First synthesis of a pentadeuterated 3′-hydroxystanozolol - An internal standard in doping analysis

Article abstract of DOI:10.1016/j.steroids.2004.10.002

The first synthesis of 16,16,20,20,20-pentadeuterio-3′- hydroxystanozolol (8) in 26% yield over nine steps is described using moderately priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC-MS or gas chromatography-mass spectrometry (GC-MS).

Full text of DOI:10.1016/j.steroids.2004.10.002

Steroids 70 (2005) 103–110
First synthesis of a pentadeuterated 3ꢀ-hydroxystanozolol—an internal
standard in doping analysis
a
b
a,∗
¨
¨
Wolfgang Felzmann , Gunter Gmeiner , Peter Gartner
a
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria
b
ARC Seibersdorf Research Ges.m.b.H., A-2444 Seibersdorf, Austria
Received 19 May 2004; received in revised form 5 October 2004; accepted 11 October 2004
Dedicated to Prof. Peter Stanetty on the occasion of his 60th birthday
Abstract
The first synthesis of 16,16,20,20,20-pentadeuterio-3ꢀ-hydroxystanozolol (8) in 26% yield over nine steps is described using moderately
priced starting materials and economic amounts of reagents. Compound 8 can be used as an internal standard in screening procedures
for anabolic steroids as well as for the quantification of stanozolol metabolites via mass spectrometric techniques, such as LC–MS or gas
chromatography–mass spectrometry (GC–MS).
© 2004 Elsevier Inc. All rights reserved.
Keywords: Stanozolol metabolite; Deuterium labelling; Diastereoselective alkylation; Doping control; Stereoselective synthesis; Steroid
1. Introduction
To be able to detect steroid metabolites under GC–MS
conditions, the extracts are persilylated with a mixture of
methylsilyltrifluoroacetic acid (MSTFA) and trimethylio-
dosilane (TMSI), leading to sharp peaks and therefore, better
limits of detection compared to the unsilanized compounds.
3ꢀ-Hydroxystanozolol (II), in particular, forms a silyl deriva-
tive, which is prone to decompose in the injection port of
the gas chromatograph, most probably due to active sites or
disturbing matrix compounds, which vary from sample to
sample. To get insight into this problem, the use of a partially
deuterated internal standard would allow for monitoring of
the detection quality of 3ꢀ-hydroxystanozolol as a marker for
stanozolol doping in each sample.
However, until now, only the synthesis of non-deuterated
3ꢀ-hydroxystanozolol has been described starting from mes-
tanolon (17␤-hydroxy-17␣-methyl-5␣-androstan-3-one) [1]
(two steps, overall yield 12%). Deuterated derivatives are
only known for stanozolol itself (d3-stanozolol has been syn-
thesized in five steps and 15% overall yield [2]). To our
knowledge, the synthesis of a pentadeuterated stanozolol
derivative, despite its usefulness, has not been described at
all in the literature. The aim of this work was to synthesize
The detection of anabolic androgenic steroids, either of
endogenous or synthetic origin, is one of the major tasks in
doping analysis. Much work has been done in the last decades
to implement fast and powerful screening procedures for the
detection of this classic group of doping substances in blood,
hair, and especially in urine. One of the most widespread
screening methods for urine uses gas chromatography–mass
spectrometry (GC–MS) after enzymatic cleavage, liquid or
solid phase extraction, and silylation of the dried extract.
3ꢀ-Hydroxystanozolol (II) is one of the main metabo-
lites of stanozolol (17-methyl-5␣-androstano[3,2-c]pyrazol-
17␤-ol) (I), found among other hydroxylated metabolites
(e.g., 16␣-hydroxystanozolol, 16␤-hydroxystanozolol, 4␤-
hydroxystanozolol) in urine after application of its parent
compound (Scheme 1).
∗
Corresponding author. Tel.: +43 1 58801 15421;
fax: +43 1 58801 15492.
E-mail address: peter.gaertner@tuwien.ac.at (P. Ga¨rtner).
0039-128X/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.10.002

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W. Felzmann et al. / Steroids 70 (2005) 103–110
Scheme 1. Stanozolole I and its main metabolite, 3-hydroxystanozolol II.
d5-3ꢀ-hydroxstanozolol starting from epiandrosterone and to
incorporate all five deuterium atoms in the D-ring by use
of deuterated organometallic reagents and a H/D exchange
process.
2.3. NMR spectroscopy
NMR spectra were recorded on Bruker AC 200 (200 and
1
50 MHz, for H and 13C, respectively) or Bruker Avance
DRX 400 (400 and 100 MHz, for 1H and 13C, respectively)
instruments and calibrated using the solvent as an internal
standard.
2. Experimental
Chemical shifts for deuterium-substituted carbons are not
given because accumulation times required to measure sig-
nificant signals were too long.
2.1. General methods and instrumentation
All reactions were carried out under a nitrogen atmosphere
in dry, freshly distilled solvents under anhydrous conditions
unless otherwise stated. Reactions were monitored by TLC
carried out on 0.25 mm Merck silica gel plates (60F-254) us-
ing UV light as a visualizing agent and molybdato phosphoric
acid (5% in ethanol) and heat as developing agents. For vac-
uum flash chromatography (VFC) and column chromatogra-
phy (CC), the amount of silica gel 60 (Merck, 0.2–0.5 mm
mesh size) and the eluent are given. Melting points were
recorded using a Kofler apparatus and are uncorrected. Ele-
mental analyses were carried out at the Institute of Physical
Chemistry, University of Vienna, under the supervision of
Mag. J. Theiner.
2.4. Reagents
Epiandrosterone was purchased from Steraloids (New-
port, RI, USA). MSTFA was supplied by Machery & Nagel
¨
(Duren, Germany). All other materials were supplied by
Sigma–Aldrich (Vienna, Austria) in standard commercial
quality. Tetrahydrofuran and diethyl ether were distilled
from sodium/benzophenone. Methylenechloride was dis-
tilled from phosphorous pentoxide. Methanol was distilled
from magnesium turnings.
2.5. Experimental
2.2. Gas chromatography
2.5.1. O3-[(1,1-Dimethylethyl)dimethylsilanyl]
epiandrosteron (2)
Unless otherwise stated, samples were derivatized with
MSTFA/NH4I/ethanethiol-TMS 1000:2:6 (v/w/v) [3], which
is equivalent to a MSTFA/trimethyliodosilane solution
1000:2(v/v)[4], byheatingfor15 minat60 ◦C. GC–MSanal-
yses were conducted on a VOYAGER MS directly interfaced
to a TRACE 2000 GC (Thermo Quest, Vienna, Austria). The
electron energy was set to 70 eV, and the ion source tempera-
ture was set to 250 ◦C. A DB-1 (17 m × 0.2 mm i.d., 0.11 ␮m
film thickness) cross-bonded dimethyl polysiloxane capillary
column (J&W Scientific, Folsom, CA, USA) was used in the
split injection mode (10:1). The column head pressure of he-
lium as the carrier gas was set to 100 kPa. The oven program
temperatureusedfor3, 4, and9was210 ◦C(0 min), 8 ◦C/min,
320 ◦C (5 min) and for 8: 100 ◦C (0 min), 25 ◦C/min, 320 ◦C
(2 min). Injector and transfer line temperatures were set at
250 and 280 ◦C, respectively. Chromatograms were recorded
in scanning mode. The mass range was m/z 50–590 at a rate of
1.5 scans/s. Data were processed with XCALIBUR software
from Thermo Quest.
Epiandrosterone (2.00 g, 6.89 mmol) was dissolved in
8 ml CH2Cl2. Imidazole (1.17 g, 17.2 mmol) and TBDMSCl
(1.25 g, 8.29 mmol) were then added, and the reaction mix-
ture was stirred for 30 min. Reaction control via TLC showed
the reaction to be complete. The reaction was quenched by
addition of 3 ml H2O and diluted with 20 ml CH2Cl2. The
layers were separated, and the organic phase was washed se-
quentially with 5% KHSO4 solution, saturated NaHCO3 so-
lution, and brine. The organic layer was dried over Na2SO4,
and the solvent was evaporated to give 2 (2.79 g) in 100%
yield as a white solid; m.p. 161–163 ◦C (cf. 166–167 ◦C [5]);
TLC petroleum ether/ethyl acetate (10:1): Rf 0.48; 1H NMR
(200 MHz, CDCl3): δ 0.04 (s, 6H, Si (CH3)2, 0.5–2.0 [m,
41H, aliphatic H, therein 0.87 (s, 9H, Si C (CH3)3), 0.84
(s, 3H, H-18) and 0.81 (s, 3H, H-19)], 2.07 (dd, 1H, H-16),
2.43 (dd, 1H, H-16), 3.54 (m, 1H, H-3␤); 13C NMR (50 MHz,
CDCl3): δ −4.6 (CH3, Si (CH3)2), 12.3 (CH3, C-19), 13.8
(CH3, C-18), 18.2 (C, Si C CH3)3), 20.4, 21.7, 25.9 (CH3,
Si C CH3)3), 28.4, 30.9, 31.6, 31.9, 35.0, 35.6, 35.8 (CH2,

W. Felzmann et al. / Steroids 70 (2005) 103–110
105
C-16), 37.1, 38.6, 45.0, 47.8, 51.4, 54.5, 71.9 (CH, C-3),
221.2 (C, C-17).
and finally for 3 h at 140 ◦C. The flask was then flushed
with argon, cooled to 0 ◦C in an ice-bath, and 10 ml THF
were added. The mixture was sonicated for 1.5 h in an ultra-
sound bath to give an opalescent suspension.The flask was
then cooled to −80 ◦C, and methyllithium in diethyl ether
(0.28 ml, 1.6 M, 0.45 mmol) was added dropwise at −80 ◦C.
After stirring for 30 min, the initially brown solution turned
yellow, and a precooled solution of 3 (100 mg, 0.25 mmol)
in 2 ml THF was added via cannula. The reaction was stirred
for 45 min at −80 ◦C and was then allowed to warm to room
temperature. After quenching with 2 ml H2O, 20 ml of diethyl
ether were added. The aqueous layer was then extracted three
times with diethyl ether. The combined organic phases were
washed sequentially with 1 M Na2S2O3 solution and brine,
dried with Na2SO4, and the solvent was removed in vacuo.
VFC (petroleum ether/ethyl acetate, 10:1) gave 9 as the main
product (76 mg) in 75% yield; m.p. 184–185 ◦C; spectra were
identical to those for 9 prepared by method A.
2.5.2. 16,16-Dideuterio-O3-[(1,1-dimethylethyl)
dimethylsilanyl ]epiandrosterone (3)
NaOMe (40% w/w in D2O, 0.17 ml) was added to a so-
lution of 2 (3.40 g, 8.40 mmol) in THF (120 ml) and D2O
(5 ml) and heated at reflux overnight. Then, the mixture was
filtered, and the solvent was evaporated. The obtained residue
was subjected to the same conditions two more times to give
compound 3 after treatment with excess of Na2SO4, filtra-
tion, and evaporation of the solvent (2.95 g). The yield was
86%; m.p. 161–163 ◦C; TLC petroleum ether/ethyl acetate
1
(10:1): Rf 0.48; GC–MS (underivatized): Rt 6.18 min; H
NMR (200 MHz, CDCl3): δ 0.04 (s, 6H, Si (CH3)2), 0.5–2.0
[m, 41H, aliphatic H, therein 0.87 (s, 9H, Si C (CH3)3),
0.84 (s, 3H, H-18) and 0.81 (s, 3H, H-19)], 3.54 (m, 1H, H-
3␤); 13C NMR (50 MHz, CDCl3): δ −4.7 (CH3, Si (CH3)2),
12.2 (CH3, C-19), 13.6 (CH3, C-18), 18.1 (C, Si C CH3)3),
20.3, 21.4, 25.8 (CH3, Si C CH3)3), 28.3, 30.8, 31.4, 31.7,
34.9, 35.5, 37.0, 38.5, 44.9, 47.6, 51.3, 54.4, 71.8 (CH, C-
3), 221.2 (C, C-17); MS(EI): [M]+ = 406; C25H42O2SiD2
(406.71): calcd. C 73.83 H 10.41 D 0.99; found C 73.60 H
11.15 (incl. amount of D in an equimolar ratio).
2.5.3.3. Method C. Methyllithium (0.22 ml, 1.6 M,
0.37 mmol) was dissolved in 10 ml diethyl ether at −80 ◦C.
HMPA (66 mg, 0.06 ml, 0.37 mmol) was added, and the
reaction was warmed to −50 ◦C for 10 min and then cooled
again to −80 ◦C. Then, 3 (100 mg, 0.25 mmol), dissolved
in 2 ml THF, was added slowly via cannula. The reaction
was kept at −80 ◦C for 1 h and was subsequently warmed to
room temperature. After quenching with 2 ml H2O, 20 ml of
diethyl ether were added. The aqueous layer was then ex-
tracted three times with diethyl ether. The combined organic
phases were washed sequentially with 1 M NaHCO3 and
brine, dried with Na2SO4, and the solvent was evaporated.
VFC (petroleum ether/ethyl acetate, 10:1) gave 9 (85 mg) in
84% yield; m.p. 183–184 ◦C; spectra were identical to those
for 9 prepared by method A.
2.5.3. 16,16-Dideuterio-3␤-[[(1,1-
dimethylethyl)dimethylsilanyl]oxy]-17␣-methyl-5␣-
androstan-17␤-ol (9)
2.5.3.1. Method A. MeMgI (0.59 ml, 2.1 M, 1.24 mmol) was
dissolved in 5 ml diethyl ether, and 3 (100 mg, 0.25 mmol),
dissolved in 2 ml THF, was added. The reaction was heated at
reflux for 9 h, then cooled to r.t., and quenched with 3 ml H2O.
For workup, the solution was diluted with 20 ml H2O and was
then extracted three times with diethyl ether. The combined
organic layers were washed with saturated NaHCO3 solution
and brine, dried over Na2SO4, and the solvent was removed
in vacuo. VFC (petroleum ether/ethyl acetate, 10:1) gave 9
as the main product (69 mg) in 65% yield; m.p. 184–185 ◦C;
TLC petroleum ether/ethyl acetate (1:1): Rf 0.74; GC–MS
(TMS-derivative): Rt 6.95 min; 1H NMR (CDCl3, 200 MHz):
δ 0.05 (s, 3H, CH3 Si), 0.07 (s, 3H, CH3 Si), 1.79–0.67 [m,
39 H, aliphatic H, therein 0.88 (s, 9H, Si C (CH3)3), 0.83 (s,
3H, H-18), 0.82(s, 3H, H-19)], 3.55(m, 1H, 3␣-H);13CNMR
(CDCl3, 50 MHz): δ −4.7 (CH3, Si (CH3)2), 12.2 (CH3, C-
19), 13.8 (CH3, C-18), 18.1 (C, Si C CH3)3), 20.7 (CH2),
22.9 (CH2), 25.5 (CH3, C-20), 25.8 (CH3, Si C CH3)3),
28.5 (CH2), 31.5 (CH2), 31.7 (CH2), 31.8 (CH2), 35.5 (C,
C-10), 36.3 (CH), 37.1 (CH2), 38.5 (CH2), 45.0 (CH), 45.4
(C, C-13), 50.6 (CH), 54.4 (CH), 72.0 (CH, C-3), 81.5 (C,
C-17); MS(EI) (TMS-derivative): [M]+ = 494.
2.5.4. Methyllithium-d3
Methyliodide-d3 (2.174 g, 15 mmol) was dissolved in
30 ml diethyl ether under an argon atmosphere and cooled
to −20 ◦C. t-Butyllithium (17.6 ml, 1.52 M, 30 mmol) was
added dropwise. After the addition was complete, the solu-
tion was warmed to 0 ◦C and stirred for 2 h. An aliquot of the
solution was titrated with a 1.91 M solution of 2-butanol in
toluene using 1,10-phenantroline-hydrochloride as an indica-
tor. After titration, the solution was used immediately (vide
infra).
2.5.5. 16,16,20,20,20-Pentadeuterio-3␤-[[(1,1-dimethyl
ethyl)dimethylsilanyl]oxy]-17␣-methyl-5␣-androstan-
17␤-ol (4)
Methyllithium-d3 (46.6 ml, 0.15 M, 7 mmol)wasplacedin
a 100 ml flask and cooled to −80 ◦C. HMPA (1.25 g, 1.21 ml,
7 mmol) was added, and the solution was stirred for 20 min. A
precooled solution of 3 (1.918 g, 4.7 mmol) in 30 ml THF was
then added slowly via cannula, the solution was stirred for
1 h at −80 ◦C, and then it was warmed slowly to 0 ◦C over
3 h. After quenching with 5 ml H2O, the reaction mixture
2.5.3.2. Method B. A three-necked, round bottom flask
equipped with septum and stopcock was flushed with
argon and charged with powdered CeCl3.7H2O (95 mg,
0.24 mmol). With magnetic stirring, the flask was heated un-
der high vacuum (0.5 mbar) for 2 h at 60 ◦C, then 2 h at 90 ◦C,

106
W. Felzmann et al. / Steroids 70 (2005) 103–110
was diluted with 50 ml diethyl ether and 250 ml H2O.The
aqueous layer was subsequently extracted three times with
diethyl ether. The combined organic phases were washed
with 1 M NaHCO3 solution and brine, dried over Na2SO4,
and the solvent was removed in vacuo. VFC (petroleum
ether/ethyl acetate, 15:1 to 10:1) produced 4 (1.735 g) in 87%
yield; m.p. 183–184 ◦C; TLC petroleum ether/ethyl acetate
rated in vacuo. VFC (petroleum ether/ethyl acetate, 5:1) gave
6 (0.405 g) in 79% yield; m.p. 202–204 ◦C (cf. nondeuterated
compound: [7,8] 197–200 ◦C); TLC petroleum ether/ethyl
acetate (1:1): Rf 0.57; 1H NMR (CDCl3, 200 MHz): δ
2.50–0.63 [m, 27 H, aliphatic H, therein 1.02 (s, 3H, H-
19), 0.86 (s, 3H, H-18)]; 13C NMR (CDCl3, 50 MHz): δ 11.5
(CH3, C-19), 13.9(CH3, C-18), 21.1 (CH2), 23.0 (CH2), 28.8,
31.4, 31.6, 35.7 (C, C-10), 36.2, 38.1, 38.6, 44.7, 45.5 (C, C-
13), 46.8, 50.5 (CH), 53.8 (CH), 81.3 (C, C-17), 212.0 (C,
C-3).
1
(1:1): Rf 0.74; GC–MS (TMS-derivative): Rt 6.95 min; H
NMR (CDCl3, 400 MHz): δ 0.04 (s, 6H, CH3 Si), 1.78–0.53
[m, 35 H, Androstran-Aliphaten-H, darunter 0.87 (s, 9H,
Si C (CH3)3), 0.82 (s, 3H, H-18), 0.81 (s, 3H, H-19)],
3.52 (m, 1H, 3␣-H); 13C NMR (CDCl3, 100 MHz): δ −4.7
(CH3, Si (CH3)2), 12.2 (CH3, C-19), 13.8 (CH3, C-18),
18.1 (C, Si C CH3)3), 20.7 (CH2), 22.9 (CH2), 25.8 (CH3,
Si C CH3)3), 28.6 (CH2), 31.5 (CH2), 31.5 (CH2), 31.7
(CH2), 35.5 (C, C-10), 36.2 (CH), 37.1 (CH2), 38.5 (CH2),
45.0 (CH), 45.4 (C, C-13), 50.5 (CH), 54.4 (CH), 72.0 (CH,
C-3), 81.3 (C, C-17); MS (EI) (TMS-derivative): [M]+ = 497,
[M (CH3)2CCH2)]+ = 440; C26H43O2SiD5 (425.77): calcd.
C 73.34 H 10.18 D 2.37; found C 73.63 H 11.66 (incl. amount
of D in an equimolar ratio).
2.5.8. 16,16,20,20,20-Pentadeuterio-3,17␤-dihydroxy-
17␣-methyl-androst-2-ene-2-carboxylic acid methyl
ester (7)
2.5.8.1. TMS-protection. Tertiary alcohol
6
(100 mg,
0.32 mmol) and imidazole (53 mg, 0.78 mmol) were dis-
solved in 1 ml CH2Cl2. TMSCl (45 mg, 53 ␮l, 0.39 mmol)
was then added, and the reaction was stirred for 2 h at r.t.
2 ml H2O were then added, and the mixture was diluted
with 20 ml CH2Cl2. After separation, the organic layer was
washed with H2O, dried over Na2SO4, and the solvent was
removed in vacuo to give TMS-protected-6 (116 mg) in 95%
yield. TLC petroleum ether/ethyl acetate (3:1): Rf 0.53;
1H NMR (CDCl3, 200 MHz): δ 0.07 (s, 9H, Si (CH3)3),
2.50–0.60 [m, 26 H, aliphatic H, therein 1.02 (s, 3H, H-19),
0.78 (s, 3H, H-18)].
2.5.6. 16,16,20,20,20-Pentadeuterio-17␣-methyl-5␣-
androstane-3␤,17␤-diol (5)
TBS-ether 4 (0.810 g, 1.90 mmol) was dissolved in 7 ml
THF and TBAF.3H2O (2.73 g, 8.60 mmol) was added. Af-
ter stirring at r.t. for 20 h, the reaction was quenched by
the addition of 5 ml 1 M NH4Cl. The mixture was then
extracted five times with ethyl acetate. The combined or-
ganic phases were dried over Na2SO4, and the solvent was
removed in vacuo to give 5 (0.514 g) in 86% yield; m.p.
208–211 ◦C (cf. nondeuterated compound: [6] 209–210 ◦C);
2.5.8.2. Alkylation. N-isopropylcyclohexylamine (48 mg,
56 ␮l, 0.34 mmol) was dissolved in 10 ml THF and cooled to
−5 ◦C. n-BuLi (0.14 ml, 2.27 M, 0.33 mmol) was then added
dropwise, and the solution stirred for 45 min. The reaction
mixture was cooled to −60 ◦C, and TMS-protected-6
(100 mg, 0.26 mmol), dissolved in 4 ml THF, was added.
The reaction was warmed to −20 ◦C, HMPA (58 mg,
57 ␮l, 0.34 mmol) was added, and the reaction was kept
for 1 h at this temperature. The reaction mixture was
cooled to −80 ◦C, and methyl cyanoformate (33 mg, 31 ␮l,
0.39 mmol) was added. The mixture was stirred for 20 min
at this temperature. The reaction was allowed to warm to r.t.
overnight. After quenching with 4 ml 1 M NH4Cl solution,
the mixture was stirred for 20 min and extracted five times
with ethyl acetate. The combined organic layers were
washed consecutively with saturated NaHCO3 solution and
brine, dried over Na2SO4, and the solvent was removed in
vacuo. VFC (petroleum ether/ethyl acetate, 5:1 to 3:1) gave
7 (67 mg) in 70% yield. TLC petroleum ether/ethyl acetate
1
TLC petroleum ether/ethyl acetate (1:1): Rf 0.25; H NMR
(CDCl3, 200 MHz): δ 1.88–0.53 [m, 29 H, aliphatic H,
therein 0.84 (s, 3H, H-18), 0.82 (s, 3H, H-19)], 3.58 (m,
1H, 3␣-H.); 13C NMR (CDCl3, 50 MHz): δ 12.3 (CH3, C-
19), 13.9 (CH3, C-18), 20.9 (CH2), 23.0 (CH2), 28.6 (CH2),
31.5 (CH2), 31.6 (CH2, C-16), 31.8 (CH2), 35.6 (C, C-10),
36.4 (CH), 37.0 (CH2), 38.2 (CH2), 45.0 (CH), 45.5 (C, C-
13), 50.7 (CH), 54.4 (CH), 71.3 (CH, C-3), 81.4 (C, C-17);
C20H29O2D5.0.75H2O (325.02): calcd. C 73.91 H 9.46 D
3.10; found C 73.88 H 11.26 (incl. amount of D in an equimo-
lar ratio).
2.5.7. 16,16,20,20,20-Pentadeuterio-17␤-hydroxy-17␣-
methyl-5␣-androstan-3-one (6)
1
(1:1): Rf 0.70; H NMR (CDCl3, 400 MHz): δ 1.73–0.65
Dess–Martin periodinane (1.40 g, 3.30 mmol) was dis-
solved in 40 ml CH2Cl2 under an argon atmosphere. 5
(0.514 g, 1.65 mmol), dissolved in 10 ml CH2Cl2, was added,
whereupon the solution turned turbid. After 2 h, TLC showed
the reaction to be complete. For workup, 20 ml 1N NaOH
were added, and the reaction was stirred for another 2 h at r.t.
Most of the CH2Cl2 was removed in vacuo, and the remaining
reaction mixture was extracted five times with ethyl acetate.
The combined organic layers were washed three times with
1N NaOH, dried over Na2SO4, and the solvent was evapo-
[m, 21 H, aliphatic H, therein 0.84 (s, 3H, H-18), 0.75 (s,
3H, H-19)], 1.77 (d, 15.6 Hz, 1H, H-1), 1.96 (m, 1H, H-4ꢀ),
2.12 (m, 1H, H-4), 2.31 (d, 15.6 Hz, 1H, H-1), 3.73 (s, 3H,
COOMe), 12.08 (s, 1H, OH); 13C NMR (CDCl3, 100 MHz):
δ 11.6 (CH3, C-19), 13.9 (CH3, C-18), 20.8 (CH2), 23.0
(CH2), 28.0 (CH2), 31.2 (CH2), 31.6 (CH2), 33.3 (CH2),
34.9 (C, C-10), 36.3 (CH), 36.9 (CH2), 41.0 (CH), 45.4 (C,
C-13), 50.5 (CH), 51.4 (CH3, C-21), 53.7 (CH), 81.5 (C,
C-17), 96.4 (C, C-2), 170.9 (C, C-3), 173.3 (C, C-21).

W. Felzmann et al. / Steroids 70 (2005) 103–110
107
2.5.9. 16,16,20,20,20-Pentadeuterio-3ꢀ-hydro-
xystanozolol (8)
tect the free alcohol in 1 as a TBDMS-ether using standard
conditions [9]. Deuterium exchange at the 16-position of 2
was achieved using sodium methoxide in D2O and THF, as
reported previously by our group [10]. Under the conditions
of HD-exchange, a small amount of TBDMS-cleavage was
also observed, lowering the yield to 86%. Preliminary exper-
iments for methylation were conducted using non-deuterated
reagents. At first, alkylation of the 17-position using methyl
magnesium iodide did not match our expectation. When
1.5 equiv. of MeMgI were used, no product formation was ob-
served, whereas only 65% yield was achieved using 5 equiva-
lents [2]. Addition of MgI2 to 2.2 equiv. of CH3MgI also only
provided 9 in 12% yield. As our goal was the use of only a
slight excess of methylating agent, we decided to increase
the nucleophilicity of the organometallic reagent by trans-
metallation from the organomagnesium to the organocerium
species [11].
␤-Ketoester 7 (67 mg, 0.18 mmol) was dissolved in 5 ml
methanol, and NH2NH2.H2O (12 mg, 12 ␮l, 0.24 mmol) was
added. The mixture was stirred at r.t. for 20 h and for 3 h
further at 50 ◦C. The solvent was removed in vacuo, and the
residue was recrystallized from methanol to furnish 8 (49 mg)
in 78% yield; m.p. 275 ◦C (decomp.) (cf. nondeuterated com-
pound: [1] 285 ◦C (decomp.)); deuterium exchange: 97%;
1
GC–MS (TMS-derivative): Rt 8.31 min; H NMR (CDCl3,
200 MHz): δ 1.88–0.55 [m, 20 H, aliphatic-H, therein 0.75
(s, 3H, H-18), 0.66 (s, 3H, H-19)], 2.02 (d, 1H), 2.33 (m, 2H),
4.00 (s, 1H), 10.2 (s, 1H, OH); 13C NMR (50 MHz, DMSO-
d6): δ 11.4 (CH3, C-19), 14.0 (CH3, C-18), 20.4, 22.9, 25.8,
28.6, 31.1, 31.3, 33.4, 35.9, 36.1, 41.5, 44.9, 50.0, 53.3, 79.4
(C, C-17), 97.8 (C, C-2), 138.5 (C, C-3), 158.7 (C, C-3ꢀ);
MS(EI) (TMS-derivative): M+ = 565, [M CH3]+ = 550.
Unfortunately, following the only rarely described process
of adding MeMgI at 0 ◦C to a solution of anhydrous CeCl3
[12,13], the reaction mixture turned brown within each
experiment, and no conversion of 3–9 could be observed.
Since the brown color of the reaction mixture disappeared
upon addition of Na2S2O3, we ascribed it to the presence of
iodine. Therefore, the active complex must have decomposed
producing iodine and a low-valent cerium species, which
probably led to further decomposition. To check the effec-
tiveness of the anhydrous CeCl3 used for this transformation,
we performed a transmetalation from salt-free methyllithium
to form the corresponding organocerium species [14–16].
When methyllithium was added to a suspension of anhy-
drous CeCl3 at −78 ◦C, the solution also turned brown,
but decolorized over the next 30 min, whereupon addition
3. Results and discussion
The described synthesis of pentadeuterated 3ꢀ-
hydroxystanozolol started from epiandrosterone, which
provided the possibility to easily introduce all functional
groups present in the product with only a minimum amount
of functional group interconversion as well as the opportu-
nity to incorporate all five deuterium atoms in the D-Ring.
The pivotal point of the synthesis was the introduction of the
CD3-unit at C-17 and its optimization with respect to yield
and equivalents of reagent used (Scheme 2).
Since it was thought that a free alcohol at C-3 could give
rise to complications in the alkylation step, we decided to pro-
Scheme 2. Synthesis of 16,16,20,20,20-pentadeuterio-3ꢀ-hydroxy stanozolol 8. (a) 1.2 eq. TBDMSCI, 2.5 eq. imidazole (quant.) (b) 3 × D2O/NaOMe, THF,
reflux (86%) (c) 1.5 eq. MeLi-d3, HMPA, THF, 1 h, −80 ◦C, 4 (87%) (d) 1.5 eq. MeLi, HMPA, THF 1 h, −80 ◦C, 9 (84%) (e) 5 eq. TBAF·3H2O, 20 h (86%)
(f) 1.5 eq. Dess–Martin periodinane, CH2Cl2 (79%) (g) 1.2 eq. TMSCI, 2.4 eq. imidazole, 1 h (95%) (h) 1.3 eq. LICA, 1.3 eq. HMPA, 1.5 eq. CNCOOMe,
THF, −80 ◦C, then 2N HCl (70%) (i) NH2NH2·H2O, MeOH, 2 h, 50 ◦C (78%).

Fig. 1. Chromatogram and mass spectra of 16,16,20,20,20-pentadeuterio-3ꢀ-hydroxy-stanozolol 8: besides the molecule peak at 8.31 min, only two peaks deriving from the derivatization reagents are visible.

W. Felzmann et al. / Steroids 70 (2005) 103–110
109
Table 1
The synthesis starts with epiandrosterone (1) and proceeds
via protection of the hydroxyl group at position 3 and com-
plete deuterium exchange at position 16. For the subsequent
introduction of a deuterated methyl group at C-17, a highly
efficient method has been developed using methyllithium-
d3 and HMPA. Deprotection and oxidation of the secondary
alcohol in position 3 led to mestanolone-d5 (6). Regioselec-
tive alkylation using Mander’s reagent and ring closure to the
pyrazole heterocycle produced 3ꢀ-hydroxystanozolol-d5 (8).
Comparison of various nucleophiles for alkylation of 3
Nucleophile
Equivalents used
Additive
Yield (%)
CH3MgI
CH3MgI
CH3MgI
CH3MgI
CH3Li
CH3Li
CH3Li
CD3Li
1.5
5
2.2
2
1.9
1.5
1.5
1.5
None
None
MgI2
CeCl3
CeCl3
None
HMPA
HMPA
0
65
12
0
75
0
84
87
of 3 yielded 9 in 75% yield. Fearing that LiI present in com-
mercially available methyllithium-d3 would also succumb to
the same redox reaction that we had observed with MeMgI
and CeCl3, we looked for another alternative. Methyllithium
in the presence of 1 equivalent of HMPA added very ef-
ficiently to 3 at −80 ◦C, yielding 9 in 84% yield. With-
out HMPA, no conversion could be observed. To our sat-
isfaction, we also found that methyllithium-d3 could be pro-
duced easily from iodomethane-d3, which is the cheapest
d3-methyl source available, by adding 2 equivalents of t-
butyllithium to a solution of iodomethane-d3 at −20 ◦C [17].
Using methyllithium-d3 prepared by this method with the
conditions developed for 9, we were able to obtain 4 in 87%
yield (Table 1).
Deprotection of the TBDMS-protected alcohol using
tetrabutylammonium fluoride [9] yielded 5 in 86% yield. Al-
cohol 5 was oxidized using the Dess–Martin periodinane
[18], yielding ketone 6 in 79%. For introduction of the
methoxycarbonyl group in the 2-position, we decided to use
Mander’s reagent, which is a highly effective means for intro-
duction of a methyl ester functionality. In a first attempt, de-
protonation of 6 at −80 ◦C using 2 equivalents of lithium iso-
propylcyclohexylamine (LICA) and treatment with methyl
cyanoformate [19] only yielded 7 in 51% yield. Apparently,
the unprotected tertiary alcohol seemed to be responsible for
the mediocre yield of this reaction, as protection of the al-
cohol with the TMS-ether and subsequent alkylation yielded
7 in 70%. Careful examination of the raw product showed
that besides the 2-alkylated product, the 4-alkylated isomer
was also formed in a ratio of 92:8 [20]. Directly after column
chromatography, NMR analysis showed 7 to exist nearly ex-
clusively in the enol-form. After standing at r.t., the enol-form
of 7 equilibrated slowly towards the keto form [21]. Forma-
tion of the pyrazole heterocyle was achieved by treating 7
with hydrazine hydrate in methanol at elevated temperatures
to give 8 in 78% yield. GC–MS analysis showed 8 to be
pure. The degree of deuterium exchange was calculated as
described in Reference [10] to be 97% (Fig. 1).
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