JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 2008; 43: 958–964

Published online in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jms.1462

Synthesis and identification of hydroxylated

metabolites of the anti-estrogenic agent cyclofenil
Peter Gärtner,1 Karin Hofbauer,1 Christian Reichel,2 Thomas Geisendorfer2 and
Günter Gmeiner2∗
1
Institute of Applied Synthetic Chemistry, Vienna University of Technology, Vienna, Austria
2
Doping Control Laboratory, Austrian Research Centers GmbH, ARC, A-2444 Seibersdorf, Austria

Received 11 April 2008; Accepted 2 May 2008

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping
Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites,
bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the
phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution
mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised
cyclofenil metabolites M1–M3 was done using gas chromatography for separation and electron ionisation
mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine
deriving from an excretion study within the WADA 2007 Educational Programme. Copyright  2008 John
Wiley & Sons, Ltd.

KEYWORDS: anti-estrogens; doping control; gas chromatography; mass spectrometry; cyclofenil

INTRODUCTION Aims of the present study are to synthesise and char-

The anti-estrogen cyclofenil (1), generally used as a stimulant
of ovarian function,1 is listed as a doping substance on the
World Anti-Doping Agency (WADA) Prohibited List2 due
to its selective estrogen receptor modulator capabilities. Up
to now there is one case reported in the official statistics
of WADA.3 Treatment of the negative effects of anabolic
androgenic steroid abuse as well as a negative feedback to
testosterone metabolism causing an indirect enhancement of
the serum testosterone concentration is the suspected reason
for the ban of this substance.4 Adverse effects of treatments
with cyclofenil include hepatitis5 and haemolytic anaemia.6
Proposed metabolites of cyclofenil – 4,40 -(cyclohexylid-
enemethylene)bis(phenyl acetate) – are the corresponding
diphenol (2), formed after hydrolysis of both acetate groups,
and further hydroxylated diphenol (3–5).1,4
GC-MS-studies lead to the assumption that the third
hydroxyl group is located in the cyclohexyl moiety, but
the exact position has not yet been confirmed.1,4 Structural
characterisation and identification of the diphenol (2) after
an administration study with cyclofenil has already been
published by Myung et al.1
For an unambiguous identification, all three hydroxyl-
bis-deacetyl-cyclofenil products (3–5) have to be synthesised
in order to compare these compounds with the substances
found in a reference collection (Fig. 1).
Ł Correspondence to: Günter Gmeiner, Doping Control Laboratory,
Austrian Research Centers GmbH, ARC, A-2444 Seibersdorf,
Austria. E-mail: guenter.gmeiner@arcs.ac.at
acterise hydroxylated metabolites of cyclofenil by chemi-
cal synthesis and mass spectrometric detection, having a
hydroxyl group in the aliphatic cyclohexyl ring in addition
to the phenolic hydroxyl groups of the aromatic rings; to
compare the synthesised target substances with excreted
metabolites in a reference collection provided by the WADA
as part of its 2007 Educational Programme.
EXPERIMENTAL
Chemical synthesis
Materials
Reactions requiring air-sensitive manipulations were carried
out under nitrogen.
All reagents purchased from commercial sources, were
of per analysis quality and were used without additional
purification. Tetrahydrofurane (THF) was dried by filtration
over an Al2 O3 cartridge, MeOH was dried by filtration over a
molecular sieve cartridge. Thin layer chromatography (TLC)
was carried out on pre-coated silica gel plates, Merck 60F254 .
TLC spots were visualised by irradiation with UV light and
by staining them with KMnO4 reagent and heating.
Analytical methods
NMR spectra were measured with a Bruker AC 200 at
200 MHz (1 H), 50 MHz (13 C) and with a Bruker AC 400
at 400 MHz (H–H–, C–H–COSY).
Elemental analysis for the determination of the carbon
and hydrogen content of the reference substances was done
on an EA 1108 elementary analyser from Carlo Erba (Milan,
Italy).

Copyright  2008 John Wiley & Sons, Ltd.


metabolism
AcO OAc HO
1 2
Figure 1. Metabolic pathway of the estrogenic substance cyclofenil.
Elemental compositions of the final products were deter-
mined using a high-resolution hybrid mass spectrometer
(LTQ Orbitrap, Thermo Electron, Bremen). The mass spec-
trometer was operated in positive ion mode with a spray
voltage of 2 kV and a capillary temperature of 160 ° C. Data
were acquired in Fourier Transform (FT) full MS mode (m/z
100–350) at a resolving power of R D 100 000 at m/z 400.
Automated gain control (AGC) target settings were 2 ð 10e5
for full MS scans in the orbitrap mass analyser. A Prox-
eon nano-ESI source (Odense, Denmark) equipped with new
objective glass emitters (1.2 mm OD, 0.94 mm ID, 4 µm tip ID)
was used for ionisation of the cyclofenil metabolites in static
electrospray (Woburn, MA). Metabolites were dissolved in
50% acetonitrile/water containing 0.1% formic acid (Merck,
Germany). Data were processed with XCALIBUR Version
2.0 software from Thermo Electron Corporation.
Synthetic pathways
4-Hydroxycyclohexanone (7)
Cyclohexane-1,4-diol (4.0000 g, 34.44 mmol) was suspended
in 14 ml Et2 O. A solution of Na2 Cr2 O7 .2H2 O (3.4938 g,
11.72 mmol) and concentrated H2 SO4 (2 ml) in 14 ml H2 O
were added dropwise. The reaction was stirred for 3 h at
room temperature (RT), extracted with Et2 O, dried over
Na2 SO4 and concentrated in vacuo. The crude product was
purified by flash chromatography (petroleum ether/EtOAc
1/1) and 1.3141 g (33.43%) colourless oil were obtained.7
3-Hydroxycyclohexanone (8)
3-Hydroxycyclohexanone was synthesised in the same way
as 4-hydroxycylohexanone, starting from cyclohexane-1,3-
diol (3.0000 g, 25.83 mmol). A total of 1.1282 g (38.30%)
colourless oil was obtained.8
4-[Bis(4-hydroxyphenyl)methylene]cyclohexanol
(3) – cyclofenil metabolite M1
Zinc (4.23352 g, 64.77 mmol) was suspended in 150 ml dry
THF and TiCl4 (7.14 ml, 64.77 mmol) was added. After
O
OH TiCl4, Zn
+
THF
HO OH
O
6 7,8
Figure 2. Synthetic pathways to the cyclofenil metabolites M1 (3) and M2 (4).
Copyright  2008 John Wiley & Sons, Ltd.
Characterisation of hydroxylated cyclofenil metabolites 959
OH
+
OH HO OH
3-5
3: Cyclofenil metabolite M1 (4-hydroxy)
4: Cyclofenil metabolite M2 (3-hydroxy)
5: Cyclofenil metabolite M3 (2-hydroxy)
refluxing the reaction mixture for 1 h, a solution of 4-
hydroxycyclohexanone (7) (0.9589 g, 8.40 mmol) and 4,40 -
dihydroxybenzophenone (6) (0.3326 g, 8.40 mmol) in 30 ml
dry THF was added and the reaction mixture was refluxed for
3 h. After cooling to RT, the reaction mixture was poured onto
saturated NaHCO3 -solution and extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na2 SO4 and concentrated in vacuo. The crude product was
purified by flash chromatography (petroleum ether/EtOAc
3/1 to 1/1) and washed with cold EtOAc. A total of 1.4613 g
(58.70%) colourless powder was obtained. M.p. 206–208 ° C.
Elemental composition of [M C H]C (HRMS, NSIC ) : m/z
calculated for C19 H21 O3 : 297.1485; found: 297.1486.7
3-[Bis(4-hydroxyphenyl)methylene]cyclohexanol
(4) – cyclofenil metabolite M2
3-[Bis(4-hydroxyphenyl)methylene]cyclohexanol was syn-
thesised in the same way as 4-[bis(4-hydroxyphenyl)methy-
lene]cyclohexanol starting from 3-hydroxycyclohexanone (8)
(0.1598 g, 1.40 mmol) and 4,40 -dihydroxybenzophenone (6)
(0.3000 g, 1.40 mmol). A total of 0.2110 g (50.84%) yellow
crystals was obtained after flash chromatography (petroleum
ether/EtOAc 5/1 to 1/1). M.p. 100–102 ° C. 1 H–NMR (d6 -
DMSO, 200 MHz): υ D 1.10–1.35 (m; 2H, CH2 ), 1.58–1.92 (m;
4H, 2 ð CH2 ), 2.24–2.69 (m; 2H, CO–CH2 –CH), 6.57–6.72
(m; 4H, 4 ð CH–C–OH), 6.75–6.91(m; 4H, 4 ð CH–C–C),
9.27 (s; 2H, 2 ð OH). 13 C–NMR (d6 -DMSO, 50 MHz):
υ D 24.33 (t; CH2 –CH2 –CH2 ), 30.98 (t; C–CH2 –CH2 ),
35.41(t; CH2 ), 41.43 (t; CH2 ), 69.57 (d; CH–OH), 114.66
(d; 4 ð CH–C–OH), 130.41(d; 2 ð CH–C–C), 130.45 (d;
2 ð CH–C–C), 133.62 (s; Ar–C–C), 133.81 (s; Ar–C–C),
133.85 (s; Ar–C–C), 135.46 (s; C–CH2 ), 155.63 (s; C–OH),
155.66 (s; C–OH). Elemental analysis: C19 H20 O3 ð 0.3 EtOAc:
calculated C 75.16%, H 6.99%; found C 75.32%, H 7.03%.
Elemental composition of [M C H]C (HRMS, NSIC ) : m/z
calculated for C19 H21 O3 : 297.1485; found: 297.1487.
OH
3, 7: 4-hydroxy-
4, 8: 3-hydroxy-
HO OH
3,4
J. Mass Spectrom. 2008; 43: 958–964
DOI: 10.1002/jms
960 P. Gärtner et al.
2-[Bis(4-hydroxyphenyl)methylene)cyclohexanol
(5) – cyclofenil metabolite M3
2-[Bis-4-(t-butyldimethylsilanyloxy)phenyl]cyclohexylmeth-
anol (12) (0.5053 g, 0.96 mmol) was dissolved in 15 ml THF
and TBAF Ð 3H2 O (1.2169 g, 3.84 mmol) was added. The reac-
tion mixture was stirred overnight at RT. After washing with
water and brine, the organic layer was dried over Na2 SO4
and concentrated in vacuo. The crude product was purified by
flash chromatography (petroleum ether/EtOAc 2/1) and a
total of 0.2390 g (83.73%) orange crystals was obtained. M.p.
87–90 ° C. 1 H–NMR (d6 -acetone, 200 MHz): υ D 1.35–2.19
(m; 6H, 3 ð CH2 ), 2.33–2.55 (m; 2H, CH2 ), 4.67 (m; 1H,
CH–OH), 6.76–6.91 (m; 4H, 4 ð CH–C–OH), 6.95–7.15 (m;
4H, 4 ð CH–C–C), 8.36 (s; 1H, OH). 13 C-NMR (d6 -acetone,
50 MHz): υ D 21.02 (t; CH2 ), 27.77 (t; CH2 ), 28.99 (t; CH2 ),
35.52 (t; CH2 ), 68.19 (d; CH–OH), 115.42 (d; CH–C–OH),
115.49 (d; CH–C–OH), 131.40 (d; CH–C–C), 131.49 (d;
CH–C–C), 134.85 (s; Ar–C–C), 135.19 (s; Ar–C–C), 136.94 (s;
DB–C–C), 139.10 (s; DB–C–CH2 ), 156.74 (s; C–OH), 156.79
(s; C–OH). Elemental analysis: C19 H20 O3 ð 0.1 CH2 Cl2 : cal-
culated C 75.25%, H 6.68%; found C 75.15%, H 6.46%.
Elemental composition of [M C H]C —H2 O (HRMS, NSIC):
m/z calculated for C19 H19 O2 : 279.1380; found: 297.1382.
2,2-Dimethoxycyclohexanecarboxylic acid ethyl ester (10)
2,2-Dimethoxycyclohexanecarboxylic acid ethyl ester (10)
was prepared according to Ref. 9.
[Bis-4-(t-butyldimethylsilyloxy)phenyl]cyclohexane-
2-one (11)
(4-Bromophenoxy)-t-butyldimethylsilane10 (10.1278 g, 35.26
mmol) was dissolved in 130 ml dry THF and cooled to

80 ° C. n-BuLi (2.5 M in hexane, 13.6 ml, 34.00 mmol) was
added and after stirring for 1 h at
80 ° C, a solution of
2,2–dimethoxycyclohexanecarboxylic acid ethyl ester (10)
2.7666 g, 12.79 mmol) in 20 ml dry THF was added. The
reaction mixture was stirred for 7 h at
80 ° C, and overnight
at RT. After adding 2 M HCl, the reaction mixture was
extracted with EtOAc. The combined organic layers were
dried over Na2 SO4 and concentrated in vacuo. The crude
product was purified by flash chromatography (petroleum
ether/EtOAc 100/1) and 1.0397 g (15.55%) colourless oil
was obtained. 1 H–NMR (CDCl3 , 200 MHz): υ D 0.19(s; 6H,
SiCH3 2 ), 0.22(s; 6H, SiCH3 2 ), 0.97(s; 9H, CCH3 3 ), 0.99
(s; 9H, CCH3 3 ), 1.69–1.85(m; 4H, 2 ð CH2 ), 1.89–2.07(m;
4H, 2 ð CH2 ), 6.73(dd; J1 D 8.41 Hz, J2 D 16.82 Hz, 4H,
4 ð CH–C–OTBDMS), 6.93(dd; J1 D 8.41 Hz, J2 D 17.4 Hz,
4H, 4 ð CH–C–C). 13 C–NMR (CDCl3 , 50 MHz): υ D
4.27(q;
2 ð SiCH3 2 ), 18.23 (s; CCH3 3 ), 18.26(s; CCH3 3 ), 25.74
(q; 2 ð CCH3 3 ), 26.46 (t; CH2 ), 26.81 (t; CH2 ), 34.53 (t;
CH2 ), 45.01(t; CH2 ), 119.39(d; 2 ð CH–C–OTBDMS), 119.45
(d; 2 ð CH–C–OTBDMS), 130.58(d; 2 ð CH–C–C), 131.64
(d; 2 ð CH–C–C), 134.15 (s; Ar–C–C), 135.47 (s; Ar–C–C),
137.45 (s; DB–C–CO), 144.35 (s; DB–C–C), 155.14 (s; C-
OTBDMS), 155.39 (s; C-OTBDMS), 207.28 (s; CO). Elemental
analysis: C31 H46 O3 Si2 ð 0.2 EtOAc: calculated 70.67%, H
8.88%; found C 70.77%, H 9.11%.
Copyright  2008 John Wiley & Sons, Ltd.
2-[Bis-4-(t-butyldimethylsilyloxy)phenyl]
cyclohexylmethanol (12)
[Bis-4-(t-butyldimethylsilyloxy)phenyl]cyclohexane-2-
one (11) (0.8697 g, 1.66 mmol) and CeCl3 .7H2 O (0.9313 g,
2.50 mmol) were dissolved in 30 ml MeOH. NaBH4 (0.1010 g,
2.67 g) was added slowly and the reaction was stirred
overnight at RT. After adding 10% NH4 Cl-solution and
extracting with Et2 O, the combined organic layers were
dried over Na2 SO4 and concentrated in vacuo. The crude
product was purified by flash chromatography (petroleum
ether/EtOAc 100/1) and a total of 0.6265 g (71.76%) colour-
less oil was obtained. 1 H–NMR (CDCl3 , 200 MHz): υ D 0.21
(s; 12H, 2 ð SiCH3 2 ), 0.99 (s; 18H, 2 ð CCH3 3 ), 1.48–1.99
(m; 6H, 3 ð CH2 ), 2.23–2.54 (m; 2H, CH2 ), 4.62 (s; 1H,
CH–OH), 6.68–6.82 (m; 4H, 4 ð CH–OTBDMS), 6.89–7.06
(m; 4H, 4 ð CH–C). 13 C–NMR (CDCl3 , 50 MHz): υ D
4.27
(q; 2 ð SiCH3 2 ), 18.25 (s; 2 ð CCH3 3 ), 20.41 (t; CH2 ),
25.77 (q; 2 ð CCH3 3 ), 27.24 (t; CH2 ), 28.21 (t; CH2 ), 34.62
(t; CH2 ), 68.41 (d; CH–OH), 119.40 (d; CH–C–OTBDMS),
119.48 (d; CH–C–OTBDMS), 130.71 (d; CH–C–C), 130.76 (d;
CH–C–C), 135.35 (s; Ar–C–C), 135.63 (s; Ar–C–C), 137.07 (s;
DB–C–C), 138.08 (s; DB–C–COH), 154.28 (s; C–OTBDMS),
154.41 (s; C–OTBDMS). Elemental analysis: C31 H48 O3 Si2 0.9
C6 H14 : calculated C 72.57%, H 10.14%; found C 72.94%, H
9.55%.
Analytical characterisation
Substrates and reagents
All solvents purchased by local suppliers were of high-
performance liquid chromatography (HPLC) grade. All
reagents and salts were of analytical grade. ˇ-Glucuronidase
from E. coli was supplied by Boehringer (Mannheim,
Germany). Methyltestosterone was purchased from Sigma-
Aldrich (Vienna, Austria).
Sample preparation
For the identification of cyclofenil metabolites an educational
sample provided within the WADA 2007 Educational
Programme was used.
Sample preparation was done according to standard
procedures for anabolic steroid screens, already published
in, e.g. Ref. 11.
In brief: 5 ml of urine was adjusted to pH D 7 using a
0.8 M phosphate buffer, and incubated for 60 min with ß-
glucuronidase from E. coli at 50 ° C. After adjusting the pH to
approximately 9.8 with sodium carbonate the analytes were
extracted with methyl-t-butyl ether (MTBE). The organic
phase was concentrated to dryness. Derivatisation was
performed by adding 100 µl MSTFA/NH4 I/ethanethiol-TMS
1000 : 2 : 6 (v/w/v), and by subsequent heating at 60 ° C for
20 min.
Methyltestosterone was used as internal standard.
Spiked blank urine samples were prepared with the
following levels for each reference substance: 10 ng/ml,
100 ng/ml and 1 µg/ml. These concentration levels were
further used to estimate the approximate concentration of
the metabolites in the reference urine.
For the estimation of unchanged excretion of hydroxy-
lated cyclofenil metabolites, the same aliquot volume was
prepared without enzymatic hydrolysis.
J. Mass Spectrom. 2008; 43: 958–964
DOI: 10.1002/jms
 Characterisation of hydroxylated cyclofenil metabolites 961

Table 1. Comparison of the relative abundances and their In each case, 5 µg of the substances were derivatised
relative deviation of the ion traces of the cyclofenil metabolites according to the procedure described above to achieve
M1–M3 as tris-TMS derivatives between the synthesised reference spectra (see Fig. 4). The derivatisation with
substances and the reference urine after sample preparation MSTFA/NH4 I/ethanethiol-TMS was chosen to meet the
conditions used for steroid screen. In case of cyclofenil
Cyclofenil metabolite 1 – tris-TMS metabolite M3, additional derivatisation was done using
Synth. substance Ref. urine MSTFA to avoid the loss of water during the derivatisation
Rel. abundance Rel. abundance Rel. deviation reaction caused by the presence of iodide.
m/z (%) (%) (%)
Gas chromatography–mass spectrometry
512 10.2 10.9 0.7 GC-MS analyses were conducted on a Trace DSQ directly
422 100 100 0.0 interfaced to a TRACE GC ultra gas chromatograph and
343 31.2 30.6 0.6 equipped with an AS 3000 autosampler (all from Thermo
Electron Corporation, Vienna, Austria). The electron energy
was set to 70 eV, and the ion source temperature to 250 ° C. A
Cyclofenil metabolite 2 – tris-TMS
RTX-1 ms (15 m ð 0.25 mm i.d., 0.1 µm film thickness) cross-
Synth. substance Ref. urine bonded dimethyl polysiloxane capillary column (Restek,
Rel. abundance Rel. abundance Rel. deviation Bellefonte, PA, USA) was used in the split injection mode
m/z (%) (%) (%) (10 : 1). The column-head pressure of helium as the carrier
gas was set to 55 kPa. The oven programme temperature
512 33.7 35.0 1.3
was 166 ° C/3 ° C per min/215 ° C/10 ° C per min/306 ° C for
422 100 100 0.0
2 min. Injector and transfer line temperatures were set at 250
381 38.3 35.4 2.9
and 280 ° C, respectively. Chromatograms were recorded in
scanning mode. The mass range was m/z 50–600 at a scan rate
Cyclofenil metabolite 3 – tris-TMS of 800 amu/s. The acquisitions in the selected ion monitoring
Synth. substance Ref. urine (SIM) were performed to calculate the relative abundance
ratio shown in Table 1, and used the same temperature
Rel. abundance Rel. abundance Rel. deviation
programme by monitoring the following ions: m/z D 179,
m/z (%) (%) (%)
204, 256, 301, 343, 381, 394, 422, 446, 469 and 512 with a mass
512 24.7 24.2 0.5 width set to 1 amu and a dwell time per mass of 60 ms. Data
469 23.8 21.9 1.9 were processed with XCALIBUR Version 1.4 software from
422 100 100 0.0 Thermo Electron Corporation.

RESULTS AND DISCUSSION

Reference substances Chemical synthesis
Stock solutions in MeOH of the synthesised reference sub- The first attempt to synthesise the desired substances was the
stances cyclofenil metabolites M1–M3 (3–5) were prepared McMurry-coupling reaction, in which 4,40 -dihydroxybenzo-
with concentrations of 1 mg/ml. phenon (6) and hydroxycyclohexanones with hydroxyl

O HC(OMe)3, OMe Li
p-TsOH OMe
TBDMSO O
O O
MeOH THF
O O
TBDMSO OTBDMS
9 10 11

CeCl3/NaBH4
MeOH

OH TBAF OH
THF

HO OH TBDMSO OTBDMS
5 12

Figure 3. Synthetic pathway to the cyclofenil metabolite M3 (5).

Copyright  2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 958–964
DOI: 10.1002/jms
 962
P. Gärtner et al.

A: Cyclofenil Metabolite M1 – tris TMS B: Cyclofenil Metabolite M2 – trisTMS

cyc_stM03 #1241 RT:17,66 AV:1 SB: 22 17,80-17,92, 17,45-17,57 NL: 9,67E5 TMS cyc_stm02 #1083 RT:15,79 AV: 1 SB: 11 15,98-16,10 NL: 3,73E5 TMS
T: + c Full ms[ 50,00-600,00] O T: + c Full ms [ 50,00-600,00]
O
422 73
100 100

80 80
422

Copyright  2008 John Wiley & Sons, Ltd.

60 60 O O
73
O O 421
TMS TMS
40 423 TMS TMS 40
343 381
423 512
257

Relative Abundance
Relative Abundance
20 421 20 179 217
512 75 513
75 179 204 381 513 129 203 343 368
256
0 0
100 200 300 400 500 600 100 200 300 400 500 600
m/z m/z

C: Cyclofenil Metabolite M3 – tris TMS D: Cyclofenil Metabolite M3 Artefact – bis TMS

cyc_stm03w #934 RT: 14,03 AV: 1 SB: 5 14,16-14,20 NL: 1,53E5 cyc_stm03w #895 RT:13,57 AV: 1 SB: 34 13,02-13,41 NL: 2,67E5
T: + c Full ms [50,00-600,00] T: + c Full ms [ 50,00-600,00]
73 TMS 422
100 O 100
422
80 80
O O
O O
60 TMS TMS 60 TMS TMS
423
40 40 73 423
512

Relative Abundance
Relative Abundance

20 179 267 421 469 20

75 257 513
147 343 347 455 179
196 241 257
394
0 0
100 200 300 400 500 600 100 200 300 400 500 600
m/z m/z

Figure 4. Mass spectra of the synthesised cyclofenil metabolites M1–M3 (3–5) as tris-TMS derivatives (A–C) as well as of the cyclofenil M3 bis-TMS artefact (D).

J. Mass Spectrom. 2008; 43: 958–964

DOI: 10.1002/jms
 Characterisation of hydroxylated cyclofenil metabolites 963

RT: 12.46 - 18.49 SM: 5G

17.72 NL:
A: Total Ion Current (TIC)
4.61E7
Cyclofenil M2 15.78 Cyclofenil M1 TIC F: MS
Genesis
10 17.87 cy_av01
Cyclofenil M3 IS: Methyltestosterone

5 14.58
13.20 13.57 15.41
14.03 15.19 18.31
12.64 12.82 15.98 16.70 17.21 17.38

0
17.72 NL:
B: Mass Trace 512 8.60E5
Relative Abundance

m/z=
15.76 512-513 F:
10
MS Genesis
17.87 cy_av01

5
14.03

0 NL:
17.72
C: Mass Trace 422 8.04E6
m/z=
422-423 F:
10
Cyclofenil M3 (-H2O) bis-TMS MS Genesis
cy_av01
Cyclofenil M3 tris-TMS
5
15.76
13.57
14.03
0
12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0
Time (min)

Figure 5. Total ion chromatogram (A) and mass traces 512 (B) and 422 (C) of a reference collection deriving from an excretion study
with cyclofenil.

groups on position 2, 3 and 4 react to the olefine in one
step. 4-Hydroxycyclofenil (3) has already been synthesised
by this method, although protecting groups were used.7 As
alcohols usually do not interfere with McMurry reaction
conditions,12 we tried to carry out the coupling without
protecting groups.
Cyclofenil metabolite M1 (3) and M2 (4) were obtained
by this reaction pathway in 51 and 59%, respectively (Fig. 2),
when starting from hydroxycyclohexanones (7) and (8)
which were synthesised by partial oxidation with Na2 Cr2 O7
and H2 SO4 of the cyclohexanediols.13
Cyclofenil metabolite M3 (5) was not obtained by this
method with the respective 2-hydroxycyclohexanone, even
by trying different protecting groups and reaction conditions.
Thus, alternative pathways were investigated.
Finally, the desired 2-hydroxycyclofenil (5) was obtained
by the pathway shown in Fig. 3. After protecting 2-
oxocyclohexanecarboxylic acid ethylester (9) as dimethoxy-
ketal (10),9 the addition of f4-[t-butyldimethylsilyl)oxy]phen-
ylglithium to the ester group was carried out to give
(11), which after reduction of the carbonyl group with
CeCl3 /NaBH4 gave (12), and final deprotection with TBAF
yielded (5).
Analytical identification
Mass spectra
Figure 4 shows the mass spectra of the three hydroxy-
lated metabolites of cyclofenil after silylation. In the case of
metabolites M1 and M2 (Fig. 4(A) and (B)) standard derivati-
sation conditions were used (MSTFA/NH4 I/ethanethiol-
TMS). Derivatisation of cyclofenil metabolite M3 showed
two peaks in equal intensity after derivatisation with this
reagent mix (mass spectra are given in Fig. 4(C) and (D));
derivatisation with MSTFA yielded predominately (about
85 area %) the desired tris-TMS product (Fig. 4(C)). Each
of the tris-TMS derivatised products shows a molecular ion
at m/z D 512 as well as a loss of TMS-OH (MC
90) at
m/z D 422.
Due to the abundant molecular ion at m/z D 422 it
was concluded that the byproduct after derivatisation of
cyclofenil metabolite M3 is an artefact deriving from the
loss of water. This elimination reaction is facilitated by
the resonance stabilisation of a positive cation in allylic
position and by the presence of iodide as a catalyst
for nucleophilic substitution of the hydroxyl group and
subsequent elimination of a water moiety.
Metabolite identification
The identification of the synthesised substances in a reference
urine sample provided within the WADA 2007 Educational
Programme was done after preparation of the sample
according to the procedure described above. The sample
was, in addition, prepared without enzymatic hydrolysis
of the glucuronidated conjugates to estimate the amount of
hydroxylated metabolites excreted unconjugated.
As can be seen in Fig. 5 and Table 1, all three metabolites
could be unambiguously identified using the standard

Copyright  2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 958–964
DOI: 10.1002/jms
964 P. Gärtner et al.
screening procedure for anabolic steroids. Identification of
each substance was done using the criteria as issued by the
WADA technical document TD2003IDCR.14 The three most
abundant mass traces were used for qualitative identification
purposes. The differences of the relative abundances of the
monitored ion traces lie well within the tolerance window
specified for EI – GC/MS in this technical document.
Approximately 1% of the metabolites monitored in the
investigated urine was excreted unconjugated. This holds for
all the three hydroxylated metabolites.
An estimation of the concentrations of these metabolites
in the reference urine using spiked samples yielded that
cyclofenil metabolite M1 (3) was present at approximately
160 ng/ml, cyclofenil metabolite M2 (4) at approximately
14 ng/ml, and cyclofenil metabolite M3 (5) at approximately
8 ng/ml.
CONCLUSION
The presented study describes the chemical synthesis as well
as the mass spectrometric characterisation of metabolites of
the anti-estrogenic substance cyclofenil, having two pheno-
lic hydroxyl groups and a hydroxyl function at the aliphatic
ring. All three synthesised substances (cyclofenil metabolites
M1–M3; (3–5)) are unambiguously identified after gas chro-
matographic separation and mass spectrometric detection in
a reference urine collection provided by the WADA within
its 2007 Educational Programme.
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