Synthesis of a human long-term oxymetholone metabolite

Article abstract of DOI:10.1016/j.steroids.2019.108430

A long-term metabolite of the doping agent oxymetholone (OXM-M2, 17β-hydroxymethyl-2,17α-methyl-18-norandrost-13-en-3-one) which has been identified by GC-MS/MS was synthesized from commercially available materials. Two efficient synthetic routes to access both C-17 epimers of tentative metabolites were developed. The identity and molecular configuration of the in vivo metabolite: 17β-hydroxymethyl-2α,17α-methyl-18-norandrost-13-en-3-one was confirmed by single crystal X-ray diffraction.

Full text of DOI:10.1016/j.steroids.2019.108430

Accepted Manuscript
Synthesis of a human long-term oxymetholone metabolite
Nicolas Kratena, Nina Biedermann, Biljana Stojanovic, Lorenz Göschl,
Matthias Weil, Valentin S. Enev, Günter Gmeiner, Peter Gärtner
PII:
S0039-128X(19)30120-5
DOI:
https://doi.org/10.1016/j.steroids.2019.108430
Article Number:
Reference:
108430
STE 108430
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 April 2019
29 May 2019
17 June 2019
Please cite this article as: Kratena, N., Biedermann, N., Stojanovic, B., Göschl, L., Weil, M., Enev, V.S., Gmeiner,
G., Gärtner, P., Synthesis of a human long-term oxymetholone metabolite, Steroids (2019), doi: https://doi.org/
10.1016/j.steroids.2019.108430
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Synthesis of a human long-term oxymetholone metabolite
Nicolas Kratena1, Nina Biedermann1, Biljana Stojanovic3, Lorenz Göschl3, Matthias Weil2,
Valentin S. Enev*1, Günter Gmeiner3, Peter Gärtner*1
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology
2 Institute for Chemical Technologies and Analytics, Vienna University of Technology
3 Doping Control Laboratory, Seibersdorf Labor GmbH
* valentin.enev@tuwien.ac.at, peter.gaertner@tuwien.ac.at,
Abstract:
A long-term metabolite of the doping agent oxymetholone (OXM-M2, 17β-hydroxymethyl-
2,17α-methyl-18-norandrost-13-en-3-one) which has been identified by GC-MS/MS was
synthesized from commercially available materials. Two efficient synthetic routes to access
both C-17 epimers of tentative metabolites were developed. The identity and molecular
configuration of the in vivo metabolite: 17β-hydroxymethyl-2α,17α-methyl-18-norandrost-
13-en-3-one was confirmed by single crystal X-ray diffraction.
1. Introduction
Oxymetholone (Anadrol, Anapolon, 1) is an anabolic androgenic steroid (AAS), more
specifically a 17α-methyltestosterone derivative. It was first described in 1959 by Ringold
and coworkers at Syntex [1] and has since been used to treat anemia, osteoporosis and to
promote muscle growth in different disorders like HIV-related wasting. It was considered to
be one of the strongest AAS available in medicine. [2-4] This lead to abuse of the substance
for performance enhancing purposes by bodybuilders and professional athletes.
Consequently, it is listed as “prohibited at all times” by the World Anti-Doping Agency
(WADA). [5]
The metabolism of oxymetholone has been thoroughly investigated. [6-16] The A-ring can be
transformed in vivo in a number of ways, some leading to oxidation and decarboxylation at
C-2, others reducing the C-2 substituent as well as the C-3 ketone. Not all of these
metabolites could be traced unilaterally to oxymetholone, and the detection timeframe for
the polyhydroxylated metabolites was generally short (max. 5 days). In doping analysis,
special interest is bestowed on specific long-term metabolites (retained for more than two
weeks) to maximize the window of detection. Two such metabolites (OXM-M1 and OXM-
M2) were discovered by Sobolevsky and Rodchenkov and reported in 2012. [16] The
assigned structures are depicted in Figure 1. The metabolite OXM-M2 (2) was selected as a
target compound for synthesis to elucidate C-2 and C-17 configuration and secure its
identity.
1

Figure 1: Molecular structures of relevant compounds
The C-17 substituents and the 13,14-double bond were planned to be introduced in the
same fashion as in earlier projects [17, 20] concerning long-term metabolites with
rearranged D-ring. Further, it was envisaged to first prepare the 2β-methyl (axial) derivatives
of 2 via cuprate addition to an epoxide. This method would grant absolute stereocontrol
over the newly formed center and does not allow immediate epimerization at that carbon to
occur. After oxidation of the C-3 hydroxy group to a carbonyl, basic or acidic [21]
epimerization
would
provide
the
more
stable
2α-methyl
derivatives.
Dehydroepiandrosterone (DHEA) acetate was chosen as a commercially available starting
material.
2. Experimental
Dehydroepiandrosterone acetate was purchased from FluoroChem. Dry toluene,
dichloromethane, dimethylformamide and m-CPBA are from Acros Organics. HPLC grade
solvents (acetonitrile, methanol, isopropanol, benzene) were from VWR. All other non-
specified chemicals were from Sigma-Aldrich. Anhydrous tetrahydrofuran was pre-dried
using an Innovative Technologies PureSolv system, degassed and stored over 3Å molecular
sieves. Potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate,
potassium hydrogen carbonate, potassium carbonate, diphosphorus pentoxide were
purchased from Merck. N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was purchased
from Macherey-Nagel, and beta-glucuronidase obtained from Roche. The internal standard
D3-Testosterone was provided by the National Measurement Institute (Sydney, Australia).
NMR spectra were recorded on a Bruker AC400 and AC600 using TMS as internal standard.
IR spectra were recorded on a Perkin Elmer Spectrum 65 as thin films (ATR FT-IR). TLC-
analysis was performed with pre-coated aluminium-backed plates (Silica gel 60 F254,
Merck). Compounds were visualized by submerging in an acidic phosphomolybdic
acid/cerium sulphate solution and heating. Melting points were determined with a Kofler
hot-stage apparatus.
Sample preparation for GC-MS/MS analysis: To 5 mL of urine 100µl of D3-Testosterone (IS), 1
mL of 0.8M phosphate buffer pH 7 and 25 µL of β-glucuronidase were added. Hydrolysis was
performed at 50°C for 2h. After hydrolysis, 1 mL of 20% potassium carbonate buffer pH 9.0
and 7 mL of MTBE were added, and a liquid-liquid extraction was performed by shaking for
10 min. After that, sample was centrifugated for 7 min with 2100 rpm, the organic layer was
transferred and evaporated. Subsequently the samples were dried for 15 min over
diphosphorus pentoxide. Derivatization was performed by adding 100 µL working solution
for derivatization and heating at 60°C for 20 min. For derivatization, a trimethylsilyl iodide
stock solution was prepared by mixing 5 mL of MSTFA with 300 µL of ethanethiol.
Subsequently, 100 mg of NH4I were dissolved in this mixture. A working solution for
derivatization was prepared by adding 1 mL of the trimethylsilyl iodide stock solution to 9 mL
of MSTFA. Solutions of internal standard (1µg/ml) and analytes (1µg/ml) were prepared in
methanol and stored at -20°C.
The GC-MS/MS measurements were performed with a Trace-1300 gas chromatograph
coupled to a TSQ-8000 Evo triple quadrupole mass spectrometer, and a TriPlus-100
2

autosampler (Thermo Fisher, Austin, USA). For chromatographic separation a Rtx-1MS fused
silica capillary column, 15 m x 0.25 mm ID, 0.11 μL film thickness was used (Restek, CP-
Analytica, Mistelbach, Austria). Injections of 2.5 µL were performed in split mode with an
injector temperature of 270°C, using a split flow of 40 mL/min. The temperature program for
the GC run was following: 170°C initial column temperature, 3°C/min to 210°C, held for 1
min, 25°C/min to 305°C, held for 3 min. High purity helium gas was used as carrier gas with a
constant pressure of 80 kPa. The temperature of the transfer line and ion source was set to
270°C. Electron impact ionization was used with an electron energy of 70 eV and data were
acquired in selected reaction monitoring (SRM) mode. Transition m/z 435 → 209 was used
for internal standard D3-Testosterone.
For single crystal X-ray diffraction analysis, crystals were embedded in perfluorinated
polyether and mounted on MITGEN loops. X-ray diffraction data were measured in a cold
stream of nitrogen at T = 100 K on a Bruker APEX-II diffractometer with Mo-Kα radiation. The
collection strategy for the measurement was optimized with APEX-3 using ω- and -scans.
After integration of the data with SAINT, a semi-empirical absorption correction was
performed with SADABS. The crystal structures were solved by Direct Methods and refined
using the SHELXTL program package. All H atoms were placed geometrically and refined in a
riding model approximation. H atoms of hydroxyl groups were taken from difference maps
and refined freely. Crystallographic data for the structure(s) reported in this paper have
been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-1900498-1900499. Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax.: (internat.) +44
1223/336-033; e-mail: deposit@ccdc.cam.ac.uk]
2.1 3β-(p-Toluenesulfonyloxy)-13α-androstan-17-one (5)
Starting material 4 (2.05 g, 6.74 mmol) was charged in a round bottom flask and dry pyridine
(15 mL) was added until complete dissolution. After addition of a catalytic amount of 4-
dimethylaminopyridine (4-DMAP, ca. 0.005 g), tosyl chloride (2.57 g, 13.48 mmol) was added
in three portions over 15 minutes. The reaction was stirred at room temperature for 18
hours, then quenched by addition of water (20 mL) followed by CH2Cl2 (50 mL). The mixture
was extracted with CH2Cl2 (3x) and the pooled organic extracts were dried over MgSO4 and
evaporated. Crude product was purified via column chromatography (75 g SiO2, LP:EtOAc =
5:1) to give 2.59 (86%) of product 5 as colorless crystals. 1H-NMR (400 MHz, CDCl3): δH = 7.79
(2H, d, J = 8.07 Hz), 7.32 (2H, d, J = 8.07 Hz), 4.42 (1H, m), 2.44 (3H, s), 2.33 (1H, m), 1.96 –
2.20 (4H, m), 1.69 – 1.85 (3H, m), 1.42 – 1.66 (5H, m), 1.02 – 1.31 (4H, m), 0.95 (3H, s), 0.82 –
13
0.93 (3H, m), 0.62 (3H, s), 0.52 – 0.72 (2H, m). C-NMR (100 MHz, CDCl3): δC = 222.33,
144.49, 134.91, 129.86, 127.71, 82.39, 51.52, 50.78, 50.22, 44.37, 37.78, 36.69, 35.49, 34.81,
33.91, 32.96, 32.17, 28.38, 28.29, 25.36, 22.73, 21.77, 21.41, 11.94. IR [cm-1]: 2916, 1730,
20
1341, 1171. αD : -84.77 (c 1.35, CH2Cl2). m.p.: 130 – 131 °C. HRMS: (M-OTs) Calcd.:
273.2213, found: 273.2229.
2.2 13α-Androst-2-en-17-one (6, 7)
Tosylate 5 (2.39 g, 5.38 mmol) was weighed into a round bottom flask and dissolved in dry
DMF (60 mL). The reaction was heated to 50 °C and LiBr (2.8 g, 32.3 mmol) and Li2CO3 (2.38
3

g, 32.3 mmol) were added at once. The reaction was further heated on an oil bath at reflux
for 1 hour, then allowed to cool. The reaction mixture was poured onto 1 M HCl (100 mL)
and the mixture diluted with CH2Cl2 (60 mL). After extraction with CH2Cl2 (2x), washing the
pooled extracts with water, drying over MgSO4 and evaporating under reduced pressure the
crude product was obtained. This could be purified by recrystallizing from MeOH/H2O = 2:1,
yielding 1.33 g (91%) of a mixture of regioisomeric products 6 and 7 in a mixture of 6:1
(determined by 1H-NMR). HRMS: (M+H) Calcd.: 273.2213, found: 273.2208.
2.3 2α,3α-Epoxy-13α-androstan-17-one (8, 9)
Product 6 (1.18 g, 4.33 mmol) was charged in a round bottom flask and dissolved in CH2Cl2
(40 mL). There was added KHCO3 (0.87 g, 8.7 mmol), and meta-chloroperoxybenzoic acid
(1.17 g, 4.76 mmol, ca. 70%) in one portion. The suspension was stirred at room
temperature for 3 hours and quenched with saturated Na2S2O3 solution. Extracted with
CH2Cl2 (3x) and washed with saturated NaHCO3 solution before drying over MgSO4 and
evaporating under reduced pressure. Purification via column chromatography (55 g SiO2,
LP:Et2O = 7:1) gives 1.04 g of epoxides 8 and 9 (83%). Compound 8 was not isolated in a pure
form. Analytical data of 9 obtained after the cuprate addition (See 2.4.) in accordance with
literature [17]. HRMS: (M+H) Calcd.: 289.2162, found: 289.2165.
2.4 3α-Hydroxy-2β-methyl-13α-androstan-17-one (10)
In a Schlenk flask there was charged dry CuI (2.36 g, 12.4 mmol) and dry Et2O (15 mL). The
mixture was chilled to -20 °C and 1.6 M methyllithium (15.5 mL, 24.75 mmol) was added
dropwise over the course of 15 minutes. After complete addition a clear solution formed and
starting material 8 (0.51 g, 1.77 mmol) was added as a solution in dry Et2O (5 mL). The
reaction was allowed to warm to room temperature, sealed and stirred for 18 hours at room
temperature. The resulting suspension was poured onto saturated NH4Cl solution (100 mL)
and treated with conc. NH3 (2 mL). The mixture was stirred vigorously for 30 minutes and
then extracted with Et2O (4x). The pooled organic phases were washed with water, dried
over MgSO4 and evaporated to dryness. Purification via column chromatography (30 g SiO2,
LP:CH2Cl2:CH3CN = 25:25:1) provided recovered 69 mg starting material (13%, unreacted 9)
as well as 272 mg of a mixture of product 10 and 3-oxo byproduct 11 (51%). This was used
directly in the next step. HRMS: (M+H) Calcd.: 305.2475, found: 305.2479.
2.5 3α-Hydroxy-2β-methyl-13α-androstan-17-one pivalate (12)
To mixture of 10 and 11 (0.54 g, 1.77 mmol) in a round bottom flask was added dry pyridine
(15 mL) and 4-DMAP (ca. 0.005 g). The clear solution was chilled to 0 °C and trimethylacetyl
chloride (0.175 mL, 1.4 mmol) was added dropwise. The mixture was stirred for 16 hours
and taken up in H2O (50 mL) and CH2Cl2 (50 mL). The mixture was shaken and extracted with
CH2Cl2 (2x), washed with 1 M NaOH, dried over MgSO4 and evaporated to dryness. Column
chromatography of the crude product (25 g SiO2, LP:CH2Cl2:CH3CN = 40:40:1) provided 337
mg pivalate 12 (65% single step, 33% over two steps) as white crystalline solid. 1H-NMR (400
MHz, CDCl3): δH = 4.68 (1H, m), 2.31 (1H, m), 2.08 – 2.20 (2H, m), 1.87 – 2.05 (3H, m), 1.80
(1H, m), 1.65 (1H, m), 1.30 – 1.58 (6H, m), 1.23 (3H, s), 1.08 – 1.28 (3H, m), 1.18 (9H, s), 1.00
13
(3H, d, J = 7.70 Hz), 0.94 (3H, s), 0.84 – 0.92 (2H, m), 0.67 (3H, s), 0.56 – 0.72 (2H, m). C-
NMR (100 MHz, CDCl3): δC = 222.51, 177.96, 74.27, 53.16, 50.94, 50.31, 41.21, 39.96, 39.01,
4

37.52, 36.07, 33.97, 33.05, 32.79, 32.39, 29.78, 28.18, 27.41, 26.70, 25.45, 22.58, 21.37,
20
20.10, 15.50. IR [cm-1]: 2916, 1733, 1720, 1284. αD : -24.04 (c 0.8, CH2Cl2). m.p.: 108 °C.
HRMS: (M+H) Calcd.: 389.3050, found: 389.3050.
2.6 2β-Methyl-17-methylene-13α-androstan-3α-ol pivalate (13)
In a Schlenk flask there was weighed in zinc (2.18 g, 33.4 mmol) and dry THF (15 mL) was
added. Dibromomethane (1.93 g, 11.12 mmol) and PbCl2 (< 0.005 g) were added and the
mixture was cooled to -25 °C. Neat TiCl4 (1.19 g, 6.25 mmol) was added dropwise under
vigorous stirring and the resulting suspension was stirred at -10 °C for 2 hours. Starting
material 12 (0.515 g, 1.33 mmol) dissolved in dry THF (5 mL) was added dropwise at 0 °C, the
vessel was then sealed and stirred for 16 hours at room temperature. The mixture was
poured onto ice/1 M HCl (150 mL) and extracted with Et2O (4x) and EtOAc (1x). The organic
phases were washed with saturated NaHCO3 solution, dried over MgSO4 and evaporated
under reduced pressure. Crude product was purified via flash chromatography (10 g SiO2,
LP:Et2O = 10:1) to give 300 mg of pivalate 13 (59%). 1H-NMR (400 MHz, CDCl3): δH = 4.80 (1H,
s), 4.70 (1H, m), 4.67 (1H, s), 2.28 – 2.52 (2H, m), 1.85 – 2.00 (2H, m), 1.79 (1H, m), 1.67 (1H,
m), 1.56 (1H, m), 1.40 – 1.51 (3H, m), 1.28 – 1.39 (2H, m), 1.15 – 1.27 (2H, m), 1.20 (9H, s),
1.11 (1H, m), 1.02 (3H, d, J = 7.61 Hz), 0.88 – 0.98 (1H, m), 0.77 – 0.87 (2H, m), 0.72 (3H, s),
13
0.62 (1H, m). C-NMR (100 MHz, CDCl3): δC = 177.98, 157.56, 102.84,74.36, 54.19, 54.15,
46.09, 41.31, 40.00, 38.96, 36.64, 36.03, 34.68, 32.91, 32.80, 31.50, 29.84, 29.48, 28.39,
20
27.35, 24.39, 21.10, 20.09, 15.65. IR [cm-1]: 2926, 1722, 1148, 873. αD : 1.03 (c 0.98, CH2Cl2).
HRMS: (M-OPiv) Calcd.: 285.2577, found: 285.2590.
2.7 2β-Methyl-17-methylene-13α-androstan-3α-ol (14)
Pivalate 13 (0.47 g, 1.22 mmol), was charged in a round bottom flask and dissolved in dry
THF (10 mL). The solution was chilled to 0 °C and LiAlH4 (0.09 g, 2.4 mmol) was added under
argon flow. The suspension was stirred for 30 minutes and then quenched by addition of
water (2 mL), followed by Et2O (50 mL). The suspension was stirred and excess MgSO4 was
added. The solids were filtered and washed thoroughly with Et2O (3x 30 mL). The filtrate was
1
evaporated to provide 370 mg pure alcohol 14 (>99%) as white solid. H-NMR (400 MHz,
CDCl3): δH = 4.80 (1H, m), 4.68 (1H, m), 3.71 (1H, m), 2.28 – 2.51 (2H, m), 1.85 – 1.95 (3H, m),
1.77 (1H, m), 1.67 (1H, m), 1.51 – 1.60 (2H, m), 1.48 (1H, m), 1.36 – 1.43 (2H, m), 1.27 – 1.35
(2H, m), 1.17 – 1.24 (2H, m), 1.12 (1H, m), 1.01 (3H, d, J = 7.47 Hz), 0.93 (3H, s), 0.80 – 0.96
13
(4H, m), 0.70 (3H, s), 0.68 (1H, m). C-NMR (100 MHz, CDCl3): δC = 157.70, 102.77, 72.12,
54.24, 54.05, 46.11, 40.61, 38.84, 36.67, 36.27, 35.93, 34.73, 32.85, 32.75, 31.52, 29.51,
20
28.49, 24.38, 21.12, 20.59, 15.66. IR [cm-1]: 3393, 2922, 997, 887. αD : 24.65 (c 0.6, CH2Cl2).
m.p.: 117 – 118 °C. HRMS: (M-OH) Calcd.: 285.2577, found: 285.2594.
2.8 2β-Methylspiro[13α-androstan-17ξ,2’-oxiran]-3α-ol (15, 16)
To alcohol 14 (0.37 g, 1.22 mmol) in CH2Cl2 (12 mL) was added KHCO3 (0.25 g, 2.5 mmol)
followed by meta-chloroperoxybenzoic acid (0.36 g, 1.47 mmol, 70%) at 0 °C. The mixture
was stirred at room temperature for 2 hours and quenched with saturated Na2S2O3 and
saturated NaHCO3 solution. After extraction with CH2Cl2 (2x) and drying over MgSO4 the
organic phases were evaporated to dryness and the residue purified via column
5

chromatography (20 g SiO2, LP:EtOAc = 12:1) giving 73 mg 17β-epoxide 15 (19%) as colorless
oil and 290 mg 17α-epoxide 16 (74%) as white foam.
1
17β-Epoxide (15): H-NMR (400 MHz, CD2Cl2): δH = 3.66 (1H, m), 2.63 (1H, d, J = 4.71 Hz),
2.55 (1H, d, J = 4.71 Hz), 2.13 (1H, dd, J = 11.12 Hz, 9.56 Hz), 2.00 (1H, s), 1.98 (1H, m), 1.84
(2H, m), 1.66 – 1.76 (3H, m), 1.53 – 1.64 (2H, m), 1.36 – 1.52 (3H, m), 1.29 (2H, m), 1.24 (2H,
m), 1.17 (1H, m), 1.10 (1H, m), 1.00 (3H, d, J = 7.67 Hz), 0.90 (3H, s), 0.86 (1H, m), 0.77 (3H,
13
s), 0.66 (1H, m), C-NMR (400 MHz, CD2Cl2): δC = 72.30, 68.66, 54.68, 53.32, 47.83, 41.67,
41.00, 39.25, 37.08, 36.67, 36.42, 33.48, 33.23, 32.43, 30.61, 28.84, 28.58, 24.62, 23.00,
20
20.74, 15.99. IR [cm-1]: 3414, 2919, 737. αD : 3.86 (c 1.32, CH2Cl2). HRMS: (M-OH) Calcd.:
301.2526, found: 301.2545.
1
17α-Epoxide (16): H-NMR (400 MHz, CD2Cl2): δH = 3.67 (1H, m), 2.77 (1H, d, J = 5.29 Hz),
2.68 (1H, d, J = 5.29 Hz), 1.93 – 2.04 (3H, m), 1.62 – 1.90 (4H, m), 1.54 – 1.61 (5H, m), 1.31 –
1.42 (2H, m), 1.25 (3H, m), 1.11 – 1.21 (2H, m), 1.07 (1H, m), 1.00 (3H, d, J = 7.80 Hz), 0.94
13
(3H, s), 0.86 (1H, m), 0.75 (3H, s), 0.70 (1H, m). C-NMR (100 MHz, CD2Cl2): δC = 72.21,
67.03, 55.55, 54.60, 53.49, 41.39, 40.91, 39.19, 37.36, 36.62, 36.37, 33.41, 33.16, 32.73,
20
29.89, 28.77, 26.74, 24.12, 22.25, 20.75, 15.90. IR [cm-1]: 3336, 2920, 1442, 1036. αD : -
7.477 (c 0.65, CH2Cl2). m.p.: 102 – 103 °C. HRMS: (M-OH) Calcd.: 301.2526, found: 301.2537.
2.9 2β-Methylspiro[13α-androstan-17ξ,2’-oxiran]-3-one (17)
To a solution of epoxide 15 (0.085 g, 0.27 mmol) in CH2Cl2 (5 mL) at room temperature Dess-
Martin periodinane (0.136 g, 0.32 mmol) was added in one portion. The mixture was stirred
for 30 minutes and quenched by addition of saturated Na2S2O3 and saturated NaHCO3
solution. After extraction with CH2Cl2 (3x) and drying over MgSO4 the crude product was
obtained after evaporation of the solvent. This was purified via column chromatography (5 g
1
SiO2, LP:EtOAc = 10:1) to give 60 mg ketone 17 (71%) as white solid. H-NMR (400 MHz,
CD2Cl2): δH = 2.64 (1H, d, J = 4.69 Hz), 2.58 (1H, m), 2.56 (1H, d, J = 4.69 Hz), 2.09 – 2.21 (2H,
m), 1.91 – 2.07 (4H, m), 1.85 (1H, m), 1.65 – 1.79 (2H, m), 1.49 (1H, m), 1.35 – 1.44 (2H, m),
1.17 – 1.34 (4H, m), 1.06 – 1.16 (2H, m), 0.98 (3H, d, J = 6.89 Hz), 0.91 (3H, s), 0.69 (3H, s),
13
0.76 – 0.95 (2H, m). C-NMR (100 MHz, CD2Cl2): δC = 215.74, 68.51, 54.28, 52.96, 47.87,
47.12, 43.56, 42.16, 41.69, 41.53, 37.66, 36.24, 33.20, 32.41, 30.62, 28.81, 28.60, 24.83,
20
23.69, 16.14, 15.12. IR [cm-1]: 2929, 1710, 931. αD : 95.54 (c 1.2, CH2Cl2). m.p.: 146 – 148 °C.
HRMS: (M+H) Calcd.: 317.2475, found: 317.2492.
2.10
17β-Hydroxymethyl-2α,17α-methyl-18-norandrost-13-en-3-one (2a)
In a Schlenk flask under argon atmosphere there was added dry CH2Cl2 (10 mL). The solvent
was chilled to -78 °C and 2,6-lutidine (0.044 ml, 0.38 mmol) and trimethylsilyl triflate (0.058
ml, 0.32 mmol) were added. Starting material 17 (0.06 g, 0.19 mmol) dissolved in dry CH2Cl2
(1 mL) was added dropwise. After 1 hour the reaction was warmed to room temperature
and methanol (2 mL) was added followed by saturated NaHCO3 solution. The reaction
mixture was extracted with CH2Cl2 (3x) and the pooled extracts were washed with water and
brine, dried over MgSO4 and evaporated to dryness. The residue was taken up in acetonitrile
(3 mL) and H2SiF6 (0.027 mL, 0.09 mmol) was added. After 45 minutes the reaction was
quenched by addition of saturated NaHCO3 solution. Extraction with CH2Cl2 (2x) followed by
drying over MgSO4 and evaporating under reduced pressure provided crude product.
6

Purification via column chromatography (6 g SiO2, LP:EtOAc 10:1) gave 45 mg of 2a (76%) as
1
white crystals. A single crystal for x-ray analysis was crystallized from methanol/H2O. H-
NMR (400 MHz, CDCl3): δH = 3.46 (1H, d, J = 10.31 Hz), 3.29 (1H, d, J = 10.37 Hz), 2.48 (1H,
m), 2.21 – 2.37 (2H, m), 2.04 – 2.20 (4H, m), 1.83 – 2.01 (5H, m), 1.47 – 1.60 (2H, m), 1.17 –
1.42 (5H, m), 1.03 (3H, s), 1.00 (3H, d, J = 6.61 Hz), 0.89 – 1.04 (2H, m), 0.93 (3H, s). 13C-NMR
(100 MHz, CDCl3): δC = 213.28, 140.71, 136.68, 69.06, 51.68, 51.37, 48.30, 47.84, 44.70,
41.18, 36.61, 36.39, 34.06, 31.00, 30.64, 29.09, 23.01, 22.57, 21.85, 14.72, 12.06. IR [cm-1]:
20
3410, 2926, 1713. αD : -3.91 (c 0.9, CH2Cl2). m.p.: 135 – 136 °C. HRMS: (M+H) Calcd.:
317.2475, found: 317.2469.
EI fragmentation MS:
The mass spectrum of the bis-TMS derivative of the title compound shown below is in
accordance with already published spectra in the literature [16]. The most abundant
fragment 357.3 m/z is suggested to result from the loss of CH2OTMS (Δ = 105). The smaller,
less abundant fragment ions were assigned and discussed by Sobolevsky and Rodchenkov
[16].
OXM_M3_1610 #3013 RT: 11.10 AV: 1 NL: 2.43E8
T: + c EI Full ms [50.000-750.000]
357.34
100
90
80
70
60
50
40
73.05
30
20
193.13
147.13
267.27
10
0
460.39
100
200
300
m/z
400
500
Figure 2: EI spectrum of TMS-2a
Crystal data (CCDC-1900498). C21H32O2, M = 316.47, orthorhombic, a = 7.7653(6), b =
9.7987(7), c = 22.7256(17) Å, Z = 4, T = 100 K, space group P212121 (No. 19), 70505 reflections
measured, 6263 unique (Rint = 0.042), which were used in all calculations. Flack parameter
using 2440 quotients is -0.2(2), the final wR(F2) is 0.090.
2.11
3β-Hydroxyandrostan-17-one acetate (18)
Dehydroepiandrosterone acetate (3 g, 9.08 mmol) was charged in a three-necked flask and
dissolved in dry EtOH (470 mL). Pd/C (10%, 0.483 g, 0.45 mmol) was added under argon flow,
the atmosphere was exchanged for hydrogen and the vessel sealed and stirred for 18 hours.
The mixture is filtrated over Celite, solids washed with EtOAc (2x 100 mL) and the filtrate
evaporated under reduced pressure. Flash chromatography (70 g SiO2, LP:Et2O = 3:1) of the
crude product gave 2.25 g of acetate 18 (75%) as colorless solid.
7

Analytical data in accordance with literature [18].
2.12
17-(1,3-Dioxolan-2-yl)-androstan-3β-ol acetate (19)
Epiandrosterone acetate (2.25 g, 6.87 mmol) was dissolved in benzene (150 mL). Ethylene
glycol (8.55 g, 137 mmol) and para-toluenesulfonic acid monohydrate (0.013 g, 0.07 mmol)
were added to the solution and the mixture was heated to reflux on a Dean-Stark trap. After
16 hours the reaction was allowed to cool to room temperature and treated with saturated
NaHCO3 solution (50 mL) and shaken in a separatory funnel. The phases were separated and
the aqueous phase extracted with EtOAc (3x). The combined organic phases were dried over
MgSO4 and evaporated to dryness. The crude product was crystallized from MeOH:EtOH =
1:1 to give 2.1 g acetate 19 (81%) as polycrystalline material.
Analytical data in accordance with literature [19].
2.13
17-(1,3-Dioxolan-2-yl)-androstan-3β-ol (20)
In a round bottom flask was charged starting material 19 (3.04 g, 8.08 mmol) and MeOH
(150 mL). The mixture was heated to reflux and K2CO3 (2.23 g, 16.17 mmol) was added to the
clear solution. After 45 minutes the reaction was concentrated and the residue taken up in
CH2Cl2 (150 mL) and water (50 mL). After separation of the phases the aqueous phase was
extracted with CH2Cl2 (2x 50 mL) and the pooled organics were washed with water, brine,
dried over MgSO4 and evaporated under reduced pressure to give 2.8 g alcohol 20 (>99%) as
1
white solid. H-NMR (400 MHz, CDCl3): δH = 3.87 (4H, m), 3.57 (1H, m), 1.95 (1H, m), 1.49 –
1.82 (9H, m), 1.15 – 1.42 (9H, m), 1.09 (1H, m), 0.85 – 1.00 (2H, m), 0.81 (3H, s), 0.79 (3H, s),
13
0.65 (1H, m). C-NMR (100 MHz, CDCl3): δC = 119.58, 71.32, 65.26, 64.63, 54.26, 50.44,
46.06, 44.95, 38.28, 37.15, 35.86, 35.62, 34.28, 31.58, 31.44, 30.81, 28.68, 22.75, 20.76,
20
14.52, 12.42. IR [cm-1]: 3508, 3254, 2931, 1310. αD : -15.74 (c 1.0, CH2Cl2). m.p.: 152 – 155
°C. HRMS: (M+H) Calcd.: 335.2581, found: 335.2595.
2.14
17-(1,3-Dioxolan-2-yl)-androstan-3β-ol tosylate (21)
Starting material 20 (2.83 g, 8.46 mmol) was dissolved in dry pyridine (30 mL) and chilled to
0 °C. To this solution DMAP (0.005 g), followed by tosyl chloride (2.02 g, 10.6 mmol) was
added and the reaction stirred for 5 hours at room temperature. The resulting suspension
was concentrated under reduced pressure and the residue dissolved in CH2Cl2 (100 mL) and
water (50 mL). Phases were separated and the aqueous phase extracted twice with CH2Cl2.
Pooled organic phases were washed with NaHCO3 solution and brine, dried over MgSO4 and
evaporated to dryness. The crude product was purified by column chromatography (200 g
SiO2, LP:Et2O = 2:1) to give 3.97 g of tosylate 21 (96%) as colorless solid. 1H-NMR (400 MHz,
CDCl3): δH = 7.77 (2H, d, J = 8.25 Hz), 7.31 (2H, d, J = 8.25 Hz), 4.40 (1H, m), 3.78 – 3.95 (4H,
m), 2.43 (3H, s), 1.94 (1H, m), 1.43 – 1.80 (8H, m), 1.28 – 1.39 (3H, m), 1.14 – 1.26 (4H, m),
1.05 (1H, m), 0.78 – 0.96 (2H, m), 0.80 (3H, s), 0.76 (3H, s), 0.61 (1H, m). 13C-NMR (100 MHz,
CDCl3): δC = 144.40, 134.86, 129.82, 127.67, 119.45, 82.52, 65.26, 64.60, 53.97, 50.31, 45.98,
44.86, 36.89, 35.70, 35.33, 34.91, 34.24, 31.23, 30.68, 28.47, 28.36, 22.69, 21.72, 20.67,
20
14.47, 12.19. IR [cm-1]: 2939, 1356, 933. αD : -18.68 (c 1.0, CH2Cl2). m.p.: 163 – 165 °C.
HRMS: (M+H) Calcd.: 489.2669, found: 489.2694.
2.15
17-(1,3-Dioxolan-2-yl)-androst-2-en (22, 23)
8

Tosylate 21 (3.97 g, 8.08 mmol) was weighed into a round bottom flask and dissolved in dry
DMF (100 mL). LiBr (4.23 g, 48.8 mmol) and Li2CO3 (3.86 g, 48.8 mmol) were added at once.
The reaction was heated on an oil bath at reflux for 1.5 hours, then allowed to cool. The
reaction mixture was poured onto 1 M HCl (150 mL) and the mixture diluted with CH2Cl2 (80
mL). After extraction with CH2Cl2 (2x), washing the pooled extracts with water, drying over
MgSO4 and evaporating under reduced pressure the crude product was obtained. This could
be purified by recrystallizing from MeOH/H2O = 3:2, yielding 2.18 g of a mixture of
1
regioisomeric products 22 and 23 (85%) in a mixture of 6:1 (determined by H-NMR). HRMS:
(M+H) Calcd.: 317.2475, found: 317.2490.
2.16
17-(1,3-Dioxolan-2-yl)-2α,3α-epoxyandrostan (24, 25)
Mixture of products 22 and 23 (1.58 g, 4.74 mmol) was charged in a round bottom flask and
dissolved in CH2Cl2 (40 mL). There was added KHCO3 (0.96 g, 9.5 mmol), and meta-
Chloroperoxybenzoic acid (1.29 g, 5.21 mmol, ca. 70%) in one portion. The resulting
suspension was stirred at room temperature for 2.5 hours and quenched with saturated
Na2S2O3 solution. Extracted with CH2Cl2 (3x) and washed with saturated NaHCO3 solution
and brine, before drying over MgSO4 and evaporating to dryness. Purification via flash
chromatography (37 g SiO2, LP:Et2O = 7:1) gives 1.47 g of epoxides 24 and 25 (94%). HRMS:
(M+H) Calcd.: 333.2424, found: 333.2450.
2.17
17-(1,3-Dioxolan-2-yl)-2β-methylandrostan-3α-ol (26)
Dry CuI (1.97 g, 10.36 mmol) was charged in a Schlenk flask (100 mL) and dry Et2O (20 mL)
was added to form a suspension. The reaction was chilled to -20 °C and 1.5 M methyllithium-
LiBr complex in Et2O (13.81 mL, 20.72 mmol) was added dropwise until a clear, colorless
solution formed. Starting material 24 and 25 (0.49 g, 1.48 mmol) was dissolved in Et2O (5 mL)
and added dropwise to the solution at -20 °C. The reaction was then allowed to warm to
room temperature, sealed and stirred for 20 hours. Reaction was quenched by pouring onto
a mixture of 30 ml saturated NH4Cl, 30 ml saturated NaHCO3, 30 ml water and 2 ml conc.
NH3 and stirring for 1 hour. The aqueous phase was extracted with Et2O (3x), the pooled
organic phases washed twice with dilute NH3 and dried over MgSO4. After evaporating the
solvent the crude product was purified via column chromatography (25 g SiO2, LP:Et2O = 2:1)
1
to give 207 mg of alcohol 26 (40%) as a white solid. H-NMR (400 MHz, CDCl3): δH = 3.81 –
3.93 (4H, m), 3.71 (1H, m), 1.84 – 2.00 (2H, m), 1.77 (1H, m), 1.62 – 1.69 (2H, m), 1.54 – 1.61
(2H, m), 1.46 – 1.53 (2H, m), 1.42 (1H, m), 1.34 – 1.41 (3H, m), 1.31 (1H, m), 1.16 – 1.28 (5H,
m), 1.03 (3H, d, J = 7.53 Hz), 0.94 (1H, m), 0.84 (3H, s), 0.83 (1H, m), 0.82 (3H, s), 0.73 (1H,
13
m). C-NMR (100 MHz, CDCl3): δC = 119.61, 72.05, 65.28, 64.67, 55.65, 50.34, 46.19, 40.62,
39.26, 36.26, 35.93, 35.56, 34.32, 32.75, 31.32, 30.90, 28.31, 22.74, 20.66, 20.44, 15.73,
20
14.61. IR [cm-1]: 2917, 1003, 737. αD : 2.18 (c 1.0, CH2Cl2). m.p.: 119 – 121 °C. HRMS: (M+H)
Calcd.: 349.2737, found: 349.2757.
2.18
3α-Hydroxy-2β-methylandrostan-17-one (27)
To a solution of starting material 26 (0.6 g, 1.71 mmol), in 1,4-dioxane (20 mL) was added 0.5
M H2SO4 (10 mL) and the solution was stirred at room temperature. After 2 hours the
reaction was neutralized with saturated NaHCO3 solution and extracted with Et2O (3x).
Pooled organic phases were washed with brine, dried over MgSO4 and evaporated under
9

reduced pressure. The crude product was crystallized from boiling n-Hexane/MTBE to give
1
427 mg of ketone 27 (82%) as white crystals. H-NMR (400 MHz, CDCl3): δH = 3.74(1H, m),
2.41 (1H, dd, J = 8.78 Hz, 18.93 Hz), 2.05 (1H, m), 1.85 – 1.96 (2H, m), 1.72 – 1.81 (2H, m),
1.65 – 1.72 (2H, m), 1.61 (1H, m), 1.46 – 1.57 (3H, m), 1.36 – 1.46 (2H, m), 1.19 – 1.34 (6H,
m), 0.97 – 1.07 (1H, m), 1.03 (3H, d, J = 7.40 Hz), 0.86 (3H, s), 0.85 (3H, s), 0.75 (1H, m). 13C-
NMR (100 MHz, CDCl3): δC = 221.58, 71.93, 55.98, 51.48, 48.03, 40.49, 39.25, 36.36, 35.97,
35.92, 34.86, 32.55, 31.75, 30.89, 28.12, 21.87, 20.61, 20.29, 15.71, 14.03. IR [cm-1]: 3546,
20
2909, 1723, 1007. αD : 94.03 (c 1.0, CH2Cl2). HRMS: (M+H) Calcd.: 305.2475, found:
305.2476.
2.19
2β-Methyl-17-methyleneandrostan-3α-ol (28)
A Schlenk flask (100 mL) was charged with zinc (2.69 g, 41.2 mmol), dry THF (35 mL), PbCl2
(0.005 g), and CH2Br2 (2.38 g, 13.72 mmol). The mixture was stirred vigorously and chilled to
-20 °C. At that temperature neat TiCl4 (1.62 g, 8.57 mmol) was added dropwise over 10
minutes. The mixture was further stirred at -10 °C for 3 hours. The reaction was then
warmed to 0 °C and starting material 27 (0.52 g, 1.71 mmol) was added in dry THF (10 mL).
The reaction was stirred for 1.5 hours at room temperature and then quenched with 1 M HCl
(50 mL) and ice (50 g). The mixture was extracted with Et2O (3x) and EtOAc (1x) and the
pooled organic phases washed with NaHCO3 solution and brine. After drying over MgSO4 and
evaporating the solvent the crude product was purified by flash chromatography (13 g SiO2,
1
LP:EtOAc = 20:1) to give 438 mg of olefin 28 (85%) as polycrystalline material. H-NMR (400
MHz, CDCl3): δH = 4.60 (2H, m), 3.72 (1H, m), 2.46 (1H, m), 2.22 (1H, m), 1.89 (1H, m), 1.78
(1H, m), 1.60 – 1.73 (4H, m), 1.47 – 1.59 (3H, m), 1.35 – 1.44 (2H, m), 1.15 – 1.33 (6H, m),
1.03 (3H, d, J = 7.68 Hz), 0.89 – 1.00 (2H, m), 0.86 (3H, s), 0.76 (3H, s), 0.72 (1H, m). 13C-NMR
(100 MHz, CDCl3): δC = 162.15, 100.68, 72.04, 56.10, 54.45, 44.34, 40.58, 39.31, 36.32, 35.92,
35.90, 35.25, 32.68, 31.89, 29.49, 28.38, 24.19, 20.90, 20.64, 18.73, 15.75. IR [cm-1]: 3358,
20
2911, 874. αD : 40.37 (c 1.0, CH2Cl2). m.p.: 126 – 128 °C. HRMS: (M-OH) Calcd.: 285.2577,
found: 285.2591.
2.20
2β-Methylspiro[androstan-17α,2’-oxiran]-3α-ol (29)
A round bottom flask was charged with olefin 28 (0.44 g, 1.45 mmol). This was dissolved in
CH2Cl2 (12 mL) and KHCO3 (0.29 g, 2.9 mmol), followed by meta-chloroperoxybenzoic acid
(0.43 g, 1.74 mmol) was added in one portion at room temperature. After 1 hour the
reaction was quenched by addition of saturated Na2S2O3 solution and NaHCO3 solution. The
mixture was extracted with CH2Cl2 (3x) and the extracts washed with water, brine and dried
over MgSO4. The solvent was evaporated and the crude product purified via column
chromatography (32 g SiO2, LP:EtOAc = 10:1) to give 185 mg desired 17α-epoxide 29 (40%).
1
The other stereoisomer (17β-epoxide) was not isolated in a pure form. H-NMR (400 MHz,
CDCl3): δH = 3.72 (1H, m), 2.72 (1H, d, J = 4.94 Hz), 2.64 (1H, d, J = 4.94 Hz), 2.25 (1H, m), 1.77
– 1.92 (2H, m), 1.65 – 1.76 (2H, m), 1.47 – 1.64 (4H, m), 1.34 – 1.44 (3H, m), 1.10 – 1.32 (8H,
m), 1.02 (3H, d, J = 7.49 Hz), 0.97 – 1.07 (1H, m), 0.84 (3H, s), 0.79 (3H, s), 0.75 (1H, m). 13C-
NMR (100 MHz, CDCl3): δC = 72.04, 70.16, 55.66, 52.69, 47.47, 41.71, 40.62, 39.26, 36.30,
35.94, 35.41, 32.72, 31.82, 30.45, 30.05, 28.35, 24.09, 20.66, 20.07, 16.41, 15.75. IR [cm-1]:
20
3385, 2922, 929, 731. αD : 16.81 (c 1.0, CH2Cl2). m.p.: 130 – 132 °C. HRMS: (M-H) Calcd.:
317.2486, found: 317.2468.
10

2.21
2β-Methylspiro[androstan-17ξ,2’-oxiran]-3-one (30)
A solution of starting material 29 (0.16 g, 0.49 mmol) in CH2Cl2 (10 mL) was chilled to 0 °C
and Dess-Martins periodinane (0.25 g, 0.59 mmol) was added at that temperature as a solid
in one portion. After 1 hour the reaction was quenched with saturated NaHCO3 and Na2S2O3
solution and extracted with CH2Cl2 (3x). Pooled organic phases were washed with NaHCO3
solution, water and brine and dried over MgSO4. After evaporation of the solvent under
reduced pressure the crude product was purified via flash chromatography (9 g SiO2,
1
LP:EtOAc = 15:1) to provide 87 mg of ketone 30 (55%) as a white solid. H-NMR (400 MHz,
CDCl3): δH = 2.73 (1H, d, J = 4.52 Hz), 2.66 (1H, d, J = 4.52 Hz), 2.59 (1H, m), 2.20 – 2.32 (2H,
m), 1.92 – 2.12 (3H, m), 1.84 (1H, m), 1.74 (1H, m), 1.45 – 1.60 (3H, m), 1.11 – 1.44 (8H, m),
0.93 – 1.06 (1H, m), 1.02 (3H, d, J = 6.75 Hz), 0.85 (1H, m), 0.80 (3H, s), 0.75 (3H, s). 13C-NMR
(100 MHz, CDCl3): δC = 215.96, 70.01, 54.97, 52.57, 47.41, 46.72, 43.33, 41.97, 41.66, 41.18,
35.71 (2C), 31.34, 30.39, 29.99, 28.32, 24.06, 20.64, 16.24, 15.99, 14.61. IR [cm-1]: 2913,
20
1705, 927. αD : 115.14 (c 1.0, CH2Cl2). m.p.: 136 – 140 °C. HRMS: (M+H) Calcd.: 317.2475,
found: 317.2483.
2.22
17β-Hydroxymethyl-2α,17α-methyl-18-norandrost-13-en-3-one (2b)
In a round bottom flask (25 mL) there was charged epoxide 30 (0.04 g, 0.13 mmol) and acetic
acid (3 mL) was added. The solution was stirred for 1 hour, then diluted with water (10 mL)
and neutralized by addition of KHCO3. The neutral aqueous solution was extracted with
CH2Cl2 (3x) and the pooled extracts washed with water and brine. After drying over MgSO4
and evaporating to dryness the crude product was purified via column chromatography (3 g
1
SiO2, LP:EtOAc = 5:1) to give 28 mg of ketone 2b (69%) as colorless crystals. H-NMR (400
MHz, CDCl3): δH = 3.39 (1H, d, J = 10.63 Hz), 3.29 (1H, d, J = 10.63 Hz), 2.61 (1H, m), 2.22 –
2.34 (2H, m), 2.02 – 2.14 (2H, m), 1.96 – 2.02 (3H, m), 1.88 – 1.95 (2H, m), 1.73 – 1.86 (2H,
m), 1.51 – 1.61 (2H, m), 1.16 – 1.40 (5H, m), 1.09 (1H, m), 1.03 (3H, d, J = 6.57 Hz), 1.01 (1H,
13
m), 0.96 (3H, s), 0.70 (3H, s). C-NMR (100 MHz, CDCl3): δC = 215.86, 140.97, 136.42, 69.13,
52.80, 51.67, 46.47, 43.14, 41.49, 40.96, 37.03, 35.57, 34.21, 30.97, 30.72, 28.69, 23.27,
20
22.78, 21.85, 15.80, 14.48. IR [cm-1]: 3429, 1703, 1037. αD : 75.24 (c 1.0, CH2Cl2). HRMS:
(M+H) Calcd.: 317.2475, found: 317.2474.
2.23
17-(1,3-Dioxolan-2-yl)-2β-methylandrostan-3α-ol 4-nitrobenzoate (31)
In a round-bottom flask (25 mL) starting material 26 (0.03 g, 0.086 mmol) was dissolved in
dry CH2Cl2 (5 mL). There was added 4-DMAP (0.012 g, 0.1 mmol), 4-nitrobenzoic acid (0.017
g, 0.1 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.019 g, 0.1
mmol) in that order at room temperature. The reaction was stirred at room temperature
and was quenched after 4 hours by addition of saturated NaHCO3 solution. The mixture was
extracted with CH2Cl2 (2x) and organic phases dried over MgSO4 and evaporated. Flash
column chromatography (2.5 g SiO2, LP:EtOAc = 10:1) provided 41 mg of ester 31 (96%). 1H-
NMR (400 MHz, CDCl3): δH = 8.30 (2H, d, J = 9.00 Hz), 8.19 (2H, d, J = 9.00 Hz), 5.06 (1H, m),
3.81 – 3.95 (4H, m), 2.15 (1H, m), 2.03 (1H, s), 1.96 (1H, m), 1.72 – 1.85 (2H, m), 1.58 – 1.70
(3H, m), 1.52 – 1.56 (3H, m), 1.50 (1H, m), 1.33 – 1.46 (3H, m), 1.26 (1H, m), 1.19 – 1.24 (2H,
m), 1.13 (3H, d, J = 8.03 Hz), 0.92 (3H, s), 0.82 – 0.95 (2H, m), 0.84 (3H, s), 0.77 (1H, m). 13C-
NMR (100 MHz, CDCl3): δC = 164.08, 150.57, 136.61, 130.73, 123.69, 119.52, 65.34, 64.65,
11

55.94, 50.44, 46.14, 41.25, 40.71, 36.06, 35.46, 34.25, 33.00, 31.29, 30.83, 29.68, 28.03,
22.67, 20.51, 20.14, 15.81, 14.64. IR [cm-1]: 2912, 1686, 881.
Crystal data (CCDC-1900499). C29H39NO6, M = 497.61, monoclinic, a = 10.9205(11). b =
11.4099(12), c = 20.867(2) Å, Z = 4 (Z’ = 2), T = 100 K, space group P21 (No. 4), 37131
reflections measured, 12209 unique (Rint = 0.077), which were used in all calculations. Flack
parameter using 2391 quotients is 0.0(7), the final wR(F2) is 0.126.
3. Results and discussion
The first synthetic route served to prepare only the 17β-hydroxymethyl isomer 2a. Synthesis
of 2a commenced from 4 (3 steps from DHEA acetate [20], 75% yield) which was converted
into tosylate 5 in excellent yield. Elimination to the desired 2,3-olefin was achieved with LiBr
and Li2CO3 in refluxing DMF [22]. The product was isolated as a 6:1 mixture of regioisomeric
1
olefins 6 and 7 (determined from H-NMR integrals of olefinic protons). The mixture was
epoxidized with m-CPBA and the α-epoxides 8 and 9 were isolated exclusively, again as a
mixture (See Scheme 1: compounds 9, 10, 11, 14, 16 not shown). Treatment of this epoxide
with lithium dimethylcopper (Me2CuLi) in Et2O [23-24] provided the desired 2β-methyl
alcohol 10 as a chromatographically inseparable mixture together with the rearranged 3-oxo
product 11. Protection of the hydroxy group (pivaloyl chloride, pyridine) allowed for isolation
as a pure product (12, 33% over two steps).
Scheme 1: Synthesis of target compound 2a
With A-ring substituents in place, the D-ring was now elaborated. Methylenation of the
ketone at C-17 with Takai-Lombardo reagent (Zn, TiCl4, CH2Br2) in THF [25] gave ester 13 in
59% yield. Cleavage of the protecting group with LiAlH4 provided alcohol 14 in quantitative
yield. The 17-exo-methylene double bond was epoxidized with m-CPBA to yield a mixture of
diastereomers, of which the minor 17β-epoxide 15 is the desired product (19% yield). The
alcohol was now oxidized to the ketone with Dess-Martins periodinane (DMP) in CH2Cl2 to
give ketone 17 in 71% yield. Finally, Wagner-Meerwein rearrangement was initiated by
12

treating 17 with TMSOTf and 2,6-lutidine in CH2Cl2 at -78 °C. In order to minimize
epimerization of the C-2 stereocenter deprotection of the resulting primary TMS ether was
effected using H2SiF6 in acetonitrile, arriving at product 2a (76% over two steps). [26] Single
crystal X-ray analysis revealed 2α-configuration of the isolated product (vide infra: Figure 3).
This indicates that during the last two steps equilibration of the C-2 carbon had already
occurred.
Figure 3: Molecular structures determined by X-ray single crystal analysis
The second synthetic route allows access to the 17α-hydroxymethyl epimer 2b and was
carried out using the same methodology. Starting from DHEA acetate which was
hydrogenated over Pd/C in ethanol in 75% yield followed by protection of the ketone moiety
by ketal formation giving acetate 19 (See Scheme 2: compounds 18, 25, 30 not depicted).
Next, the ester was cleaved using K2CO3/methanol (99% yield) and alcohol 20 was tosylated,
eliminated and the formed olefin epoxidized as described before (77% over three steps).
Opening of the epoxide using excess Me2CuLi in Et2O gave a 1:1 mixture of rearranged 3-oxo
product and desired 2β-methyl-3α-alcohol 26, the latter could be isolated in 40% yield after
column chromatography. The configuration of A-ring substituents was secured by X-ray
analysis of the 4-nitrobenzoate 31 (Figure 2) of this alcohol. After deprotection of the cyclic
ketal (H2SO4, dioxane) the synthesis proceeded as shown before. Methylenation of the 17-
ketone using Takai-Lombardo reagent was successful and delivered alcohol 28 in 85% yield
without the need for a protecting group. Epoxidation with m-CPBA gives the desired 17β-
epoxide 29 (40% yield), which was again oxidized with DMP and rearranged in acetic acid
(38% over two steps) [27] to furnish 2b.
13

Scheme 2: Synthesis of target compound 2b and nitrobenzoate 31
Only the 2α-isomers of the series of putative metabolites could be accessed by this route
since the 2β-methyl-3-oxo isomers equilibrated rapidly under acidic conditions and
reasonably fast under basic conditions (ca. 8 hours in KOH/CD3OD/D2O). With these
observations in mind it is conceivable that over a long time in an in vivo system the
metabolite would also equilibrate to its most stable isomers: 2a or 2b.
Due to the method chosen for analytical comparison with excretion studies (GC-MS/MS),
TMS-derivatization is required, possibly destroying the stereochemical information at C-2 by
enolization at that carbon ([16] suggests enolization at C-4). The comparative measurements
including a positive sample, a blank urine sample and a spiked blank sample are shown
below in Figure 4. For metabolite 2a transitions: m/z 357 → 161, m/z 357 → 193 and m/z
357 → 207 were selected.
14

B
C
A
11.07
11.06
11.06
10.38
10.70
10.74
11.05
100
100
100
50
50
50
1
2
0
0
0
10.84
11.05
11.05
100
100
100
10.89
50
50
50
0
0
0
10.85
100
100
100
10.82
11.08
50
50
50
3
4
0
0
0
10.71
10.70
10.70
100
100
100
50
0
50
0
50
0
10.5
11.0
11.5
10.5
11.0
11.5
10.5
11.0
11.5
Time (min)
Time (min)
Time (min)
Figure 4: Detection of metabolite 2a in positive urine sample by GC-MS/MS; Row 1-3: mass
transitions for metabolite 2a (357 → 161, 357 → 193, 357 → 207); Row 4: mass transition for
internal standard D3-Testosterone (435 → 209); A: positive urine sample; B: blank urine
sample; C: blank urine sample spiked with 5 ng/mL metabolite 2a
4. Conclusion:
To summarize, we accomplished the first chemical synthesis of compounds 2a and 2b in 13
and 12 steps respectively (from DHEA acetate). These two putative metabolites were
examined and showed accordance of 2a with the in vivo formed metabolite 2.
Acknowledgements:
We are grateful to Prof. Christian Hametner for NMR measurements. The World Anti-Doping
Agency is acknowledged for providing funding of the project. The X-ray Centre of TU Wien is
acknowledged for providing access to the X-ray diffractometer. We thank Sandra Pfeffer for
obtaining the EI(MS) and HRMS data.
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https://www.wada-ama.org/en/content/what-is-prohibited/prohibited-at-all-
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16

Synthesis of a human long-term oxymetholone metabolite
Nicolas Kratena1, Nina Biedermann1, Matthias Weil2, Valentin S. Enev*1, Günter Gmeiner3,
Peter Gärtner*1
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology
2 Institute for Chemical Technologies and Analytics, Vienna University of Technology
3 Doping Control Laboratory, Seibersdorf Labor GmbH
* valentin.enev@tuwien.ac.at, peter.gaertner@tuwien.ac.at,
Highlights:
-) First chemical synthesis of reported metabolite OXM-M2
-) Two epimeric putative metabolites investigated
-) Identity and structure of in vivo metabolite secured
17

Synthesis of a human long-term oxymetholone metabolite
Nicolas Kratena1, Nina Biedermann1, Matthias Weil2, Valentin S. Enev*1, Günter Gmeiner3,
Peter Gärtner*1
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology
2 Institute for Chemical Technologies and Analytics, Vienna University of Technology
3 Doping Control Laboratory, Seibersdorf Labor GmbH
* valentin.enev@tuwien.ac.at, peter.gaertner@tuwien.ac.at,
Graphical abstract:
18