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Home , Desoxymethyltestosterone, Metribolone, Mibolerone, Tetrahydrogestrinone

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

C. Ayotte, D. Goudreault, D. Cyr and J. Gauthier1, P. Ayotte and C. Larochelle2, and D. Poirier3

Characterisation of chemical and pharmacological properties of new related to doping of athletes

1INRS-Institut Armand-Frappier, Pointe Claire, Canada 2Institut de Santé publique de Québec, U. Laval, Québec, Canada 3Centre de recherche en chimie médicinale, U. Laval, Québec, Canada

In the USA, a female cyclist, T. Thomas, was banned for the use of norbolethone, a which has been studied by the pharmaceutical industry in the 60s but not commercialised1. Later in 2004, following the characterisation of THG2 from a oily residue given to the American testing authorities, and in collaboration with the international athletic federation, several elite athletes’ samples were tested and found to contain that “designer” steroid. The properties of were subsequently studied; it appears that THG is a potent and progestin3. Public comments made by individuals involved in the “Balco” scandal were adding to the information coming from other sources, mainly informants, to the effect that other “designer”, potent and undetecTable steroids have been prepared and would be available to certain athletes. This has become a certainty in December 2003, following a seizure at the Canadian border of hGH, and two steroid-products one of which found to be THG. The identification of the second one, DMT (17α-methyl-5α-androst-2-en-17β-ol major isomer along with the 3-en isomer), is the subject of the first part of this work; picture of the bottle seized is shown at Figure 1.

Figure 1: Plastic bottle seized at the Canadian border containing 3g per 100 mL of a steroid identified as 17α-methyl-5androst-2 (-3)-en-17-ol (DMT) in vegetable oil.

151 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Experimental: Cryopreserved hepatocytes from a single donor or a pool of five were purchased from In vitro Technologies. In a typical experiment, the hepatocytes preserved at - 150°C are thawed in a water bath at 37°C. The cells are suspended in In vitro GRO HT Medium and centrifuged at 50 g for 5 min. The supernatant is discarded, the cells are suspended in 1 mL of KHB buffer. Cells are counted using Trypan Blue exclusion method and concentration is adjusted to 2 millions cells per mL with KHB buffer. Incubations are done for 5 hours; the cells are precipitated and the steroids are extracted after solid phase

extraction on Sep Pak plus C18 cartridges, liquid-liquid extraction with diethyl ether and TMS- ether, TMS-enol formation.

Assessment of hormonal properties: All compounds were dissolved in 95% ethanol and the final concentration of the vehicle in the cell culture medium was 0.1%. For each assay, we included a standard curve with the following : (DHT) for the androgenic assay, for the progestagenic assay and for the estrogenic assay. A minimum of 5 concentrations were used to construct dose response curves for each substance. Measurements were made in triplicates and at least three independent experiments were conducted. The specificity of the response in each system was tested by using specific antagonists: (anti-androgen), ICI 182 720 (anti-oestrogen) and (antiprogestin).

Dose-response relations were fitted to a four parameter sigmoid curve using the Slide Write Plus for Windows software (version 6.00, Advanced Graphics Software Inc., Encinitas, CA).

EC50 values (the concentration that yields a response equal to 50% of the maximum response induced by the of reference) were intrapolated from these modeled curves. The efficacy of compounds in each assay was also calculated relative to that of the reference agonist.

Characterisation:

The chemical synthesis of DMT (3a and 3b) from (1) is summarised in Figure 2, mass spectra of the TMS-derivatives of the two isomers present in the “designer” product are shown at Figure 3.

152 O In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

O H 2 a 1) Tosyl chloride O 2) Collidine / 150°C HO H

1 H 2b Methyl Lithium

OH OH

H H 3a 3b Figure 2: Scheme of chemical synthesis of 17α-methyl-5androst-2 -en-17-ol (DMT) (3a) from epiandrosterone (1) via 5α-androst-2-en-17-one (2a; major)

Abundance

TIC: 3372TDIL.D 2400000

2200000

2000000 Clandestine steroids 1 1800000

1600000

1400000

1200000

1000000

2 800000

600000

400000

200000

12.00 12.20 12.40 12.60 12.80 13.00 13.20 13.40 13.60 13.80 14.00 14.20 14.40 Time-->

Abundance Abundance

Scan 956 (13.687 min): KF444DER.D 3000000 143 Scan 968 (13.762 min): KF444DER.D 2800000 1100000 143

2600000 1000000 2400000 900000 2200000 800000 2000000

1800000 700000

1600000 600000 1400000 500000 1200000 400000 1000000 345 73 800000 300000 270 73 600000 200000 345 400000 255 100000 91 91 115129 255270 228 200000 105 129 228 360 55 161175 199213 360 55 159 327 175 201 291306 327 0 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 m/ z--> m/ z--> Figure 3: GC/MS analysis of the clandestine steroid product (upper panel) and mass spectra of the TMS-derivatives of the authentic synthesised standards of the two isomers (peaks 1 and 2) of DMT 3a (2-en major at left) and 3b.

Another 17α-alkylated steroid, 2α,17α-dimethyl-5α-androstan-3-one-17-ol, 17- methyldrostanolone (5), was commercialised under the trade names of Superdrol, . The GC/MS analysis of the content of one capsule of Superdrol seized at the

153 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Canadian customs is shown at Figure 4 along with the mass spectrum of the TMS-ether,TMS-

enol (d9-TMS in parenthesis) derivative. The spectrum is characterised by the presence of

at m/z 462 (molecular ), 419 (-43, C1-C2-CH3; A-ring), 157 and 141 (A-ring), 143 and 130 (D-ring).

Abundance

Abundance 141 #80: TMS-énol 141 448 462 (480) Scan 1572 (17.667 min): SUPERDRO.D 9500 OTMS 143 OTMS 9000 5000000

8500 141 (150) -43 141 (150) 4500000 8000 448 130 448 7500 TMSOH 143(152) TMSO -CH 4000000 7000 H 3 TMSO H 6500 73 157 (166) 3500000 6000 157 157 (166) 5500 3000000 462 5000

4500 2500000

4000

3500 2000000

3000 1500000 2500 141 2000 405405 1000000 1500 209 1000 105 158 187 500000 55 157 332 500 357 123 233253270289 319 419 0 0 60 80 100120140160180200220240260280300320340360380400420440 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 m/ z--> m/z-->

Figure 4: GC/MS analysis of the content of a capsule of commercial product, Superdrol; mass spectrum of the TMS-derivative of main peak (lower right) compared to that of drostanolone (4) (lower left).

A summary of the steps involved in the synthesis of 17-methyldrostanolone (5) from drostanolone (4) is shown at Figure 5 while Figure 6 presents the mass spectra of underivatised and TMS-ether derivative of the synthesised steroid (5). The chromatographic properties, NMR and mass spectrum were found to be identical to that of the steroid isolated from the commercial product.

OH OH 1) Protection

2) Oxidation 3) Methylation O 4) Deprotection H O H

Drostanolone (4) Methyldrostanolone (5) Figure 5: Synthesis of 17-methyldrostanolone (5) from drostanolone (4).

154 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Abundance Abundance

Scan 1334 (16.089 min): 2561NDER.D 261 143 85000 Scan 1497 (17.188 min): 2561ETHE.D 80000

450000 75000 390 70000 303 400000 OTMS 65000 350000 130 60000 55000 300000 258 (r2H) 50000

143 (rH) 45000 250000 O H 40000 318 200000 305 (rH) 375 35000 30000 150000 320 25000 100000 20000 15000 50000 300 390 258 10000 0 5000 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 0 60 80 100 120 140 160 180 200 220 240 260 280 300 320 m/z--> m/z-->

Figure 6: Mass spectrum of synthesised 2α,17α-dimethyl-5α-androstan-3-one-17-ol, TMS- ether (left) and underivatised (right) and proposed fragmentation pathways.

Identification of urinary metabolites: For this purpose we have used cryopreserved human hepatocytes which were reported to be a suiTable model and to produce results comparable to in vivo experimentation. More specifically, human hepatocytes have been shown to produce phase I and phase II metabolites similar, qualitatively to those produced in vivo for , norandrostenedione4 and THG5. Recently, freshly isolated human hepatocytes were successfully cryopreserved, thus maintaining their viability and showing comparable drug-metabolizing enzyme activities to freshly isolated ones6.

We were not able to get phase II metabolites in significant amounts under those conditions. Using the molecule of 17-methyldrostanolone as a model compound, we have nonetheless isolated reduced and hydroxylated metabolites in the supernatant. The in vitro experiments lead to clean extracts which were easier than urine to analyse; the reconstructed total ion chromatogram of a representative result is shown at Figure 7. The mass spectrum of the 3α- (major) reduced metabolite is shown as well along with the spectra of 16-hydroxylated metabolites of parent compound and reduced in C-3.

155 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Abundance

TIC: S5HL0803.D 8500000

8000000

7500000

70000003 alpha-OH Cholesterol

6500000 3-OH mono-OH 6000000

5500000

5000000

4500000 Parent

4000000 3-OH, di-OH 3500000

3000000

2500000

2000000 3 OH, 16-OH ISTD3B-OH 16-OH 1500000

1000000

500000

14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 Time--> Abundance Abundance Scan 1167 (14.138 min): S5HL0803.D Scan 1439 (15.983 min): S5HL0803.D 143 218 552 500000 65000 OTMS 60000 450000 OTMS 464 55000 OTMS 218 400000 50000 231 - TMSOH 45000 TMSO - CH 350000 H 3 552 40000 300000 143 TMSOH 35000 - TMSO - CH 231 250000 H 3 30000

25000 200000

20000 462 150000 15000 372 449 100000 10000

50000 269 5000 374 464 0 0 50 100 150 200 250 300 350 400 450 500 550 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 m/ z--> m/z--> Figure 7: GC/MS Analysis of supernatant fraction of hepatocyte with 17-methyldrostanolone (5) (upper): mass spectra of 3α-reduced and 16-OH hydroxylated metabolites (lower).

Hormonal properties: Some of the steroids offered to athletes via this underground market have been synthesised and studied by researchers of pharmaceutical companies in the early 60s. Their androgenic/anabolic properties have been estimated using techniques available at the time. DMT (17α-methyl-5α-androst-2-en-17β-ol) for example, was reported in two studies to possess 2 to 5 times the anabolic activity of and half of its androgenicity7. Employing different bioassays, another group reported for the same steroid, 12 times the anabolic activity and less than twice the androgenic activity of methyltestosterone8. Recently, the hormonal properties of THG have been assayed in vitro using yeast-based bioassays3 and novel microarray approaches9. Using mammalian-cell based reporter gene bioassays, we investigated the hormonal properties of tetrahydrogestrinone and other designer steroids that are suspected of being used to enhance

156 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

performance and included steroids of known activity for comparison: (DMT), , , methyldrostanolone, methyltestosterone, , and .

Androgenic activity: The androgenic activity of the designer steroids was determined using CAMA-1 cells transiently transfected with a reporter plasmid, which comprises the androgen-responsive mouse mammary tumor virus promoter in front of the luciferase gene (MMTV-luc). CAMA-1 cells express a functional .

The EC50 and efficacy values are listed in Table 1. Mibolerone, metribolone, mestanolone, tetrahydrogestrinone and methyldrostanolone were potent full agonists in this assay. Methyltestosterone and desoxymethyltestosterone were somewhat less potent . Ethylestrenol was 400-fold less potent than mibolerone and only showed a 50% efficacy. Progesterone did not display any androgenic activity. We tested the potential of hydroflutamide (1 µM), a potent , to block the androgenic response induced by steroids in this assay. We used concentrations of steroids that yielded near maximal response. In those conditions, hydroxyflutamide reduced the response of all steroids by more than 85%, indicating that our assay was specific for androgens (data not shown).

Table 1. EC50 and efficacy values of designer steroids in CAMA-1 cells transiently transfected with the MMTV-luc plasmid.

Compound EC50 (nM) Efficacy (%) Dihydrotestosterone 0,30 ± 0,03 100 Mibolerone 0,10 ± 0,02 90± 6 Metribolone 0,26 ± 0,09 82± 19 Mestanolone 0,40 ± 0,14 84± 15 Tetrahydrogestrinone 0,54 ± 0,10 103 ± 10 Methyldrostanolone (5) 0,79 ± 0,17 88 ± 10 Methyltestosterone 1,67 ± 0,40 67± 16 DMT (1) 2,50 ± 0,87 92 ± 3 Ethylestrenol 40,8 ± 6,8 52± 7 Progesterone >1000 3 ± 1 Estrogenic activity: The estrogenic activity of the compounds was assessed using MCF-7 breast cancer cells transiently transfected with a reporter vector that contains two copies of the

157 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

vitellogenin consensus response elements fused to a 105-bp thymidine kinase promoter and the luciferase gene [(ERE)2-vit-TK-luc]. MCF-7 cells express a functional estrogen receptor (ER-α). In both androgenic and estrogenic cell transfection assays, a second plasmid containing another reporter gene (Renilla) was co-transfected to control for the efficacy of the transfection.

As shown in Table 2, listing the EC50 and efficacy values, all steroids exhibited estrogenic activity, albeit at concentrations greater than 10 nM. The most potent compounds were tetrahydrogestrinone and mibolerone, followed by metribolone, desoxymethyltestosterone and

mestanolone. EC50 values for ethylestrenol, methyltestosterone, and methyldrostanolone were greater than 250 nM, hence some 30000-fold less potent than estradiol. We tested the potential of ICI 182 720 (1 µM), a selective estrogen receptor blocker, to antagonise the estrogenic response induced by steroids in this assay. We used concentrations of steroids that yielded near maximal response. ICI 182 720 reduced the response of all steroids by more than 95%, indicating that our assay was specific for (data not shown).

Table 2. EC50 and efficacy values of designer steroids in MCF-7 cells transiently transfected with the (ERE)2-vit-TK-luc plasmid.

Compound EC50 (nM) Efficacy (%) Estradiol 0,008 ± 0,002 100 Tetrahydrogestrinone 36,1 ± 5,5 104 ± 3 Mibolerone 38,7 ± 8,5 71± 6 Metribolone 94,0 ± 9,3 105± 13 DMT (1) 96,2 ± 13,6 103 ± 12 Mestanolone 118,3 ± 13,3 98± 6 Ethylestrenol 257,7 ± 34,1 84± 10 Methyltestosterone 304,0 ± 99,2 43 ± 2 Methyldrostanolone (5) 383,9 ± 55,2 64 ± 19

Progestagenic activity: The progestagenic activity of the steroids was evaluated by measuring the activity of alkaline phosphatase (AP) in T47D breast cancer cells, a breast

158 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

cancer cell line that express a functional . T47D cells express AP when exposed to a progestagen10.

Metribolone, mibolerone and tetrahydrogestrinone were potent progestins in this assay, with

EC50 values two-fold lower or more compared to that of progesterone (see Table 3). However, these compounds were only partial agonists (efficacy values of 57%, 68% and 28%, respectively). Ethylestrenol was 6.8-fold less potent than progesterone but had a similar efficacy. Mestanolone, methyltestosterone, and desoxymethyltestosterone were also full agonists in this assay but were an order of magnitude less potent than ethylestrenol. We tested the potential of mifepristone (1 µM), a selective progesterone receptor blocker, to antagonise the progestagenic response induced by steroids in this assay. We used concentrations of steroids that yielded near maximal response. Mifepristone reduced the response of all steroids by more than 85%, indicating that our assay was specific for progestins (data not shown).

Table 3. EC50 and efficacy values of designer steroids for the induction of alkaline phosphatase expression in T47D cells

Compound EC50 (nM) Efficacy (%) Progesterone 0,40 ±0,09 100 Metribolone 0,13 ±0,03 57 ± 10 Mibolerone 0,13 ±0,01 68 ± 4 Tetrahydrogestrinone 0,19 ± 0,03 28 ± 5 Ethylestrenol 2,73 ±0,33 112 ± 15 Mestanolone 28,5 ±11,4 99 ± 20 Methyltestosterone 31,5 ± 8,1 104 ± 16 DMT (1) 50,4 ± 6,6 104 ± 7 Methyldrostanolone (5) 457,4 ± 42,0 54 ± 5

159 In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (14). Sport und Buch Strauß - Köln 2006

Acknowledgments

The invaluable contribution of the scientists, Cathy Copeland, Paul Loo and Philippe St- Amour of the Laboratory and Scientific Services Directorate of the Canada Border Services Agency, is gratefully acknowledged.

This work is supported by a grant of the World Anti-Doping Agency; we also thank Dr Olivier Rabin for having connected both Canadian Laboratories and for his support.

Reference

1 Catlin et al., Detection of norbolethone, an never marketed, in athlete’s urine. Rapid Communication Mass Spectrom., (2002) 16, 1273-5 2 Catlin et al., Tetrahydrogestrinone : discovery, synthesis, and detection in urine, Rapid Commun. Mass Spectrom., 18, 1245 (2004) 3 Death AK, McGrath KC, Kazlauskas R, Handelsman DJ. Tetrahydrogestrinone is a potent androgen and progestin. Rapid Communications, J Clin Endocrinol Metab. 2004;89:2498- 500 4 Lévesque J.-F. et al., Evaluation of human hepatocyte incubation as a new tool for metabolism study of androstenedione and norandrostenedione in a doping control perspective, J. Chromatogr. B., 780, 145 (2002) 5 Levesque JF, Templeton E, Trimble L, Berthelette C, Chauret N., Discovery, biosynthesis, and structure elucidation of metabolites of a doping agent and a direct analogue, tetrahydrogestrinone and , using human hepatocytes. Anal Chem. 2005 15;77(10):3164-72. 6 Li. A. P. et al., Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel, Chem.-Biol. Interactions, 121:1, 117 (1999) 7 Bowers A. et al., Steroids. CCV. Ring A modified hormone analogs. Part I. Some ring A olefins, J. Med. Chem., 6, 156 (1962). 8 Counsell R. E. et al., Anabolic agents. 19-nor- and 19-substituted 5 -androst-2-ene derivatives, J. Med. Chem., 9, 685 (1966) 9 Labrie F. et al., Tetrahydrogestrinone (THG) induces a genomic signature typical of a potent anabolic steroid, J. Endocrinol., 184, 427–433 (2005) 10 Dong Y, Roberge JY, Wang Z, Wang X, Tamasi J, Dell V, Golla R, Corte JR, Liu Y, Fang T, Anthony MN, Schnur DM, Agler ML, Dickson JK Jr, Lawrence RM, Prack MM, Seethala R, Feyen JH. Characterization of a new class of selective progesterone receptor agonists. Steroids. 2004;69:201-17

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