960
P. Ga¨rtner et al.
2-[Bis(4-hydroxyphenyl)methylene)cyclohexanol
(5) – cyclofenil metabolite M3
2-[Bis-4-(t-butyldimethylsilyloxy)phenyl]
cyclohexylmethanol (12)
[Bis-4-(t-butyldimethylsilyloxy)phenyl]cyclohexane-2-
one (11) (0.8697 g, 1.66 mmol) and CeCl3.7H2O (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% NH4Cl-solution and
extracting with Et2O, the combined organic layers were
dried over Na2SO4 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. 1H–NMR (CDCl3, 200 MHz): υ D 0.21
(s; 12H, 2 ð SiꢀCH3ꢁ2), 0.99 (s; 18H, 2 ð CꢀCH3ꢁ3), 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). 13C–NMR (CDCl3, 50 MHz): υ D ꢀ4.27
(q; 2 ð SiꢀCH3ꢁ2), 18.25 (s; 2 ð CꢀCH3ꢁ3), 20.41 (t; CH2),
25.77 (q; 2 ð CꢀCH3ꢁ3), 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: C31H48O3Si2 0.9
C6H14: calculated C 72.57%, H 10.14%; found C 72.94%, H
9.55%.
2-[Bis-4-(t-butyldimethylsilanyloxy)phenyl]cyclohexylmeth-
anol (12) (0.5053 g, 0.96 mmol) was dissolved in 15 ml THF
and TBAF Ð 3H2O (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 Na2SO4
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. 1H–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). 13C-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: C19H20O3 ð 0.1 CH2Cl2: cal-
culated C 75.25%, H 6.68%; found C 75.15%, H 6.46%.
Elemental composition of [M C H]C —H2O (HRMS, NSIC):
m/z calculated for C19H19O2: 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.
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).
[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
Sample preparation
°
added and after stirring for 1 h at ꢀ80 C, a solution of
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.
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 Na2SO4 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
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
1
was obtained. H–NMR (CDCl3, 200 MHz): υ D 0.19(s; 6H,
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
performedbyadding100 µl MSTFA/NH4I/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.
SiꢀCH3ꢁ2), 0.22(s; 6H, SiꢀCH3ꢁ2), 0.97(s; 9H, CꢀCH3ꢁ3), 0.99
(s; 9H, CꢀCH3ꢁ3), 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). 13C–NMR (CDCl3, 50 MHz):υ D ꢀ4.27(q;
2 ð SiꢀCH3ꢁ2), 18.23 (s; CꢀCH3ꢁ3), 18.26(s; CꢀCH3ꢁ3), 25.74
(q; 2 ð CꢀCH3ꢁ3), 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: C31H46O3Si2 ð 0.2 EtOAc: calculated 70.67%, H
8.88%; found C 70.77%, H 9.11%.
°
Copyright 2008 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2008; 43: 958–964
DOI: 10.1002/jms