CZ306553B6 - Biotinylated derivatives of testosterone, boldenone, trenbolone and methandrolone at positions and C-3 and O-17β for an immunoanalytical kit - Google Patents

Abstract

The subject of solution are new compounds of Formulae I-VIII. They are steroid derivatives in which the residue of testosterone, boldenone, trenbolone and methandrolone is either conjugated via the position of the steroid carbon number 3 or 17; while the conjugation via position 3 is carried out by the classical CMO coupling which is extended by 2-[2-(2-{2-[4-(aminomethyl)-1H-1,2,3-triazol-1-yl]-ethoxy}-ethoxy)ethoxy]ethanamine and through it, via an amide bond, it is attached to the carboxyl group of biotin, whereas, otherwise, the hydroxyl group in position 17 is ester-extended by a bridge consisting of 1-[4-(aminomethyl)-1H-1,2,3-triazol-1-yl]-3,6,9,12-tetraoxapentadekan-15-oic acid whose N-terminus is connected to biotin via an amide bond. For classically performed ELISA assays, there is used a protein, preferably avidin, which sorbs directly to the surface of the pits, and then, with the advantage of high affinity of avidin, a steroid is immobilized to biotin. The result is a stable and well-defined system useful in developing detection systems based on different ELISA formats.

Description

Biotinylated derivatives of testosterone, boldenone, trenbolone and methandrolone in positions and C-3 and Ο-17β for immunoassay kits

Field of technology

The invention relates to newly developed components of immunochemical kits for the detection of testosterone (173-hydroxyandrosten-3-one), boldenone (1,4-androstadien-3-one-17β-ol), trenbolone (17β-hydroxyestra-4,9,1 l). -trien-3-one) and methandrolone (1,4-androstadien-17a-methyl-3-on-17-ol), in which the link between biotin and the steroid is realized by an arm containing an optimized length of oligoethylene glycol units (PEG). Immunochemical detection of an analyte is based on the development of an indirect competitive ELISA method, where immobilized biotinylated steroids serve as a competitor of the analyte for an epitope on the antibody. The essence of the analyte determination is the conversion of the chromogenic substrate and the spectrophotometric determination of the product, which is formed by the enzymatic reaction.

Prior art

ELISAs (Enzyme-Linked Immunosorbent Assays) are platelet assays designed to detect and quantify substances such as peptides, proteins, antibodies and hormones. In the ELISA, the antigen is immobilized on the well surface of a microtiter plate and complexed with an antibody that is associated with the enzyme. Subsequent detection is performed based on the assessment of enzyme activity after reaction with the substrate, when a spectrophotometrically measurable product is formed. The most important part of this method is the detection of a highly specific interaction between the antibody and the antigen.

ELISAs can be designed with a number of modifications to the basic procedure. The key step is the immobilization of the analyzed antigen. This can be accomplished by direct adsorption to the microtiter plate or indirectly by capturing the antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most commonly used format for indirect ELISA is the "sandwitch" test, in which the analyte to be determined is bound between two primary antibodies - the capture and detection antibodies. This method is one of the most sensitive and robust ELISA formats. Compared to direct ELISA, the indirect format is much more advantageous especially because: there is a wide range of commercially available labeled secondary antibodies; is universal; has maximal immunoreactivity of the primary antibody that is retained because it is not labeled; has increased sensitivity because each primary antibody contains several epitopes that can bind labeled secondary antibodies, allowing an increase in signal; different visualization labels with the same primary antibody can be applied.

Current immunoassay methods for the determination of anabolic steroids - testosterone, boldenone, trenbolone and methandrolone, in indirect ELISA, LFIA (Lateral-Flow Immunochromatographic Assay) and other formats, use competition between the steroid present in the sample and the hapten conjugate derived from the respective steroid with carrier protein immobilized on microtiter plates, membranes, particles and others. Examples of methods for indirect ELISA determination of trenbolone are described in the literature (Fitzpatrick J, Manning B, O'Kennedy R. Enzyme-linked immunosorbent assay-based detection of free trenbolone in bovine bile. J. Agric. Food Chem. 2004; 52: 4351 ^ 1 .; Zhang YY, He FY, Wan YP, Meng M, Xu J, Yi JA, et al. Generation of anti-trenbolone monoclonal antibody and establishment of an indirect competitive enzyme-linked immunosorbent assay for detection of trenbolone in animal tissues , feed and urine. Taianta. 2011; 83: 732-7.) and testosterone (Lu HH, Conneely G, Crowe MA, Aheme M, Pravda M, Guilbault GG. Screening for testosterone, methyltestosterone, 19-nortestosterone residues and their metabolites in bovine urine with enzyme-linked immunosorbent assay (ELISA). Anal. Chim. Acta. 2006; 570: 1 16-23.), however, this method has not yet been described for methandrolone or boldenone.

- 1 CZ 306553 B6

As carrier proteins, bovine serum albumin, ovalbumin, or other proteins with suitable properties. Conjugates are prepared by reacting an activated form of hapten (reactive anhydrides or esters) with ε-amino groups of lysine residues of the protein to form amide bonds (Yatsimirskaya EA, Gavrilova EM, Egorov AM, Levashov AV. Preparation of Conjugates of Progesterone with Bovine Serum-Albumin in the Reversed Micellar Medium. Steroids. 1993; 58: 547-50.). Conjugation results in a statistically random occupancy of the lysyls present in the protein. The properties of the conjugates from different batches differ and are to some extent heterogeneous, which may adversely affect the parameters of the kits, the reproducibility of the assay and the possible duration of their use.

The essence of the invention

The present invention provides biotinylated derivatives of testosterone, boldenone, trenbolone and methandrolone (Figure 1), such as compounds I-VIII in which the remainder of testosterone, boldenone, trenbolone and methandrolone is conjugated via either steroid carbon position 3 or 17, and their use. . The steroid-biotin conjugate is preferably linked by a bridge containing ethylene glycol units, which eliminates possible interactions between the two functional parts of the molecule. Conjugation via the 3-position is accomplished via a carboxymethyloxime linker (steroid {[3-ylidene] amino) oxy} acetamide), which upon extension by amide condensation, preferably 11azido-3,6,9-trioxaundecanamine, provides azidothermal derivatives. 17β-Hydroxyl of testosterone, trenbolone and boldenone is preferably esterified with 15-azido-4,7,10,13-tetraoxapentaundecanoic acid. The tertiary β-hydroxy group of methandrolone is acylated with 5-azidopentanoic acid. Biotinylation of anabolic steroids is achieved by 1,3-dipolar cycloaddition between synthesized anabolic steroid derivatives and alkyne-terminated biotin derivatives.

The prepared derivatives are preferably used in the construction of analytical ELISA kits, as described, for example, in Example IX. The mechanism of the assay in the competitive ELISA system is shown in Figure 2; the biotinylated conjugate is preferably immobilized on anchored avidin (B); upon addition of a specific antibody and analyte, competition occurs between the immobilized and free steroids in system (C); a secondary antibody - goat anti-specific rabbit antibody (D) is added to quantify bound specific antibodies; a substrate for peroxidase (3,3 ', 5,5'-tetramethylbenzidine) is added and an enzymatic reaction occurs (F); the reaction is stopped by adding sulfuric acid (D). The dependence of the absorbance at 450 nm is inversely proportional to the decimal logarithm of the analyte concentration.

The advantage of the method of immobilizing steroids in the biotin-avidin system is especially the stability of both partners and thus much higher reproducibility of the analyte determination.

Preparation method

Compounds of formula I-IV are prepared by converting a 3-oxosteroid to 3-ylidene (amino) oxy acetic acid. Reaction with azido-PEG ₃ amine (11-azido-3,6,9-trioxaundecanamine) yields derivatives containing an azido group-terminated PEG linker. 1,3Dipolar cycloaddition of products catalyzed by in situ generated copper ions (Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise Huisgen cycloaddition process: Copper (I) catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chern. Int. Ed. 2002; 41: 2596-2599), with biotinylated propargylamine (5 - ((335,45,6a7R) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) -AHprop-2-yn-1-yl) pentanamide) (Poole LB, Klomsiri C, Knaggs SA, Furdui CM, Nelson KJ, Thomas MJ, et al., Fluorescent and Affinity-Based Tools To Detect Cysteine Sulfenic Acid Formation in Proteins. Bioconjugate Chern., 2007; 18: 2004-17.) Will provide products in high yields.

-2EN 306553 B6

Compounds of formula V-VII are prepared by esterifying a υβ-hydroxy group with azido-PEG ₄ acid (1-azido-3,6,9,12-tetraoxapentadecane-15-acid). 1,3-Dipolar cycloaddition of products with biotinylated propargylamine gives products in high yields.

The compound of formula VIII is prepared by converting a tertiary 17β-hydroxy group to an ester by reaction with 5-azidopentanoic anhydride. 1,3-dipole cycloaddition product with biotinylated _{propargyl-4-amino} PEG (5 - [(3a S, 4 ^S,, 6a7? Ý-2-oxo-hexahydro-l // - thieno [3,4- <7] imidazol-4-yl] -N- (3,6,9,12-tetraoxapentadec-14-yn-1-yl) pentanamide) the product is obtained in high yield.

The process for the preparation of biotinylated anabolic steroids is illustrated by the following examples, without limiting these preparations in any way.

Explanation of drawings

Figure 1 Examples of patented substances

Figure 2 Scheme of indirect competitive ELISA

Examples of embodiments of the invention

Example I

Testosterone (0.5 g, 1.73 mmol) was dissolved in MeOH (20 mL) and pyrrolidine (284 μL, 3.46 mmol) was added dropwise via syringe. After 15 minutes, O- (carboxymethyl) hydroxyamine hemihydrochloride (246 mg, 2.25 mmol) was added and the reaction was allowed to proceed for 60 minutes at room temperature. Toluene (10 mL) was added and the solvents were evaporated. The product was purified twice on a silica gel column (CH ₂ C 12 -MeOH, gradient 50/1 -> 10/1) and finally filtered. The product ({[(17P) -1,7-hydroxyestr-4-en-3-ylidene] amino} oxyacetic acid (486 mg, 1.34 mmol) was isolated in 78% yield.

({[(17P) -17-Hydroxyestr-4-en-3-ylidene] amino} oxy) acetic acid (64 mg, 0.178 mmol) and 11-azido-3,6,9-trioxaundecanamine (51 mg, 0 , 23 mmol) were dissolved in dry N, N-dimethylformamide (2 mL). 4- (Dimethylamino) pyridine (28 mg, 0.23 mmol), N-hydroxybenzotriazole hydrate (14 mg, 0.11 mmol) and N- (3-dimethylaminopropyl) N-ethylcarbodiimide (36 mmol, 0) were added sequentially to the mixture. , 23 mmol). The mixture was stirred at room temperature for 60 minutes. The solvent was evaporated and the product was purified by column chromatography (CH ₂ Cl ₂ -MeOH, gradient 100/1 -> 25/1). The product N- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17β> -17-hydroxyandrost-4-ene-4-en-3-ylidene) was isolated ] amino} oxy) acetamide (88 mg, 0.55 mmol) in 88% yield.

N- (2- {2- [2- (2-Azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17β) -17-hydroxyandrost-4-en-3-ylidene] amino} oxy) acetamide (50 mg , 0.09 mmol) and 5 - [(3a, S ^, 4, S ', 6a7R) -2-oxohexahydro-1H-thieno [3,4-b] imidazol-4-yl] -N- (prop-2-yn-1-yl) pentanamide (28 mg, 0.1 mmol) was weighed into a microwave reaction vessel and dissolved in dry in N-dimethylformamide (2 mL). Sodium ascorbate (3 mg, 0.015 mmol), tris [(1-benzyl-177-1,2,3-triazol-4-yl) methyl] amine (5.3 mg, 0.01 mmol) and CuSO _{4 were added.} -5H ₂ O (2.5 mg, 0.01 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was purified by column chromatography on silica gel (CHCl ₃ —MeOH, gradient 25/1 → 10/1). The product-containing fractions were combined and evaporated to dryness. The residue was dissolved in CH ₂ Cl ₂ and filtered. The product N - ({1- [14 - ({[(17β) -17-hydroxyandrost-4-en-3-ylideneamino} oxy) -13-oxo-3,6,9-tri-3] was isolated. oxa-12-azatetradec-1-yl] -1H-1,2,3-triazol-4-yl} methyl) -5 - ((3R, 4R, 6R) -2-oxohexahydro-17β -thieno [3,4-d] imidazol-4-yl] pentanamide (59 mg, 0.07 mmol) in 79% yield 1 H NMR (300 MHz, CDCl 3) δ ppm: 0.75 (s, 3 H, CH-J, Ο, ΊΊ to 1.01 (m, 4 H, steroid envelope), 1.05 (s, 3 H, CH 2 1.09 (s, 3 H, CH 2) 1.18 to 2, 36 (m, 23 H, steroid envelope), 2.68 to 2.78 (m, 1H), 2.82 δ to 3.02 (m, 2 H, biotin SCS), 3.04 to 3.15 (m , 1H, biotinSC / 7), 3.43 to 3.51 (m, 2H, PEG //),

3.52 to 3.64 (m, 12 H, 6xPEG 2), 3.83 (t, J = 4.98 Hz, 2 H, PEG //), 4.27 to 4.39 (m, 2 H , 2xbiotin C / 7), 4.42 to 4.58 (m, 6 H, propargylC //), CMOC // & PEG //), 5.71 (s, 0.6 H, = CH-), 6 .36 (s, 0.4 H, = CH-) 6.55 (br. S., 1H), 6.67 to 6.77 (m, 1H, N //), 7.10 (br s., 1H), 7.72 (s, 1H, triazole C / 7), 7.89 (t, J = 4.98 Hz, 1H, N / 7). ¹³ C NMR (75 MHz, CDCl ₃ ) δ ppm: 11.36, 18.01, 10 18.25, 19.83, 20.97, 21.18, 23.60, 24.72, 25.69, 28.18, 28.40, 30.49, 31.94, 32.30, 32.70, 33.22,

34.52, 34.83, 35.93, 35.97, 36.00, 36.33, 36.72, 36.79, 38.23, 38.90, 38.94, 39.29, 40, 88, 43.01, 43.05, 50.42, 50.79, 50.82, 54.06, 54.33, 56.03, 60.49, 61.93, 69.60, 70.06, 70.46, 70.67, 70.73, 70.76, 72.81, 73.00, 81.66, 81.72, 110.72, 116.76, 155.51, 157.38, 158, 39, 161.52, 164.85, 170.37, 170.59, 173.72. and _D ²⁰ = + 82 (c = 1, CHCl ₃ -MeOH, 1/1). IR (ATR, KBr): 3289 (s), 3081 (w), 15 2930 (5), 2870 (s), 2240 (w), 1696 (vs), 1663 (vs) 1538 (m), 1455 (m) ) 1434 (m), 1374 (w), 1434 (m), 1336 (w), 1250 (w), 1113 (m), 1077 (m), 1020 (w), 912 (m). HRMS-ESI: monoisotopic mass: 842.47243 Da, found (m / z) 843.48006 (calculated 843.47971) corresponds to ion [M + H] ⁺ 865.46200 (calculated 865.46165) corresponds to ion [M + Na ] ⁺ and 881.43333 (calculated 881.43599) correspond to the ion [M + K] ^{+ of the} prepared substance.

Example //

Boldenone (300 mg, 1 mmol) and 9 9 - (carboxymethyl) hydroxyamine hemihydrochloride (142 mg, 25 1.3 mmol) were dissolved in MeOH (10 mL) and pyridine (100 μL) was added dropwise via syringe. The reaction was run for 24 hours at room temperature. Toluene (10 mL) was added and the solvents were evaporated. The product was purified on a silica gel column (CH ₂ Cl ₂ -MeOH, gradient 50/1 -> 10/1). The product ({[(17P) -17-hydroxyandrosta-1,4-dien-3-ylidene] amino} oxy) acetic acid (350 mg, 0.97 mmol) was isolated in 93% yield.

({[(17P) -17-Hydroxyestr-4-en-3-ylidene] amino} oxy) acetic acid (64 mg, 0.178 mmol) and 11-azido-3,6,9-trioxaundecanamine (51 mg, 0 , 23 mmol) were dissolved in dry N, N-dimethylformamide (2 mL). 4- (Dimethylamino) pyridine (28 mg, 0.23 mmol), A-hydroxybenzotriazole hydrate (14 mg, 0.11 mmol) and N- (3-dimethylaminopropyl) 35 N-ethylcarbodiimide (36 mmol, 0.23 mmol). The mixture was stirred at room temperature for 60 minutes.

The solvent was evaporated and the product was purified by column chromatography (CH ₂ C 12 -MeOH, gradient 100/1 -> 25/1). The product N- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl} -2 ({[(17P) -17-hydroxyandrost-4-en-3-ylidene] amino} oxy) was isolated. acetamide (88 mg, 0.55 mmol) in 88% yield.

N- (2- {2- [2- (2-Azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17 [3) -17-hydroxyandrost-4-en-3-ylidene] amino} oxy) acetamide ( 50 mg, 0.09 mmol) and 5 - [(3a5,4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl] -N- (prop-2- In-1-yl) pentanamide (28 mg, 0.1 mmol) were weighed into a microwave reaction vessel and dissolved in dry N-dimethylformamide (2 mL). Sodium ascorbate (3 mg, 0.015 mmol), tris [(1-benzyl-1 H -1,2,3-triazol-4-yl) methyl] amine (5.3 mg, 0.01 mmol) were added. and CuSO ₄ -5H ₂ O (2.5 mg, 0.01 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was purified by column chromatography on silica gel (CHCl ₃ -MeOH, gradient 25/1 -a- 10/1). The product-containing fractions were combined and evaporated to dryness. The residue was dissolved in CH ₂ Cl ₂ and filtered. The product 50 - A - ({1- [14 - ({[(17P) -17-hydroxyandrost-1,4-dien-3-ylidene] amino} oxy) -13-oxo-3,6,9-trioxa-12 was isolated. -azatetradec-1-yl] -1H-1,2,3-triazol-4-yl} methyl) -5 - [(3S, 4S, 6aR) -2-oxohexahydro-1H-thieno [3,4-J] imidazol-4-yl] pentanamide (59 mg, 0.07 mmol) in 79% yield. 1 H NMR (300 MHz, CDCl ₃ ) δ ppm: 0.78 (s, 3 H, Cl 2), 0.82 to 1.08 (m, 3 H), 1.16 (s, 3 H, CH ₃ ) 1.20 to 1.75 (m, 16 H, steroid envelope), 1.77 to 2.53 (m, 5 H, steroid envelope), 2.67 to 2.94 (m,

-4GB 306553 B6

H, biotinSC 4), 3.11 (d, 1H, biotinSC 4), 3.49 (br. S., 2 H, PEGCl 2), 3.57-3.65 (m, 12 H, δxPEGC) //), 3 ^ 83 (t, J = 4.10 Hz, 2 H, PEGC / L), 4.26 to 4.40 (m, 2 H, 2xbiotinCH), 4.43 to 4.59 (m , 6 H, propargyl (C ₂ H 2, CMOC 2 & PEGCl 2), 5.29 (d, J = 2.05 Hz, 0.5 H, = CH-), 5.89 (s, 0.5 H, = CH-), 6.09 (d, J = 10.25 Hz, 0.5 H, = CH), 6.30 (d, J = 10.54 Hz, 0.5 H, = CH-), 6 .45 (d, J = 10.25 Hz, 0.5 H, = CH-), 6.58 (br. S „1H), 6.68 to 6.81 (m, 1H), 7, 13 (br. S., 1H), 7.72 (s., 1H, triazoleCH), 7.90 (br. S., 1H, NH). ¹³ C NMR (75 MHz, CDCl 3) δ ppm: 11.47, 19.67, 19.95, 21.99, 23.68, 25.67, 28.18, 28.35, 29.92, 30. 39, 32.56, 33.21, 33.32, 34.52, 35.95, 36.65, 38.94, 40.89, 43.26, 43.36, 50.45, 50.55, 52.82, 53.05, 55.98, 60.51, 61.89, 69.61, 70.04, 70.52, 70.70, 70.74, 73.17, 81.56, 109, 55, 113.78, 115.47, 120.49, 123.75, 143.69, 148.05, 151.17, 151.27, 155.48, 160.51, 164.85, 170.35, 170.43, 173.67. αD ²⁰ = + 43 (c = 1, CHCl 3 -MeOH, 1/1). IR (ATR, KBr): 3289 (5), 3081 (w), 2927 (5), 2868 (5), 2856 (s), 2239 (w), 1696 (vi), 1659 (vs), 1538 (m) ), 1456 (m), 1377 (w), 1339 (w), 1248 (w), 1110 (m), 1075 (m), 1018 (w), 913 (w). HRMS-ESI: monoisotopic mass: 840.45678 Da, found (m / z), 841.46361 (calculated 841.46406) corresponds to ion [M + H] ⁺ , 863.44565 (calculated 863.44600) corresponds to ion [M + Na] ⁺ and 879.41879 (calculated 879.41994) correspond to the ion [M + K] ^{+ of the} prepared substance.

Example III

Trenbolone (300 mg, 1.11 mmol) and O- (carboxymethyl) hydroxyamine hemihydrochloride (157 mg, 1.44 mmol) were dissolved in MeOH (10 mL) and pyridine (100 μL) was added dropwise via syringe. The reaction was run for 60 minutes at room temperature. Toluene (8 mL) was added and the solvents were evaporated. The product was purified on a silica gel column (CH ₂ Cl ₂ -MeOH, gradient 50/1 -> 10/1). The product ({[(17β) -17-hydroxyestra-4,9,1-trien-3-ylidene] amino} oxy) acetic acid (301 mg, 0.87 mmol) was isolated in 79% yield.

({[(173) -17-Hydroxyestra-4,9,11-trien-3-ylidene] amino} oxy) acetic acid (63 mg, 0.183 mmol) and 11-azido-3,6,9-trioxaundecanamine (52 mg, 0.238 mmol) was dissolved in dry N, N-dimethylformamide (2 mL). To the mixture were added 4- (dimethylamino) pyridine (29 mg, 0.24 mmol) A-hydroxybenzotriazole hydrate (15 mg, 0.11 mmol) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (37 mg) sequentially. , 0.24 mmol). The mixture was stirred at room temperature for 60 minutes. The solvent was evaporated and the product was purified by column chromatography (CH ₂ Cl ₂ MeOH, gradient 100/1 -> 25/1). The product N- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17P) -174-hydroxyestra-4,9,1-trien-3-ylideneamino] was isolated } oxy) acetamide (85 mg, 0.16 mmol) in 85% yield.

N- {2- [2- (2-Azidoethoxy) ethoxy] ethoxy} ethyl) -2 - {[(17β) -17-hydroxyestra-4,9,11-trien-3-ylidene] amino} oxy) acetamide ( 50 mg, 0.09 mmol) and 5 - [(3aS ', 45', 6a2R) -2-oxohexahydro-1H-thieno [3,4-imidazol-4-yl] -N- (prop-2- In-1-yl) pentanamide (28 mg, 0.1 mmol) were weighed into a microwave reaction vessel and dissolved in dry N, N-dimethylformamide (2 mL). Sodium ascorbate (3 mg, 0.015 mmol), tris [(1-benzyl-177-1,2,3-triazol-4-yl) methyl] amine (5.3 mg, 0.01 mmol) and CuSO _{4 were added.} -5H ₂ O (2.5 mg, 0.01 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl ₃ -MeOH, gradient 25/1 -> 15/1). The product-containing fractions were combined and evaporated to dryness. The residue was dissolved in CH ₂ Cl ₂ and filtered. The product N - ({1- [14 - ({[(17P) -17-hydroxyestra-4,9,1-trien-3-ylidene] amino} oxy) -13oxo-3,6,9-trrioxa was isolated -12-azatetradec-1-yl] -1H-1,2,3-triazol-1-yl} methyl) -5 - [(3aS ', 45,6') - 2-oxohexahydro-1 H -thieno [3,4-e7] ] imidazol-1-yl] pentanamide (59 mg, 0.07 mmol) in 79% yield. 1 H NMR (300 MHz, CDCl ₃ ) δ ppm: 0.86 (s, 3 H, Cl 2), 1.13 to 1.72 (m, 14 H, steroid envelope), 1.78 to 1.93 (m, 2H), 2.00 to 2.80 (m, 10H, steroid envelope), 2.82 to 3.01 (m, 2H) 3.04 to 3.18 (m, 1H, biotinSCH), 3.45 to 3.52 (m, 2 H, PEGCZ / J, 3.56 to 3.64 (m, 12 H, 6xPEGCl 2), 3.83 (t, ./=4.98 Hz , 2 H, PEGC // ₂ ), 4.33 (d, J = 3.81 Hz, 2 H, 2xbiotinCH 2), 4.44 to 4.59 (m, 6 H, propargylC // ₂ , CMOCW ₂ & PEGCl 2), 5.84 (s, 1H), 6.24 to 6.44 (m, 2H), 6.52 (br. S., 1H), 6.66 to 6.77 (m 1 H), 7.10 (br. S., 1 H), 7.68 to 7.95 (m, 2 H) ¹³ C NMR (75 MHz,

-5GB 306553 B6

CDCl ₃ ) δ ppm: 13.80, 22.39, 23.04, 23.30, 25.67, 27.52, 28.18, 28.37, 29.91, 30.52, 31.53, 34.61, 35.98, 37.89, 38.86, 40.89, 45.58, 48.20, 50.51, 55.98, 60.53, 61.91, 69.59, 70, 02, 70.50, 70.69, 70.74, 73.13, 77.46, 112.04, 118.46, 123.65, 127.41, 137.71, 139.61, 140.27, 144.64, 147.43, 158.47, 170.24, 173.66. and _D ²⁰ = + 27 (c = 1, CHCl ₃ -MeOH, 1/1). IR (ATR, KBr): 3303 (5), 3086 (w), 2929 (s), 2869 (m), 2240 (vw), 1693 (vs), 1661 (vi), 1538 (m), 1454 (m) ), 1345 (w), 1335 (w), 1250 (w), 1098 (m), 1074 (m), 1018 (w). HRMS-ESI: monoisotopic mass: 824.42548 Da, found (m / z), 847.41486 (calculated 847.41470) corresponds to ion [M + Na] ⁺ and 863.38672 (calculated 863.37796) corresponds to ion [M + K] ⁺ prepared substances.

Example IV

Methandrolone (300 mg, 0.99 mmol) and O- (carboxymethyl) hydroxyamine hemihydrochloride (141 mg, 1.29 mmol) were dissolved in MeOH (10 mL) and pyridine (100 μL) was added dropwise via syringe. The reaction was run for 24 hours at room temperature. Toluene (8 mL) was added and the solvents were evaporated. The product was purified on a silica gel column (CH 2 Cl 2 -MeOH, gradient 50/1 -> 10/1). The product ({[(17β) -17-hydroxy-17-methylandrosta-1,4-dien-3-ylidene] amino} oxy) acetic acid (296 mg, 0.79 mmol) was isolated in 80% yield.

({[(17β) -17-Hydroxy-17-methylandrosal-1,4-dien-3-ylidene] andno} oxyacetic acid (65 mg, 0.174 mmol) and 11-azido-3,6,9 -Trioxaundecanamine (49 mg, 0.226 mmol) was dissolved in dry N / A% dimethylformamide (2 mL), and 4- (dimethylamino) pyridine (28 mg, 0.23 mmol) A-hydroxybenzotriazole hydrate (14 mg 0.01 mmol) and N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (35 mg, 0.23 mmol) The mixture was stirred at room temperature for 60 minutes, the solvent was evaporated and the product was purified by column chromatography (CH ₂ Cl 2 -MeOH , gradient 100/1 -> 25/1) The product N- (2- {2- [2- (2-azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17β) -17) was isolated]. -hydroxy-17-methylandrosta-1,4-dien-3-ylidene] amino} oxy) acetamide (93 mg, 0.162 mmol) in 93% yield.

N- (2- {2- [2- (2-Azidoethoxy) ethoxy] ethoxy} ethyl) -2 - ({[(17β) -17-hydroxy-17-methylandrosta-1,4-dien-3-ylidene) ] amino} oxy) acetamide (50 mg, 0.09 mmol) and 5 - [(3a5 ', 45', 6a R) -2-oxohexahydro-1 H -thieno [3,4- b] imidazole-4-yl. yl] -N ^N - (prop-2-yn-l-yl) pentanamide (28 mg, 0.1 mmol) were weighed into a microwave reaction vessel and dissolved in dry Λζ / V-dimethylformamide (2 mL). Sodium ascorbate (3 mg, 0.015 mmol), tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (5.3 mg, 0.01 mmol) and CuSO 4 - were added. 5H 2 O (2.5 mg, 0.01 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl 3 -MeOH, gradient 25/1 -> 15/1). The product-containing fractions were combined and evaporated to dryness. The residue was dissolved in dichloromethane and filtered. The product 1 - ({1- (14 - ({[(17β) -17-hydroxy-17-methylandrosta-1,4-dien-3-ylidene] amino} oxy) -1,3-oxo-3,6) was isolated , 9-trioxa-12-azatetradec-1-yl] -1H-1,2,3-triazol-1-yl} methyl) -5 - [(3a5,45,6aR) -2-oxohexahydro-1 / N-thieno [3,4-d] imidazol-4-yl] pentanamide (59 mg, 0.07 mmol) in 79% yield 1 H NMR (300 MHz, CDCl 3) δ ppm: 0.89 (s, 3 H, (s, 3 H, CH 2, 0.92 to 1.10 (m, 2 H), 1.16 (d, J = 2.93 Hz, 6 H, 2xCl 3), 1.23 (s, 3 H, CHf), 1.25 to 1.84 (m, 19 H, steroid envelope & 4xbiotinCl), 2.19 (t, J = 6.74 Hz, 2 H, biotin C // 2), 2.23 to 2.53 (m, 4 H), 2.70 to 2.78 (m, 1H, biotin SCl), 2.84 to 2.93 (m, 1H, biotin SCl), 3.04 to 3 , 15 (m, 1H, biotinSC //), 3.45 to 3.51 (m, 2H, PEGC / L), 3.51 to 3.62 (m, 12 H, 6x PEGSC ^), 3 , 82 (t, J = 4.83 Hz, 2 H, PEGC // 2), 4.27 to 4.41 (m, 1 · H, 2xbiotin C //), 4.42 to 4.56 (m, Δ H, 3xC / ε), 5.88 (s, 0.5 H, = CH-), 6.06 to 6.11 (m, 0.5 H, = CH-), 6.30 (d, J = 10.25 Hz, 0.5 H, = CH-), 6.45 (d, J = 10.25 Hz, 0.5 H, = CH-), 6.52 to 6.59 (m, 1H), 6.65 to 6.81 (m, 1H), 7.12 (br. S., 1H, N / 7 ), 7.72 (s, 1H, triazoleIC //), 7.87 (br. s., 1 Η, N //). ¹³ C NMR (75 MHz, CDCl 3) δ ppm: 14.28, 14.30, 16.03, 19.65, 19.94, 21.95, 22.01, 22.07, 23.54, 25. 66, 26.06, 28.17, 28.36, 29.90, 31.63, 32.60, 33.24, 33.35, 33.46, 34.51, 35.98, 36.74, 36.76, 38.89, 38.94, 38.99, 40.88, 43.27, 43.36, 45.80, 45.83, 50.22, 50.26, 50.44, 52, 71, 52.97, 56.00, 60.49, 61.91, 69.59, 70.02, 70.47, 70.49, 70.68, 70.76, 72.67, 73.16, 81.55, 109.55, 113.77, 115.47, 120.47,

-6GB 306553 B6

143.68, 148.04, 151.17, 15128, 155.47, 160.50, 164 ₍ 83, 170.40, 173.61) and _D ²⁰ = + 33 (c = 1, CHCl ₃ MeOH, 1 IR (ATR, KBr): 3289 (s), 3081 (w), 2927 (5), 2868 (s), 2240 (w), 1695 (vs), 1682 (vs), 1538 (m) , 1454 (m), 1371 (w), 1337 (w), 1294 (w), 1261 (w), 1246 (w), 1103 (m), 1085 (m), 915 (w) HRMS-ESI: monoisotopic mass: 854.47243 Da, found (m / z) 877.46106 (calculated 877.46165) corresponds to the ion [M + Na] ⁺ and 893.43402 (calculated 893.43614) corresponds to the ion [M + K] ⁺ prepared substances.

Example V

Testosterone (51 mg, 0.18 mmol) and 1-azido-3,6,9,12-tetraoxapentadecan-15-acid (68 mg, 0.23 mmol) were dissolved in benzene (3 mL). 'Α'-Dicyclohexylcarbodiimide (57 mg, 0.28 mmol) and 4- (dimethylamino) pyridine (34 mg, 0.28 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The dicyclohexylurea was filtered off and the solvent was evaporated and the mixture was purified on a silica gel column (hexane-AcOEt, 3/1 -> 1/1). The product (17β) -3-oxoandrost-4-en-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (73 mg, 0.13 mmol) was isolated in 72% yield.

(17β) -3-Oxoandrost-4-en-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (40 mg, 0.07 mmol) and 5- (3a5 ', 4. S, 6a /?) - 2-oxo-hexahydro-L.R. / '- thieno [3,4- <7] imidazol-4-yl] ^-N-L - (prop-2-yn-l-yl) pentanamide (26 mg , 0.09 mmol) were dissolved in N, N-dimethylformamide (3 mL). Sodium ascorbate (2.7 mg, 0.14 mmol), tris [(1-benzyl-1H-1,2,3-triazol-1-yl) methyl] amine (4.9 mg, 0.09 mmol) were added. ) and CuSO 4 -5H 2 O (2.3 mg, 0.09 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl 3 -MeOH, gradient 50/1 -> 20/1). The product was isolated (l7p) -3-oxo-androsta-4-ene-L7-yl l- {4 - [({5 - ^[(3aS, 45 ', 6a7?) - 2-oxohexahydro17 / thieno [3,4- - [(imidazol-1-yl] pentanoyl} amino) methyl] -1H-1,2,3-triazol-1-yl} -3,6,9,12-tetraoxapentadecan-15-oate (49 mg, 0.06 mmol) in 83% yield. 1 H NMR (300 MHz, CDCl 3) δ ppm: 0.81 (s, 3 H, CA 3), 0.85 to 1.14 (m, 5 H, steroid envelope), 1.17 ( s, 3 H, CA ₃ ), 1.21 to 1.92 (m, 15 H, steroid envelope & biotin CCT), 1.96 to 2.48 (m, 8 H, steroid envelope & biotin CCT), 2 , 57 (t, J = 6.59 Hz, 2 H, -OOCC // ₂ ), 2 ', 69 to 2.78 (m, 1H), 2.85 to 3.00 (m, 2H, biotinSC 4), 3.06 to 3.15 (m, 1H, biotinSCA), 3.44 (s, 1H), 3.58 to 3.62 (m, 12 H, 6xPEGCl 2), 3.72 (t, j = 6.59 Hz, 2 H, PEGCl ₂ ), 3.83 (t, J = 5.13 Hz, 2 H, PEGCl 2), 4.28 to 4.41 (m, 2 H, 2xbiotinCA), 4.46 to 4.51 (m, 2H, PEGC / A), 4.51 to 4.55 (m, 1H, biotinCH), 4.60 (dd, J = 8, 93, 8.05 Hz, 1H, biotinCg), 5.71 (s, 1H, = 1H ~), 6.56 (s, 1H), 7.18 (s, 1H), 7.72 (s, 1H, triazolo / 7), 7.88 (t, J = 5.71 Hz, 1 H, CON77). ^I3 C NMR (75 MHz, CDC13) δ ppm : 12.29, 17.62, 20.74, 23.69, 25.65, 27.68, 28.18, 28.40, 31.69, 32.94, 34.14, 34.49, 35 , 45, 35.59, 35.89, 36.00, 36.82, 38.82, 40.86, 42.75, 50.42, 53.88, 56.02, 60.50, 61.91 , 66.93, 69.59, 70.56, 70.66, 70.69, 70.77, 82.75, 123.65, 124.13, 145.21, 164.87, 171.23, 171 , 73, 173.56, 199.71. αD ²⁰ = + 58 (c = 1, CHCl 3 -MeOH, 1/1). IR (ATR, KBr): 3280 (ms), 3146 (w), 3081 (w), 2934 (s), 2873 (s), 2241 (w), 1728 (ms), 1698 (vs), 1669 (vs) ), 1539 (m), 1455 (m), 1434 (m), 1375 (w), 1351 (w), 1332 (w), 1267 (m), 1233 (m), 1188 (m), 1115 (m) ), 942 (nj, 917 (w). HRMS-ESI: monoisotopic mass: 842.46120 Da, found (m / z), 843.46895 (calculated 843.46847) corresponds to the ion [M + H] ⁺ , 865, 45061 (calculated 865.45042) corresponds to the ion [M + Na] ⁺ and 881.42267 (calculated 881.42436) corresponds to the ion [M + K] ^{+ of the} prepared substance.

Example VI

Boldenone (50 mg, 0.17 mmol) and 1-azido-3,6,9,12-tetraoxapentadecan-15-acid (66 mg, 0.22 mmol) were dissolved in benzene (4 mL). N, N-Dicyclohexylcarbodiimide (55 mg, 0.26 mmol) and 4- (dimethylamino) pyridine (32 mg, 0.26 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The dicyclohexylurea was filtered off and the solvent was evaporated. The mixture was purified on a silica gel column (petroleum ether-AcOEt, 3/1 -> 1/1). He was isolated for

(173) -3-Oxoandrost-1,4-dien-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (71 mg, 0.13 mmol) in yield 73%.

(173) -3-Oxoandrost-1,4-dien-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (42 mg, 0.075 mmol) and 5 - [(3aS, 4S, 6αA) -2-Oxohexahydro-1H-thieno [3,4-b] imidazol-4-yl] -N- (prop-2-yn-1-yl) pentanamide (27 mg, 0.096 mmol) was dissolved in A , N-dimethylformamide (3 mL). Sodium ascorbate (2.9 mg, 0.014 mmol), tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (5.1 mg, 0.001 mmol) and CuSO ₄ -5H ₂ O (2.4 mg, 0.001 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl ₃ -MeOH, gradient 50/1 -> 20/1). The product (17P) -3-oxoandrosta-1,4-dien-17-yl 1- {4 - [({5 - [(3a5,45,6aR) -2-oxohexahydro-1H-thieno) was isolated [3,4-d] imidazol-1-yl] pentanoyl} amino) methyl] -1H-1,2,3-triazol-1-yl} -3,6,9,12-tetraoxapentadecan-15-oate (49 mg , 0.06 mmol) in a yield of 78%. 1 H NMR (300 MHz, DMF) δ ppm: 1.05 (s, 3 H, CHC, 1.11 to 1.39 (m, 4 H, steroid envelope), 1.43 (s, 3 H, C // ₃ ), 1.48 to 2.66 (m, 29 H, steroid envelope & 4xbiotinC7f ₂ ), 2.74 (t, J = 6.00 Hz, 2 H, OOCCH ₂ ), 3.04 to 3 , 26 (m, 3 H), 3.37 (br. S., 1H), 3.67 to 3.77 (m, 12 H, 6xPEGC 3), 3.87 (t, J = 6.00 Hz, 2 H, PEGCl ₂ ), 4.05 (t, J = 4.83 Hz, 2 H, PEGCl 3), 4.10 to 4.17 (m, 1H, biotinCl 2), 4, 41 to 4.49 (m, 1H, biotime), 4.54 to 4.67 (m, 2H, CH 2 OC & propargyl), 4.71 to 4.81 (m, 2H, PEGCl 2), 6.17 (s, 1H, = CH-), 6.31 (d, J = 9.96 Hz, 1H, = CH-), 6.53 (br. S., 1H), 6, 61 (br. S., 1H), 7.39 (d, J = 9.96 Hz, 1H, = CH-), 8.19 (s, 1H, triazole C / 7), 8.42 ( br. s., 1 H, CON ^). ^I3 C NMR (75 MHz, DMF) δ ppm: 12.13, 18.83, 22.61, 23.80, 25.94, 26.01, 27, 69, 28.34, 32.65, 33.53, 34.05, 35.48, 35.78, 35.86, 36.84, 40.58, 43.20, 43.90, 50.01, 50.14, 52.91, 56.28, 60.37, 61.99, 66.99, 69.64, 70.48, 70.58, 70.69, 72.23, 82.46, 123, 76, 124.01, 127.40, 156.70, 163.52, 169.86, 171.61, 172.52, 172.72, 185.6 3. αD ²⁰ = + 38 (c = 1, CHCl 3 -MeOH, 1/1). IR (ATR, KBr): 3289 (s), 3082 (w), 2926 (s), 2868 (m), 1694 (vs), 1659 (vs), 1539 (m), 1455 (m), 1371 (w) ), 1338 (w), 1246 (w), 1081 (m). HRMS-ESI: monoisotopic mass: 840.44555 Da, found (m / z), 841.45257 (calculated 841.45282) corresponds to ion [M + H] ⁺ , 863.43446 (calculated 863.43477) corresponds to ion [M + Na] ⁺ and 879.40772 (calculated 879.40871) correspond to the [M + K] ⁺ ion of the prepared substance.

Example VII

Trenbolone (40 mg, 0.15 mmol) and 1-azido-3,6,9,12-tetraoxapentadecan-15-acid (56 mg, 0.19 mmol) were dissolved in dichloromethane (2 mL). ΛζΑ'-Dicyclohexylcarbodiimide (47 mg, 0.23 mmol) and 4- (dimethylamino) pyridine (28 mg, 0.23 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The dicyclohexylurea was filtered off and the solvent was evaporated. The mixture was purified on a silica gel column (petroleum ether-AcOEt, 3/1 -> 1/1). The product (173) -3-oxoestra-4,9H-1-trien-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (54 mg, 0.1 mmol) was isolated in a yield of 68 %.

(17β) -3-Oxoestra-4,9,11-trien-17-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (35 mg, 0.064) and 5 - [(3aS, 45.6aR) -2-Oxohexahydro-1H-thieno [3,4-d] imidazol-1-yl] -N- (prop-2-ynyl) pentanamide (23 mg, 0.082 mmol) was dissolved in tetrahydrofuran (4 mL). Sodium ascorbate (2.4 mg, 0.012 mmol), tris [(1-benzyl-1 H -1,2,3-triazol-1-yl) methyl] amine (4.3 mg, 0.008 mmol) and CuSO ₄ -5H were added. ₂ O (2 mg, 0.008 mmol) and the mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl ₃ -MeOH, gradient 50/1 -> 15/1). The product (173) -3-oxoestra-4,9,11-trien-17-yl-1- {4 - [({5 - [(3a5,45,6aA) -2-oxohexahydro-1H] was isolated. thieno [3,4-j] imidazol-4-yl] pentanoyl} amino) methyl] -1H-1,2,3-triazol-1-yl} -3,6,9,12-tetraoxapentadecan-15-oate ( 44 mg, 0.05 mmol) in 82% yield. 1 H NMR (300 MHz, DMF) δ ppm 1.12 (s, 3 H, CH 2), 1.24 to 2.18 (m, 12 H, steroid envelope & 3xbiotinCl ₂ ), 2.31 to 2, 86 (m, 13 H, steroid envelope), 3.01 (dd, J = 14.94, 7.91 Hz, 2 H, biotinSC 4), 3.27-3.42 (m, 1 H, biotinSCH 2) , 3.63 to 3.81 (m, 12 H, 6XPEGCl 3), 3.83 to 3.96 (m, 2 H PEGCH 3). 3.99 to 4.11

-8CZ 306553 B6 (m, 2H, PEGCT 2), 4.40 to 4.50 (m, 1H, biotinCl 2), 4.55 to 4.62 (m, 1H, biotin Cl 2), 4, 74 (t, J = 5.27 Hz, 1H, -OOCH), 6.45 to 6.64 (m, 2H, = CH-), 6.73 (d, J = 9.96 Hz, H, = CH-), 6.81 to 7.02 (m, 1H), 8.13 (s, 1H, triazole C / f), 8.43 (dt, J = 10.10, 5.20 Hz, 1H, N. ¹³ C NMR (75 MHz, DMF) δ ppm: 14.47, 23.30, 24.57, 26.03, 27.39, 27.58, 27.70, 28. 85, 34.21, 35.85, 37.00, 37.67, 40.59, 45.54, 48.11, 50.10, 56.30, 60.36, 61.97, 67.00, 69.38, 69.67, 70.60, 70.69, 71.22, 78.60, 80.98, 123.56, 123.81, 124.10, 127.74, 139.23, 142, 11, 145.82, 156.30, 164.34, 171.83, 172.73, 197.97 αD ²⁰ = + 37 (c = 1, CHCl 3 -MeOH, 1/1) IR (ATR, KBr ): 3279 (w), 3080 (w), 2926 (5), 2869 (m), 1698 (vs), 1652 (vs), 1570 (m), 1556 (m), 1540 (m), 1456 (m) ), 1376 (w), 1349 (w), 1335 (w), 1263 (m), 1252 (m), 1188 (m), 1113 (s), 1055 (m), 1022 (m). HRMS-ES1 : monoisotopic mass: 824.41425 Da, found (m / z), 825.42169 (calculated 825.42152) corresponds to ion [M + H] ⁺ , 847.40356 (calculated 847.40402) corresponds to [M + Na] ⁺ and 863.37610 (calculated 863.37610) correspond to the [M + K] ⁺ ion of the prepared substance.

Example VIII

5-Azidopentanoic acid (189 mg, 1.32 mmol) was dissolved in distilled dichloromethane (4 mL) and N, N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol) was added. The mixture was stirred at room temperature for 60 minutes and the dicyclohexylurea was filtered off. Methandrolone (100 mg, 0.33 mmol) was dissolved in distilled dichloromethane (4 mL) and freshly prepared 5-azidopentanoic anhydride was added dropwise with a pasteur pipette. The mixture was stirred at room temperature for 6 hours. Subsequently, the solvent was evaporated and the product was purified on a silica gel column (petroleum ether-AcOEt, 4/1 -> 1/1). The product (17P) -17-methyl-3-oxoandrostal, 4-dien-17-yl 5-azidopentanoate (108 mg, 0.25 mmol) was isolated in 77% yield. (173) -17-Methyl-3-oxoandrosta-1,4-dien-17-yl 5-azidopentanoate (40 mg, 0.09 mmol) and 5 - ((33.5, 4S, 6α) -2 -oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) -N- (3,6,9,12-tetraoxapentadec-14-yn-1-yl) pentanamide (56 mg, 0.12 mmol) were dissolved in tetrahydrofuran (4 mL). Sodium ascorbate (3.6 mg, 0.018 mmol), tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (6.4 mg, 0.012 mmol) and CuSO ₄ -5H ₂ O (3 mg, 0.012 mmol). The mixture was placed in a microwave reactor and stirred at 55 ° C for 60 minutes. The solvent was evaporated and the product was repeatedly purified by silica gel column chromatography (CHCl ₃ -MeOH, gradient 50/1 -> 15/1). The product (17β) -17-methyl-3-oxoandrosta-1,4-dien-17-yl-5- (4- {15-oxo-19 - [(3a £, 4 £, 6a.5) was isolated. ) -2-oxohexahydro-1H-thieno [3,4-c (] imidazol-4-yl] -2,5,8,11-tetraoxa-14-azanonadec-1-yl} -1H-7- 1,2,3-triazol-1-yl) pentanoate (61 mg, 0.1 mmol) in 74% yield 1 H NMR (300 MHz, CDCl ₃ ) δ ppm: 0.89 (s, 3 H, CH 2) 0.96 to 1.21 (m, 6 H, steroid envelope), 1.24 (s, 8 H, 2xCH 2 CH 2), 1.36 (s, 3 H, CH 2), 1.39 to 2.02 (m, 19 H, steroid envelope, 3xbiotin CJ E), 2xVAC ^), 2.02 to 2.52 (m, 8 H, steroid envelope, 2sNKCHf, 2.68 to 2.80 (m, 1 H , biotinSC / Z?) & 2.90 (br. s., 1H, biotinSC ^), 3.05 to 3.20 (m, 1H, biotinCTT), 3.42 (br. s., 2 H , PEGCl 2), 3.53 to 3.58 (m, 2 H, PEGCl 2, 3.59 to 3.77 (m, 12 H, δxPEGCZQ), 4.29 to 4.41 (m, 2 H, VAC ^), 4.50 (br. S., 2 H, 2xbiotin C77), 4.68 (s, 2 H, PEGCH ₂ ), 6.06 (s, 1 H, = CH-), 6.23 ( dd, J = 10.10, 1.90 Hz, 1H, = CH-), 6.86 (br. s., 1H), 7.06 (d, J = 10.25 Hz, 1H, = CH-), 7.62 (s, 1 H triazoIC //) · ^I3 C NMR (75 MHz, CDC13) δ ppm: 14.76, 19.00, 21.66, 22.18, 22.68 , 24.05, 25.79, 28.32, 29.93. 33.01, 33.56, 34.47, 36.02, 36.25, 36.71, 39.42, 40.96, 43.89, 47.26, 48.54, 50.30, 52, 46, 56.09, 60.42, 62.27, 64.84, 66.79, 69.92, 70.25, 70.70, 81.17, 121.10, 124.14, 127.77, 145.05, 156.01, 166.08, 169.17, 173.59, 173.79, 186.94. αD ²⁰ = + 23 (c = 1, CHCl 3 -MeOH, 1/1). IR (ATR, KBr): 3297 (m), 3145 (w), 3094 (w), 2924 (s), 2857 (s), 1699 (vs), 1659 (vs), 1622 (m), 1556 (wm ), 1455 (m), 1373 (m), 1260 (m), 1145 (m), 1122 (m), 1092 (s), 1049 (m). HRMS-ESI: monoisotopic mass: 883.49250 Da, found (m / z), 905.49108 (calculated 905.48172) corresponds to the ion [M + Na] ^{+ of the} prepared substance.

-9EN 306553 B6

Example IX (use of conjugates for ELISA)

The advantage of this is the stability of the system and the possibility of increasing the affinity of individual antibodies, and thus improving detection (improving the detection limit).

A commercially available Costar 9018 microtiter plate was coated with a 5 pg / mL avidin solution (in redistilled H ₂ O) and incubated at 37 ° C for 2 h. After incubation, the avidin solution was knocked out and a steroid-biotin conjugate was applied to the plate. The conjugate stock solution was appropriately diluted in 0.01 M PBS - 0.05% Tween 20 (100 mL of 0.1 M PBS; 0.5 mL Tween 20; 900 mL of redistilled water) and pipetted into the wells of a microtiter plate at 100 μL per hole. The concentrations for the individual conjugates are listed in Table I. Immobilization was performed for 1 h at room temperature. Unbound conjugate was then removed by automatic washing four times with 0.01 M PBS - 0.05% Tween 20 wash solution (1000 mL 0.01 M PBS pH 7.4; 0.5 mL Tween 20).

After washing the unbound conjugates, the competing components were pipetted into the wells of a microtiter plate in the order of 50 μL of antigen solution (calibration standard) diluted in 0.01 M PBS (pH 7.4; 100 mL 0.1 M PBS; 900 mL deionized water) to various concentrations. (500 ng / mL, 50 ng / mL, 5 ng / mL, 0.5 ng / mL, 0.05 ng / mL and 0.005 ng / mL) and 50 μL of a solution of polyclonal rabbit specific antibody diluted also in 0.01 M PBS (Table I). Incubation was with gentle shaking at room temperature for 1 hour. Unreacted immunoreactants were removed by repeatedly rinsing the wells with wash solution (0.01 M PBS-Tween 20, 4x300 pL, washer).

The so-called secondary antibody GAR-Po (from Goat Anti-Rabbit - goat anti-peroxidase-labeled anti-rabbit antibodies) was used to quantify the bound rabbit antibodies to the conjugate. The secondary antibody was diluted 1: 10,000 in 0.01 M PBS-Tween 20 and pipetted at 100 μL per well. Incubation was with gentle shaking at 37 ° C for 90 minutes. Unbound antibody was then removed by repeatedly rinsing the wells with wash solution (0.01M PBS-Tween 20, 4x300 pL, washer).

100 μL of freshly prepared peroxidase substrate solution (1 mg TMB (3,3 ', 5,5'-tetramethylbenzidine); 1 mL DMSO; 9 mL 0.05M citrate-phosphate buffer, pH 5 was then added to each well of the microtiter plate. .0 (1 tablet of citrate-phosphate buffer in 100 mL of deionized water); 2 μL of 30% H ₂ O ₂ ). The enzyme reaction was performed with gentle shaking at room temperature for 10 minutes.

Addition of 2M sulfuric acid at 50 μL per well was used to stop the enzyme reaction. The absorbance of the reaction mixture was measured in the wells of a microtiter plate at a wavelength of 450 nm. From an analytical point of view, the concentration of the competing substance (analyte) that causes 50% inhibition of antibody binding (so-called 50% intercept, I ₅₀ , see Table I) is significant.

- 10GB 306553 B6

Conjugate of steroids with biotin (designation) Conjugate of steroids with biotin Concentration I50 (ng / mL) Conjugate (ng / mL) Specific antibody (pg / mL) * Testosterone-3-Biotin AND 5 5 0.68 Testosterone-17-Biotin IN 5 10 9.10 BoIdenon-3-Biotin II 5 10 0.24 Boldenone-17-Biotin VI 1 5 0.36 Methandienone-3-Biotin IV ¥ 5 0.14 Methandienone-17-Biotin VIII 5 5 0.75 Trenbolone-3-Biotin III 50 5 1.73 Trenbolone-17-Biotin VII 5 5 0.68

Table I Summary of ELISA parameters for individual conjugates; * Specific antibody against the steroid immunogen with BSA

Industrial applicability

Applications in bioanalytical methods for the determination of steroids testosterone, boldenone, trenbolone 10 and methandrolone, in indirect ELISA, LFIA, FIA and other formats using competition between hapten and steroid present in the sample for antibody or other specific binding protein binding sites, eg SHBG (sex hormone -binding globulin) etc. Avidin, streptavidin, neutravidin or other biotin-specific binding proteins are used to immobilize or label the hapten.

Claims (3)

PATENT CLAIMS 1. Biotinylated derivatives of testosterone, boldenone, trenbolone and methandrolone in positions C-3 and 0-17β of formulas I to VIII: 2. Use of compounds of formulas I to VIII as components of immunoassay kits for classical ELISA assays using a protein, preferably avidin, which is sorbed directly onto the surface of the wells and then a conjugate of steroids with biotin to achieve a stable and well-defined system. 3. Use of compounds of general formulas I to VIII for immunoassay methods in ELISA, LFIA or F1A formats.