Testosterone derivatives with a carboxyalkyl substitution in position 3 and use thereof for the production of labelled steroids for determining the concentration of testosterone in a biological sample

Abstract

A testosterone derivative of formula (I): ##STR00001##
where n is an integer in a range of from 1 to 10, and Y represents an activated or ready-to-be-activated group allowing formation of an amide bond with a primary amine of a molecule. Conjugates including the testosterone derivatives and a marker, methods for determining the concentration of testosterone in a biological sample, and methods for preparing the testosterone derivatives are also provided.

Claims

1. A testosterone derivative of formula (I): ##STR00087## wherein: n is an integer in a range of from 1 to 10; and Y is selected from the group consisting of OH, NH(CH.sub.2).sub.mCOOH, N.sub.3, ##STR00088## where m is an integer in a range of from 1 to 10.

2. The testosterone derivative as claimed in claim 1, wherein n is in a range of from 1 to 5.

3. The testosterone derivative as claimed in claim 1, wherein Y is selected from the group consisting of: OH, NH(CH.sub.2).sub.mCOOH, and ##STR00089## where m is in a range of 1 to 10.

4. The testosterone derivative as claimed in claim 1, in a form of a derivative at the carbon in position 3.

5. The testosterone derivative as claimed in claim 1, in a form of an derivative at the carbon in position 3.

6. A conjugate comprising the testosterone derivative as claimed in claim 1 and a marker, wherein the marker is conjugated through Y of the testosterone derivative of formula (I).

7. A method for determining a concentration of testosterone in a biological sample, the method comprising: bringing into contact, within the sample: an anti-testosterone antibody or fragment thereof; and a compound selected from the group consisting of the testosterone derivative as claimed in claim 1 and a conjugate comprising the testosterone derivative and a marker; one of the anti-testosterone antibody or fragment thereof and the compound being adapted to emit a signal; and measuring an intensity of the signal.

8. A method for determining a concentration of testosterone by competitive immunoassay in a biological sample, comprising: a) bringing into contact, within said sample: (i) an anti-testosterone antibody or fragment thereof; and (ii) a compound selected from the group consisting of the testosterone derivative as claimed in claim 1 and a conjugate comprising the testosterone and a marker, one of the anti-testosterone antibody or fragment thereof (i) and the compound (ii) being adapted to emit a signal, b) optionally waiting a sufficient time to allow a competition reaction, and c) measuring an intensity of the signal and determining the concentration of testosterone by comparing the measured intensity of the signal with a calibration curve establishing a relationship between the measured intensity of the signal and the testosterone concentration.

9. The method as claimed in claim 8, wherein the compound (ii) is the conjugate.

10. A diagnostic kit for implementing the method as claimed in claim 8, comprising the testosterone derivative or the conjugate.

11. A method for preparing the testosterone derivative of formula (I) as claimed in claim 1, comprising preparing a compound of formula (Ia): ##STR00090## where n is an integer in a range of from 1 to 10.

12. A method for preparing a testosterone derivative, in a form of a isomer in position 3, of formula (Ia): ##STR00091## where n is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with acetic anhydride to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (II): ##STR00092## where Ac represents COCH.sub.3, and reacting the compound of formula (II) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain the testosterone derivative of formula (Ia).

13. A method for preparing a testosterone derivative, in a form of a isomer in position 3, of formula (Ib): ##STR00093## where: n is an integer is a range of from 1 to 10, and m is an integer in a range of 1 to 10, the method comprising: reacting testosterone with acetic anhydride to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (II): ##STR00094## where Ac represents COCH.sub.3, reacting the compound of formula (II) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00095## reacting the compound of formula (Ia) with N-hydroxysuccinimide, in the presence of a carbodiimide derivative, to produce a compound of formula (IV): ##STR00096## reacting the compound of formula (IV) with a compound of formula (V): H.sub.2N(CH.sub.2)m-COOR.sub.1, where R.sub.1 is an alkyl or aryl group, to produce a compound of formula (VI): ##STR00097## and reacting the compound of formula (VI) with a base, in the presence of a solvent, to obtain the testosterone derivative of formula (Ib).

14. A method for preparing a testosterone derivative, in a form of a isomer in position 3, of formula (I): ##STR00098## where: n is an integer in a nine of from 1 to 10, and Y represents a moiety selected from the group consisting of: ##STR00099## the method comprising: reacting testosterone with acetic anhydride to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (II): ##STR00100## where AC represents COCH.sub.3, reacting the compound of formula (II) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00101## and reacting the compound of formula (Ia), in the presence of a carbodiimide derivative, with a reagent to obtain the testosterone derivative of formula (I), wherein the reagent is selected depending on Y from the group consisting of: ##STR00102##

15. A method for preparing a testosterone derivative, in a form of a isomer in position 3, of formula (I): ##STR00103## where: n is an integer in a range of from 1 to 10; and Y represents a moiety selected from the group consisting of: ##STR00104## where m is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with acetic anhydride to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (II): ##STR00105## where Ac represents COCH.sub.3, reacting the compound of formula (II) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00106## reacting the compound of formula (Ia) with N-hydroxysuccinimide, in the presence of a carbodiimide derivative, to produce a compound of formula (IV): ##STR00107## reacting the compound of formula (IV) with a compound of formula (V): H.sub.2N(CH.sub.2)m-COOR.sub.1, where R.sub.1 is an alkyl or aryl group, to produce a compound of formula (VI): ##STR00108## reacting the compound of formula (VI) with a base, in the presence of a solvent, to obtain a compound of formula (Ib): ##STR00109## and reacting the compound of formula (Ib), in the presence of a carbodiimide derivative, with a reagent to obtain the testosterone derivative of formula (I), wherein the reagent is selected depending on Y from the group consisting of: ##STR00110##

16. A method for preparing a testosterone derivative, in a form of a isomer in position 3, of formula (Ic): ##STR00111## where n is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with acetic anhydride to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (II): ##STR00112## where Ac represents COCH.sub.3, reacting the compound of formula (II) with a compound of formula (III): N.sub.2CH(CH).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00113## reacting the compound of formula (Ia), in the presence of a carbodiimide derivative, with H.sub.2NNH.sub.2, to obtain a compound of formula (VII): ##STR00114## and reacting the compound of formula (VII) with HONO to obtain the testosterone derivative of formula (Ic).

17. A method for preparing a testosterone derivative, in a form of an isomer in position 3, of formula (Ia): ##STR00115## where n is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with t-butyldimethylchlorosilane to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (VIII): ##STR00116## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with triphenylphosphine, benzoic acid and diethyl azodicarboxylate, and then reacting with a base, in the presence of a solvent, to transform, in position 3, the isomer into an isomer and obtain a compound of formula (VIII): ##STR00117## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, and reacting the compound of formula (VIII) with a compound of formula (III): N.sub.2CH(CH).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain the testosterone derivative of formula (Ia).

18. A method for preparing a testosterone derivative, in a form of an isomer in position 3, of formula (Ib): ##STR00118## where: n is an integer in a range of from 1 to 10, and m is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with t-butyldimethylchlorosilane to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (VIII): ##STR00119## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with triphenylphosphine, benzoic acid and diethyl azodicarboxylate, and then reacting with a base, in the presence of a solvent, to transform, in position 3, the isomer into an isomer and obtain a compound of formula (VIII): ##STR00120## where SiMe.sub.2Bu t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00121## reacting the compound of formula (Ia) with N-hydroxysuccinimide, in the presence of a carbodiimide derivative, to produce a compound of formula (IV): ##STR00122## reacting the compound of formula (IV) with a compound of formula (V): H.sub.2N(CH.sub.2)m-COOR.sub.1, where R.sub.1 is an alkyl or aryl group, to produce a compound of formula (VI): ##STR00123## and reacting the compound of formula (VI) with a base, in the presence of a solvent, to obtain the testosterone derivative of formula (Ib).

19. A method for preparing a testosterone derivative, in a form of an isomer in position 3, of formula (I): ##STR00124## where: n is an integer in a range of from 1 to 10, and Y represents a moiety selected from the group consisting of: ##STR00125## the method comprising: reacting testosterone with t-butyldimethylchlorosilane to protect the OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (VIII): ##STR00126## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with triphenylphosphine, benzoic acid and diethyl azodicarboxylate, and then reacting with a base, in the presence of a solvent, to transform, in position 3, the isomer to an isomer and obtain a compound of formula (VIII): ##STR00127## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00128## reacting the compound of formula (Ia), in the presence of a carbodiimide derivative, with a reagent to obtain the testosterone derivative of formula (I), wherein the reagent is selected depending on Y from the group consisting of: ##STR00129##

20. A method for preparing a testosterone derivative, in a form of an isomer in position 3, of formula (I): ##STR00130## where: n is an integer in a range of from 1 to 10, and Y represents a moiety selected from the group consisting of: ##STR00131## where m is an integer in range of from 1 to 10, the method comprising: reacting testosterone with t-butyldimethylchlorosilane to protect an OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a of formula (VIII): ##STR00132## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with triphenylphosphine, benzoic acid and diethyl azodicarboxylate, and then reacting with a base, in the presence of a solvent, to transform, in position 3, the isomer into an isomer and obtain a compound of formula (VIII): ##STR00133## where SiMe.sub.2Bu-t t-butyldimethylsilanyl, reacting the compound of formula (VIII) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00134## reacting the compound of formula (Ia) with N-hydroxysuccinimide, in the presence of a carbodiimide derivative, to produce a compound of formula (IV): ##STR00135## reacting the compound of formula (IV) with a compound of formula (V): H.sub.2N(CH.sub.2)m-COOR.sub.1, where R.sub.1 is an alkyl or aryl group, to produce a compound of formula (VI): ##STR00136## reacting the compound of formula (VI) with a base, in the presence of a solvent, to obtain a compound of formula (Ib): ##STR00137## and reacting the compound of formula (Ib), in the presence of a carbodiimide derivative, with a reagent to obtain the testosterone derivative of formula (I), wherein the reagent is selected depending on Y from the group consisting of: ##STR00138##

21. A method for preparing a testosterone derivative, in a form of an isomer in position 3, of formula (Ic): ##STR00139## where n is an integer in a range of from 1 to 10, the method comprising: reacting testosterone with t-butyldimethylchlorosilane to protect an OH function of position 17, and then reacting with a reducing agent, in the presence of a solvent, to reduce the carbonyl in position 3 and obtain a compound of formula (VIII): ##STR00140## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with triphenylphosphine, benzoic acid and diethyl azodicarboxylate, and then reacting with a base, in the presence of a solvent, to transform, in position 3, the isomer into an isomer and obtain a compound of formula (VIII): ##STR00141## where SiMe.sub.2Bu-t represents t-butyldimethylsilanyl, reacting the compound of formula (VIII) with a compound of formula (III): N.sub.2CH(CH.sub.2).sub.n-1COOC.sub.2H.sub.5, and then reacting with a base, in the presence of a solvent, to obtain a compound of formula (Ia): ##STR00142## reacting the compound of formula (Ia), in the presence of a carbodiimide derivative, with H.sub.2NNH.sub.2, to obtain a compound of formula (VII): ##STR00143## and reacting the compound of formula (VII) with HONO to obtain the testosterone derivative of formula (Ic).

Description

(1) The invention will be better understood from the following examples, which are given for purposes of illustration and are nonlimiting, as well as from the figures, in which:

(2) FIG. 1 shows a graph giving the ratio B/B0 as a percentage as a function of the concentration of the points of the standard range (logarithmic scale), for a reference assay (REF) using a conjugate of the prior art, a 2nd assay using a conjugate of the prior art (Ab1+VIDAS conjugate) and two assays using a testosterone derivative of the invention (Ab2+Conjugate 1 of the invention, and Ab3+Conjugate 1 of the invention),

(3) FIG. 2 shows a graph giving the ratio B/B0 as a percentage as a function of the concentration of the points of the standard range (logarithmic scale), for an assay using Ab2+Conjugate 1 of the invention and an assay using Ab3+Conjugate 2 of the invention.

EXAMPLES

(4) Analysis and control of the reaction in thin-layer chromatography (TLC) are carried out on a Merck silica gel plate (type 60, F254, thickness 0.2 mm), visualized using a UV lamp at 254 nm and developed with heat after spraying phosphomolybdic acid at 5% in ethanol (95%). The products are purified by flash chromatography on Merck silica gel, 40-63 m, pH 6.5 to 7.5.

(5) The NMR spectra are recorded on the Broker Avance 250 apparatus. The chemical shifts are expressed in ppm on the basis of references: deuterated chloroform CDCl.sub.3, =7.26 ppm for .sup.1H and 77.14 ppm for .sup.13C; deuterated methanol CD.sub.3OD, =4.90 ppm for .sup.1H and 49.0 for .sup.13C; tetramethylsilane (TMS): =0 ppm for .sup.1H and .sup.13C. The IR spectra are recorded on the Thermo-Nicolet FT-IR Avatar 360 apparatus and the Golden Gate option is used. The LC-MS analyses are carried out with the PE-Sciex API 100 LC-MS system, column C.sub.4.

Example 1

Preparation of a Testosterone Derivative of the Invention of Formula (Ia)

(6) 17-hydroxy-3-carboxymethoxyandrost-4-ene or compound of formula (I) in which n=1 and Y=OH

(7) ##STR00074##

1.1 Preparation of 3-hydroxy-17-acetoxy-4-androstene (II)

(8) ##STR00075##

(9) A solution of 4.23 g (14.7 mmol) of testosterone in 8 ml of pyridine that has been dried over potassium hydroxide (KOH) overnight (5 g of KOH to 250 ml of pyridine) is prepared in a 250-ml flask equipped with magnetic stirring. The solution is cooled to 0 C. with a water/ice mixture. 4 ml of acetic anhydride is added to it dropwise. The reaction mixture is left to return to room temperature. The solution is stirred overnight at room temperature. The starting product is no longer found in analytical TLC (eluent: ethyl acetate/petroleum ether, 3/1, v/v). 30 ml of distilled water and 30 ml of ethyl acetate are poured into the flask and the mixture is stirred. The phases are separated with a separating funnel and the aqueous phase is extracted with 320 ml of ethyl acetate. The organic phases are combined and the solution obtained is washed with 20 nil of distilled water, dried over anhydrous magnesium sulfate (MgSO.sub.4) and evaporated in a rotary evaporator. The product thus obtained is dried under reduced pressure using a vacuum pump for 4 hours to remove all the solvents. 4.8 g (yield=99.0%) of the intermediate 17-acetoxy-4-androstene is obtained.

(10) .sup.1H NMR (CDCl.sub.3): 5.70 (1H, H.sub.4), 4.60 (1H, H.sub.17), 2.07 (3H, s), 1.16 (3H, s), 0.80 (3H, s). .sup.13C NMR (CDCl.sub.3): 199.5, 171.0 (2C), 123.9, 82.5, 53.7, 50.2, 42.4, 38.6, 36.6, 35.7, 35.4, 33.9, 32.8, 31.5, 27.5, 23.5, 21.2, 20.5, 17.4, 12.1.

(11) In a 250-ml flask equipped with magnetic stirring, 4.7 g (14.2 mmol) of 17-acetoxy-4-androstene is dissolved in 20 ml of methanol and 3 ml of tetrahydrofuran (THF). The solution is then cooled to 0 C. with a water/ice mixture. 0.8 g (21.1 mmol) of sodium borohydride (NaBH.sub.4) is added to the solution. The reaction is monitored on a TLC plate (eluent: ethyl acetate/petroleum ether, 1/1, v/v), checking for disappearance of the starting product. Once all of the starting product has been consumed, 10 ml of acetone is added to stop the reaction. The solution is left to return to room temperature and the solution is evaporated in the evaporator at 30 C. 30 ml of distilled water and 30 ml of ethyl acetate are poured into the flask. The mixture is stirred and the phases are decanted. The aqueous phase is extracted with 330 ml of ethyl acetate. The organic phases are combined, washed with 30 ml of distilled water, dried over anhydrous MgSO.sub.4 and finally evaporated to dryness under reduced pressure at 30 C. 4.6 g (yield=97.3%) of the product (II) is obtained after purification by silica gel chromatography (eluent: ethyl acetate/petroleum ether, 111, v/v).

(12) .sup.1H NMR (CDCl.sub.3): 5.30 (1H, H.sub.4), 4.61 (1H, t, H.sub.17), 4.18 (1H, H.sub.3), 2.04 (3H, s), 1.05 (3H, s), 0.80 (3H, s). .sup.13C NMR (CDCl.sub.3): 171.3, 147.3, 123.7, 82.8, 67.9, 54.4, 50.5, 42.6, 37.4, 36.8, 35.8, 35.5, 32.6, 32.1, 29.5, 27.5, 23.6, 21.3, 20.6, 19.0, 12.1.

1.2 Preparation of the Testosterone Derivative of the Invention of Formula (Ia)

(13) The dry reaction solvent dichloromethane (CH.sub.2Cl.sub.2) is prepared by distillation of 250 ml of CH.sub.2Cl.sub.2 on 5 g of phosphorus pentoxide (P.sub.2O.sub.5).

(14) A 250-ml three-necked flask equipped with magnetic stirring, a dropping funnel and an inert gas supply is purged with dry nitrogen for 10 min. 2.2 g (6.6 mmol) of 3-hydroxy-17-acetoxy-4-androstene (II), 10 ml of dry dichloromethane and 20 mg (0.045 mmol) of rhodium(II) acetate dimer [Rh(OAc).sub.2].sub.2 are introduced under nitrogen. The mixture obtained is cooled to 0 C. A solution of 2.5 ml (2.71 g, 23.8 mmol) of ethyl diazoacetate (N.sub.2CHCOOEt) in 5 ml of dry dichloromethane is prepared and introduced into the dropping funnel. The solution is added to the three-necked flask under nitrogen, with stirring, over the course of 70 min. The reaction mixture is left to return to room temperature and stirring is maintained for 4 hours. The presence of a small amount of the starting product can still be found in TLC (eluent: ethyl acetate/petroleum ether, 1/1, v/v). So that the reaction goes to completion, 2.5 mg (0.006 mmol) of [Rh(OAc).sub.2].sub.2 is added. A solution of 0.5 ml (4.8 mmol) of N.sub.2CHCOOEt in 2 nil of dry dichloromethane is introduced into the three-necked flask via the dropping funnel over the course of 10 min at room temperature. 20 ml of distilled water and 20 ml of dichloromethane are poured in and the mixture is stirred for 10 min. The phases are separated and the aqueous phase is extracted with 220 nil of dichloromethane. The organic phases are collected, dried over anhydrous MgSO.sub.4 and evaporated to dryness using a rotary evaporator at 25 C. to give the crude product in the form of yellow oil. Purification is carried out by silica gel chromatography (eluent: ethyl acetate/petroleum ether, 1/3, v/v). 2.4 g (yield=86.6%) of the product 3-carboxymethoxy-17-acetoxy-4-androstene ethyl ester is obtained.

(15) .sup.1H NMR (CDCl.sub.3): 55.39 (1H), 4.54 (1H, t), 4.23 (2H, q), 4.13 (1H), 2.03 (s), 1.29 (t), 1.05 (s), 0.80 (s). .sup.13C NMR (CDCl.sub.3): 171.2, 170.9, 148.1, 120.4, 82.7, 76.0, 65.5, 61.2, 54.3, 50.4, 42.5, 37.5, 36.8, 35.6, 35.2, 32.5, 32.1, 27.5, 25.2, 23.5, 21.2, 20.4, 18.8, 14.0, 12.0. MS (LC-MS): 347.0 (MH).sup..

(16) 2.0 g (4.8 mmol) of 3-carboxymethoxy-17-acetoxy-4-androstene ethyl ester and 20 ml of methanol are put in a 250-ml flask equipped with magnetic stirring. The mixture is cooled to 0 C. 10 ml (1.0M, 10 mmol) of sodium hydroxide solution (NaOH) is added. Stirring is maintained at 0 C. for 3 hours. The reaction mixture is left to return to room temperature and the saponification reaction is completed after 20 hours of stirring. The reaction mixture is neutralized with hydrochloric acid (HCl, 1.0N) at pH 6 and then evaporated at 25 C. with a rotary evaporator. The residue is purified on a silica column gel to give 1.3 g (yield=78.0%) of the product (Ia).

(17) .sup.1H NMR (CD.sub.3OD): 5.32 (1H, H.sub.4), 3.83 (2H), 3.50 (1H, t), from 2.40 to 1.10 (m), 1.01 (3H, s), 0.69 (3H, s). .sup.13C NMR (CD.sub.3OD): 174.5, 148.9, 119.8, 81.9, 76.3, 64.8, 54.5, 50.7, 42.9, 37.6, 36.6, 35.9, 35.2, 32.6, 32.2, 30.3, 25.2, 23.4, 20.6, 18.9, 11.1. IR (cm.sup.1): 3383, 2928, 2869, 2847, 1721, 1529, 1432, 1377, 1336, 1243, 1213, 1110, 1050.

Example 2

Preparation of a Testosterone Derivative of the Invention of Formula (I)

(18) 17-hydroxy-3-carboxymethoxy-androst-4-ene N-hydroxysuccinimide ester or compound of formula (I) in which n=1 and Y=

(19) ##STR00076##

(20) The procedure described in example 1 was repeated, then continuing as follows.

(21) 10 ml of 1,2-dimethoxyethane (DME) distilled on calcium hydride (CaH.sub.2), 100 mg (0.287 mmol) of the compound of formula (Ia) prepared in example 1, and 33 mg (0.287 mmol) of N-hydroxysuccinimide (HOSu) are put in a 25-ml flask equipped with magnetic stirring. The mixture is cooled to 0 C. 59 mg (0.287 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) is added. Stirring is maintained at temperature for 15 hours. TLC (eluent: chloroform/acetone/acetic acid, 70/25/1, v/v/v) shows almost complete disappearance of the starting product. The reaction mixture is filtered and the filtrate is evaporated at room temperature under reduced pressure. 6.0 ml of anhydrous dichloromethane is added to the residue and the mixture thus obtained is then filtered. The solution is evaporated to dryness at room temperature with a rotary evaporator. The product is dried under vacuum for 4 hours. 64 mg (50.0%) of the product (IV, n=1) is obtained.

(22) .sup.1H NMR (CDCl.sub.3): 5.38 (1H, H.sub.4), 4.47 (2H), 4.40 (1H, t, H.sub.17), from 4.20 to 3.40 (4H, m), 2.85 (4H, s), from 2.60 to 1.10 (m), 1.05 (3H, s), 0.74 (3H, s). .sup.13C NMR (CDCl.sub.3): 168.9, 166.6, 149.2, 119.7, 81.9, 76.9, 63.2, 54.5, 50.7, 42.9, 37.6, 36.6, 36.0, 35.1, 32.6, 32.2, 30.5, 25.7, 25.2, 23.4, 20.6, 18.9, 11.2. IR (cm.sup.1): 3537, 3323, 2929, 2849, 1784, 1732, 1203, 1071. IR (cm.sup.1): 3536, 3507, 3322, 2928, 2849, 1783, 1731, 1624, 1573, 1442, 1426, 1376, 1203, 1071. MS (LC-MS): 446 (M+H).sup.+, 908 (2M+H.sub.2O).sup.+.

Example 3

Preparation of a Testosterone Derivative of the Invention of Formula (Ib)

(23) Compound of formula (I) in which n=1 and Y=NHCH.sub.2COOH

(24) ##STR00077##

(25) The procedure described in example 1 was repeated, then continuing as follows.

3.1 Preparation of the Compound of Formula (VI) in which m=n=1

(26) ##STR00078##

(27) The product from example 1 (Ia) (500 mg, 1.43 mmol) and 30 ml of anhydrous DME are put in a 100-ml flask. N-Hydroxysuccinimide (HOSu, 247.7 mg, 2.15 mmol) is added. The mixture is stirred and 1,3-dicyclohexylcarbodiimide (DCC, 440 mg, 2.15 mmol) is then added. Stirring is maintained at room temperature overnight. The reaction mixture is filtered, and the filtrate containing the compound (IV, n=1) is used directly for the rest of the synthesis.

(28) Sodium bicarbonate solution (NaHCO.sub.3, 1.0N, 10 ml) and 1.0 ml of product tert-butyl glycine ester (H.sub.2NCH.sub.2COOBu.sup.t) are added to the filtrate. The mixture formed is stirred at room temperature for 12 hours. 70 ml of distilled water and 50 ml of ethyl acetate are mixed with the reaction mixture. The phases are decanted and the phase is extracted with ethyl acetate (350 ml). The combined organic solution is dried over anhydrous MgSO.sub.4 and then evaporated using a rotary evaporator at 30 C. The residue is purified by chromatography (eluent: ethyl acetate 100%). 401 mg (yield=60.2%) of the product (VI, m=n=1) is formed in the form of white crystals.

(29) .sup.1H NMR (CDCl.sub.3): 7.19 (1H), 5.32 (1H), 4.35 (1H), 4.01 (2H, s), 3.94 (2H, d), 3.64 (1H, t), from 2.30 to 0.80 (m), 1.45 (s), 1.04 (s), 0.73 (s). .sup.13C NMR (CDCl.sub.3): 170.6, 168.8, 148.8, 120.0, 82.3, 81.8, 76.3, 67.0, 54.5, 50.7, 42.9, 41.4, 37.6, 36.6, 36.0, 35.1, 32.6, 32.2, 30.5, 28.1, 25.5, 23.4, 20.6, 18.9, 11.1.

3.2 Preparation of the Testosterone Derivative of Formula (Ib) in which m=n-=1

(30) 400 mg (0.87 mmol) of the tert-butyl ester (VI, n=1) prepared is dissolved in 30 ml of methanol. 2.0 ml of sodium hydroxide solution (NaOH, 1.0N) is added. Stirring is maintained at room temperature for 15 hours. The reaction mixture is neutralized with hydrochloric acid (HCl, 1.0N) at pH=6. The solution thus formed is evaporated at 30 C. under reduced pressure. The crude product obtained is purified by chromatography with a methanol/chloroform eluent (20/80, v/v). 340 mg (yield=96.8%) of the derivative of formula (Ib, m=n=1) is obtained in the form of white crystals.

(31) .sup.1H NMR (CD.sub.3OD): 5.35 (1H), 3.96 (2H, s), 3.86 (2H, s), 3.50 (1H, t), 3.25 (1H), from 2.30 to 0.70 (m), 1.02 (3H, s), 0.69 (3H, s). .sup.13C NMR (CD.sub.3OD): 173.5, 173.4, 149.5, 121.5, 82.3, 77.5, 67.7, 56.1, 52.0, 44.0, 42.0, 38.6, 37.9, 37.3, 36.4, 34.7, 33.9, 33.2, 30.5, 24.3, 21.7, 19.3, 11.6. IR (cm.sup.1): 3371, 3322, 2922, 2848, 1714, 1622, 1537, 1437, 1420, 1230, 1077. MS (LC-MS): 406.6 (M+H).sup.+, 405.4 (MH).sup..

Example 4

Preparation of a Testosterone Derivative of the Invention of Formula (I)

(32) Compound I of formula (I) in which m=n=1 and Y=

(33) ##STR00079##

(34) The procedure described in example 3 was repeated, then continuing as follows.

(35) The product of formula (Ib) obtained in example 3 (220 mg, 0.54 mmol) is dissolved in 15 ml of anhydrous tetrahydrofuran (THF) in a flask equipped with magnetic stirring. 62.5 mg (0.54 mmol) of N-hydroxysuccinimide (HOSu) is introduced and a transparent solution is formed. 112 mg (0.54 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) is added. The mixture is stirred at room temperature for 4 hours 30 min. The reaction mixture is filtered and the filtrate is evaporated without heating using a rotary evaporator. The residue obtained is washed with 6.0 ml of anhydrous dichloromethane, and then dried under vacuum. 190 mg (yield=70.1%) of the ester (I) is produced. .sup.1H NMR (CDCl.sub.3): 7.20 (1H), 5.35 (1H), 4.45 (H, d), 4.05 (2H), 3.98 (1H), 3.64 (1H, t), 2.84 (4H, m), from 2.60 to 0.80 (m), 1.04 (3H, s), 0.73 (3H, s). .sup.13C NMR (CDCl.sub.3): 171.0, 168.7, 165.6, 149.1, 119.8, 81.8, 76.5, 66.8, 54.5, 50.7, 42.9, 38.3, 37.6, 36.6, 36.0, 35.0, 33.8, 32.6, 32.3, 30.4, 25.6, 23.4, 20.6, 18.9, 11.1. MS (Fab, Na): 501 (M1).sup.+, 525 (M+Na).sup.+.

Example 5

Preparation of a Testosterone Derivative of the Invention of Formula (Iaa)

(36) 17-hydroxy-3-carboxymethoxy-androst-4-ene or compound of formula (I) in which n=1 and Y=OH

(37) ##STR00080##

5.1 Preparation of 3-hydroxy-17-O-(t-butyldimethylsilyl)-4-androstene (VIII)

(38) 2.88 g (10 mmol) of testosterone, 12 ml of anhydrous N,N-dimethylformamide (DMF) and 1.0 g (14.7 mmol) of imidazole are put in a 250-ml flask under nitrogen. 2.26 g (15 mmol) of tert-butyldimethylchlorosilane (ClTBDMS) is added. The mixture is stirred under nitrogen at room temperature for 4 hours 30 min. 100 ml of distilled water and 100 ml of ethyl acetate are poured into the reaction mixture and the mixture is stirred. The phases are separated and the aqueous phase is extracted with ethyl acetate (370 ml). The combined organic solution is washed with 100 ml of distilled water, dried on anhydrous MgSO.sub.4 and evaporated. The residue is purified by silica gel chromatography (eluent: ethyl acetate/petroleum ether, 1/5, v/v) to give 3.67 g of the reaction product in the form of white crystals.

(39) 3.66 g of this product obtained is mixed with 40 ml of methanol, 10 mg of hydrated cerium(III) chloride (CeCl.sub.3.7H.sub.2O) in a 250-ml flask equipped with magnetic stirring. A suspension is formed. 0.40 g (10.6 mmol) of sodium borohydride (NaBH.sub.4, 98%) is added in 3 portions over the course of 5 min. Stirring is maintained at temperature for 10 min. The reaction mixture is evaporated at 30 C. using a rotary evaporator under reduced pressure. The residue is mixed with 50 ml of ethyl acetate and 50 ml of distilled water. The phases are decanted and the aqueous phase is extracted with ethyl acetate (250 ml). The organic solution collected is dried over anhydrous MgSO.sub.4 and evaporated to give the crude product, which is then purified by silica gel chromatography (eluent: ethyl acetate/petroleum ether, 1/4, v/v). 3.5 g (yield=86.5%) of the product (VIII) is formed in the form of white crystals.

(40) .sup.1H NMR (CDCl.sub.3): 5.29 (1H, H.sub.4), 4.18 (1H, H.sub.3), 3.56 (1H, t, H.sub.17), from 2.40 to 0.80 (m), 1.08 (3H, s, H.sub.19), 0.89 (9H, s, t-Bu), 0.74 (3H, s, H.sub.18), 0.02 (6H, s, Me.sub.2Si). .sup.13C NMR (CDCl.sub.3): 147.8, 123.5, 81.8, 68.0, 54.8, 50.5, 43.3, 37.2, 37.1, 36.1, 35.5, 32.8, 32.2, 31.0, 29.6, 25.9, 23.6, 20.8, 19.1, 18.9, 11.4, 4.3, 4.6.

5.2 Preparation of 3-hydroxy-17-O-(t-butyldimethylsilyl)-4-androstene (IX)

(41) ##STR00081##

(42) A 500-ml flask is charged under nitrogen with 4.46 g (11.0 mmol) of the product (VIII) prepared, 50 ml of anhydrous benzene, 5.78 g (22.0 mmol) of triphenylphosphine and 2.69 g (22.0 mmol) of benzoic acid. 3.84 g (22.0 mmol) of diethyl azodicarboxylate (DEAD) is dissolved in 10 ml of anhydrous benzene and the solution obtained is added dropwise to the flask under nitrogen shielding for 10 min. The reaction mixture is stirred at room temperature for 1 hour. 50 ml of sodium bicarbonate solution (NaHCO.sub.3, 1.0 M) is poured into the mixture and the phases are separated. The aqueous phase is extracted with ethyl acetate (330 ml) and the combined organic solution is washed with 50 ml of NaHCO.sub.3 solution (1.0 M), then with 50 ml of distilled water, dried over anhydrous MgSO.sub.4 and evaporated. The residue is taken up in 150 ml of ethanol, and 30 ml of sodium hydroxide solution (NaOH, 1.0 N) is added. The mixture is stirred at room temperature overnight and at 70 C. for 3 hours. There is no longer any starting product on TLC (eluent: ethyl acetate/petroleum ether, 1/9, v/v). The reaction mixture is evaporated to 50 ml with a rotary evaporator. 50 ml of distilled water and 100 ml of ethyl acetate are added. The phases are separated after stirring. The aqueous phase is extracted with ethyl acetate (270 ml). The organic solution obtained is washed with distilled water (70 ml), dried over anhydrous MgSO.sub.4 and evaporated under reduced pressure to give the crude product. Purification is carried out by silica gel column chromatography (eluent: ethyl acetate/petroleum ether, 1/7, v/v). 2.19 g (yield=47.1%) of the product (Ix) is obtained.

(43) .sup.1H NMR (CDCl.sub.3): 5.45 (1H, H.sub.4), 4.07 (1H, H.sub.3), 3.52 (1H, t, H.sub.17), from 2.40 to 0.80 (m), 0.97 (3H, s, H.sub.19), 0.86 (9H, s, t-Bu), 0.71 (3H, s, H.sub.18), 0.00 (6H, s, Me.sub.2Si). .sup.13C NMR (CDCl.sub.3): 150.2, 120.8, 81.7, 64.2, 54.3, 50.4, 43.3, 37.6, 37.1, 35.9, 32.4 (2C), 31.7, 30.9, 27.9, 25.9, 23.6, 21.2, 18.2, 18.1, 11.4, 4.3, 4.7.

5.3 Preparation of the Testosterone Derivative of Formula (Ia)

(44) In a 250-ml three-necked flask equipped with magnetic stirring, 3.95 g (9.8 mmol) of the product (Ix) is dissolved in 65 ml of anhydrous dichloromethane under nitrogen. 100 mg of rhodium(II) acetate dimer is added. The solution is cooled to 0 C. A solution of 4.0 g (35 mmol) of ethyl diazoacetate in 25 ml of anhydrous dichloromethane is prepared and added dropwise to the three-necked flask under nitrogen for 1 hour. The reaction mixture is left to return to room temperature and stirring is maintained for 4 hours. 100 ml of distilled water is added and the phases are separated. The aqueous phase is extracted with ethyl ether (3100 ml). The combined organic solution is dried over anhydrous MgSO.sub.4 and evaporated. The residue obtained is purified on a silica gel chromatographic column (eluent: ethyl acetate/petroleum ether, 1/6, v/v) to give 3.2 g of product, which is used directly for the rest of the synthesis.

(45) 3.0 g of the product thus obtained is dissolved in 20 ml of anhydrous tetrahydrofuran (THF) and the solution is cooled to 0 C. 15 ml of solution of tetra-n-butylammonium fluoride (Bn.sub.4NF, 1.0 M in THF) is added under nitrogen. The reaction mixture is left to return to room temperature and stirring is maintained for 22 hours. The reaction mixture is evaporated using a rotary evaporator. 100 ml of distilled water and 100 ml of ethyl ether are mixed with the residue. The phases are decanted and the phase is extracted with ethyl ether (350 ml). The organic solution is dried over anhydrous MgSO.sub.4 and evaporated. Purification of the residue formed by silica gel chromatography (eluent: ethyl acetate/petroleum ether, 1/1, v/v) gives 1.0 g of product (transparent oil), which is then dissolved in 30 nil of ethanol. 6.0 ml of sodium hydroxide solution (NaOH, 1.0 N) is added to the solution at room temperature and the mixture is stirred for 30 min. TLC (eluent: chloroform/acetone/acetic acid, 70/25/2, v/v/v) is used for monitoring the evaluation of saponification. The reaction mixture is neutralized with hydrochloric acid (HCl, 1.0 N) at pH 6 and is evaporated to dryness at room temperature under reduced pressure. The crude product thus formed is purified on a silica gel chromatographic column (eluent: chloroform/methanol, 4/1, v/v). 950 mg of the product (Ia) is obtained.

(46) .sup.1H NMR (CDCl.sub.3): 5.44 (1H, H.sub.4), 4.11 (2H, s), 3.84 (1H, H.sub.3), 3.63 (1H, t, H.sub.17), from 2.40 to 0.80 (m), 1.01 (3H, s, H.sub.19), 0.78 (3H, s, H.sub.18). .sup.13C NMR (CD.sub.3OD): 175.8, 152.2, 119.2, 82.4, 73.5, 66.7, 55.3, 52.0, 44.0, 38.7, 37.9, 37.2, 33.6, 33.4, 33.1, 30.5, 25.0, 24.2, 22.2, 18.7, 11.6. IR (cm.sup.1): 3375, 2925, 2869, 2846, 1725, 1589, 1434, 1213, 1105, 1072. MS (LC-MS): 347.2 (MH).sup..

Example 6

Preparation of a Testosterone Derivative of the Invention of Formula (I)

(47) 17-hydroxy-3-carboxymethoxy-androst-4-ene N-hydroxysuccinimide ester or compound of formula (I) in which n=1 and Y=

(48) ##STR00082##

(49) The procedure described in example 5 was repeated, then continuing as follows.

(50) 100 mg (0.29 mmol) of the product (Ia) synthesized in example 5, 34.1 mg (0.29 mmol) of N-hydroxysuccinimide (HOSu) and 18 ml of anhydrous tetrahydrofuran (THF) are put in a 50-ml flask equipped with magnetic stirring. A suspension is obtained. 59.8 mg (0.29 mmol) of 1,3-dicyclohexylearbodiimide (DCC) is added. The mixture is stirred at room temperature overnight. The reaction mixture is filtered and the filtrate is evaporated at 25 C. 5 ml of anhydrous dimethoxymethane is added and the mixture is filtered. The filtrate is evaporated at 25 C. using a rotary evaporator. The residue is dried using a vacuum pump, washed with 5 ml of anhydrous hexane and dried under reduced pressure. 86 mg (66.5%, purity: 90% in HPLC) of the testosterone derivative (I) is obtained.

(51) .sup.1H NMR (CDCl.sub.3): 5.50 (1H, H.sub.4), 4.44 (2H, s), 3.85 (1H, H.sub.3), 3.61 (1H, t, H.sub.17), 2.84 (4H, s), from 2.40 to 0.80 (m), 0.97 (3H, s, H.sub.19), 0.74 (3H, s, H.sub.18). .sup.13C NMR (CDCl.sub.3): 169.0, 166.7, 152.2, 117.2, 81.9, 73.3, 63.6, 53.9, 50.6, 43.0, 37.7, 36.6, 35.8, 32.4, 32.2, 31.9, 30.4, 25.6, 24.2, 23.4, 21.1, 18.1, 11.1. IR (cm.sup.1): 3517, 3324, 2917, 2848, 1780, 1729, 1703, 1427, 1379, 1204, 1066. MS (LC-MS): 447.2 (M+H).sup.+.

Example 7

Preparation of a Testosterone Derivative of Formula (Ib)

(52) Compound of formula (I) in which n=1 and Y=NHCH.sub.2COOH

(53) ##STR00083##

(54) The procedure described in example 5 was repeated, then continuing as follows.

7.1 Preparation of the Compound (VI) in which m=n=1

(55) ##STR00084##

(56) 380 mg (1.09 mmol) of the product (Ia), 125.5 mg (1.09 mmol) of N-hydroxysuccinimide (HOSu) and 30 ml of anhydrous tetrahydrofuran (THF) are mixed in a 100-ml flask under nitrogen shielding. 225 mg (1.09 mmol) of 1,3-dicyclohexylcarbodiirnide (DCC) is added. The mixture is stirred at room temperature for 4 hours. TLC (eluent: chloroform/methanol, 5/1, v/v) is used for monitoring the reaction. The mixture containing the product (IV, n=1) is filtered.

(57) 6 ml of sodium bicarbonate solution (NaHCO.sub.3, 1.0 M) and 142 mg (1.09 mmol) of glycine tert-butyl ester are quickly added to the filtrate. The mixture is stirred at 25 C. for 30 min, and then concentrated to 10 ml under reduced pressure. 50 ml of distilled water and 50 ml of ethyl acetate are added. The phases are decanted and the aqueous phase is extracted with ethyl acetate (350 ml). The organic solution is collected and dried over MgSO.sub.4 and evaporated. The crude product is then purified on a silica gel chromatographic column (eluent: ethyl acetate 100%) to give 440 mg of the product (VI, m=n=1).

(58) .sup.1H NMR (CDCl.sub.3): 7.12 (1H, NH), 5.48 (1H, H.sub.4), 4.01 (2H, m), 3.80 (1H, H.sub.3), 3.66 (1H, t, H.sub.17), from 2.40 to 0.70 (m), 1.48 (s, t-Bu), 0.99 (3H, s, H.sub.19), 0.76 (3H, s, H.sub.18). .sup.13C NMR (CDCl.sub.3): 170.8, 168.6, 151.6, 117.5, 82.2, 81.6, 72.8, 67.6, 53.8, 50.5, 42.9, 41.3, 37.6, 36.6, 35.8, 32.3, 32.2, 32.0, 30.3, 28.0, 24.3, 23.3, 21.0, 18.1, 11.1.

7.2 Preparation of the Testosterone Derivative of Formula (Ib)

(59) 440 mg (0.95 mmol) of the product synthesized (VI, m=n=1) is dissolved in 30 ml of methanol and a transparent solution is formed. 3 ml of sodium hydroxide solution (NaOH, 1.0 N) is added at room temperature. Stirring is maintained for 4 hours. There is no longer presence of the starting product (VI) in TLC (eluent: chloroform/methanol, 4/1, v/v). The reaction mixture is neutralized with hydrochloric acid (HCl, 1.0 N) at pH=6 and evaporated at 25 C. under reduced pressure. The residue is purified with a silica gel chromatography column (eluent: chloroform/methanol, 2.5/1, v/v). 380 mg (yield=98.6%) of the product (I) is obtained.

(60) .sup.1H NMR (CD.sub.3OD): 7.88 (1H, t, .sup.2J=3.13 Hz, NH), 5.50 (1H, d, .sup.2J=4.06 Hz, H.sub.4), 4.00 (2H, d, .sup.2J=3.13 Hz), 3.90 (2H, s), 3.80 (1H, t, H.sub.3), 3.56 (1H, t, H.sub.17), from 2.40 to 0.70 (m), 1.02 (3H, s, H.sub.19), 0.76 (3H, s, H.sub.18). .sup.13C NMR (CD.sup.3OD): 174.2, 173.5, 152.5, 119.0, 82.4, 74.0, 68.3, 55.4, 52.0, 44.0, 42.5, 38.7, 37.9, 37.2, 33.7, 33.4, 33.2, 30.6, 25.3, 24.3, 22.2, 18.6, 11.6. IR (cm.sup.1): 3369, 2921, 2847, 1715, 1624, 1538, 1436, 1422, 1233, 1078. MS (LC-MS): 404.4 (MH).sup., 809.9 (2MH).sup..

Example 8

Preparation of a Testosterone Derivative of the Invention of Formula (I)

(61) compound of formula (I) in which m=n=1 and Y=

(62) ##STR00085##

(63) The procedure described in example 7 was repeated, then continuing as follows.

(64) The product of formula (Ib) prepared in example 7 (120 mg, 0.295 mmol) is dissolved in 10 ml of anhydrous 1,2-dimethoxyethane (DME) in a flask equipped with magnetic stirring. 35.1 mg (0.295 mmol) of N-hydroxysuccinimide (HOSu) is added and a transparent solution is formed. 61.7 mg (0.295 mmol) of 1,3-dicyclohexylcarbodiimide (DCC) is added. The mixture is stirred at room temperature for 4 hours 30 min. The reaction mixture is filtered and the filtrate is evaporated without heating using a rotary evaporator. The residue obtained is washed with 6.0 ml of anhydrous dichloromethane, and then dried under vacuum. 90.4 mg (yield=61.0%, purity 85% in HPLC) of the product (la) is obtained. The product is stored at 20 C. under nitrogen.

(65) .sup.1H NMR (CDCl.sub.3): 7.17 (1H, NH), 5.42 (1H, H.sub.4), 4.45 (2H, d), 4.03 (2H, s), 3.76 (1H, H.sub.3), 3.62 (1H, t, H.sub.u), 2.84 (4H, s), from 2.30 to 0.80 (m), 1.05 (3H, s, H.sub.19), 0.75 (3H, s, H.sub.18). .sup.13C NMR (CDCl.sub.3): 171.2, 168.7, 165.7, 152.0, 117.4, 81.9, 73.1, 67.5, 53.9, 50.6, 43.0, 38.3, 37.7, 36.6, 35.8, 32.4, 32.3, 32.1, 30.5, 25.6, 24.3, 23.4, 21.1, 18.2, 11.1.

Example 9

Preparation of a Conjugate 1 of the Invention 17-Hydroxy-3-Carboxymethoxyandrost-4-ene/Alkaline Phosphatase

(66) 0.3 ml of a solution of recombinant alkaline phosphatase (ALP) at 20 mg/ml (Roche, Ref. 03-535-452) is dialyzed in Spectra/Por tubing (cutoff threshold 6000-8000 Da, Spectrum Laboratories, USA) against 300 ml of borate buffer 50 mM pH 7.6, with magnetic stirring, overnight at 2-8 C. At the end of dialysis, the concentration of the protein is determined by reading the optical density at 280 nm and this concentration is adjusted to 8 mg/ml.

(67) The ester 17--hydroxy-3--carboxymethoxyandrost-4-ene-N-hydroxysuccinimide (or NHS) obtained in example 2 is taken up in dimethylformamide (DMF) at a concentration of 1 mg/ml.

(68) For coupling of type (1-3) (1 mole of alkaline phosphatase-3 moles of testosterone derivative), 312.5 l of the solution of ALP is mixed with 30 l of the solution of ester 17--hydroxy-3--carboxymethoxyandrost-4-ene-NHS. For coupling of type (1-5), 312.5 pd of the solution of ALP is mixed with 50 l of the solution of ester. The mixtures are incubated for 1 h on a water bath at 30 C., with gentle magnetic stirring.

(69) Next, the reaction is blocked by adding 1 mM lysine diluted in water. The amount of lysine added is equimolar with the amount of ester used for coupling. Therefore 68 l of the solution of lysine is added for the coupling of type (1-3) and 112 l for the coupling of type (1-5). The mixtures are incubated for 20 min on a wheel, at 18/25 C.

(70) After stopping the reaction, the conjugates are dialyzed in Spectra/Por tubing (cutoff threshold about 7000 Da) for 1 h at 18/25 C. against 250 ml of Tris buffer 50 mM pH 7.4, NaCl 9 g/l, MgCl.sub.2 5 mM, ZnCl.sub.2 0.1 mM, azide 0.9 g/1, with magnetic stirring. After 1 h, the tubings are transferred to new baths, always containing 250 ml of the same buffer. Dialysis is continued overnight at 2/8 C., with magnetic stirring.

(71) At the end of dialysis, the volume of the conjugates is made up to 1 ml with dialysis buffer. The conjugates are then purified by hydrophobic-interaction chromatography using a RESOURCE Phenyl column (Cat No. 17-1186-01, GE Healthcare Lifesciences) installed in an KTA chromatography chain. The pump flow rate is set at 0.5 ml/min. The buffer TA is Tris 50 mM pH 7.4, NaCl 9 g/l, MgCl.sub.2 5 mM, ZnCl.sub.2 0.1 mM, azide 0.9 g/l, (NH.sub.4).sub.2SO.sub.4 0.8M. The buffer TB is Tris 50 mM pH 7.4, NaCl 9 g/l, MgCl.sub.2 5 mM, ZnCl.sub.2 0.1 mM, azide 0.9 g/1, this is the dialysis buffer.

(72) The RESOURCE Phenyl column is equilibrated in buffer TA. The conjugate to be purified is mixed volume for volume (475 l conjugate and 475 l buffer) with the buffer Tris 50 mM pH 7.4, NaCl 9 g/1, MgCl.sub.2 5 mM, ZnCl.sub.2 0.1 mM, azide 0.9 g/l, (NH.sub.4).sub.2SO.sub.4 1.6 M. This step gives the conjugate in buffer TA. Injection of the conjugate is followed by washing of 30 ml in buffer TA. Then a gradient from 0 to 100% of TB is applied for 30 ml, then washing in buffer TB for 10 ml and washing in water for 10 ml. The progress of chromatography is monitored by measuring the optical density at 280 nm. The fractions starting from 42 ml of elution and up to 51 ml (i.e. 10 ml in total) are recovered, combined and then concentrated by diafiltration using an Amicon cell (Amicon stirred cells, Millipore), an Amicon PM membrane with a cutoff threshold of 10 000 Da and the buffer TB. During this step, the volume of the solution of conjugate is reduced to about 1 ml. The conjugates are stored at 2/8 C. until they are used in an immunoassay.

Example 10

Determination of Testosterone Using Conjugate 1 of the Invention 17--Hydroxy-3--Carboxymethoxyandrost-4-Ene/Alkaline Phosphatase and Comparison with Assays Using a Conjugate of the Prior Art

(73) The VIDAS kit (bioMrieux) for testosterone assay (Cat. No 30418) was used as the reference immunoassay, it is a commercial kit with a CE mark. This assay is described in the instructions in the box ref. 09345 F-fr-2010/08 and it is carried out using the VIDAS automatic immuno analyzer.

(74) The disposable cone serves both as solid phase for the reaction and as pipetting system. The cartridge is made up of 10 wells covered with sealed and labeled aluminum foil. The first well comprises a precut part to facilitate introduction of the sample. The last well is an optical cuvette in which the fluorescence of the substrate is measured. The various reagents required for the analysis are contained in the intermediate wells.

(75) The cone contained in the VIDAS kit for testosterone assay (bioMerieux Cat. No 30418) has been sensitized with a sheep anti-rabbit IgG polyclonal antibody, and then with a rabbit anti-testosterone polyclonal antibody. The surface is passivated and it is ready to use.

(76) The sample to be assayed is put in the first well of the cartridge. Then all the steps of the assay reaction are performed automatically by the VIDAS analyzer. The sample to be assayed is mixed with the conjugate, which is a testosterone derivative labeled with alkaline phosphatase, but in which position 3 is not modified as in the present invention. This conjugate will be called VIDAS Conjugate or Conjugate of the prior art.

(77) Competition then takes place between the testosterone present in the sample and the testosterone derivative of the conjugate for the sites of the anti-testosterone antibody fixed on the cone. The washing steps are carried out with the buffer Tris-NaCl (0.05 mol/l) pH 7.4 or with diethanolamine (1.1 mol/1) pH 9.8 and make it possible to remove the compounds that are not fixed. During the final development step, the substrate 4-methylombelliferyl phosphate is aspirated and then introduced into the cone; the enzyme of the conjugate catalyzes the reaction of hydrolysis of this substrate in 4-methylombelliferone, and the fluorescence emitted from the latter is measured at 450 nm. The value of the fluorescence signal is inversely proportional to the concentration of the testosterone present in the sample. This concentration is calculated relative to a calibration curve.

(78) For the other assays, we used the reagents of the VIDAS testosterone kit (Cat. No 30418) and the assay protocol given in the instructions with the kit, with the following modifications: Cones were sensitized with other anti-testosterone antibodies: 2 mouse monoclonal immunoglobulins G (IgG), the clones 19E10H6 and 15H9H10 (bioMerieux), which will be called Ab1 and Ab2 respectively, and 1 sheep monoclonal IgG, the clone testo3.6A3 (Bioventix), which will be called Ab3. As in the cone of the format of the reference assay, the anti-testosterone antibodies were captured by anti-species polyclonal antibodies adsorbed on the cone beforehand. It is a sheep anti-mouse IgG polyclonal antibody for Ab1 and Ab2, and a donkey anti-sheep IgG polyclonal antibody for Ab3. The anti-species antibodies are diluted to a concentration between 1 and 12 g/ml in the sensitizing solution. After incubation for 6 h at +18/25 C., washing is carried out with saline solution. Then the solution of anti-testosterone antibodies containing a saturating agent of the protein or peptide type is added (concentration of the antibody: 0.01-0.2 g/ml). Sensitization/passivation is continued at +18/25 C. overnight. Then the cones are emptied and dried. The cones sensitized with the antibody Ab1 are tested with the VIDAS conjugate, diluted to 1/2.5 for the tests with the monoclonal antibody. The cones sensitized with the antibodies Ab2 and Ab3 are tested with conjugate 1 of the invention prepared in example 9. This last-mentioned conjugate is used at a concentration of 3 ng/ml for the tests with the cones sensitized with the antibody Ab2 and 2.5 ng/ml for the cones sensitized with the antibody Ab3. The conversion to dose is performed based on a standard 10-point range manufactured by supplementing woman's serum with testosterone in ethanolic solution. The nominal concentrations of each of the points are: 0.001 ng/ml-0.03 ng/ml-0.18 ng/ml-0.41 ng/ml-0.76 ng/ml-1.29 ng/ml-1.91 ng/ml-3.82 ng/ml-7.48 ng/ml-11.55 ng/ml. The sample volume taken for each assay is 200 l.

(79) The standard range was measured with each assay format. Table 1 below summarizes the results obtained as RFV signal (relative fluorescence value) and B/B0% ratio, for the dilutions of the standard range. The ratio B/B0 is the signal obtained for the range point tested divided by the signal obtained for the range point 0.001 ng/ml of testosterone, multiplied by 100.

(80) TABLE-US-00001 TABLE 1 [c] REF = VIDAS Ab1 + VIDAS Ab2 + Conjugate Ab3 + Conjugate Testos- kit Conjugate 1 of the invention 1 of the invention terone Signal Signal Signal Signal (ng/ml) (RFV) B/B0% (RFV) B/B0% (RFV) B/B0% (RFV) B/B0% 0.001 4202 100 4799 100 4077 100 3209 100 0.03 3630 86 4412 92 3490 86 2492 78 0.18 3056 73 3537 74 2935 72 1700 53 0.41 2645 63 3093 64 2527 62 1403 44 0.76 2440 58 3320 69 2238 55 1286 40 1.29 2057 49 2940 61 1880 46 960 30 1.91 1939 46 2836 59 1729 42 990 31 3.82 1493 36 2391 50 1264 31 644 20 7.48 1183 28 2163 45 905 22 380 12 11.55 880 21 1849 39 632 16 205 6

(81) The B/B0% ratio values are shown in FIG. 1 in a graph that gives these values as a function of the logarithm of the concentration of the points of the standard range for each format, namely two formats using a conjugate of the prior art ((i) REF which is the reference assay of the prior art which corresponds to the VIDAS commercial kit for testosterone assay (bioMerieux, Cat. No 30418), and (ii) Ab1+VIDAS Conjugate), and two formats using a conjugate 1 of the invention (Ab2+Conjugate 1 of the invention, and Ab3+Conjugate 1 of the invention).

(82) The graph in FIG. 1 shows that the two assay formats that are the most sensitive are those using the conjugate of the invention. In fact, the most sensitive assay format is the format combining the antibody Ab3 and Conjugate 1 of the invention. A 50% decrease in the signal is observed starting from about 0.2 ng/ml of testosterone. For the format combining the antibody Ab2 and Conjugate 1 of the invention, as well as the reference assay on VIDAS, about 1 ng/ml of testosterone is required to reach a B/B0 ratio of 50%. The format Ab2+Conjugate 1 of the invention is slightly more sensitive than the reference format. Finally, with the format Ab1+VIDAS Conjugate, 3.82 ng/ml of testosterone is required to reach 50%. It is the least sensitive format.

(83) Secondly, to confirm that the format Ab2+Conjugate 1 of the invention is far more sensitive than the reference format REF, VIDAS Testosterone kit, for which the measurement range recommended in the instructions extends from 0.1 to 13 ng/ml, these two formats were compared against serum samples obtained from the Etablissements Franais du Sang (the French National Blood Service). These were 4 samples collected from women (codes with F), for whom the serum testosterone level is below 1 ng/ml, and 2 samples collected from men (codes with M). The results are presented in Table 2. The theoretical concentration was determined by a third-party laboratory, by a technique that gives accurate measurement of concentrations below 1 ng/ml such as ID/GC-MS mass spectrometry. The ratio [c] measured/[c] theoretical100 presented in Table 2 for each assay provides an assessment of the accuracy of each assay, [c] signifying concentration. The closer this ratio is to 100%, the more accurate the assay.

(84) TABLE-US-00002 TABLE 2 Ab2 + Conjugate 1 of REF = VIDAS Kit the invention [c] me/ [c] me/ Sample [c] [c] [c] th [c] [c] th code theoretical measured x100 measured x100 F37 0.11 0.25 223 0.13 118 F38 <0.025 0.09 340 0.03 100 F45 0.07 0.20 279 0.07 100 F46 0.21 0.41 193 0.30 140 M29 2.38 3.20 134 2.97 125 M34 3.94 3.35 85 4.05 103

(85) The results in Table 2 show that the format using a conjugate of the invention is more accurate than the reference assay on VIDAS, especially for assay of the low levels of serum testosterone found in women.

Example 11

Preparation of Conjugate 2 of the Invention 17--Hydroxy-3--Carboxymethoxyandrost-4-ene/Biotin

(86) The molecule will be abbreviated here to testosterone-3-EMC-DAP-biotin, DAP being diaminopropyl, and has the following formula.

(87) ##STR00086##

(88) In a 50-ml flask equipped with magnetic stirring, 100 nag (0.287 mmol) of 17--hydroxy-3-carboxymethoxyandrost-4-ene acid obtained in example 1 is dissolved in 7 ml of dimethoxyethane (DME) anhydride. 50 mg of N-hydroxysuccinimide (NHS) is added and stirring is maintained at room temperature for 3 min. 69 mg (0.334 mmol) of N,N-dicyclohexylcarbodiiniide (DCC) is added and a transparent solution is obtained. The reaction mixture is stirred at room temperature overnight.

(89) The reaction mixture is filtered and the filtrate thus obtained is used directly. 100 mg (0.241 mmol) of N-(+)-biotinyl-3-aminopropylammonium trifluoroacetate (biotin-NH-DAP, TFA salt, Sigma-Aldrich Cat. No. 71776) is mixed with 1.5 ml of 1N NaHCO.sub.3 solution and then added to the filtrate. Stirring is maintained at room temperature for 14 h.

(90) The reaction mixture is dried at room temperature under reduced pressure. The residue obtained is purified by chromatography on silica gel 60 (0.040-0.063 mm, Merck Cat. No. 109385) with the eluent: dichloromethane/methanol, 8/1, v/v to start and dichloromethane methanol, 5/1, v/v to finish the purification. 125 mg of product is obtained, which corresponds to a yield of 82%. Testosterone-313-EMC-DAP-biotin is a white powder.

(91) The product is analyzed by high-performance liquid chromatography (HPLC). The column used is a Thermokromasil C18, 1504.6 mm and the eluent is a mixture of acetonitrile, water (0.1% trifluoroacetic acid) in a gradient. The chromatography is monitored by measuring the absorbance at 214 nm. The product purity determined in this way is 90.1%.

Example 12

Determination of Testosterone Using Conjugate 2 of the Invention Testosterone-3-EMC-DAP/Biotin

(92) 100 l/well of the mouse anti-goat Fe IgG monoclonal antibody (clone 9A4A5, bioMerieux) diluted to 10 g/ml in PBS buffer 1 is distributed in a 96-well microplate (Nunc Maxisorp F96). The microplate is incubated overnight at room temperature in order to obtain adsorption of the antibody. The microplate is emptied, and then 300 l/well of passivating buffer (buffer Tris 0.2 M pH 6.2) containing a saturating agent of the protein or peptide type, and the sheep anti-testosterone monoclonal antibody clone 3.6A3 (Ab3-bioVentix) diluted to 0.2 g/ml are added. The microplate is incubated for 1 h at 37 C. Three washings are carried out with TBS (Tris buffered saline)-Tween-20 0.05%. The range points used in example 10 are diluted to 1/2 in Tris buffer; 100 l of these dilutions are distributed in the wells of the plate. After incubation for 1 h at 37 C., the wells are emptied and 0.2 ng/well of Conjugate 2 of the invention testosterone-3-EMC-DAP-biotin in Tris buffer is added. The reaction with the conjugate is carried out for 15 minutes at 37 C., and is followed by 3 washings. 100 l/well of streptavidin-peroxidase solution (Jackson Immunoresearch Cat. No. 016-30-084) diluted to 1/20 000 in TBS-Tween-20 0.05%, BSA 2% is added and the microplate is incubated for 30 min at 37 C. After 3 washings, 100 l/well of the 1-step Ultra TMB substrate (Thermo Scientific, Cat. No. 34028) is added and the microplate is incubated for 5 min at room temperature, away from the light. The reaction is then stopped by adding 100 l of 2M sulfuric acid. The optical density (OD) at 450 nm and at 630 nm is measured in a microplate reader. For each well, the optical density at 630 nm is subtracted from the optical density at 450 nm. The results obtained on applying this protocol are presented in Table 3.

(93) TABLE-US-00003 TABLE 3 [c] Testosterone (ng/ml) OD.sub.450-OD.sub.630 B/B0% 0.001 2.52 100 0.03 2.10 83 0.18 1.88 74 0.41 1.61 64 0.76 1.36 54 1.29 1.00 40 3.82 0.42 17 11.55 0.17 7

(94) The values of the B/B0% ratio from Table 3 are shown in FIG. 2 as a graph that gives these values as a function of the logarithm of the concentration of the points of the standard range. FIG. 2 gives the values obtained in example 10 for the format using Ab2+Conjugate 1 of the invention.

(95) This graph shows that the character of the testosterone assay range obtained with the antibody Ab3+Conjugate 2 of the invention testosterone-3-EMC-DAP-biotin on the microplate is comparable to that obtained with the format Ab2+Conjugate 1 of the invention on VIDAS in example 10. This microplate assay with a conjugate of the invention is therefore more sensitive than the VIDAS assays using the VIDAS Conjugate.

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