3? Ro protein toporphyrin,</td><td>found e (pmole / mg 20 hours)</td>

<td>Steroid A</td><td>4-2 / ​​tg / ml</td><td> 756,0 +</td><td> 27,6</td>

<td>Steroid A</td><td>4 - 10 / kg / ml</td><td> 814,8 +</td><td> 40,7</td>

<td>Steroid b</td><td>4 - 2 / dg / ml</td><td> 541,1 +</td><td> 52,0</td>

<td>Steroid b</td><td>4 - 10 / ^ g / ml</td><td> 454,5 +</td><td> 45,9</td>

<td>Steroid C</td><td>4 - 2 / è / ml</td><td> 647,6 +</td><td> 87,1</td>

<td>Steroid 0</td><td>4 - 10 / e / ml</td><td> 751,4 +</td><td> 105,4</td>

<td>Steroid D</td><td>4 - 2 / g / ®l</td><td colspan="2">About half of</td>

<td>Steroid 1</td><td>.4 - 10 / g / ml</td><td>values ​​of</td><td>VS</td>

the invention is illustrated by the following nonlimiting examples:

Bxenrole 1

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Pharmaceutical forms of administration in ampoules î

<img file="LU75901A1_D0007.tif" />

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Pharmaceutical forms of administration are prepared by mixing 500 g of nor-19 etiocholanolone with 2 liters of sterile sesame oil containing about 500 ml of benzyl alcohol and the mixture obtained is heated to about 80 ° C to obtain a solution.

The solution is allowed to cool to room temperature and the volume is brought to 10 liters by adding sesame oil. The solution is filtered through a sterilizing filter membrane and conditioned, for example in 2 or 5 ml bottles or in 1 ml ampoules. the concentration of the steroid solution is approximately 50 mg / ml.

Similarly, oily solutions of the following compounds are prepared, intended to be packaged in 1 ml ampoules:

nor-19 etiocholanolone propionate-5, (4-methyl-pentanoate) -5 nor-19 etiocholanolone-5, nor-19 etiocholanolone decanoate-5, hydroxy-5ß 5ß-oestranone-17, l · '

r. · l · '·.

if rfe^: p 'ί,. · é

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<img file="LU75901A1_D0008.tif" />

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çundécanoyloxy-3ß 5ß-oestranone-17, phenylpropionyloxy-3ß 5ß “Qestranone-17 ·

Example 2

tablets

<td> 5</td><td>Nor-19 cyclopentyloxy type derivative</td><td>etiocholanolone</td><td>25 mg</td>

<td></td><td>Potato starch</td><td></td><td>25 mg</td>

<td></td><td>polyvinylpyrrolidone</td><td></td><td>10 mg</td>

<td></td><td>Magnesium stearate</td><td></td><td>2 mg</td>

<td></td><td>tocopherol</td><td></td><td>0.1mg</td>

<td> 10</td><td>lactose, qs</td><td></td><td>200 mg</td>

<td></td><td>Tetrahydropyrannyloxy-3ß 5ß-oestranone-17</td><td></td><td>10 mg</td>

<td></td><td>Potato aid</td><td></td><td>10 mg</td>

<td></td><td>polyvinylpyrrolidone</td><td></td><td>5 mg</td>

<td></td><td>Magnesium sterate</td><td></td><td>1 mg</td>

<td> 15</td><td>tocopherol</td><td></td><td>0.05mg</td>

<td></td><td>lactose, qs</td><td></td><td>100 mg</td>

<td></td><td colspan="2">'(2-Kethyl decanoate) -3 of nor-19 etiocholanolone</td><td>10 mg</td>

<td></td><td>Decanoic acid</td><td></td><td>20 mg</td>

<td></td><td>tocopherol</td><td></td><td>0.1mg</td>

<td> 20</td><td>Potato starch</td><td></td><td>80 mg</td>

<td></td><td>lactose, qs</td><td></td><td>250 mg</td>

Example 5

Soft gelatin capsules

A sterile 83.33 g / l solution of (19-methyl .cyclohexyl-3 propionate) -3 nor-19 etiocholanolone is encapsulated in peanut oil at the rate of 0.12 ml so that each capsule contains 10 mg of active ingredient.

the combination of a branched ester chain and an oily vehicle increases oral activity.

E.

<img file="LU75901A1_D0010.tif" />

Example 4

suppositories

Suppositories are usually prepared from cocoa butter. The finely divided nor-19 etiocholanolone is dispersed in the base, the mixture is melted, poured into molds and cooled; suppositories weighing 1 g are obtained, containing 50 mg of nor-19 etiocholanolone.

i “!

35'

Example 5

To a suspension of 10 g of 3-hydroxy-5β-estranone-17 in 100 ml of pentane and 10 ml of pyridine, 10 ml of decanoyl chloride at 15 ° C. are added over 1 hour. After stirring for 2.5 h, water is added to the reaction mixture to decompose the excess acid chloride. The reaction mixture is extracted with pentane. The pentane extract is dried over sodium sulfate and evaporated to dryness. The residue is crystallized from pentane. 14.1 g of decanoyloxy-ρα 5ß-oestranone-17 are obtained; mp 39-43 ° W / α / ρθ = + 30 ° (in chloroform).

Similarly, the following esters are prepared from the steroid and the corresponding acid chloride: propionyloxy-3a 5β-estranone-17;

phenylpropionyloxy-3 "5ß ~ estranone ~ 1_7¹, ;

octanoyloxy-3 "5β-estranone-17;

(2-methyl-3-cyclohexyl propionyloxy) -3a 5β-estranone-17; heptanoyioxy-5a 5ß-oestranone-17; decanoyloxy-3ß 5ß-estranone-17;

(2-methyl decanoyloxy) -3ß 5ß-oestranone-17 ·

Example 6

To a solution of 2.3 g of hydroxy-3a 5ß-estranone-17 in 20 ml of 2,3-dihydro-pyran, 0.15 ml of phosphoryl chloride is added, then the mixture is stirred at room temperature for 3 hours. The mixture is poured into wa ter and extracted with meth ylene chloride. The extract is washed with water until neutral and dried over sodium sulfate. The solvent is evaporated in vacuo and the residue is crystallized to obtain 2.4 g of (tetrahydropyranny 1-2 oxy) -3oc 5β-estranone-17.

Similarly, (tetrahydropyrannyl-2 oxy) 3ß 5ß-oestranone-17 is prepared

Sxemnle 7

A mixture of 0.96 g of sodium hydride (suspension at 50 / un in oil) in 15 ml of dimethyl sulfoxide is stirred under a nitrogen atmosphere for 1.5 hours at 75 ° C. After cooling to room temperature, a solution of 3.2 g of hydroxy-3a 5βoestranone-17 in 40 ml of dimethylsulfoxide is added and the mixture is stirred for 1 hour;

After adding 3.7 ml of methyl iodide, the reaction mixture is stirred for 3 hours at room temperature. We £

· L '

<img file="LU75901A1_D0011.tif" />

Cl.

usual treatment of the reaction mixture to obtain 3.1 g of methoxy-Jo 5ß-estranone-17 "

Similarly, the following ethers are prepared: cyclope ntyloxy-3 "5β-estranone-17;

ethoxy-3ß 5ß-oestranone-17 and cyclopentyloxy-3ß 5ß-oestranone-17.