FR2548192A1 - Iodine-labelled radioactive estradiene derivatives, process for preparing them and their application in radioimmunological assays - Google Patents

Abstract

The subject of the invention is iodine-labelled radioactive estradiene derivatives of the general formula: in which the wavy line on the oxime nitrogen means that the oxime functional group may be in syn or anti position, in which formula R represents hydrogen, an alkyl group having 1 to 6 carbon atoms or an acyl group of a carboxylic acid having from 2 to 12 carbon atoms and R1 represents the residue of an amino acid R1NH2 or the residue of a derivative of the latter, this residue possessing an iodine-accepting group and being labelled with <125> or <131>iodine, the process for preparing them and their application in radioimmunological assays.

Description

The present invention relates to new radioactive estradienic derivatives labeled with iodine, their preparation process and their application to radioimmunological assays.

dans laquelle la ligne ondulée sur l'azote oximinique signifie que la fonction oxime peut être en position syn ou anti, fcrmule dans laquelle et dans ce qui suit R représente de l'hydrogene, un groupement alcoyle ayant de 1 a 6 atomes de carbone ou un groupement acyle d'un acide carboxylique ayant de 2 a 12 atomes de carbone et R1 représente le reste d'un acide aminé R1NH2 ou le reste d'un dérivé de ce dernier, ce reste étant accepteur d'iode et étant marqué a l'iode 125 ou 134. More particularly, it relates to estradienic derivatives labeled with iodine, in the form of a mixture of syn and anti isomers or in the form of syn isomers or in the form of anti isomers, of general formula (I)

in which the wavy line on the oximine nitrogen means that the oxime function can be in the syn or anti position, in which and in the following R represents hydrogen, an alkyl group having from 1 to 6 carbon atoms or an acyl group of a carboxylic acid having from 2 to 12 carbon atoms and R1 represents the residue of an amino acid R1NH2 or the residue of a derivative of the latter, this residue being an acceptor of iodine and being labeled with l iodine 125 or 134.

Among the values of R, there may be mentioned a) the methyl, ethyl, propyl, isopropyge, butyl, sec-butyl, tert-butyl, pentyl, iso-pentyl, sec-pentyl, tert-pentyl, neo-pentyl, hexyl radicals , iso-hexyl, sec-hexyl, tert-hexyl; b) formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, hexanoyl, benzoyl, capryl, methoxy-carbonyl and ethoxy carbonyl radicals.

A more particular subject of the invention is the radioactive estradienic derivatives labeled with iodine of general formula (I) in which R1 represents the residue of an amino acid P1NH2 or the residue of a derivative of the latter, chosen from the group consisting of histidine, tyrosine, histidinol, histamine, tyramine and methyl tyrosinate labeled with iodine 125 or 131.

(Produit A) l'iode pouvant etre en position 2 ou 5.Among the products of general formula (I), there will be mentioned in particular 3-carboxymethyloxime of 17 ss -hydroxy-11 / - (4-dimethylaminophenyl) -17r - (prop-1-ynyl) estra-4,9-dien-3- one in the form of a mixture of syn isomer and anti isomer, in the form of syn isomer and in the form of coupled anti isomer of 1'125 I histamine (product A) of formula

(Product A) iodine can be in position 2 or 5.

A subject of the invention is also a process for preparing the products corresponding to the general formula above.

dans laquelle et dans ce qui suit R a les significations précitées avec avec un halogénure de carboxymethoxylamine en présence d'une base et obtient un produit de formule (III) ::

dans laquelle la ligne ondulee sur l'azote indique que ce produit est sous forme de mélange d'isomères syn et anti, puis - fixe sur la fonction acide de ce produit un groupement activateur de la fonction carbonyle et obtint le produit de formule générale (IV)

dans laquelle R2 représente un groupement activateur de la fonction carbonyle et la ligne ondulée sur l'azote indique que ce produit est sous forme de melange d'isomères syn et anti, que l'on fait réagir avec un acide aminé R1NH2 possédant un groupe accepteur d'iode marqué a l'iode 125 ou 131 ou avec un dérivé d'un tel acide et obtient le produit cherché de formule générale (I), que l'on isole sous forme de mélange d'isomères syn et anti ou que l'on sépare, si désiré, en ses isomères.This process is characterized in that - a product of general formula (II) is made:

in which and in what follows R has the aforementioned meanings with with a carboxymethoxylamine halide in the presence of a base and obtains a product of formula (III):

in which the wavy line on the nitrogen indicates that this product is in the form of a mixture of syn and anti isomers, then - fixes on the acid function of this product an activating group of the carbonyl function and obtains the product of general formula ( IV)

in which R2 represents an activating group of the carbonyl function and the wavy line on the nitrogen indicates that this product is in the form of a mixture of syn and anti isomers, which is reacted with an amino acid R1NH2 having an acceptor group of iodine labeled with iodine 125 or 131 or with a derivative of such an acid and obtains the desired product of general formula (I), which is isolated in the form of a mixture of syn and anti isomers or that the It is separated, if desired, into its isomers.

This process is further characterized in that: the amino acid acceptor of iodine or the amino acid derivative is chosen from the group consisting of histidine, tyrosine, histidinol, histamine, tyramine or methyl tyrosinate; - the carboxymethoxylamine halide is caboxymethoxylamine hemihydrochloride and the operation is carried out under an inert atmosphere and in the presence of sodium hydroxide; - An activating group of the carbonyl function is fixed to the acid function by causing an alkyl halo formate to act in the presence of a tertiary base, in an anhydrous medium and under an inert atmosphere.

Under the preferred operating conditions, the process is characterized by the following points: - the alkyl haloformate is isobutyl chloroformate and the operation is carried out in the presence of tri-n-butylamine; - the amino acid or its derivative, which is reacted with the product of general formula (IV) in which Re represents an activating group of the carbonyl function, is histamine labeled with iodine 125 or 131 or its derivative, and the operation is carried out under an inert atmosphere.

Activation of the carbonyl function of the product of general formula (III) can also be carried out by causing N-hydroxy succinimide or dicyclohexylcarbodiimide to act thereon, which generates activating groups of said carbonyl function.

The etherification or esterification of the hydroxyl function at 17 of the product of general formula (I), in which R represents hydrogen, can be carried out according to the usual methods.

The etherification can be carried out, for example, by means of an etherifying agent such as an alkyl or aryl halide, or a dialkyl sulfate.

Esterification can be carried out, for example, by means of an esterifying agent such as a functional acid derivative, such as an acid chloride or an anhydride.

To obtain the syn and anti isomers of the products of general formula (I) separated from the object of the invention, one can proceed, for example, in the manner described below: a product of general formula (III) is subjected in the form of a mixture of syn isomer and anti isomer, R has the above meanings, has the action of an esterifying agent to esterify the carboxyl function, separates the syn isomer from the anti isomer from the resulting ester puts saponifies separately said ester function of each of the syn and anti isomers, obtains the syn isomer and the anti isomer of the products of general formula (III), in which R keeps the aforementioned meanings and then proceeds separately, according to the process of the invention , on the attachment to the acid function released from the syn and anti isomers, of an activating group of the carbonyl function, the corresponding mixed anhydrides are obtained, which are reacted with an iodine acceptor amino acid labeled with iodine 125 or 131, or with a drift of this t acid * to obtain products of general formula (I), object of the invention, in the form of syn isomer and in the form of separated anti isomer.

A subject of the invention is also, as already stated, the application of the products of general formula (I) and in particular of 3-carboxymethyloxime 17ss -hydroxy-11 ss - (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) estra-4,9-dien-3-one, as a mixture of syn isomer and anti isomer, as syn isomer and as anti isomer coupled to 1 '125I histamine (product A), under study and in the radioimmunological assay of 17ss -hydroxy-11ss - (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) non-radioactive estra-4,9-dien-3-one (product B) in biological fluids, in humans or in animals.

dans lesquelles la ligne ondulée sur l'azote oximinique signifie que la fonction oxime peut être en position syn ou anti et dans laquelle R représente de l'hydrogènes un groupement alcoyle ayant de 1 à 6 atomes de carbone ou un groupement acyle d'un acide carboxylique ayant de 2 à 12 atomes de carbone, produits de formule (IV) où R2 représente un groupement activateur de la fonction carbonyle.A subject of the invention is also, as new industrial products and in particular as intermediate products necessary for carrying out the process of the invention, the products of general formulas (III) and (IV) in the form of a mixture of isomers. syn and anti, or as syn isomer or as anti isomer

in which the wavy line on the oximine nitrogen means that the oxime function can be in the syn or anti position and in which R represents hydrogen, an alkyl group having from 1 to 6 carbon atoms or an acyl group of an acid carboxylic acid containing from 2 to 12 carbon atoms, products of formula (IV) where R2 represents an activating group of the carbonyl function.

Among these products, the following products will be mentioned: - 3-carboxymethyl bxi me of 17? -hydroxy-1- (4-dimethyl ami nophenyl) -17 - (prop-1-ynyl) estra-4,9-dien-3-one as a mixture of syn and anti isomers, as syn isomer and as anti isomer, - the mixed anhydride of 3-carboxymethyloxime of 17ss-hydroxy-11ss - (4- dimethylaminophenyl) -17 q - (prop-1-ynyl) estra-4,9-dien-3- one with isobutyl formate in the form of a mixture of syn and anti isomers, under
as syn isomer and as anti isomer

The products of general formula (III) are starting products useful for the preparation of antigens, also necessary for the radioimmunological assays of the above-mentioned product B.

Their application as such is also the subject of the present invention and is characterized in that one of these products is conjugated with bovine serum albumin (BSA) or with human serum albumin (HSA) and obtained the antigen sought.

dans laquelle la ligne ondulée et R ont les significations précitées en vue de l'activation de la fonction carbonyle, avec un halogénoformiate d'alcoyle, en présence d'une base tertiaire, en milieu anhydre et sous atmosphère Inerte, obtient l'anhydride mixte correspondant, de formule (IV) ::

dans laquelle R garde la signification précitée et R2 représente un radical de formule -CO-O-alc, alc étant un radical alcoyle ayant au plus 6 atomes de carbone, - que l'on conjugue avec l'albumine sérique bovine (BSA) ou humaine (HSA) et obtent l'antigène cherché.Under preferential conditions of implementation, the application of the products of general formula (III) is characterized by the following points - a product of general formula (III) is reacted in the form of a mixture of syn and anti isomers, as syn isomer or as anti isomer

in which the wavy line and R have the aforementioned meanings for the purpose of activating the carbonyl function, with an alkyl haloformate, in the presence of a tertiary base, in an anhydrous medium and under an inert atmosphere, obtains the mixed anhydride corresponding, of formula (IV) ::

in which R keeps the aforementioned meaning and R2 represents a radical of formula -CO-O-alc, alc being an alkyl radical having at most 6 carbon atoms, - which is conjugated with bovine serum albumin (BSA) or human (HSA) and obtain the desired antigen.

The application of the products of general formula (III) is further characterized by the following points - the halogeno ~ alkyl formate is isobutyl chloroformate and the operation is carried out in the presence of tri-n-butylamine and under an inert atmosphere, - the mixed anhydride of general formula (IV) is reacted with bovine (BSA) or human (HSA) serum albumin, having previously dissolved the latter in a water-dioxane mixture under an inert atmosphere.

The application described makes it possible to obtain, respectively, starting from 17 ss 3-carboxymethyloxime -hydroxy-11ss- (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) estra-4,9-dien-3-one, as a mixture of syn isomer and anti isomer, as syn isomer or as anti isomer, antigen of general formulas (V) and (VI> in which R has the

<tb> aforementioned <SEP> meanings <SEP>: <SEP> 1
<tb><SEP> H3C3 <SEP> R
<tb><SEP>(> Dt <SEP> C <SEP> ~ <SEP> C-CH3
<tb><SEP> (v)
<tb><SEP> 3
<tb><SEP> o
<tb><SEP> CH2
<tb><SEP> I <SEP> I
<tb><SEP> n
<tb><SEP> BSA
<tb> in which n = 20 to 30 and the oxime part of steroid is in the form of syn isomer, anti isomer and in the form of mixture of syn isomers and

<tb> anti <SEP> and <SEP>: <SEP> 7
<tb><SEP> IH3C-N1 <SEP> R
<tb><SEP> CH3 <SEP>
<tb><SEP> C-CH3
<tb><SEP> 3 <SEP> (VI)
<tb><SEP> 0
<tb><SEP> CH2
<tb><SEP> I <SEP> to- <SEP> I
<tb><SEP>N'H<SEP> n
<tb><SEP> HSA
<tb> in which n = 20 to 30 and the oxime part of sterord is in the form of syn isomer, in the form of anti isomer and in the form of mixture of syn and anti isomers.

A subject of the invention is also, as new industrial products, the antigens obtained during the application described above, and in particular the products of general formulas (V) and (VI).

A subject of the invention is also the application of the above antigens to the preparation of the antibodies.

The products of general formula (I), object of the invention and in particular 17 ss 3-carboxymethyloxime -hydroxy-11 ss- (4dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one coupled to (125I) histamine (product A), as a mixture of syn isomer and anti isomer, in the form of 'syn isomer or in the form of anti isomer, are used as already mentioned during the studies and during the radioimmunological assay of 17 ss -hydroxy-11 ss- (4-dimethylaminophenyl) -17 &alpha; Non-radioactive (prop-1-ynyl) -estra-4,9-dien-3-one (product 8) in biological fluids in humans or animals.

In addition, product A, after its administration, can be followed in its evolution and in its behavior with regard to biological fluids in humans and in animals.

During these studies, product A allows in particular an easy specific dosage of quantities of the order of a few tens of picograms per ml of biological fluid, without having to resort to isolation and control methods. purification by chromatography, before proceeding with the actual assay.

To study and assay by radioimmunological methods 17 - hydroxy-11 ss- (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one non-radioactive (product B), as already said from the latter, antigens with bovine serum albumin (BSA) or with human serum albumin (HSA).

The antigens resulting from formulas (V) and (VI), also subjects of the invention can be used for the development of antibodies when they are injected into animals in the presence of an adjuvant. They thus make it possible to obtain sera containing antibodies.

These antibodies then serve as receptors for radioactive products and / or non-radioactive products, namely receptors for products of general formula (I) and in particular 17-hydroxy11 ss- (4-dimethylaminophenyl) -17 & alpha 3-carboxymethyloxime ; - (prop-1-ynyl) -estra-4,9-dien-3-one in dark mixture of syn isomer and anti isomer, in the form of syn isomer and in the form of anti isomer, coupled to 1 '(125I) histamine (product A), as well as 17 ss-hydroxy-11 ss- (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) - non-radioactive estra-4,9-dien-3-one (product B).

The presence of these antibodies is demonstrated by means of a product of general formula (I) and in particular by means of 3-carboxymethyloxime of 17 ss-hydroxy-11 ss- (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one in the form of mixture of syn isomer and anti isomer, in the form of syn isomer and in the form of anti3 isomer coupled to the '(125I) radioactive histamine (product A).

The product B is then assayed according to conventional radioimmunological methods, such as those described by S.A. BERGSON and R.S. YALOW, HORMONE 4, p. 557 (1964) and - G.E. ABRAHAM J. of CHEM. ENDOCRINAL METAB., 29, p. 866 (1969).

The following examples illustrate the invention without, however, limiting it.

EXAMPLE 1: 17 ss 3-carboxymethyloxime -hydroxy-11 ss- (4-dimethylaminophenyl) 17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one coupled to (125I) histamine.

STAGE A: 17 ss 3-carboxymethyloxime -hydroxy-11ss (4-dimethylaminophenyl) -17 &alpha; (prop-1-ynyl) -estra-4,9-dien-3-one.

To a solution of 1 g of 17ss-hydroxy-11ss- (4-dimethylaminophenyl) - 17 04 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one in 35 ml of ethanol, 665 mg of carboxymethoxylamine halfhydrochloride are added, the reaction mixture is stirred for about an hour at room temperature, add 1.6 ml of N sodium hydroxide, then concentrate under vacuum, take up the residue in chloroform, filter off, evaporate the filtrate to dryness, chromatography on silica (eluent chloroform / methanol 7: 3), isolate the sought product which is impasted with isopropyl ether, filtered off and dried under vacuum to obtain 1.13 g of said desired product in the form of a mixture of syn and anti isomers. elution with the chloroform / methanol system (8: 2) gives Rf = 0.45.

STAGE B: 17 4 -hydroxy-11- (4 di methyl ami nophenyl) -17 - (prop-1-ynyl) -estra-4,9-dien-3-one 3-carboxymethyloxime coupled to (125I) histamine as a mixture of syn and anti isomers.

Preparation of mixed 17ss-hydroxy-11ss - (4-dimethylaminophenyl) -17 q - (prop-l-ynyl) -estra-4,9-dien-3-one 3-carboxymethyloxime anhydride as a mixture of 'syn and anti isomers, with isobutyl formate.

3.5 mg of 17p -hydroxy-11 ss - (4-dimethylaminophenyl) -17 (prop-1-ynyl-estra-4,9-dien-3-one prepared in step above in 50 1 of dioxane, add 10 1 of tri-n-butylamine / tetrahydrofuran mixture (1: 5) then 10 1 of isobutyl chloroformate / tetrahydrofuran mixture (1:10), maintain this reaction mixture under stirring for half a -hour at about 4 ° C., then add 3.4 ml of tetrahydrofuran and obtain a solution of the desired product, which is used as it is for the following stage of the synthesis.

Histamine iodination.

To 10 l of a solution of 2 mM histamine in a buffered solution of 0.5 M sodium phosphate of pH 8, one successively adds 1 mCi of sodium iodide 125 and 50z * ç9 g of chloramine T in 10 1 1 d 'distilled water, the reaction mixture is stirred for approximately 90 seconds, 300 µg of sodium metabisulphite dissolved in 10 l of distilled water is added and an aqueous solution of the desired product Rf = 0.1 is obtained (thin layer chromatography of silica, methanol / triethylamine (98: 2) solvent system, which is used as for the remainder of the synthesis.

Condensation
Is added, under an inert atmosphere, to the iodinated histamine solution obtained above, 50 l of mixed anhydride solution, previously prepared, stirred and left to stand at about 40C under an inert atmosphere for about 1 hour and a half.

The reaction mixture is then diluted with 400 1 @ 1 of a 0.1 M sodium bicarbonate solution, extracted with 1.6 ml of methylene chloride, the organic phase is separated and evaporated to dryness under an inert atmosphere. The residue is taken up in 250 l of ethyl acetate, the product is purified by high performance liquid chromatography eluting with the chloroforin / iethanol system (98.5: 1.5). The solution corresponding to 2 roughly separated peaks is isolated and evaporated to dryness under an inert atmosphere. The residues are taken up in methanol so as to obtain a concentration of radioactive product with an activity of 25 Ci / ml. We get about 100, *
This product in all.

The entire preparation of the iodine product is carried out in inactinic light.

Chromatography on a thin layer of silica with the solution by the cyclohexane / ethanol / triethylamine system (70: 30: 1) gives Rf = 0.28.

EXAMPLE 2: antigen from 17ss -hydroxy-11-ss 3-carboxymethyloxime (4-dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one under a mixture of syn and anti isomers and bovine serum albumin (BSA).

STAGE A: 17ss -hydroxy-11ss (4-dimethylaminophenyl) -17 & alpha 3-carboxymethyloxime mixed anhydride; - (prop-1-ynyl) -estra-4,9-dien-3-one in the form of a mixture of syn and anti isomers with isobutyl formate.

63 mg of 17ss -hydroxy-11 ss - (4-dimethylaminophenyl) -17 & alpha 3carboxymethyloxime are dissolved with stirring and under an inert atmosphere; (prop-1-ynyl-estra-4,9-dien-3-one, prepared in Stage A of Example 1 in 1.2 μl of dioxane, add 57.5 l of tri-n-butylamine, then lower the temperature at about 13 ° C., add 16/1 of isobutyl chloroformate, maintain this reaction mixture under stirring at a temperature of approximately 134 ° C. for half an hour. A solution of the desired product is obtained, which is used as it is for the next stage of synthesis.

STAGE B: Dissolution of bovine serum albumin.

Is introduced at 40C with stirring and under an inert atmosphere 175 mg of bovine serum albumin in 0.5 ml of water, stirred for about 20 minutes, added successively after dissolution 3 ml of
dioxane and 17Ol of NaOH N.

STAGE C: Conjugation.

Slowly poured into the solution of bovine serum albumin obtained in stage B, the mixed anhydride solution obtained in stage A, bring the pH to 8.9 using N hydrochloric acid and stir the reaction mixture for 5 hours at approximately 10 C. The organic phase is then separated from the precipitate formed, diluted with 30 ml of distilled water, the pH is brought to 4.1 by the addition of N hydrochloric acid, the precipitate formed is separated, taken up in 25 ml of a solution. 1% aqueous sodium bicarbonate. The resulting solution is left to stand, then subjected to uterrafiltration. The solution is then depleted of molecular weight molecules.
(10-15000 per 510 ml of distilled water then adjust the final volume to 17 ml.

This solution is lyophilized for about 30 hours and 169 mg of the desired antigen is obtained.

Analysis
UV spectrum - in water: max 265 nm E @ 1 = 53
max 290 nm E'1 = 62 - in N / 10 HCl: max 290 nm E1 '= 61
Circular dichroTsmus - in water: max 222 nm # E1 = -0.305
max 260 nm # E'1 = -0.070
max 289 nm # E'1 = -0.050 - in N / 10 HCl: max 222nm # E'1 = -0.250
max 290 nm E1 1: +0.027
This product contains 20-22 bonded steroid groups per mole of protein.

EXAMPLE 3: antigen from 17ss-hydroxy-11ss - (4-dimethylaminophenyl) -17 & alpha 3-carboxymethyloxime; - (prop-1-ynyl) -estra-4,9-dien-3-one in the form of a mixture of syn and anti isomers and human serum albumin (HSA).

By proceeding as described in Stage C of Example 2, but starting from the mixed anhydride solution of Stage A and the human serum albumin solution of Stage B, 180 mg of the desired antigen is obtained.

This product contains 34-36 bonded steroid groups per mole of protein.

Analysis
UV spectrum - in water: max 265 ni El 1 = 79
max 291 nm El1 = 94 - in HCl N / 10: max 290 ni E'1 = 93
Circular dichroism - in water: infl. 218 nm # E'1 = -0.308
max 259 nm # E'1 = -0.115 max 288 nm # E'1 = + 0.085 - in N / 10 HCl: max 223 nm # E'1 = -0.300
max 283 ni # E'1 = + 0.037

Claims (16)

1) Estradienic products labeled with iodine 125 or 131 in the form of a mixture of syn and anti isomers or in the form of syn or anti isomers of general formula (I)

in which the wavy line on the oximine nitrogen means that the oxime function can be in the syn or anti position, formula in which R represents hydrogen, an alkyl group having 1 to 6 carbon atoms or an acyl group of a carboxylic acid having from 2 to 12 carbon atoms and R1 represents the residue of an amino acid R1 NH2 or the residue of a derivative thereof, this residue having an iodine acceptor group and being labeled with iodine 125 or 131 2) radioactive estradienic products labeled with iodine of general formula (I) according to claim 1 wherein R1 represents the residue of an amino acid R1 NH2or the residue of a derivative of the latter selected from the group consisting of histidine, tyrosine, histidinol, histamine, tyramine and methyl tyrosinate labeled with iodine 125 or 131 3) 17 o -hydroxy-11 ss- (4-dimethylaminophenyl) -17 α 3-carboxymethyloxime; - (prop-1-ynyl) -estra-4,9-dien-3-one, in the form of mixture of syn isomer and anti isomer, coupled to (125I) histamine the iodine being able to be in position 2 or 5 of formula

4) Process for preparing the products defined in claims 1 to 3, characterized in that - a product of general formula (II) is reacted:

in which and in that which follows R has the meanings indicated in claim 1 with a non-amino carboxymethoxyl halide in the presence of a base and obtains a product of formula (III)

in which the wavy line on the nitrogen indicates that this product is in the form of a mixture of syn and anti isomers then, - fixes on the acid function - of this product an activating group of the carbonyl function and obtains the product of general formula (IV)

in which R2 represents an activating group of the carbonyl function and the wavy line on the nitrogen indicates that this product is in the form of a mixture of syn and anti isomers, which is reacted with an amino acid, R1 NH2 having a iodine acceptor group labeled with iodine 125 or 131 or with a derivative of this acid and obtains the desired product of general formula (I), which is isolated in the form of a mixture of syn and anti isomers or that the It is separated, if desired, into its isomers. 5) Method according to claim 4, characterized in that the amino acid acceptor of iodine or the amino acid derivative is selected from the group consisting of histidine, tyrosine, histidinol, histamine, tyramine and methyl tyrosinate. 6) Method according to claim 4 or 5, characterized in that - the carboxymethoxylamine halide is carboxymethoxylami hemihydrochloride and the operation is carried out under an inert atmosphere and in the presence of sodium hydroxide, - an activating group of the carbonyl function by causing an alkyl haloformate to act in the presence of a tertiary base, in an anhydrous medium and under an inert atmosphere, - the alkyl haloformate is isobutyl chloroformate and the operation is carried out in the presence of tri-n- butylamine. 7) A method according to any one of claims 4 -a 6, characterized in that the amino acid or its derivative which is reacted with the product of general formula (IV) in which R2 represents an activating group of carbonyl function, is histamine labeled with iodine 125 or 131 or its derivative and which is carried out under an inert atmosphere. 8) A method according to any one of claims 4 to 7, characterized in that the starting material being 17ss -hydroxy-11ss- (4 dimethylaminophenyl) -17 &alpha; - (prop-1-ynyl) -estra-4,9-dien-3-one, we obtain the product of formula

as a mixture of syn and anti isomers, or as syn isomer or as anti isomer 9) Application of the products as described in claims 1 to 3, to studies and radioimmunological assays of 17ss -hydroxy-11ss- (4- dimethylaminophenyl-17 &alpha; - (prop-1-ynyl) -estra-4,9- non-radioactive dien-3-one. 10) As intermediate products necessary for the preparation of the products of general formula (I) according to claims 1 to 3, the products of general formulas (III) and (IV), in the form of a mixture of syn and anti isomers, as syn isomer or as anti isomer

in which the wavy line on the oximine nitrogen means that the oxime function can be in the syn or anti position and in which R represents hydrogen, an alkyl group having from 1 to 6 carbon atoms or an acyl group of a carboxylic acid having from 2 to 12 carbon atoms1 products of formula (IV) where R2 represents an activating group of the carbonyl function. 11) Products according to claim 10, namely - 17ss-hydroxy-11ss- (4-dimethylaminophenyl) -17 & alpha 3-carboxymethyloxime; - (prop-1-ynyl) -estra-4,9-dien-3-one in the form of a mixture of syn and anti1 isomers in the form of the syn isomer and in the form of the anti isomer, - the mixed anhydride of 17ss-hydroxy-11ss- (4-dimethylaminophenyl) -17 &alpha;3-carboxymethyloxime; - (prop-1-ynyl) -estra-4,9-dien-3-one with isobutyl formate as a mixture of syn and anti isomers, as svn isomer and as anti isomer

12) Application of the products of general formula (III) according to claims 10 and 11, to the preparation of antigens, characterized in that one of these products is conjugated with bovine serum albumin (BSA) or with human serum albumin (HSA) and obtains the desired antigen. 13) Use according to claim 12, characterized in that a product of general formula (III) is reacted in the form of a mixture of syn and anti isomers, in the form of the syn isomer or in the form of the anti isomer

in which the wavy line and R have the aforementioned meanings, for the purpose of activating the carbonyl function, with an alkyl haloformate, in the presence of a tertiary base, in an anhydrous medium and under an inert atmosphere, the anhydride is obtained corresponding mixed, of formula (IV)

in which R keeps the aforementioned meaning and R2 represents a radical of formula -CO-O-alc, alc being an alkyl radical having at most 6 carbon atoms, which is conjugated with bovine serum albumin (BSA) or human (HSA) and obtains the desired antigen. 14) Use according to claim 13, characterized in that - the alkyl haloformate is isobutyl chloroformate and the operation is carried out in the presence of tri-n-butylamine and under an inert atmosphere - the mixed anhydride is reacted of general formula (IV) with bovine (BSA) or human (HSA) serum albumin, having previously dissolved the latter in a water-dioxane mixture under an inert atmosphere. 15) -Antigens consisting of the conjugation of one of the products of general formula (III) according to claim 10, with bovine serum albumin (BSA) or with human serum albumin (HSA), namely the antigens of formula (V) and (VI) in which R has the meanings given in claim 1

in which n = 20 to 30 and the oxime part of steroid is in the form of syn isolates, of anti isomer and in the form of a mixture of syn isomers and

<tb> wherein n = 20 to 30 and the oxime portion of the steroid is as the syn isomer, as the anti isomer, and as a mixture of syn and anti isomers. <tb> <SEP> HSA <tb> <SEP> NH <SEP> n <tb> <SEP> I <SEP> '<SEP> i <SEP> 1 <tb> <SEP> CH2 <tb> <SEP> o <tb> <SEP> 3 <SEP> (vi) <tb> <SEP> CH3 <SEP> C-CH3 <tb> anti <SEP> st 16) Application of the antigens according to claim 15, to the preparation of antibodies.