Synthesis of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: A long-term metandienone metabolite

Article abstract of DOI:10.1016/j.steroids.2016.08.013

The goal of this work was a good-yielding chemical synthesis of a metandienone metabolite which is of interest in doping analysis. 20βOH-NorMD (IUPAC: 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-triene-3-one) has been identified as a long-term urinary metabolite which can be detected and attributed to metandienone up to almost 3?weeks after exposure. The chemical synthesis of its epimer 20αOH-NorMD has been described before, as was an enzymatic synthesis of 20βOH-NorMD, but no chemical synthesis was published.

Full text of DOI:10.1016/j.steroids.2016.08.013

Accepted Manuscript
Synthesis of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-
one: a long-term metandienone metabolite
Nicolas Kratena, Valentin S. Enev, Günter Gmeiner, Peter Gärtner
PII:
S0039-128X(16)30109-X
http://dx.doi.org/10.1016/j.steroids.2016.08.013
STE 8017
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 June 2016
11 August 2016
14 August 2016
Please cite this article as: Kratena, N., Enev, V.S., Gmeiner, G., Gärtner, P., Synthesis of 17β-
hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a long-term metandienone metabolite, Steroids
(2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.08.013
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Synthesis of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a long-term
metandienone metabolite
Nicolas Kratena1, Valentin S. Enev*1, Günter Gmeiner2, Peter Gärtner*1
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9,
1060 Vienna, Austria
2Doping Control Laboratory, Seibersdorf Labor GmbH, 2444 Seibersdorf, Austria
* valentin.enev@tuwien.ac.at, peter.gaertner@tuwien.ac.at
Abstract:
The goal of this work was a good-yielding chemical synthesis of a metandienone metabolite
which is of interest in doping analysis. 20ßOH-NorMD (IUPAC: 17ß-hydroxymethyl-17--
methyl-18-norandrosta-1,4,13-triene-3-one) has been identified as a long-term urinary
metabolite which can be detected and attributed to metandienone up to almost 3 weeks
after exposure. The chemical synthesis of its epimer 20αOH-NorMD has been described
before, as was an enzymatic synthesis of 20ßOH-NorMD, but no chemical synthesis was
published.
Graphical abstract:
O
O
OH
WM-rearrangement
elimination
8 steps
O
O
O
O
commercially available precursor
DHEA acetate
20 OH-NorMD
Keywords: metandienone, metabolism, wagner-meerwein rearrangement,
1. Introduction
Metandienone (1) is a synthetic anabolic androgenic steroid (AAS) that was first synthesized
in 19551 and is still being used by professional athletes today. It is one of the highly abused
synthetic AAS together with Nandrolone and Stanozolol judging from adverse analytical
findings (AAF) from 2004-20072. It is also reportedly used by amateur athletes.3 Since the
discovery of the first metabolite in 19634 the metabolism has been extensively
investigated5,6,7,8,9
.
As reported by Schänzer et al. in 1992 the 17-sulphate conjugate (1a) of metandienone can
epimerize and/or rearrange (Wagner-Meerwein-Rearrangement) to give primarily NorMD
(17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) (1b). This intermediate is further
hydroxylated by Cytochrome P450 enzymes CYP21 and CYP3A4 to 20βOH-NorMD (1c).10
Hydroxylation can also take place at C-16 (CYP21) and on the 17α-methyl group (CYP3A4)
giving the epimer.11 Both 17-epimers have identical detection limits. Additionally it was
reported that the metabolite is also formed by C-18 hydroxylation via CYP11B2 and
subsequent rearrangement.12
1

Scheme 1: Suspected in vivo formation of the metabolite
Compound 1c (IUPAC: 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-triene-3-one,
also called “nightwatch” and “20βOH-NorMD”) has been identified as a long-term urinary
metabolite which can be detected and attributed to metandienone up to 19 days after
administration of a single dose.13 This constitutes a significantly longer window of detection
than with metandienone metabolites used before. When screening routines using this new
metabolite were implemented in 2006 the number of AAF rose from 15 to 68 for
metandienone (450% increase).14
In 2011 a small quantity (10 mg) of this metabolite was synthesized from NorMD using
recombinant strains of the fission yeast Schizosaccharomyces pombe expressing CYP21.11
The epimer of this metabolite: 17α-hydroxymethyl-17β-methyl-18-norandrosta-1,4,13-
triene-3-one has been reportedly synthesized in 2012 in 5 steps with an overall yield of
about 0.1%.12 Thus far there is no report on the chemical synthesis of this compound. We
hereby report the first synthesis of this metabolite which is needed as a reference material
for metandienone abuse in antidoping laboratories around the world.
2. Experimental
Dehydroepiandrosterone acetate and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone were
purchased from FluoroChem. MSTFA was supplied by Machery & Nagel (Düren, Germany).
Silica gel (0–63 μm, 60Å) for chromatography was from Merck. Titanium tetrachloride,
anhydrous dimethylformamide and anhydrous dimethylsulfoxide were from Acros Organics.
Sodium sulfate was from VWR. All other non-specified chemicals were from Sigma Aldrich.
Anhydrous methylene chloride, diethyl ether, toluene and 1,4-dioxane were retrieved from
an Innovative Technologies PureSolv system. Anhydrous tetrahydrofuran was pre-dried
using an Innovative Technologies PureSolv system, refluxed over sodium/benzophenone and
freshly distilled.
NMR spectra were recorded on a Bruker AC400. IR spectra were recorded on a Perkin Elmer
Spectrum 65. TLC-analysis was performed with precoated aluminium-backed plates (Silica
gel 60 F254, Merck). Compounds were visualized by submerging in an acidic phosphomolybdic
acid / cerium sulfate solution and heating. Melting points were determined with a Kofler
hot-stage apparatus.
Preparative HPLC was carried out on a Reveleris® Prep system by Grace using a Luna® 10 μm,
C18 (TMS endcapping) 100 Å, LC Column (250 x 21.2 mm).
GC-EI-MS analyses were performed using a Thermo Trace GC coupled to a Thermo Trace MS
instrument (Thermo Quest, Austin, USA) equipped with a Restek RTX-5ms GC column (length
15 m, inner diameter 0.25 mm, film thickness 0.25 µm). The GC oven temperature program
started at 150°C was increased at 20°/min to 320°C using Helium as carrier gas 100 kPa,
2

constant pressure). The injector temperature was set to 270°C, the interface temperature to
280°C and the ion source temperature to 250°C. Ionization was accomplished using EI (70
eV), and full scan analysis (m/z 60-600) was employed at 2 scans/s. A volume of 1 µL of a
derivatization mixture consisting of 5 µg of the target product in 200 µl of a mixture of
MSTFA/ammonium iodide/ethanethiol- TMS 1000:2:6 (v/w/v) was injected in split mode (1 /
40) into the GC-MS system.
High resolution/high accuracy mass spectra were recorded on a Thermo Q Exactive Focus
mass spectrometer (Thermo Scientific, Austin, Texas) by direct injection of a 10 µg/ml
solution of the target product in acetonitrile.
The mass spectrometer was operated in electrospray ionization mode at a spray voltage of 4
kV at 400°C. The instrument was calibrated using the manufacturers calibration mixture
allowing or mass accuracies < 3 ppm. Full scan mass spectra were recorded in profile mode
at a resolution of 70.000 at a scan range of m/z 100 – 350.
2.1 3β-Hydroxy-13α-methylandrost-5-en-17-one acetate (2)
A solution of dehydroepiandrosterone acetate (10 g, 30.26 mmol) in acetic acid (125 mL) and
1,2-phenylenediamine (5.43 g 50.2 mmol) was refluxed for 24 hours. The color changed from
light brown to dark green in about 2 h and then proceeded to turn very dark with blue
complexion which disappeared after cooling the reaction. The solution was then flooded
with 150 mL deionised water upon which a beige precipitate formed. This was extracted
with 150 mL ethyl acetate and the organic phase washed ten times with small portions of
water and four times with saturated NaHCO3 solution, dried over Na2SO4 and the solvent
evaporated in vacuo. The crude product was recrystallized from diisopropylether, giving 6 g
of product, and the mother liquors concentrated and separated on a 180 g silica gel column
with 5/1 v/v petroleum ether/diethyl ether as eluent to give 1.16 g starting material and
in total 8.2 g (82%) of the title compound 2 as white solid, m.p. 141 – 143 °C.
1
TLC: Rf: (petroleum ether/diethyl ether 3/1 v/v) 0.55. H-NMR (400 MHz, CDCl3): δ = 5.33
(1H, tt, J = 2.56), 4.53 (1H, h, J = 5.4), 2.18 – 2.38 (4H, m), 2.0 – 2.19 (3H, m), 1.96 (3H, s),
1.68 – 1.87 (3H, m), 1.45 – 1.63 (3H, m) 1.15 (1H, td, J = 13.44, 3.61), 0.98 – 1.1 (2H, m), 0.93
(3H, s), 0.92 (1H, m), 0.80 (3H, s), 0.8 (1H, m). 13C-NMR (100MHz, CDCl3): δ= 222.09, 170.43,
139.19, 121.88, 73.72, 50.98, 50.02, 47.86, 37.86, 36.81, 36.61, 34.12, 34.05, 33.03, 31.56,
27.56, 25.12, 22.91, 22.04, 21.39, 19.05. IR [cm-1]: 2935, 1731, 1235, 1024. [α]20D = -154.3 (c
0.78, dichloromethane)
2.2 3β-Hydroxy-13α-methylandrost-5-en-17-one (3)
A solution of 2 (1.15 g, 3.48 mmol) and potassium carbonate (1 g, 7.24 mmol) in methanol
(50 mL) was refluxed for an hour and TLC showed full conversion. The reaction mixture was
diluted with deionised water and extracted with dichloromethane. The pooled extracts were
washed with brine, dried over Na2SO4 and evaporated to give 982 mg (98%) of the title
compound as white crystals, m.p. 180 – 181 °C.
1
TLC: Rf: (petroleum ether/ethyl acetate 5/1 v/v) 0.24. H-NMR (400 MHz, CDCl3): δ = 5.35
(1H, tt, J = 5.10, 2.55), 3.50 (1H, h, J = 5.28), 2.25 – 2.41 (3H, m), 2.03 – 2.24 (4H, m), 1.71 –
3

1.91 (4H, m), 1.41 – 1.67 (4H, m), 1.19 (1H, td, J = 13.56, 4.04), 1.0 – 1.14 (2H, m), 0.97 (3H,
s), 0.94 (1H, td, J = 11.94, 2.1), 0.85 (1H, td, J = 12.9, 3.55), 0.83 (3H, s). 13C-NMR (100MHz,
CDCl3): δ= 222.50, 140.44, 121.03, 71.74, 51.18, 50.18, 48.07, 42.12, 36.98, 36.85, 34.29,
34.21, 33.17, 31.73, 31.57, 25.26, 23.09, 22.17, 19.25. IR [cm-1]: 3456, 2932, 1722. [α]20 = -
D
167.4 (c 0.81, dichloromethane)
2.3 3β-Hydroxy-13α-methylandrost-5-en-17-one pivalate (4)
To a solution of 3 (1.63 g, 5.64 mmol) in pyridine (10 mL) there was added trimethylacetyl
chloride (815 mg, 6.77 mmol) dropwise at 0°C. The reaction was stirred for 3 hours. The
solvent was evaporated in vacuo and the residue dissolved in dichloromethane and
saturated NaHCO3 solution. After phase separation the aqueous layer was extracted three
times with small portions of dichloromethane, the pooled organic phases washed with
deionised water and brine and dried over Na2SO4 to give 1.94 g (92%) as off-white solid, m.p.
167 – 170 °C.
1
TLC: Rf: (petroleum ether /ethyl acetate 7/1 v/v) 0.55. H-NMR (400 MHz, CDCl3): δ = 5.38
(1H, tt, J = 2.58), 4.56 (1H, h, J = 5.42), 2.04 – 2.42 (7H, m), 1.74 – 1.93 (3H, m), 1.49 – 1.7
(5H, m), 1.21 (1H, td, J = 13.37, 3.59), 1.17 (9H, s), 1.04 – 1.13 (1H, m), 0.98 (3H, s), 0.98 (1H,
td, J = 11.54, 2.73), 0.87 (1H, td, J = 13.29, 3.53), 0.85 (3H, s). 13C-NMR (100MHz, CDCl3): δ =
222.45, 178.17, 139.49, 121.86, 73.48, 51.15, 50.20, 47.99, 38.75, 37.89, 36.97, 36.74, 34.30,
34.20, 33.18, 31.70, 27.58, 27.28, 25.26, 23.07, 22.16, 19.23. IR [cm-1]: 2957, 1732, 1717,
1480, 1284, 1161. [α]20D = -131 (c 0.96, dichloromethane).
2.4 17-Methylene-13α-methylandrost-5-en-3β-ol pivalate (5)
Nysted reagent suspension (30.6 g, 20 wt%, 13.42 mmol) in THF (20 mL) was stirred in a
Schlenk flask at 0 °C and TiCl4 (8.05 mmol, 8 mL, 1M solution) in dichloromethane was
added dropwise. The white milky suspension turned yellow and then light brown over
the course of 10 minutes. Ketone 5 (1 g, 2.7 mmol) was added after formation of the
reagent and the cooling bath was removed. The reaction was complete after stirring at
room temperature for 18 hours and was quenched with 100 mL 2M hydrochloric acid
and extracted with diethyl ether. The pooled organic phases were washed with brine,
dried over Na2SO4 and evaporated to dryness. A yellow oil (1.28 g) was obtained as
crude product. This was purified over a 60 g silica gel column using 15/1 v/v petroleum
ether/ethyl acetate as eluent to recover 230 mg (23%) starting material and give 586 mg
(59%) of product as colorless crystals, m.p. 108 - 111°C.
1
TLC: Rf: (petroleum ether/ethyl acetate 15/1 v/v) 0.34. H-NMR (400 MHz, CDCl3): δ = 5.37
(1H, t, J = 4.65), 4.84 (1H, t, J = 2.02), 4.69 (1H, t, J = 2.02), 4.57 (1H, h, J = 4.65), 2.18 – 2.54
(5H, m), 1.78 – 1.95 (4H, m), 1.39 – 1.63 (5H, m), 1.36 (1H, dd, 13.4, 3.24), 1.27 (1H, dd, 9.64,
6.2), 0.98 – 1.20 (3H, m), 1.17 (9H, s), 0.95 (3H, s), 0.89 (3H, s). 13C-NMR (100MHz, CDCl3): δ =
178.15, 157.51, 139.36, 122.47, 103.05, 73.66, 54.52, 48.93, 46.08, 38.75, 37.95, 37.02,
36.86, 34.12, 33.42, 33.19, 31.54, 29.85, 27.70, 27.30, 25.30, 21.55, 19.36. IR [cm-1]: 2942,
1724. [α]20 = -131 (c 0.96, dichloromethane)
D
2.5 17-Methylene-13α-methylandrost-5-en-3β-ol (6)
4

A solution of 5 (506 mg, 1.37 mmol) in THF (15 mL) was stirred at room temperature and
lithium aluminium hydride (129 mg, 3.41 mmol) was added at once. After 15 minutes TLC
analysis indicated complete consumption of starting material. The grey suspension was
quenched with 1 mL deionised water, dried over Na2SO4 and the solids were filtered off and
thoroughly washed with diethyl ether. Evaporation of the pooled organic phases gave 392
mg (100%) product as colorless solid, m.p. 150 – 152 °C.
1
TLC: Rf: (petroleum ether/ethyl acetate 5/1 v/v) 0.17. H-NMR (400 MHz, CDCl3): δ = 5.35
(1H, tt, J = 2.51), 4.84 (1H, t, J = 2.23), 4.69 (1H, t, J = 2.23), 3.51 (1H, h, J = 5.28), 2.13 – 2.53
(5H, m), 1.77 – 1.95 (4H, m), 1.38 – 1.6 (6H, m), 1.35 (1H, dd, J = 13.40, 3.28), 0.99 – 1.29
(5H, m), 0.94 (3H, s), 0.93 (1H, m), 0.86 (3H, s). 13C-NMR (100MHz, CDCl3): δ = 157.47,
140.30, 121.69, 121.86, 103.06, 71.92, 54.57, 49.03, 46.05, 42.23, 37.11, 36.90, 34.17, 33.44,
33.18, 31.70, 31.55, 29.84, 25.30, 21.57, 19.37. IR [cm-1]: 3229, 2936, 2893. [α]20D = -168.9 (c
0.7, dichloromethane).
2.6 17-Methylene-13α-methylandrost-4-en-3-one (7)
To a solution of 6 (707 mg, 2.47 mmol) in toluene (50 mL) there were added cyclohexanone
(4.9 g, 50 mmol) and the solution was heated on a Dean-Stark trap for 1 h. At this point
aluminium iso-propoxide (1.26 g, 6.17 mmol) was added and the reaction mixture quickly
turned yellow. After 2 hours the reaction was complete. It was then washed with water (15
mL) and 0.1 M sulfuric acid (100 mL), dried over Na2SO4 and the now clear solution
evaporated in vacuo. Upon standing over night at room temperature the product crystallized
as white needles. The crystals were washed with cold petroleum ether and the filtrate
concentrated and purified via column chromatography on 25 g silica gel with 90/10 v/v
petroleum ether/ethyl acetate as eluent. There were obtained in total 670 mg (95%) of the
title compound as white solid, m.p. 166 – 167 °C.
TLC: Rf: (petroleum ether/ethyl acetate 5/1 v/v) 0.33. 1H-NMR (400 MHz, CDCl3): δ = 5.7 (1H,
t, J = 0.68), 4.86 (1H, t, J = 2.34), 4.71 (1H, t, J = 2.34), 2.21 – 2.57 (6H, m), 2.0 – 2.1 (2H, m),
1.96 (1H, dt, J = 13.54, 3.19), 1.8 – 1.92 (1H, m), 1.52 – 1.75 (3H, m), 1.44 (1H, dq, J = 12.3,
3.15), 1.35 (1H, td, J = 19.73, 3.55), 1.13 – 1.27 (2H, m), 1.05 (3H, s), 0.97 – 1.03 (1H, m), 0.94
(3H, s), 0.91 – 0.97 (1H, m). 13C-NMR (100MHz, CDCl3): δ = 199.78, 171.59, 156.87, 123.78,
103.35, 53.35, 52.71, 45.90, 38.90, 37.40, 35.67, 34.30, 34.06, 33.28, 32.95, 31.28, 29.46,
24.76, 21.35, 17.85. IR [cm-1]: 2931, 1662. [α]20 = 72.9 (c 1.01, dichloromethane).
D
HRMS(ESI+) 285.221 [M+H]+ (Calcd. 285.2213).
2.7 17-Methylene-13α-methylandrost-1,4-dien-3-one (8)
To compound 7 (232 mg, 0.82 mmol) dissolved in 1,4-dioxane (5 mL) was added tert-
Butyldimethylsilyl chloride (6 mg, 0.04 mmol) and then the mixture was cooled to 0 °C. To
the solidified solution there was added DDQ (204 mg, 0.9 mmol) in two portions. The
mixture was slowly allowed to reach room temperature and was stirred for 96 hours. The
solvent was evaporated, the residue dissolved in dichloromethane and washed with
saturated aqueous Na2S2O3, NaHCO3 and brine. The organic phase was dried over Na2SO4
and evaporated in vacuo to give 227 mg (99%) of a yellow solid which was used in the next
step without further purification, m.p.: 108 – 110 °C.
5

1
TLC: Rf: (petroleum ether/ethyl acetate 3/1 v/v) 0.75. H-NMR (400 MHz, CDCl3): δ = 7.07
(1H, d, J = 10.12), 6.23 (1H, dd, J = 10.14, 1.86), 6.06 (1H, t, J = 1.52), 4.89 (1H, t, J = 2.06),
4.75 (1H, t, J = 2.38), 2.3 – 2.59 (4H, m), 2.14 – 2.22 (1H, m), 1.95-2.01 (1H, m), 1.81 – 1.94
(1H, m), 1.6 – 1.67 (1H, m), 1.35 – 1.42 (2H, m), 1.13 – 1.28 (2H, m), 1.1 (3H, s), 0.97 – 1.13
(3H, m), 0.95 (3H, s). 13C-NMR (100MHz, CDCl3): δ = 186.54, 169.46, 156.63, 155.81, 127.66,
123.80, 103.55, 53.30, 50.82, 46.09, 43.58, 37.24, 34.59, 34.12, 33.14, 31.10, 29.33, 24.84,
22.83, 18.93. [α]20D = -9.1 (c 1.02, dichloromethane)
2.8 Spiro(13α-methylandrost-1,4-dien-17,2’-oxirane)-3-one (9)
To a solution of 8 (200 mg, 0.71 mmol) in CHCl3 (3 mL) and buffer solution (di-sodium
hydrogen phosphate/potassium dihydrogen phosphate) pH 6.88 (2 mL) at 0 °C there was
added meta-Chloroperoxybenzoic acid (218 mg, 0.88 mmol) in 1 mL chloroform dropwise.
The temperature was held for 3 hours. The reaction was then quenched with saturated
aqueous NaHCO3 and Na2S2O3 solutions and washed twice with saturated aqueous NaHCO3.
After back extraction of the aqueous phases the pooled organic phases were dried over
Na2SO4 and evaporated in vacuo to give 209 mg (99%) of 9 as a 54:46 mixture of
diastereomers. These can be used directly without further purification. An analytical sample
was obtained via column chromatography (2 g silica, 5/1 v/v petroleum ether/ethyl acetate).
Major diastereomer:
m.p. 128 – 130 °C. TLC: Rf: (petroleum ether/ethyl acetate 3/1 v/v) 0.33. 1H-NMR (400 MHz,
CDCl3): δ = 7.06 (1H, d, J = 10.12), 6.22 (1H, dd, J = 10.12, 1.88), 6.07 (1H, t, J = 1.56), 2.73
(1H, d, J = 4.56), 2.64 (1H, d, J = 4.64), 2.32 – 2.52 (2H, m), 2.19 – 2.3 (2H, m), 1.87 – 1.97 (1H,
m), 1.59 – 1.86 (4H, m), 1.43 (1H, td, J = 16.88, 3.67), 1.19 – 1.41 (3H, m), 1.17 (3H, s), 0.95 –
1.13 (2H, m), 0.92 (3H, s). 13C-NMR (100 MHz, CDCl3): δ = 186.55, 169.60, 155.96, 127.59,
123.69, 68.35, 53.16, 49.75, 47.54, 43.58, 41.43, 37.21, 34.77, 32.96, 31.50, 30.00, 27.96,
24.68, 24.34, 18.98. IR [cm-1]: 3402, 2924, 1652, 1610, 1597. [α]20 = -65.8 (c 0.6,
D
dichloromethane). HRMS(ESI+) 299.201 [M+H]+ (Calcd. 299.2006).
Minor diastereomer:
m.p. 132 – 133 °C. TLC: Rf: (petroleum ether/ethyl acetate 3/1 v/v) 0.18. 1H-NMR (400 MHz,
CDCl3): δ = 7.04 (1H, d, J = 10.12), 6.24 (1H, dd, J = 10.14, 1.9), 6.08 (1H, t, J = 1.50), 2.83 (2H,
dd, J = 18.54, 4.94), 2.34 – 2.5 (2H, m), 2.18 – 2.27 (1H, m), 2.02 – 2.14 (2H, m), 1.88 – 2 (1H,
m), 1.68 – 1.77 (1H, m), 1.56 – 1.67 (2H, m), 1.18 – 1.41 (5H, m), 1.15 (3H, s), 1 – 1.13 (1H,
m), 0.99 (3H, s). 13C-NMR (100MHz, CDCl3): δ = 186.38, 168.91, 155.28, 127.88, 123.94,
66.58, 55.19, 53.14, 49.70, 43.33, 41.11, 37.62, 34.65, 32.88, 31.79, 29.36, 26.23, 24.34,
23.58, 18.92. IR [cm-1]: 2930, 2900, 1667, 1602. [α]20D = -11 (c 0.6, dichloromethane)
2.9 17β-Hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-triene-3-one (1c)
To a mixture of epoxides 9 (210 mg, 0.7 mmol) in THF/H2O 1/1 (5 mL) were added 5 drops of
concentrated phosphoric acid and the solution was heated to reflux for 2 hours. After
completion of the reaction THF was evaporated and the aqueous phase extracted four times
with 20 ml dichloromethane, washed with saturated NaHCO3 solution and brine and dried
over Na2SO4.The organic solvent was evaporated to yield 209 mg crude product as oily
6

crystals. This was separated on the preparative HPLC (water + 0.1 % TFA / methanol = 2/3) to
give 47 mg (22%) of the title compound as light yellow oily solid.
1
TLC: Rf: (petroleum ether/ethyl acetate 1/1 v/v) 0.44. H-NMR (400 MHz, CDCl3): δ = 7.14
(1H, d, J = 10.16), 6.26 (1H, dd, J = 10.16, 1.88), 6.09 (1H, t, J = 1.6), 3.48 (1H, d, J = 10.54),
3.34 (1H, d, J = 10.54), 2.52 (1H, tdd, J = 20.09, 5.03, 1.51), 2.37 – 2.47 (2H, m), 2.14 – 2.34
(3H, m). 1.86 – 2.1 (4H, m), 1.5 – 1.72 (4H, m). 1.30 (1H, td, J = 11.3, 2.06), 1.21 (3H, s), 0.92
(3H, s). 13C-NMR (100MHz, CDCl3): δ = 186.44, 168.76, 155.62, 138.95, 137.60, 127.83,
124.51, 69.02, 51.67, 49.51, 43.45, 36.74, 33.96, 33.36, 32.62, 30.82, 24.03, 22.48, 21.82,
18.65. IR [cm-1]: 3395, 2923, 2855, 1655, 1615, 1600. [α]20D = 17.4 (c 0.88, dichloromethane).
EI fragmentation MS: The mass spectra of the bis-TMS derivative of the title compound
equals to already published spectra by Schänzer et al13, having abundantly discussed the
relevant fragments after deuteration as well as multi-dimensional mass spectrometry
experiments.
73
100
133
90
80
70
60
50
40
30
339
103
100
193
20
10
0
243
229
134
150
191
179
337
340
350
442
244
281
300
427
400
200
250
450
500
m/z
Figure 1: EI mass spectrum of bis-TMS-20βOH-NorMD
ESI(+)-MS: Elemental composition of the target compound was obtained by high
resolution/high accuracy MS analysis, yielding the mass of the pseudomolecular ion as
299.2002 u with a mass accuracy < 2 ppm (theoretical mass: 299.2006, error = 1.2 ppm) as
well as the elemental composition (C20H27O2).
In addition the mass spectrum shows a sodium adduct as m/z 321.1820. Only a few
fragments are visible at 281.1897 (loss of water) and 269.1898 (loss of formaldehyde); m/z
147.0803 is a typical A ring fragment indicating a 3-keto-1,4-diene structural element.13
7

mdion_m9_std05 #1418-1444 RT: 4.96-5.04 AV: 13 SB: 344 5.32-6.47 , 3.60-4.79 NL: 1.33E9
T: FTMS + p ESI Full ms [100.0000-1000.0000]
299.2002
100
90
80
70
60
50
40
30
147.0803
321.1820
320
20
281.1897
280
10
0
269.1898
260
100
120
140
160
180
200
220
m/z
240
300
340
Figure 2: ESI(+) mass spectrum of 20βOH-NorMD
3. Results and discussion
Keeping in mind the biological transformation that leads to the formation of the target
molecule our retrosynthetic strategy for introduction of the C-17 substituents is closely
related. The C-13 methyl group can migrate when a carbocation is formed on the adjacent
carbon C-17. A spiroepoxide on C-17 can form a carbocation and upon opening directly
furnishes the hydroxymethyl group that is needed.
Scheme 2: Retrosynthetic analysis of the key-step
8

Scheme 3: Synthetic route and conditions: (a) 1,2-phenylendiamine, AcOH, reflux 24h. (b) K2CO3, MeOH, reflux
2h. (c) (CH3)3CCOCl, DMAP, pyridine, 0°C, 4h. (d) Nysted reagent, THF, 0°C then TiCl4, rt o/n. (e) LiAlH4, THF, 0°C,
15 min. (f) Al(O-iPr)3, cyclohexanone, toluene, reflux, 4h. (g) DDQ, TBSCl, dioxane, rt, 96h. (h) m-CPBA,
CHCl3/buffered H2O, rt, 2h. (i) THF/H2O (1:1), phosphoric acid, reflux, 3h.
The synthesis started with epimerization of the angular methyl group on C-13. Only if the
methyl group is in the α-position it should give the correct stereochemistry after the
rearrangement. The C-13-epimerization which is suspected to proceed via an ion-radical
mechanism gives about 85% of 13α-methyl (2) and 15% 13β-methyl when it reaches
equilibrium.15a,b,First we used Corey-Chaykovsky16 conditions for the direct transformation of
the ketone to the spiroepoxides. Since this gave a complex mixture and low yields instead an
exo-cyclic double bond was installed on the C-17.
Since cleavage of the acetate was observed in some of the screened reactions, a change in
protecting groups was necessary and was easily realized by cleaving the acetate under mild
basic conditions and subsequent protection of the alcohol as a pivalate (4). Peterson
olefination17 using (trimethylsilyl)methyllithium as well as the Tebbe reagent18 proved
unsuccessful. Most likely the low reactivity towards a nucleophilic attack at the ketone stems
from the steric hindrance due to the cis-configuration of the C and D ring as well as
enolization taking place due to basic reaction conditions. After intensive investigation, the
Nysted reagent19 was able to methylenate the substrate efficiently and selectively to provide
5.
Deprotection of the pivalate protecting group was accomplished under reductive conditions
with lithium aluminium hydride and gave alcohol 6 in quantitative yield. In order to oxidize
the C-3 and isomerize the double-bond to form an enone we chose Oppenauer oxidation
conditions which smoothly furnished enone 7. Dehydrogenation to the dienone 8 was
accomplished via a tert-Butyldimethylsilyl chloride catalyzed DDQ oxidation.20
Finally selective epoxidation of the exocyclic electron-rich double was achieved using meta-
Chloroperoxybenzoic acid in chloroform/water (buffered). When using only a non-polar
solvent system considerable (>10%) amounts of Baeyer-Villiger oxidation products were
observed. Both diastereomers of the epoxides were used in the next reaction.
9

The epoxide opening / rearrangement reaction was first tested under anhydrous conditions
using boron trifluoride etherate in tetrahydrofurane. This gave only miniscule amounts of
product, but switching to an aqueous acidic system increased yields greatly. In the end
phosphoric acid in tetrahydrofurane/water was used. The complex mixture was separated
on RP-HPLC. The crude mixture consisted of approximately 40% product (estimated from 1H-
NMR data); after purification 22% of pure product 1c could be isolated. Analytical data of the
synthesized material is in accordance to previously published data on this metabolite.11,13
4. Conclusions.
The first chemical synthesis of 20βOH-NorMD was achieved in nine steps and 8.9% overall
yield of The main features of our synthesis include epimerization of the C-13 methyl group,
introducing of exo-cyclic double bond at C-17, regioselective epoxidation of exomethylen
double bond followed by acid mediated epoxide opening and subsequent rearrangement to
create the desired 20βOH-NorMD. The described synthesis can provide doping laboratories
with an essential reference material for their antidoping performance.
(1) Meystre, C.; Vischer, E.; Wettstein, A. Helv. Chim. Acta 1955, 38 (2), 381.
(2) https://www.nada.at/files/doc/Statistiken/WADA-Statistik-2007.pdf.
(3) Basaria, S. J. Clin. Endocrinol. Metab. 2010, 95 (4), 1533.
(4) Rongone, E. L.; Segaloff, A. Steroids 1963, 1 (2), 179.
(5) Pozo, O. J.; Lootens, L.; Van Eenoo, P.; Deventer, K.; Meuleman, P.; Leroux-Roels, G.;
Parr, M. K.; Schänzer, W.; Delbeke, F. T. Drug Test. Anal. 2009, 1 (11-12), 554.
(6) Schänzer, W. Clin. Chem. 1996, 42 (7), 1001.
(7) Gómez, C.; Pozo, O. J.; Garrostas, L.; Segura, J.; Ventura, R. Steroids 2013, 78 (12-13),
1245.
(8) Schänzer, W.; Geyer, H.; Donike, M. J. Steroid Biochem. Mol. Biol. 1991, 38 (4), 441.
(9) Schänzer, W.; Delahaut, P.; Geyer, H.; Machnik, M.; Horning, S. J. Chromatogr. B.
Biomed. Sci. App. 1996, 687 (1), 93.
(10) Schänzer, W.; Donike, M.; Opfermann, G. Steroids 1992, 57, 537.
(11) Zöllner, A.; Parr, M. K.; Drăgan, C.-A.; Dräs, S.; Schlörer, N.; Peters, F. T.; Maurer, H. H.;
Schänzer, W.; Bureik, M. Biol. Chem. 2010, 391 (1).
(12) Parr, M. K.; Zöllner, A.; Fußhöller, G.; Opfermann, G.; Schlörer, N.; Zorio, M.; Bureik,
M.; Schänzer, W. Toxicol. Lett. 2012, 213 (3), 381.
(13) Schänzer, W.; Geyer, H.; Fußhöller, G.; Halatcheva, N.; Kohler, M.; Parr, M.-K.; Guddat,
S.; Thomas, A.; Thevis, M. Rapid Commun. Mass Spectrom. 2006, 20 (15), 2252.
(14) Geyer, H.; Schanzer, W.; Thevis, M. Br. J. Sports Med. 2014, 48 (10), 820.
(15) (a) Yaremenko, F. G.; Khvat, A. V. Mendeleev Commun. 1994, 4 (5), 187,
(b) Wölfling, J.;
Chemie - Chemical
Szájli, Á.; Vörös, L.; Gáspár, M.; Schneider, G. Monatshefte für
Monthly 2006, 137 (8), 1099.(16) Corey, E.-J.; Chaykovsky,
M. J. Am. Chem. Soc. 1965, 87 (6), 1345.
(17) Peterson, D. J. J. Org. Chem. 1968, 33 (2), 780.
(18) Tebbe, F. N.; Parshall, G. W.; Reddy, G. S. J. Am. Chem. Soc. 1978, 100 (11), 3611.
(19) Li, J. J. In Name Reactions; Springer-Verlag: Berlin, 2009; p 403.
(20) Chen, K.; Liu, C.; Deng, L.; Xu, G. Steroids 2010, 75 (7), 513.
10

Synthesis of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a long-term
metandienone metabolite
Nicolas Kratena1, Valentin S. Enev*1, Günter Gmeiner2, Peter Gärtner*1
1 Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9,
1060 Vienna, Austria
2Doping Control Laboratory, Seibersdorf Labor GmbH, 2444 Seibersdorf, Austria
*valentin.enev@tuwien.ac.at, peter.gaertner@tuwien.ac.at
Highlights:
-) First chemical synthesis of compound 20βOH-NorMD
-) Efficient introduction of 17β-hydroxymethyl-17α-methyl substitution pattern
-) The crucial Wagner-Meerwein rearrangement is initiated by acidic epoxide opening
11