17α- and 17β-boldenone 17-glucuronides: Synthesis and complete characterization by 1H and 13C NMR

Article abstract of DOI:10.1016/j.steroids.2008.11.012

Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals destined for meat production and as a performance enhancer in athletics. Therefore its use is officially banned either in animals intended for consumption or in humans. Because most anabolic steroids are completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to detect the illegal use of anabolic steroids either in humans or in livestock. 17α- and 17β-boldenone 17-glucuronides were synthesized, purified and characterized in order to provide suitable standards for the identification and quantification of these metabolites.

Full text of DOI:10.1016/j.steroids.2008.11.012

Steroids 74 (2009) 250–255
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
17␣- and 17␤-boldenone 17-glucuronides: Synthesis and complete
characterization by 1H and 13C NMR
Silvana Casati, Roberta Ottria, Pierangela Ciuffreda∗
Dipartimento di Scienze Precliniche LITA Vialba, Università degli Studi di Milano, Via G.B. Grassi, 74-20157 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Boldenone is an androgenic anabolic steroid intensively used for growth promoting purposes in animals
destined for meat production and as a performance enhancer in athletics. Therefore its use is officially
banned either in animals intended for consumption or in humans. Because most anabolic steroids are
completely metabolized and usually no parent steroid is excreted, metabolite identification is crucial to
detect the illegal use of anabolic steroids either in humans or in livestock. 17␣- and 17␤-boldenone 17-
glucuronides were synthesized, purified and characterized in order to provide suitable standards for the
identification and quantification of these metabolites.
Received 10 September 2008
Accepted 14 November 2008
Available online 24 November 2008
Keywords:
17␣-Boldenone
17␤-Boldenone
Glucuronides
© 2008 Elsevier Inc. All rights reserved.
Boldenone metabolite
Synthetic androgenic steroid hormone
metabolite
1. Introduction
urine or faeces. Conjugation with glucuronic and sulphate acid is a
common route of metabolism for steroids and may make up to 90%
17␤-Boldenone (androsta-1,4-dien-17␤-ol-3-one, 1) is a syn-
thetic androgenic steroid hormone with anabolic properties,
obtained by dehydrogenation of the male hormone testosterone
[1]. As for other anabolic steroids, its illegal use to increase body
mass and enhance physical conditioning is widely demonstrated.
17␤-Boldenone is included in the 2008 list of banned substances
in sports by the World Anti-Doping Agency (WADA) and at the
same time its illegal administration for growth promotion purposes
in animals destined for meat production (cattle fattening) is be
coming particularly widespread. Its use is forbidden by European
directives 96/22/EC and 03/74/EC [2,3] to protect the consumer’s
health.
The forensic chemistry of natural and/or synthetic steroids as
illegal abuse substances calls for the development of analytical
methods fit for the identification of both the parent drugs and their
metabolites, in order to demonstrate its effective administration.
Biotransformation reactions of drugs are generally divided in
two classes. In phase I reactions, polar functional groups are
inserted in a drug molecule via typical oxidation, reduction and
hydrolysis. In phase II reactions, the drug and/or its phase I metabo-
lites are conjugated with endogenous species such as glucuronic
acid, sulphate, amino acids, biliary acids and so on, forming soluble
conjugate products which are readily excreted from the body via
of the excreted metabolites. They are therefore an important class
of compounds for drug screening in sports [4].
The human metabolism of 17␤-boldenone was investigated in
1971 [5] and it has been reported that 17␤-boldenone is excreted in
human urine as free and conjugate. Its main metabolites are 5␤-
androst-1-en-17␤-ol-3-one, 5␤-androst-1-en-3␣-ol-17-one and
5␤-androst-1-en-3␣,17␤-diol [6] while 17␣-boldenone (androsta-
1,4-dien-17␣-ol-3-one, 2) is considered to be the main metabolite
in human and equine urine [7,8]. The detection of 17␣-boldenone
would become meaningful evidence for the abuse of 17␤-
boldenone if at the same time additional metabolites, such as
glucuronide of 17␤-boldenone (3) and 17␣-boldenone (4), were
identified (Fig. 1).
At present, the analytical methods for boldenone glucuronide-
conjugated detection are based on application of liquid chromato-
graphic (LC) separation interfaced by soft ionization techniques,
such as electrospray ionization (ESI), with tandem mass spectrome-
try (MS/MS) [4,9]. However, a significant drawback of the conjugate
analysis is the lack of reference material, essential for the develop-
ment and application of analytical methods. In addition to doping
control analytical purposes, the conjugated reference standards are
also needed for steroid metabolism research in pharmaceutical
industry and research institutes.
A
complete characterization of boldenone glucuronides-
conjugated is apparently not reported in literature. In this paper, the
chemical synthesis of 17␣- and 17␤-boldenone 17-glucuronides are
reported together with the complete assignment of 1H and 13C data
∗
Corresponding author. Tel.: +39 02 50319695; fax: +39 02 50319694.
E-mail address: Pierangela.Ciuffreda@unimi.it (P. Ciuffreda).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.11.012

S. Casati et al. / Steroids 74 (2009) 250–255
251
Fig. 1. Structural formula of 17␣- and 17␤-boldenone, and corresponding 17-glucuronides.
and the anomericity of the glucuronic acid moiety in the steroid
glucuronides of these compounds by 1D and 2D NMR experiments.
However, new synthesis improving the yield of 17␣-boldenone17-
␤-glucuronide (4a) has been reported.
experiments were performed through standard pulse sequences.
gCOSY-45 experiments were acquired with 512 t1 increments; 2048
t2 points; spectral/spectrum width 10.0 ppm. The acquisition data
for gHSQC and gHMBC experiments were obtained with 512 t1
increments; 2048 t2 points; spectral/spectrum width 10.0 ppm for
1
1H and 220 ppm for 13C. Delay values were optimized for JC,H
2. Experimental
n
140.0 Hz and JC,H 3.0 Hz. Zero filling in F1 to 1 K, ꢀ/2 shifted
sine-bell squared (for gHSQC) or sinebell (for gHMBC) apodization
functions were used for processing. For overlapped signals of hydro-
gen atoms important for the identification of compounds, the 1D
HOHAHA technique [11] was used to obtain the chemical shifts and
coupling constants.
2.1. General
17␤-boldenone (1) was obtained from Steraloids (Newport, RI,
USA). 17␣-Boldenone (2) was synthesized from 17␤-boldenone by
a modified Mitsunobu reaction [10].
Acetobromo-␣-d-glucuronic acid methyl ester (5) was pur-
chased from Sigma–Aldrich.
2.3. Chemistry
Melting points were recorded on a Stuart Scientific SMP3 instru-
ment and were uncorrected. All reagents were purchased from
Sigma–Aldrich.
2.3.1. Methyl (androsta-1,4-dien-17ˇ-ol-3-one)-17ˇ-O-2,3,4-tri-
O-acetyl-ˇ-d-glucuronate (3a)
CdCO3 (0.76 mmol; 130 mg) was added to a solution of ␤-
boldenone (1) (0.35 mmol, 100 mg) in anhydrous toluene (5 ml)
under vigorous stirring. A portion of the toluene (0.5–1 ml) was
distilled off under azeotropic conditions to remove all traces of
water from the system. A solution of acetobromo-␣-d-glucuronic
acid methyl ester (5) (0.43 mmol, 170 mg) in toluene (2 ml)
was then added over 5 min to the reaction mixture, further
stirred and heated under azeotropic conditions for an addi-
tional 6 h. A mixture of inorganic salts was removed by filtration,
and the solvent was evaporated from the filtrate, affording a
dry residue. The product was purified by column chromatog-
raphy on silica gel: petroleum ether/ethyl acetate (60:40, v/v)
to give the methyl (androsta-1,4-dien-17␤-ol-3-one)-17␤-O-2,3,4-
tri-O-acetyl-␤-d-glucuronate (3a, white solid, 168 mg, 80%): mp
116–118 ◦C; [˛]D20 + 32.2 (c = 1.00, CHCl3); ESI-MS (positive) m/z:
625 (M + 23). C32H42O11 : calcd. C, 63.77; H, 7.02. Found. C, 63.84;
H, 7.08.
Optical rotations were measured on
a PerkinElmer 241
polarimeter (sodium D line at 25 ◦C). Mass spectra were recorded
on a Finnigan LCQdeca (TermoQuest) in ESI positive-ion mode, kV
5.00, 220 ◦C, 15 V. Mass spectral data are given as m/z (relative abun-
dance).
All reactions were monitored by TLC on silica gel 60 F254
precoated plates with a fluorescent indicator (Merck). Flash chro-
matography was performed using silica gel 60 (230–400 mesh,
Merck).
2.2. NMR experiments
NMR spectra were done on a Bruker AVANCE 500 spectrom-
eter equipped with a 5-mm broadband reverse probe with field
z-gradient operating at 500.13 and 125.76 MHz for 1H and 13C,
respectively. All NMR spectra were recorded at 298 K in DMSO-
d6 (isotopic enrichment 99.95%) or CDCl3 (isotopic enrichment
99.95%) solution and the chemical shifts were reported on a ı
(ppm) scale. The central peak of DMSO-d6 signals (2.50 ppm for
1H and 39.50 ppm for 13C) and of CDCl3 signals (7.26 ppm for 1H
and 77.7 ppm for 13C) were used as internal reference standard.
Acquisition parameters for 1D were as follows: 1H spectral width
of 5000 Hz and 32 K data points providing a digital resolution of
ca. 0.305 Hz per point, relaxation delay 2 s; 13C spectral width of
29412 Hz and 64 K data points providing a digital resolution of
ca. 0.898 Hz per point, relaxation delay 2.5 s. The experimental
error in the measured 1H–1H coupling constants was 0.5 Hz. The
2.3.2. Methyl (androsta-1,4-dien-17˛-ol-3-one)-17ˇ-O-2,3,4-tri-
O-acetyl-ˇ-d-glucuronate (4a)
2.3.2.1. Synthesis of the glucuronide donor: methyl-(2,3,4-tri-
O-acetyl-˛-d-glucopyranosyl-trichloroacetimidate)-glucuronate (7).
H2O (50 ␮l) and acetobromo-␣-d-glucuronic acid methyl ester (5)
(1.5 mmol, 600 mg) were added in sequence to a suspension of
CdCO3 (1.5 mmol, 500 mg) in acetonitrile. The reaction mixture was
stirred and heated to 70 ◦C for 1 h under nitrogen, filtered through
Celite, washed with 5 ml of acetonitrile, and concentrated in vacuo.

252
S. Casati et al. / Steroids 74 (2009) 250–255
Purification of the crude residue by flash chromatography on sil-
ica gel: ethyl acetate/hexane (50:50, v/v) afforded the hydrolyzed
product (white solid, 393 mg, 78%), 1-hydroxy-methyl 2,3,4-tri-O-
acetyl-␣/␤-d-glucopyranuronate that was subsequently dissolved
in dichloromethane (12 ml); trichloroacetonitrile (8 mmol, 1.1 g,
0.8 ml) was then added under nitrogen. After cooling to 0 ◦C, 1,8-
diazabicyclo [5.4.0] undec-7-ene (DBU, 0.2 mmol, 36 ␮l) was added.
The reaction mixture was allowed to stir for 1 h and then con-
centrated to afford a sticky, dark-brown residue. The product was
purified by flash chromatography on florisil: petroleum ether/ethyl
acetate (70:30, v/v) supplying compound 7 (off-white solid, 359 mg,
64%). 1H NMR (CDCl3, 500 MHz) ı 8.71 (s, 1H, NH), 6.66 (d, 1H, J1,2
3.6 Hz, H-1), 5.65 (dd, 1H, J3,2 9.8 Hz, J3,4 9.8 Hz, H-3), 5.29 (dd, 1H,
J4,3 9.8 Hz, J4,5 9.8 Hz, H-4), 5.17 (dd, 1H, J2,1 3.6 Hz, J2,1 9.8 Hz, H-
2), 4.52 (d, 1H, J5,4 9.8 Hz, H-5), 3.77 (s, 3H, COOMe), 2.08 (s, 3H,
OAc), 2.07 (s, 3H, OAc), 2.04 (s, 3H, OAc). 13C NMR ı 169.78 (CO),
169.73 (CO), 169.48 (CO), 167.15 (CO), 160.59 (CN), 92.63 (C(Cl3)),
70.49 (C-5), 69.47 (C-1), 69.10 (C-3), 68.96 (C-4), 53.06 (COOCH3),
20.68 (OCOCH3), 20.50 (OCOCH3), 20.42 (OCOCH3).
2.3.4.1. Androsta-1,4-dien-17ˇ-ol-3-one-17ˇ-O-glucuronide
(3b).
The product was purified by chromatography on silica gel column:
dichloromethane/methanol/water (70:27:3, v/v/v) (3b, white solid,
110 mg, 97%): mp 173–174 ◦C; [˛]D20 + 6.2 (c = 1.00, CHCl3); ESI-MS
(negative) m/z: 461 (M-1), 923 (M + M-1). C25H34O8: calcd. C,
64.92; H, 7.41. Found. C, 65.04; H, 7.28.
2.3.4.2. Androsta-1,4-dien-17˛-ol-3-one-17ˇ-O-glucuronide
(4b).
The product was purified by chromatography on silica gel:
dichloromethane/methanol/water (70:27:3, v/v/v) (4b, white solid,
109 mg, 95%): mp 188–190 ◦C; [˛]D20 − 6.3 (c = 1.00, CHCl3); ESI-MS
(negative) m/z: 461 (M-1), 923 (M + M-1). C25H34O8 calcd. C, 64.92;
H, 7.41. Found. C, 64.81; H, 7.59.
3. Results and discussion
3.1. Chemistry
In the synthesis of 17␣- and 17␤-boldenone 17-glucopyrano-
siduronic acid derivatives (referred to as glucuronides) (3b, 4b), the
starting compounds were 17␤-boldenone (1) and its 17␣-derivative
2, which had been synthesized earlier from 17␤-boldenone by a
modified Mitsunobu reaction [10].
The synthesis of glucuronides is most frequently carried
out via the Koenigs–Knorr reaction or its modifications [12] in
which the electrophilic character of the anomeric bromide 5
is enhanced by a halophilic agent such as cadmium carbon-
ate. The main drawbacks of these procedures are the instability
of the bromo derivatives [13], the need for elevated reaction
temperatures and frequently prolonged reaction times. These
events, moreover, could lead to undesirable modifications of agly-
cones.
When this reaction was applied to 17␤-boldenone (1) the cor-
responding glucuronide 3a was obtained in 80% yields. Instead
when the same reaction was applied to the glucuronidation of 17␣-
boldenone (2) byproducts were mainly obtained. A TLC separation
of the reaction mixture revealed a lot of spots, one of which cor-
responded to the unreacted sterol and one more was found to be
glucuronide 4a. The same difficulties, probably due to steric hin-
drance of 18-methyl group on the 17␣ position, have been also
reported by Bowers and Bowers [14] in the synthesis of epitestos-
terone glucuronide.
2.3.2.2. Synthesis of 17˛-boldenone-17-ˇglucuronide (4a). A solu-
tion of methyl-(2,3,4-tri-O-acetyl-␣-d-glucopyranosyl-trichloro-
acetimidate)-glucuronate (7) (0.9 mmol, 400 mg) in dry
dichloromethane (3 ml) was slowly added to
a solution of
17␣-boldenone (2) (0.35 mmol, 100 mg) in dry dichloromethane
(3 ml) and trimethylsilyl trifluoromethanesulfonate (TMSOTf,
100 ␮l of a solution 0.06 M) under nitrogen at −15 ◦C. After 48 h the
reaction was quenched with triethylamine. Solvent evaporation
under reduced pressure, and purification by flash chromatogra-
phy on silica gel: dichloromethane/acetone (95:5, v/v) afforded
the methyl (androsta-1,4-dien-17␣-ol-3-one)-17␤-O-2,3,4-tri-
O-acetyl-␤-d-glucuronate (4a, white solid, 143 mg, 68%): mp
124–125 ◦C; [˛]D20 − 0.5 (c = 1.00, CHCl3); ESI-MS (positive) m/z:
625 (M + 23). C32H42O11 : calcd. C, 63.77; H, 7.02. Found. C, 63.64;
H, 7.18.
2.3.3. Methyl (androsta-1,4-dien-17˛-ol-3-one)-17ˇ-O-cyclic-
1,2-(hydrogen-orthoacetyl-O-3,4-di-acetyl)-ˇ-d-glucuronate (8)
100 ␮l of a solution 0,06 M of trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) in dry dichloromethane were added in
three portions to a solution of 17␣-boldenone (2) (0.35 mmol,
100 mg)
and
methyl-(2,3,4-tri-O-acetyl-␣-d-glucopyranosyl-
trichloroacetimidate)-glucuronate (7) (0.9 mmol, 400 mg) in dry
dichloromethane (5 ml) under nitrogen at 0 ◦C. After usual work-up,
methyl(androsta-1,4-dien-17␣-ol-3-one)-17␤-O-cyclic1,2-
(hydrogen-orthoacetyl-O-3,4-di-acetyl)-␤-d-glucuronate (8, white
solid, 100 mg; 48%) was obtained. mp 103–105 ◦C; [˛]D20 + 18.5
(c = 1.00, CHCl3).
These difficulties were circumvented by modifying the leav-
ing group. The conversion of bromide
5 to the imidate 7,
followed by the activation through TMSOTf, allowed stere-
ospecific reactions to obtain the corresponding ␤-glucuronides
(Fig. 2).
The glucuronic acid imidate donor 7 was synthesized in 50%
yield in two steps from the commercially available acetobromo-␣-
d-glucuronic acid methyl ester (5). In turn, 5 was converted to its
corresponding free emiacetal 6 by using CdCO3/H2O, subsequently
converted to the ␣-imidate derivative 7 through trichloroace-
tonitrile and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) [15].
2.3.4. General procedure for hydrolysis of acetylated glucuronides
NaOH 1% in methanol was slowly added to a solution of glu-
curonides 3a or 4a in methanol (150 mg, 0.25 mmol). After 24 h
the solution was neutralized with dowex 50WX8-200, filtered and
evaporated.
Reaction of 17␣-boldenone (2) and imidate
7 under stan-
Fig. 2. Synthesis of imidate donor 7. Reagents and conditions: (a) CdCO3, H2O (2 equiv.), CH3CN, 70 ◦C, 1 h, yield = 78% and (b) CCl3CN, 1,2-dichloroethane, 1,8-DBU, 0 ◦C, 1 h,
yield = 64%.

S. Casati et al. / Steroids 74 (2009) 250–255
253
Fig. 3. Structural formula of the orthoester 8 and the transacylation derivative 9.
dard conditions (dichloromethane, TMSOTf, 0 ◦C) afforded almost
exclusively the orthoester 8 together with the transacylation
derivative 9 (Fig. 3).
catalyst led to minimal contact between the imidate donor and the
catalyst, thereby reducing decomposition and side reactions of the
imidate.
Structure of the orthoester 8 was unequivocally established by
NMR techniques (Table 1).
Our findings confirm that both the use of imidate and the inverse
addition of the reagent gave advantages for the formation of glu-
curonide 4a.
The following deprotection of glucuronides 3a and 4a with 1%
NaOH in methanol afforded the target compounds 3b (97%) and 4b
(95%).
However, we observed that when imidate 7 was added slowly
at −15 ◦C to a mixture of ␣-boldenone 2 and TMSOTf, rather than
adding TMSOTf to premixed 2 and 7 in dichloromethane, the con-
jugate 4a was isolated in 68% yield after chromatography.
Our findings agree with other authors [16,17] who demonstrated
that the reaction was subjected to a profound order-of-addition
effects.
3.2. NMR spectroscopy
Schmidt and Toepfer [18] considered that, in the inverse-
addition mode, prior complexation of acceptor component and
As the resulting steroid substrates 17␣- and 17␤-boldenone
contain only one possible site for glucuronidation there was no
Table 1
1H NMR chemical shifts (ppm) assignment for compounds 3a, 3b, 4a, 4b and 8.
3a CDCl3
3b DMSO
4a CDCl3
4b DMSO
8 CDCl3
Aglycon moiety
1
2
4
6 ␣
6 ␤
7 ␣
7 ␤
8 (␤)
9 (␣)
11 ␣
11 ␤
12 ␣
12 ␤
14 (␣)
15 ␣
15 ␤
16 ␣
16 ␤
17 ␣
17 ␤
18-CH3
19-CH3
7.06
6.25
6.09
2.38
2.48
0.95
1.96
1.68
1.04
1.73
1.61
1.17
1.83
0.91
1.55
1.31
2.09
1.65
3.58
–
7.20
6.12
5.89
2.32
2.47
0.91
1.85
1.70
0.95
1.69
1.62
1.13
1.92
0.93
1.66
1.25
1.87
1.49
3.57
–
7.09
6.26
6.09
2.37
2.48
1.11
1.55
1.64
1.09
1.78
1.66
1.11
1.96
1.11
1.54
1.31
1.98
1.57
–
7.26
6.11
5.98
2.33
2.49
1.48
1.65
1.71
0.99
1.75
1.63
1.00
1.98
1.43
1.63
1.24
1.90
1.63
–
7.08
6.25
6.08
2.38
2.48
1.50
1.61
1.59
1.14
1.78
1.68
1.13
1.98
1.36
1.63
1.20
1.61
1.47
–
3.81
0.78
1.25
3.76
0.73
1.21
3.68
0.78
1.25
0.81
1.25
0.82
1.20
Sugar moiety
1ꢀ
4.58
5.03
5.26
5.22
4.01
3.77
4.23
2.97
3.15
3.30
3.55
4.54
4.95
5.28–5.22
4.03
2.86
3.05
3.11
3.08
5.80
4.36
5.23
5.16
4.31
3.78
2ꢀ
3ꢀ
4ꢀ
5ꢀ
4.01
3.77
COOCH3
OCOCH3
2.07
2.04
2.03
2.04
2.04
2.02
2.12
2.11
a
OCOCH3
1.71
a
Orthoester.

254
S. Casati et al. / Steroids 74 (2009) 250–255
Table 2
13 C NMR chemical shifts (ppm) assignment for compounds 3a, 3b, 4a, 4b and 8.
3a CDCl3
3b DMSO
4a CDCl3
4b DMSO
8 CDCl3
Aglycon moiety
1
2
3
4
5
6
7
8
155.70
127.55
186.33
124.00
167.20
32.72
33.08
35.29
52.41
43.55
22.45
36.99
43.18
49.80
23.51
28.56
89.74
11.66
18.69
157.00
127.18
185.46
123.51
170.00
32.40
33.30
35.18
52.51
43.71
22.38
36.79
43.28
49.66
23.44
28.89
87.93
11.89
18.95
155.82
127.52
186.39
123.96
169.38
32.89
31.21
35.79
51.76
45.14
22.30
33.84
43.57
48.68
24.77
28.86
85.83
16.91
18.83
157.10
127.13
185.44
123.44
170.15
32.59
31.66
35.66
52.64
44.89
22.44
34.44
43.78
48.59
24.96
28.73
83.90
17.40
18.95
155.76
127.54
186.28
123.88
169.34
32.87
31.69
35.88
51.86
45.20
22.64
33.79
43.56
48.74
24.90
31.34
81.01
17.30
18.74
9
10
11
12
13
14
15
16
17
18
19
Sugar moiety
1ꢀ
101.41
71.43
72.87
69.47
72.50
–
104.05
73.84
76.11
71.99
76.57
170.92
–
98.51
71.32
72.07
69.50
72.64
–
100.52
73.86
74.35
72.68
77.38
173.15
–
96.03
73.40
69.09
68.67
68.25
–
2ꢀ
3ꢀ
4ꢀ
5ꢀ
COOH
COOCH3
COOCH3
170.20
52.84
170.23
52.81
169.29
52.72
–
–
OCOCH3
169.36
169.03
169.03
–
169.38
168.95
168.88
–
169.02
168.89
–
OCOCH3
20.72
20.64
20.51
20.68
20.58
20.53
20.79
20.70
a
OCOCH3
OCOCH3
122.61
22.37
a
a
Orthoester.
ambiguity about the position of the glycosidic linkage in the cor-
responding glucuronides.
at the anomeric centre of the carbohydrate conjugate is typically
based on the magnitude of the vicinal J1,2 coupling constants for the
anomeric proton. The magnitude of the coupling constant (Table 3)
is characteristic of a diaxial configuration for 1ꢀ-H and 2ꢀ-H protons
on the Karplus plot. Thus, a ␤-conformation can be assigned to the
anomeric centre on the glucuronic acid for each glucuronide.
Integration of the signals provided a 1:1 glucuronic acid/
aglycone ratio indicating the correct stoichiometry.
Confirmation of the anomericity of the glucuronic acid moi-
ety in the steroid glucuronides and of the stereochemistry of the
17-hydroxyl function was obtained by one-dimensional 1H NMR.
Unambiguous assignments of the carbohydrate and steroid pro-
tons of the compounds 3a, 3b, 4a, 4b and 8 were obtained using
1D and 2D NMR spectra (Tables 1 and 2). Table 3 reports the cou-
pling constants of the sugar moiety since the coupling constants of
the steroid moiety have already been shown [19].
The 13C NMR spectral data also showed that glucuronide 3a con-
tained one anomeric carbon at ı 101.41 (C-1) that correlated to the
proton signal at 4.58 (1H, d, J = 7.7 Hz) and that the compound 4a
contained one anomeric carbon at ı 98.51 (C-1) that correlated to
the proton signal at 4.54 (1H, d, J = 7.7 Hz) indicating the presence
of one sugar unit in the ␤-form.
Each glucuronide spectrum contained the five methine signals
arising from the glucuronic acid. Assignment of the configuration
In 17␣- and 17␤-boldenone 17-glucuronides (3a, 3b and 4a, 4b),
17-H resonates downfield to the steroid bulk region and can be
distinguished from each other protons due also to their different
coupling patterns. In compounds of ␤ series 17␣-H signal of steroid
aglycone (ı 3.58 for 3a and ı 3.57 for 3b) was a double doublet of the
same coupling constant with H-16␣ and H-16␤ (8.6 Hz). Otherwise,
in compounds of ␣ series (4a and 4b) the 17␤-H signal at ı 3.81 for
4a and at ı 3.76 for 4b was a broad doublet resulting from a cou-
pling constant of 6.0 Hz with H-16␤ and a small coupling constant
(<1 Hz) between H-17␤ and H-16␣, due to dihedral angle close to
90◦ between them. These NMR data confirmed that no isomeriza-
tion at 17-position took place either in glucuronidation or in the
saponification reactions.
Table 3
J(H,H) (Hz) for compounds 3a, 3b, 4a, 4b and 8.
JH,H
3a CDCl3
3b DMSO
4a CDCl3
4b CDCl3
8 CDCl3
Aglycon moiety
17␣, 16␣
17␣, 16␤
17␤, 16␣
17␤, 16␤
8.6
8.6
–
8.6
8.6
–
–
–
<1
5.4
–
–
<1
5.4
<1
5.4
–
–
Sugar moiety
1ꢀ, 2ꢀ
7.7
8.6
8.6
9.2
7.7
8.6
8.6
9.2
7.7
8.6
7.7
8.6
8.0
9.5
4.7
2.7
2.7
8.3
2ꢀ, 3ꢀ
3ꢀ, 4ꢀ
Not detectable
9.5
4ꢀ, 5ꢀ

S. Casati et al. / Steroids 74 (2009) 250–255
255
The orthoacetate 8 can be easily detected by its NMR spectra
which show a signal for the methyl group of CCH3 at ı 1.71 ppm
and the orthoester C at ı 122.71 ppm.
Additionally compound 8 has H-1 resonance less shielded than
in compound 4a (4.54 ppm versus 5.80 ppm), whereas H-2 reso-
nance of orthoester 8, is more shielded than the same signal in the
glucuronide 4a (4.95 ppm versus 4.36 ppm). Finally compound 8
shows the coupling constant between H-1 and H-2 (4.7 Hz) smaller
than that of compound 4a (7.7 Hz).
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The abundant natrium adduct ion [M + Na]+ was detected in pos-
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In negative ion ESI an intensive deprotonated molecule [M − H]−
was observed as the base peak for glucuronides 3b and 4b. In nega-
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carboxylic acid moiety of the glucuronic acid, as the steroid agly-
cones were not ionized in negative ion ESI at all and gave evidence
on the corrected molecule weights of the synthesized steroid glu-
curonides. Either the positive or the negative ion MS spectra clearly
indicate the presence of glucuronide moiety.
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Acknowledgement
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This work was financially supported by Università degli Studi di
Milano (Fondi FIRST).
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