Novel derivative of alpha substituted 17 alpha substituted 17 beta-oh-19-norsteroid,its production, use thereof as drug and pharmaceutical composition containing the same

(54) (56) (57)

INVENTOR INVENTOR

Lucien Nedelec, Le Raincy Daniel

REPRESENTATIVE TITLE

Roussel-Uclaf, Paris FR Martine Moguilewsky, Paris FR, FR, Francois Nique, Pavillons-sous-Bois FR, Philiber, La Varenne Saint-Hilaire FR H Albihns patent office AB New 17Beta-0H-19-nor-steroids substituted in 17 alpha position , process for the preparation thereof, their use as pharmaceuticals and pharmaceutical compositions containing them

CALLED PUBLICATIONS:

EP A2 0 057 115 (C07J 41/00), EP Al 0 110 434 (C07J 41/00) Chemical Abstracts, Vol. 106 (1987) abstract NO. 129 694r, Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1987, C 43 (2), 319-22 (Eng)

SUMMARY:

The invention relates to new compounds of the formula:

<img file="SE503267C2_D0001.tif" />

wherein R 'represents a propyl, propenyl, iodoethenyl, iodoethynyl group or -crc-CH 2 Hal, wherein Hal 3 represents a chlorine, fluorine or bromine atom. and salts thereof.

The invention also relates to the preparation process for the compounds (I), their use as a drug, in particular anti-glucocorticoids. androgens, anti-androgens and anti-progestoretics and pharmaceutical compositions containing them.

The numbers in brackets indicate international identification code, INID code. Letters in clamps indicate international document code.

cz π _ Ό /

μ. · '' /.

^·· »

The invention relates to novel 170-OH-19-nor-steroids substituted at the 17a position, process for their preparation, use thereof as pharmaceuticals and pharmaceutical compositions containing them.

The invention relates to the compounds of formula (I):

<img file="SE503267C2_D0002.tif" />

5 wherein R 'represents a propyl group, a 1-propenyl group, an iodoethylene group, an iodoethynyl group or a group

-CSC-C ^ Hal ^, wherein Hal ^ represents a chlorine, fluorine or bromine atom, and their addition salts with mineral acids or organic acids.

When R 'represents a iodoethylene group, the double bond has E or Z configuration.

When R 'is a propenyl group, the double bond has a Z configuration.

Thus, the invention relates in particular to any of the compounds of formula (I), as defined above, with the following names:

- 17a- (3-chloro-1-propynyl) -113- [4- (dimethylamino) -phenyl] -173-hydroxy-estra-4,9-dien-3-one.

503 267

- 110- (4- (dimethylamino) -phenyl] -17α- (3-fluoro-1-propynyl) -

-170-hydroxy-estra-4,9-dien-3-one.

- 110- (4- (dimethylamino) -phenyl] -170-hydroxy-21-iodo-19-nor-17a-pregna-4,9-dien-20-yn-3-one,

- (E) 110- [4- (dimethylamino) phenyl] -170-hydroxy-21-iodo-19-nor-17a-pregna-4,9,2O-trien-3-one,

- (2) 110- (4- (dimethylamino) phenyl] -170-hydroxy-21-iodo-19-nor-17a-pregna-4,9,20-trien-3-one,

- 110- [4- (dimethylamino) -phenyl] -170-hydroxy-17a - ((Z) -1-propenyl) -estra-4,9-dien-3-one.

- 110- (4- (dimethylamino) phenyl)] - 170-hydroxy-17α-propyl-estra-4,9-dien-3-one and

- 17a- (3-bromo-1-propynyl) -110- [4- (dimethylamino) -phenyl] -170-hydroxy-estra-4,9-dien-3-one. and their addition salts with mineral or organic acids.

Among the preferred compounds of the invention, one may in particular mention:

- 110- [4- (Dimethylamino) -phenyl)] -17a- (3-fluoro-1-propynyl) -170-hydroxy-estra-4,9-dien-3-one.

- (Z) 110- (4- (dimethylamino) -phenyl] -170-hydroxy-21-iodo-19-nor-17a-pregna-4,9,20-trien-3-one,

- 110- [4- (dimethylamino) -phenyl] -170-hydroxy-17a - [(Z) -1-propenyl] -estra-4,9-dien-3-one.

As addition salts of the compounds of formula (I) with acids, in particular, chlorohydrates and methanesulfonates can be mentioned.

The products of the invention as well as their addition salts with pharmaceutically acceptable acids are particularly interesting products in the pharmacological sense. In particular, they have a remarkable anti-glucocorticoid activity.

The products of the invention also have androgenic or anti-androgenic properties.

Investigation of the products has also admitted that it was possible to demonstrate a remarkable anti-progestomimetic activity.

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Thus, the compounds of the invention true their addition salts down to pharmaceutically acceptable acids can be used for the control in substantially noteworthy side effects of glucocortioids. They also downplay the note disorders caused by hypersecretion of glucocortioids and in particular note aging in general and in particular note hypertension, arteriosclerosis, osteoporosis, diabetes, obesity, true reduction of innuendo and insomnia.

The products of formula (I) of the invention true their addition salts down to pharmaceutically acceptable acids, which have anti-progestoninetic properties, can be used to produce original contraceptive agents. They can also be used to note hornonal disorders and, moreover, they may have an interest in the treatment of hornon-dependent cancers.

The products of formula (I) according to the invention true their addition salts down to pharmaceutically acceptable acids may also have progestoninetic properties and thus can be used in the treatment of anenorrhoea, dysrenorrhea and luteal insufficiency.

Thus, the invention relates to the so-called healing portion of the pharmaceutically acceptable compounds down to formula (I), i.e. non-toxic at the doses used, true their addition salts down pharmaceutically acceptable acids.

In particular, the present invention relates to medicinal products, the preferred products mentioned above are, in particular, the compounds of Examples 2, 5 and 6 (product A), which are pharmaceutically acceptable.

The useful dose varies as a function of the ailment to be treated and the mode of administration. For example, they can ranging from 10 mg to 1 g and preferably from 100 mg to 1 g / day in the adult upon oral administration.

The products of the present invention as well as their addition salts with pharmaceutically acceptable acids, which have anti-progestomimetic activities, can also be used as abortion agents in the veterinary field and have a special interest in dogs and cats.

The invention thus relates, as a veterinary drug, to the compounds of formula (I) and their addition salts with pharmaceutically acceptable acids.

The invention relates, in particular, as a veterinary drug, to the preferred products mentioned above, and in particular to 110- [4- (dimethylamino) -phenyl-173-hydroxy-17a - [(Z) 1-propenyl] -ester-4.9-diene. 3-one.

The daily dose varies as a function of the mode of administration and the animal to be treated.

It can be from 3 to 5 mg / kg live weight in 1, 2 or 3 injections at 24 hour intervals and e.g. 5 mg / kg live weight in 2 injections for 24 hour intervals in dogs with the product of Example 6 (product A).

The compounds of formula (I) of the present invention and their salts as defined hereinabove can be used to prepare pharmaceutical compositions containing as active substance at least one of said products.

The compounds of formula (I) of the present invention and their salts may be used by administration via the digestive tract, parenterally or locally. They can be prescribed in the form of tablets, simple or coated, capsules, granules, suppositories, injectable preparations, pomadas, creams, gels, which are prepared by the usual methods.

The active substance (s) may be incorporated therein with excipients commonly used in pharmaceutical compositions such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, greases of animal or vegetable origin, paraffin derivatives, paraffin derivatives, , dispersing or emulsifying agents, preservatives.

r. 7 **&gt; Z 7 '-J 4-- -J /

The invention thus relates to pharmaceutical compositions containing as active substance at least one compound of formula (I) according to the present application or one of its addition salt with pharmaceutically acceptable acids.

Thus, the invention also relates to pharmaceutical compositions for veterinary use containing as active substance at least one compound of formula (I) of the present invention or one of its addition salts with pharmaceutically acceptable acids.

In particular, the invention relates to veterinary pharmaceutical compositions containing as active substance 110- [4- (dimethylamino) phenyl] -17β-hydroxy-17α - [(Z) 1-propenyl] -ester-4,9-diene-3 one.

The invention also relates to a preparation process for the compounds of formula (I) characterized in that:

<img file="SE503267C2_D0003.tif" />

wherein Hal represents a bromine, chlorine or fluorine atom, a brominating or chlorinating reagent such as triphenylphosphine is reacted with carbon tetrabromide or carbon tetrachloride in the presence of a solvent such as tetrahydrofuran or methylene chloride of the compound of formula (P

<img file="SE503267C2_D0004.tif" />

503 267 to obtain the compound of formula (ΙΙ) wherein Hal, be2A 1 represents a bromine or chlorine atom, and then the latter product is reacted with a bromine or chlorine exchange reagent with fluorine such as cesium fluoride or potassium fluoride in the presence of 18-crown ether -6 in acetonitrile,

b) for the preparation of the compounds of formula (I):

2d

<img file="SE503267C2_D0005.tif" />

wherein the double bond has E or Z configuration, a product of formula (P) is reacted₂):

<img file="SE503267C2_D0006.tif" />

(P₂) wherein K represents a protected ketone group, e.g. an ethylene dioxide group, with a reducing agent such as tributyltin hydride or under radical conditions in the presence of e.g. azoisobutyric acid nitrile to obtain a double bond E. or in a polar aprotic solvent such as hexamethylphosphorous triamide to obtain a double bond Z, then an iodine agent such as N-iodosuccinimide to obtain, after elimination of the protecting group. the compounds of formula (I) wherein the double 2d bond has E or Z configuration,

(c) to prepare the product of formula (Ι)₂θ):

<img file="SE503267C2_D0007.tif" />

<img file="SE503267C2_D0008.tif" />

<img file="SE503267C2_D0009.tif" />

(IN

<img file="SE503267C2_D0010.tif" />

2C, a product of formula (P₃):

<img file="SE503267C2_D0011.tif" />

(P₃) with an iodine reagent such as N-iodo-succinimide in the presence of a silver salt such as silver carbonate or silver nitrate to obtain the product of formula (I),

<img file="SE503267C2_D0012.tif" />

wherein the dotted line denotes the presence of a single bond or a double bond (Z) between the carbons bearing it.

<img file="SE503267C2_D0013.tif" />

&lt;^p4&gt;

<img file="SE503267C2_D0014.tif" />

<img file="SE503267C2_D0015.tif" />

267 with hydrogen in the presence of a catalyst such as palladium on barlum sulfate partially poisoned with an amine such as pyridine or triethylamine to obtain as the main product the formula (Ι_2β), ^where* the dashed line indicates the presence of a second bond (Z) and, as a by-product, the product wherein the dashed line indicates the presence of a single bond.

The product is described in the French patent 2,566,779, the products P₂ and P₃ is disclosed in European Patent 0 057 115, the product P is disclosed in French Patent 2,497,807.

The products of formula (I) are included in the general formula of European patent 0 057 115 without, however, being described therein.

The following examples describe the invention without limiting it in any way.

Example 1: 17a- (3-Chloro-1-propynyl) -118- [4-dimethylamino-phenyl] -178-hydroxy-estra-4,9-di-en-3-one

800 mg of 118- [4- (dimethylamino) -phenyl] -178-hydroxy-17a- (3-hydroxy-1-propynyl) -ester-4,9-dien-3-one disclosed in French patent BF 2,566,779 is dissolved in 8 ml of tetrahydrofuran and 8 ml of carbon tetrachloride. 950 mg of triphenylphosphine is added and stirred at 90 ° C for 3 hours. A slightly insoluble material is filtered and the filtrate is evaporated to dryness. 1.40 g of crude product is recovered. The product is purified by chromatography on silica column eluting with the mixture petroleum ether (boiling point: 40-70 ° C) ethyl acetate (50/50). 420 mg of pure chlorinated product is obtained in the form of solid crystalline substance.

Mp. = 238 ° C.

IR spectrum (CHC1):

-1 ³ -1

OH 3600 cm C = 0 conjugated: 1655 cm

C = C conjugated and aromatic 1612/1562/1518 cm¹ r .-. 9 ϋ&gt;. &lt;J 4_ «· /

Example 2: 118-4- (dimethylamino) -phenyl 1-17α- (3-fluoro-1-propynyl) -178-hydroxy-estra-4,9-dien-3-one

313 mg of 17a- (3-chloro-1-propynyl) -118- [4- (dimethylamino) -phenyl] -178-hydroxy-estra-4,9-dien-3-one is stirred in 10 ml of dry acetonitrile with 600 mg of potassium fluoride and 600 mg crown ether 18 - 6. Heat to 90 ° C under inert atmosphere. After 23 hours of reflux, the solvent is driven off, the residue is taken up with water and extracted with ethyl acetate. After washing with brine and drying over magnesium sulfate, the organic phase is evaporated. 322 mg of crude fluorinated product are recovered. The crude product is purified by chromatography on silica column eluting with the mixture methylene chloride / ethyl acetate 90/10. 79 mg of pure product is recovered, which is recrystallized in a mixture of methylene chloride and isopropyl ether to obtain 55 mg of desired product. Mp. = 234 - 235 ° C.

IR spectrum (CHC1):

-1 ³ -1

OH 3600 cm C = 0 conjugated: 1655 cm

C = C conjugated and aromatic 1612/1562/1568 cm¹

Example 3: 118-1,4- (Dimethylamino) -phenyl-1,178-hydroxy-21-iodo-19-nor-17a-preqna-4,9-dien-20-yn-3-one

975 mg of 118- [4- (dimethylamino) phenyl] -178-hydroxy-19-nor-17a-pregna-4,9-diene-20-yn-3-one is dissolved in 20 ml of acetone, 640 mg of silver carbonate is added. then 450 mg of N-iodosuccinimide. The mixture is stirred for 4 hours, poured into a 10% aqueous solution of sodium thiosulfate and extracted with methylene chloride. The organic phase is washed with water, dried and evaporated to dryness. 1.2 g of crude product is obtained. The latter is chromatographed on silica under elution first with a mixture of cyclohexane-ethyl acetate (6-4) to obtain 390 mg of desired product and 760 mg of mixture which is chromatographed on silica, eluting with a mixture of hexane ether (3-7 ) to obtain 530 mg of desired product. The 920 mg of product obtained is crystallized in ether and 730 mg of products are obtained. Mp. = 210 ° C) then 79 mg of additional product starting from the mother liquors.

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IR spectrum (CHC1):

OH 3598 cm^-1

CgC 2174 cm^-1

Dienon 1654 cm '¹

Aromatic 1612/1562/1518 cm¹

Example 4: (E) 118- (4- (Dimethylamino) -phenyl) -178-hydroxy-21-iodo-19-nor-17α-preqna-4,9,20-trien-3-one Step A: (E) (1,2-Ethanediyl) -cyclic acetal of 5α, 178-dihydroxy-118- (4- (dimethylamino) -phenyl] -21-iodo-19-nor-17a-pregna-9.20-dien-3-one.

1) Dissolve 1.5 g of (1,2-ethanediyl) -cyclic acetal of 5α, 178-dihydroxy-118- (4- (dimethylamino) phenyl] -19-nor-17a-pregna-9-en-2O -yn-3-one in 30 ml of anhydrous tetrahydrofuran, add 8 ml of tributyltin hydride and 300 mg of azoisobutyric acid nitrile and reflux for 50 minutes. Concentrate under reduced pressure and chromatograph the remaining oil diluted in methylene chloride on silica. Elute with a mixture of cyclohexane-ethyl acetate (7-3) to obtain 2.96 g of intermediate tributylstannyl vinyl derivative.

2) It is dissolved in 30 ml of anhydrous tetrahydrofuran, 900 mg of N-iodosuccinimide is added. After 25 minutes of reaction, the reaction mixture is poured into a 10% aqueous solution of sodium thiosulfate and extracted with methylene chloride. The organic phase is washed with water, dried and evaporated to dryness. The residue is stirred in isopropyl ether under reflux. is then cooled and centrifuged. 1.66 g of desired product is obtained. Mp. = 246 ° C.

IR spectrum (CHC1):

³ -1

Free OH: 3600 cm

OH in 5 position: 3500 cm ¹

Aromatic: 1613/1517 cm ¹

Step B: (E) 118- [4- (Dimethylamino) -phenyl] -178-hydroxy-21-iodo-19-nor-17a-pregna-4,9,20-trien-3-one λ / η

Ο / r η -&gt;

ύ j ο

1.66 g of the product obtained under A is dissolved in 16 ml of methanol and 16 ml of 2N hydrochloric acid. The solution is allowed to stand for one hour at room temperature, the reaction mixture is poured into aqueous sodium bicarbonate solution, filtered and dissolved in the methylene chloride. The organic phase is dried and evaporated under reduced pressure. Chromatograph the residue on silica, elute with a mixture of cyclohexane-ethyl acetate (7-3) and recover 1.28 g of crude desired product. After dissolving in methylene chloride, then concentrating after crystallization by addition of ether, 1.15 g of the desired product is obtained.

Mp. = 236 ° C after recrystallization in ethanol.

IR spectrum (CHC1):

Dienon C = O: 1654 cm

C = C) 1612 cm

<td>Aromatic(</td><td> 1518</td><td>-1 cm</td>

<td rowspan="2">OH:</td><td></td><td> -1</td>

<td> 3600</td><td>cm</td>

Example 5: (Z) 11B- [4- (Dimethylamino) -phenyl] -1-176-hydroxy

<td>Step A:</td><td>-21-iodo-19-nor-17-pregna-4,9,2O-triene-3-one (Z) (1,2-ethanediyl) -cyclic acetal of 5α, 176-</td>

-dihydroxy-HB [4- (dimethylamino) phenyl] -21-tributylstannyl-19-nor-17-pregna-9,20-dien-3-one.

477 mg (1,2-ethanediyl) -cyclic acetal of 5α, 17β-dihydroxy-116- (4- (dimethylamino) -phenyl) -19-nor-17α-pregn-9-en-20-yn-3 is dissolved -one in 5 ml of hexamethylphosphorous triamide, add 2.6 ml of tributyltin hydride under inert atmosphere and heat at 70 ° C for 25 hours, cool, dilute with water and extract with ethyl acetate, wash with the organic phase with water, dry and concentrate to dryness. The residue is chromatographed on silica, eluted with a mixture of petroleum ether (boiling point: 40-70) ether (6-4), and 214 mg of E isomer is obtained, 135 mg of mixture of isomers E and Z and 346 mg of the desired isomer (Z ).

503 267

Step B: (Z) 110- [4- (Dimethylamino) -phenyl] -17B-hydroxy-21-iodo-19-nor-17a-pregna-4,9,20-trien-3-one.

1.47 g (Z) isomer obtained as in A in 30 ml of tetrahydrofuran is dissolved. with stirring, add 520 mg of N-iodosuccinimide. After 30 minutes at room temperature, the reaction mixture is poured into aqueous sodium thiosulphate and extracted with ethyl acetate. The organic phase is washed with water, dried and concentrated to dryness. The resulting residue is quenched with 10 ml of methanol with 10 ml of 2N hydrochloric acid and allowed to react for one hour. Alkaline is stirred with a sodium bicarbonate solution and extracted with methylene chloride. The organic phase is washed with water, then dried it concentrates to dryness to obtain 1.36 g of crude product. The residue is chromatographed on silica, eluted with a mixture of cyclohexane-ethyl acetate and separated by 600 mg of the desired product. Mp. 178 178 ° C after ether crystallization.

<td>IR spectrum</td><td colspan="2">(CHC1)</td>

<td>Region C = O:</td><td> 1650</td><td>cm^-1 -1</td>

<td>C = C</td><td> 1612</td><td>cm -1</td>

<td>Aromatic:</td><td> 1518</td><td>cm -1</td>

<td>OH:</td><td> 3590</td><td>cm</td>

Example 6: (Ζ) 118-Γ4-Dimethylamino) -phenyl1-17B-hydroxy-17a - [(Z) 1-propenyl] -stra-4,9-dien-3-one (product A) and 118-r4- ( dimethylamino) -phenyl-1,176-hydroxy-17a-propyl-estra-4,9-dien-3-one (product B) g 11B- [4- (dimethylamino) -phenyl] -17β-hydroxy-17α (13 - propynyl) -ester-4,9-dien-3-one is dissolved in 50 cm 2 of ethanol and 1 cm of triethylamine and 75 mg of 10% palladium hydroxide is added to barium sulphate. The mixture is hydrogenated at normal temperature and stopped after absorption of 55 cm of hydrogen. . The catalyst is filtered, rinsed with ethanol then the solvents are evaporated. 1.076 g of crude product is recovered which is chromatographed on a silica column (eluent cyclohexane-ethyl acetate: 70-30). You obtain successively:

^{n 7} 2 7

- 92 mg of (Z) lip- (4- (dimethylamino) -phenyl] -178-hydroxy-17a - [(Z) 1-propenyl] -estra-5 (10) -en-3-one (Rf: 0.40 )

- 568 mg of (Z) 118- (4- (dimethylamino) -phenyl] -178-hydroxy-17a - [(Z) 1-propenyl] -estra-4,9-dien-3-one (Rf: 0, 27) (product A),

- 59 mg of 118- (4- (dimethylamino) -phenyl] -178-hydroxy-17a-propyl-estra-4,9-dien-3-one (Rf: 0.24) (product B).

Physico-chemical constants of product A

Analysis: C_2ghrs_3?NO₂ = 431,63

Calculated: C% 80.70 Η% 8.64 N% 3.25

Found: 80.34 8.7 3.2

IR spectrum (CHCl

OH 3608 cm^-1

C = O at C = C conjugated 1654/1612 cm^-1

Aromatic: 1580/1518 cm^-1

UV Spectrum (EtOH)

Max 258 nm = 17 900

Max 303 nm = 22 700 + HCl 0, IN max = 301 nm 21 300

Physico-chemical constants of product B IR spectrum (CHC1)

-1 ³ -1

OH at 3615 cm C = O / C = C conjugated 1654/1612 cm

Aromatic 1560/1518 cm¹

Example 7: 17β- (3-Bromo-1-propynyl) -118-4- (dimethylamino) -phenyl-1,178-hydroxy-estra-4,9-dien-3-one

600 mg of 118- [4- (dimethylamino) -phenyl] -178-hydroxy-17a- (3-hydroxy-1-propynyl) -estra-4,9-dien-3-one is dissolved in 6 ml of methylene chloride with 491 mg of carbon tetrabromide. The solution is cooled to -10 ° C. In addition, a drop of solution is then added drop by drop

531 mg of triphenylphosphine in 3 ml of methylene chloride. The mixture is stirred for 20 minutes at -10 ° C. It is then placed as it is on a column of 15 g silica and eluted with the mixture petroleum ether (boiling point 40 - 70 ° C) / ethyl acetate 50/50.

503 267

384 mg of pure brominated product is obtained in the form of a solid crystallized substance.

Pharmaceutical compositions

Tablets have been prepared having the following composition:

- Product of Example 6 (Product A) ........... 200 mg

- diluent qs for a tablet up to .... 350 mg (Examples of diluents: talc, starch, magnesium stearate).

veterinary composition

An injectable solution having the following composition has been prepared:

- Product according to Example 6 (Product A) ..........

100 mg

- ethanol .........................................

0.3 ml

- peanut oil

gsp 3 ml

Pharmacological examination of the invention

I) Investigation of the activity of the product of the invention on hormonal receptors:

Rabbit progestogen receptor:

Non-adult rabbits weigh about 1 kg from a cutaneous administration of 25 g of estradiol. Five days after this treatment, the animals are killed, euthanized, weighed, and homogenized at 0 ° C using a Potter Teflon glass in a buffered solution TS (Tris 10 nM, sucrose 0.25 M, HCL pH 7.4) (1 g of tissue per 50 ml of TS). The homogenate is then ultracentrifuged (105,000 gx 90 min) at 0 ° C. Samples of the supernatant thus obtained are incubated at 0 ° C. Samples of the supernatant thus obtained are incubated at 0 ° C for a time t with a constant concentration (T) of tritium-treated product R (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) in

-9 presence of increasing concentrations (0 - 2500 x 10 M) or cold R, or cold progesterone, or the cold product to be examined. The concentration of bound tritium-treated R (B) is then measured in each incubate with the technique of carbon dextran.

/-)/-7

X- ί

7ι 7

· .. J

Rat mouse glucocorticoid receptor:

Male Sprague-Daeley EOPS rats with a weight of

160 - 200 g of ectomized across the kidneys. Four to eight days after this cut, the animals are killed, and the thymus is taken out and homogenized at 0 ° C in a buffer Tris 10 mM, sucrose 0.25 M, dithiothreitol 2 mM, HCl pH 7, 4, using a Potter polytetrafluoroethylene buffer. glass (1 g of tissue per 10 ml of TS). The homogenate is then ultracentrifuged (105,000 gx 90 min) at 0 ° C. Samples of the supernatant thus obtained are incubated at 0 ° C for a time (t) with a constant concentration (T) of tritium-treated dexamethasone in the presence of increasing concentrations (0 - 2500 x 10 M) or cold dexamethasone, or the cold product to be examined. The concentration of bound tritium-treated dexamethasone (B) is then measured in each incubate with the technique of carbon dextran.

Calculation of relative binding affinity: The calculation of relative binding affinity (RBA) is identical for all receptors.

The following two curves are drawn: the percentage of tritium-treated horB as ~ as the function of the logarithm of the concentration of

B the cold reference hormone and ψ as the function logarithm of the concentration of the cold tested product.

BB

The right equation I = (~ max + ~ min) / 2 g ψ max = percent bound tritium-treated hormone is determined for the incubation of this tritium-treated hormone at concentration (T).

β ψ = percent bound tritium-treated hormone for the incubation of this tritium-treated hormone at a concentration (T) in the presence of a large excess of cold hormone (2500 x 10⁹M).

<img file="SE503267C2_D0016.tif" />

<img file="SE503267C2_D0017.tif" />

<img file="SE503267C2_D0018.tif" />

267

The intersections between lines I and the curves allow evaluation of the cold reference hormone (KH) and cold tested product (KX) concentrations which inhibit 50% of the binding of the tritium-treated hormone to the receptor.

The relative binding affinity (RBA) of the product under investigation is determined by the equation:

RBA = 100 (KH) (KX)

The results obtained are as follows:

<td rowspan="2">Product in the examples</td><td rowspan="2">Incubation time at 0 ° C</td><td colspan="2">progestogen</td><td colspan="2">Glukokortioid</td>

<td>2 H</td><td>24 H</td><td>4 H</td><td>24 H</td>

<td> 1</td><td></td><td> 72 :</td><td> 286</td><td> 139 :</td><td> 159</td>

<td> 5</td><td></td><td> 41 :</td><td> 184</td><td> 145 :</td><td> 113</td>

<td>6 (product A)</td><td></td><td> 96 :</td><td> 491</td><td> 147 :</td><td> 115</td>

Abortive effect on dog

Production. the_solution_ of that product. to be examined 250 mg of product to be tested is dissolved in 1 ml of methylene chloride, then diluted 1 ml of this solution in sesame oil until 250 ml of final volume is obtained.

Ecography determines the state of pregnancy in dogs aged 16 months to 4 years and notes the gestation period ranging from 25 to 40 days.

The product to be tested in a solution corresponding to the above preparation is administered by subcutaneous administration at the dose of 5 mg / kg and two injections are performed at 24 hour intervals.

r η τ ζ ”

C-C ·.) J. Ο /

A control ecography is performed 3 to 10 days after the last injection.

days after the injection of the product of Example 6 (product A), abortion is noted in 66% of cases.

days after the injection of the product in Example 6 (product A), a complete abortion is noted in all the animals in the group.

Abortion effect on rabbit

Rabbits receive an injection subcutaneously 10 days after mating of the product to be tested in solution corresponding to the above preparation at the dose of 4 mg / kg and 5 mg / kg.

days after this treatment, the animals are killed and the number of abortions noted with autopsy is noted.

A complete abortion is noted in all animals in the group with the product of Example 6 (product A) administered at the dose of 4 mg / kg and at the dose of 5 mg / kg.

503 267