Regio- and stereoselective reductions of dehydrocholic acid

Article abstract of DOI:10.1016/j.steroids.2006.01.004

Dehydrocholic acid (DHCA), an unnatural bile acid, is manufactured by oxidation of cholic acid. Its biotransformation by two basidiomycetes (Trametes hirsuta and Collybia velutipes) is reported. These mycelia showed different affinities for the substrate and selectivities of attack: T. hirsuta in particular regio- and stereoselectively reduced the 3-keto group to yield 3α-hydroxy-7,12-diketo-5β-cholan-24-oic acid (7,12-diketolithocolic acid) as the main product. A number of different chemical reductions were carried out on DHCA; among them hydrogenation with Raney Nickel in water under high-intensity ultrasound proved highly regio- and stereoselective, yielding 7,12-diketolithocolic acid exclusively. ¹H and ¹³C resonances were assigned in details thanks to a series of 1D and 2D NMR runs including DEPT, NOESY, H-H COSY, gHSQC and gHMBC.

Full text of DOI:10.1016/j.steroids.2006.01.004

steroids 7 1 ( 2 0 0 6 ) 469–475
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Regio- and stereoselective reductions of dehydrocholic acid
Giancarlo Cravottoa,∗, Arianna Binelloa, Luisa Boffaa,
Ornelio Rosatib, Marco Boccalinic, Stefano Chimichic,∗
a
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita di Torino, via P. Giuria 9, 10125 Torino, Italy
b
c
`
Dipartimento di Chimica e Tecnologia del Farmaco, Universita di Perugia, via del Liceo, 06123 Perugia, Italy
Dipartimento di Chimica Organica, Universita di Firenze, via della Lastruccia 13, 50019 Sesto F.no (FI), Italy
`
a r t i c l e i n f o
a b s t r a c t
Article history:
Dehydrocholic acid (DHCA), an unnatural bile acid, is manufactured by oxidation of cholic
acid. Its biotransformation by two basidiomycetes (Trametes hirsuta and Collybia velutipes)
is reported. These mycelia showed different affinities for the substrate and selectivities of
attack: T. hirsuta in particular regio- and stereoselectively reduced the 3-keto group to yield
3␣-hydroxy-7,12-diketo-5␤-cholan-24-oic acid (7,12-diketolithocolic acid) as the main prod-
uct. A number of different chemical reductions were carried out on DHCA; among them
hydrogenation with Raney Nickel in water under high-intensity ultrasound proved highly
regio- and stereoselective, yielding 7,12-diketolithocolic acid exclusively. 1H and 13C reso-
nances were assigned in details thanks to a series of 1D and 2D NMR runs including DEPT,
NOESY, H–H COSY, gHSQC and gHMBC.
Received 7 December 2005
Received in revised form 14 January
2006
Accepted 16 January 2006
Published on line 28 February 2006
Keywords:
Basidiomycetes
Biotransformation
Dehydrocholic acid
Reduction
© 2006 Elsevier Inc. All rights reserved.
Raney nickel
13C NMR
1.
Introduction
also be recovered from complex mixtures of bile acids (left-
overs from the industrial production of ursodeoxycholic acid)
Cholic acids and their derivatives hold a great interest for
pharmaceutical industry owing to their numerous therapeu-
tic employs, as in cholestatic liver disease and for dissolving
cholesterol gallstones [1], to protect hepatocytes and other cell
types from apoptosis [2–4] and as antimicrobials [5,6]. With
the aim to increase the solubility of cholesterol in the bile
a new family of compounds, fatty-acid/bile acid conjugates
(FABACs), has recently been synthesized [7–9]. As they are able
to prevent the formation of cholesterol gallstones in hamsters
and mice, they hold a potential for use in humans.
that would otherwise be useless. DHCA finds therapeutic
applications as a cholagogue, hydrocholeretic, diuretic, lax-
ative and as a diagnostic aid. Within 6–12 h of oral adminis-
tration it acts on the smooth muscle of the intestinal wall to
cause a vigorous bowel movement. Besides this laxative effect,
it increases the water content and volume of bile. Unlike natu-
ral bile acids and their conjugates, DHCA does not readily form
with fats and phospholipids the micelles that play an essential
role in the absorption of these nutrients.
Considering that DHCA can easily be obtained at low
cost, we investigated the possibility of modifying its structure
to yield pharmacologically interesting derivatives. Published
work has shown that the biotransformation of bile acids has a
potential as an aid to synthesis [11–15]. This method has been
Dehydrocholic acid (DHCA) (1) [10] is usually obtained
from cholic acid by oxidation of the C-3, C-7 and C-12
hydroxy groups, a process carried out industrially with sodium
hypochlorite under heating. In this way a pure product can
∗
Corresponding authors. Tel.: +39 011 6707684; fax: +39 011 6707687.
E-mail address: giancarlo.cravotto@unito.it (G. Cravotto).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.01.004

470
steroids 7 1 ( 2 0 0 6 ) 469–475
spectra were recorded at 399.95 MHz for 1H, and at 100.58 MHz
for 13C with a Varian Mercuryplus 400 instrument in pyridine-
d5, CDCl3 or CD3OD solutions; chemical shifts (ı) are reported
in ppm from tetramethylsilane as secondary reference stan-
dard, coupling constants in Hz. Low-resolution mass spectra
(LRMS) were taken on a Finnigan-MAT TSQ70 (chemical ion-
ization) with isobutane as the reactant gas. Gas chromato-
graphic analyses were performed on a Shimadzu GC-14B chro-
matograph equipped with a flame ionization detector. MW-
promoted reactions were carried out in a MILESTONE ATC-
FO 300 MicroSYNTH oven. Sonochemical reactions were per-
formed in a high-power reactor developed in the authors’ lab-
oratory (frequency 19 kHz) [22].
employed to prepare a number of useful cholic acid deriva-
tives [16,17], some of which are impossible or very difficult to
obtain by ordinary chemical reactions.
For the present study we chose the basidiomycetes Tram-
etes hirsuta and Collybia velutipes that in our hands had afforded
some interesting regio- and stereoselective modifications of
terpene hydrocarbons [18]. In order to ascertain whether bio-
catalysis would yield DHCA derivatives that cannot be easily
obtained by chemical synthesis, we also carried out several
chemical reductions on DHCA. Methods employed were cat-
alytic hydrogenation (Pd/C, Raney Nickel) and reduction with
NaBH4 [19] or with HCOO− NH4 under microwaves (MW)
+
[20,21].
2.2.
Mycelia
2.
Experimental
The basidiomycetes selected for the present study belong
to the following families: Tricholomataceae (C. velutipes) and
Polyporaceae (T. hirsuta). Mycelia, obtained from the Depart-
ment of Plant Biology, University of Turin, Italy, are registered
by MUT (Mycotheca Universitatis Taurinensis) with the follow-
ing MUT accession numbers: C. velutipes 3638, T. hirsuta 3639.
2.1.
General procedures
DHCA was provided by PCA S.p.A. (Prodotti Chimici Alimentari,
Basaluzzo, AL, Italy).
Other reagents and solvents were from Carlo Erba Reagenti
and Acros Organics. Reactions were monitored by TLC on
Fluka F254 0.25 mm plates, which were visualized by spraying
with molybdic acid and heating. For column chromatography
silica gel 60 Merck was used.
2.3.
Culture, fermentation and incubation procedure
Mycelia were grown on Petri dishes for 14 days at 24 ◦C to pro-
vide precultures. The culture medium contained 20 g glucose,
20 g malt extract and 20 g agar (Merck) per litre of distilled
water. pH was not adjusted.
300-ml Erlenmeyer flasks containing 100 ml of liquid cul-
ture medium (20 g/l glucose and 20 g/l malt extract) were inoc-
ulated with fragmented disks (16 mm in diameter) of mycelia
taken from 7-day old precultures.
IR spectra were recorded on
a Shimadzu FT-IR 8001
spectrophotometer. 1H NMR, 13C NMR, DEPT (distortionless
enhancement by polarisation transfer), 1H–1H COSY (corre-
lation spectroscopy), NOESY 1D (nuclear Overhauser effect
spectroscopy), gHSQC (heteronuclear single-quantum corre-
lation), and gHMBC (heteronuclear multiple bond correlation)
Scheme 1 – Bioelaboration of DHCA (1) by Trametes hirsuta (2, 60%) and Collybia velutipes (2, 40% and 3, 20%).

steroids 7 1 ( 2 0 0 6 ) 469–475
471
In order to isolate fungal metabolites in sufficient amounts
for identification (at least 15 mg), ten 300-ml Erlenmeyer flasks
containing fragmented mycelia suspended in 100 ml of liquid
medium were inoculated with 50 mg of substrate and kept at
24 ◦C for 7 days on a rotary shaker (Dubnoff BSD) at 90 rpm.
Better yields were obtained in this way rather than using one
1-l Erlenmeyer flask. As DHCA (1) is poorly soluble in water,
its sodium salt was used for better bioavailability. Each exper-
iment was repeated three times. Blanks without mycelium
were run in parallel to rule out the occurrence of sponta-
neous chemical transformations. Substrate toxicity was eval-
uated for each mycelium by monitoring the variation of fungal
biomass during incubations. Dry weight determinations on 7-
day-old cultures showed that mycelial growth was not affected
by the presence of DHCA sodium salt (Scheme 1).
or CHCl3), and NaBH4 (56.5 mg, 1.49 mmol) was added. The
reaction was magnetically stirred for 10 h at room tempera-
ture and monitored by TLC using CHCl3/MeOH (9:1) as elu-
ent. The reacted mixture was diluted with H2O, acidified with
0.5 N HCl and extracted with CHCl3. The organic phase was
washed with H2O and brine, dried over anhydrous Na2SO4
and evaporated to dryness. Products were purified by col-
umn chromatography using a CHCl3/MeOH elution gradient
(99:1 → 8:2).
2.6.
Hydrogenation of DHCA in PARR reactor
In the reaction vessel (160 ml) of a PARR reactor a solu-
tion of DHCA (200 mg, 0.50 mmol) in MeOH (30 ml) and Pd/C
10% (50 mg) were added. The reaction was carried under
pressure (H2, 30 bar) and vigorous stirring at 105 ◦C for 15 h.
The reaction was monitored by TLC, eluent CHCl3/MeOH
(9:1). Crude product was purified by column chromatography
(gradient 99:1 → 8:2), beside the recovered starting material
(94 mg) a diastereomeric mixture of 2 (12%) and 3 (11%) was
isolated.
2.4.
Extraction and isolation of metabolites
At the end of incubations, cultures were filtered and filtrates
were centrifuged in the cold (7000 rpm for 20 min at 5 ◦C),
then acidified with HCl 1 N and extracted with CHCl3. The
solvent was evaporated to yield crude extracts that were chro-
matographed on a silica gel column with a CHCl3/MeOH gradi-
ent. Elution was monitored by TLC, eluent CHCl3/MeOH (9:1).
2.7.
Reduction of DHCA under MW
2.4.1. 3˛-Hydroxy-7,12-diketo-5ˇ-cholan-24-oic acid
(7,12-diketolithocholic acid) (2)
In a 50-ml two-necked round-bottomed flask DHCA (0.78 g,
1.94 mmol), HCOO− NH4+(0.7 g, 11.10 mmol) and a catalytic
amount of Pd/C were added to a mixture of CH2Cl2 (10 ml)
and propylene glycol (5 ml). The mixture was refluxed 1.5 h
under MW irradiation (50 W). The reaction was monitored by
TLC (CHCl3/MeOH 8:2). The reacted mixture was filtered on
a celite pad over a sintered-glass funnel (G3) and evaporated
to dryness. To improve extraction yields the crude product
was subjected to Fischer esterification by heating under reflux
(3 h) with absolute EtOH and a catalytic p-toluenesulfonic acid,
then transferred to a separatory funnel and extracted with
CHCl3. The organic phase was washed with H2O and saturated
brine, dried over anhydrous Na2SO4 and evaporated to dryness
yielding 72 mg of ethyl cholate (85%).
White solid; mp: 178–180 ◦C (Lit. mp: 185–187 ◦C) [23]; IR (KBr)
3350 (OH), 2950, 2864, 1728 (COOH), 1702 (CO), 1699 (CO), 1333,
1263, 1186, 1051, 802 cm−1 1H NMR (400 MHz, pyridine-d5): ı
;
3.77 (m, 1H, H-3), 2.93 (dd, J = 12.6, 5.5 Hz, 1H, H-6␣), 2.90 (pt,
J = 12.0 Hz, 1H, H-8), 2.70 (pt, J = 12.6 Hz, 1H, H-11␣), 2.65 (m, 1H,
H-23), 2.54 (m, 1H, H-23), 2.45 (m, 1H, H-15), 2.37 (m, 1H, H-9),
2.19 (m, 1H, H-17), 2.18 (dd, J = 12.6, 1.8, 1H, H-11␣), 2.12 (m, 1H,
H-22), 2.04 (dd, J = 12.6, 2.2 Hz, 1H, H-6␣), 1.95 (m, 1H, H-16), 1.91
(m, 1H, H-4␤), 1.82 (m, 1H, H-5), 1.81 (m, 2H, H-14 and H-2␤),
1.64 (m, 1H, H-1␣), 1.60 (m, 1H, H-22), 1.56 (m, 1H, H-4␣), 1.46
(m, 1H, H-2␣), 1.44 (m, 1H, H-20), 1.34 (m, 1H, H-16), 1.26 (m, 1H,
H-15), 1.23 (s, 3H, Me-19), 1.13 (m, 1H, H-1␤), 1.08 (d, J = 6.8 Hz,
3H, Me-21), 1.00 (s, 3H, Me-18); 13C NMR (100.58 MHz, pyridine-
d5): ı 212.21 (C12), 209.61 (C7), 176.48 (C24), 69.96 (C3), 57.09
(C13), 52.25 (C14), 48.98 (C8), 46.22 (C17), 45.82 (C9), 45.79 (C6),
45.53 (C5), 38.67 (C11), 38.32 (C4), 36.09 (C10), 36.03 (C20), 34.52
(C1), 32.24 (C23), 31.46 (C22), 30.60 (C2), 28.00 (C16), 25.40 (C15),
22.48 (C19), 19.21 (C21), 11.88 (C18). CIMS (m/z): 405 (M + H)+,
387 (M + H)+-H2O.
2.8.
Hydrogenation of DHCA sodium salt with Raney
Nickel
In a 100-ml two-necked round-bottomed flask equipped with
a gas inlet, 100 mg of sodium dehydrocholate were dissolved
in 25 ml of water (resulting pH = 8.8), Raney Nickel (20 mg) was
added and the mixture was vigorously stirred under H2 atmo-
sphere (gummy balloon) at 30 ◦C for 20 h. Under high-intensity
ultrasound at the same temperature the reaction time was
cut down to 2 h. The reaction was performed under H2 atmo-
sphere in a 1-mm thick Teflon® tube inserted in a sonochemi-
cal reactor (19 kHz, 70 W) thermostatted by two Peltier mod-
ules and was monitored by TLC, eluent CHCl3/CH3OH 9:1.
The reacted mixture was filtered, acidified with HCl 1 N and
extracted with CHCl3. The organic layer was washed with sat-
urated brine, dried over anhydrous Na2SO4 and evaporated
to dryness. The only product isolated by column chromatog-
raphy (CHCl3/CH3OH 97:3) was 3␣-hydroxy-7,12-diketocholic
acid (2) (76 mg, yield = 80%).
2.4.2. 3ˇ-Hydroxy-7,12-diketo-5ˇ-cholan-24-oic acid (3)
White solid; mp 260–262 ◦C (Lit. mp: 270–272 ◦C) [24]; IR (KBr)
3400 (OH), 2932, 1728 (COOH), 1708 (CO), 1698 (CO), 1541, 1385,
1103, 1087, 914, 731 cm−1; 1H NMR (400 MHz, pyridine-d5): ı 4.13
(m, 1H, H-3), 1.29 (s, 3H, 19-Me), 1.06 (d, J = 8.8 Hz, 3H, 21-Me),
1.02 (s, 3H, 18-Me); 13C NMR (100,58 MHz, pyridine-d5): ı 212.68
(C12), 210.21 (C7), 176.48 (C24), 65.10 (C3), 23.09 (C19), 19.23
(C21), 11.92 (C18 ). CIMS (m/z): 405 (M + H)+, 387 (M + H)+-H2O.
2.5.
NaBH4 reductions of DHCA (general procedure)
In a 50-ml round-bottomed flask DHCA (200 mg, 0.50 mmol)
was dissolved in a sufficient amount of solvent (MeOH, EtOH

472
steroids 7 1 ( 2 0 0 6 ) 469–475
2.9.
Derivatization and GLC analyses
3.
Results and discussion
Esterification (methyl ester): 50 mg of bile acid were dis-
solved in methanol (1 ml), then methansulfonic acid (80 ␮l)
and anhydrous Na2SO4 were added. The mixture was irra-
diated with MW for 2 min at 50 W. The reacted mixture was
diluted with H2O, brought to alkaline pH with 2 N NaOH and
extracted with CHCl3. The organic layer was washed with sat-
urated brine, dried over anhydrous Na2SO4 and evaporated
to dryness. All the methyl esters were obtained in high yield
(90–96%).
Silylation: 1 mg of bile acid methyl ester was dis-
solved in CHCl3 (500 ␮l) and an excess of N-methyl-N-
trimethylsilyltrifluoroacetamide (3 eq.) was added. The mix-
ture was stirred about 1 h at 60 ◦C, the reaction being moni-
tored by TLC, eluent CHCl3/CH3OH mixtures.
The products were used for GC analysis without any further
purification.
Analyses: the derivatized samples were analyzed by GLC on
fused capillary column as previously described by Lepercq et
al. [12]. A pool of standards was chromatographed for products
identification and quantification.
Biotransformation results for DHCA sodium salt (Scheme 1)
showed that both mycelia were able to reduce the keto groups,
particularly at the position 3. T. hirsuta afforded 3␣-hydroxy-
7,12-diketo-5␤-cholan-24-oic acid (7,12-diketolitocholic acid)
(2) as main product (about 60% yield). C. velutipes showed a
lesser stereospecificity (40% yield for 2), also producing 3␤-
hydroxy-7,12-diketo-5␤-cholan-24-oic acid (3) in 20% yield.
Bioelaboration by C. velutipes led to other products of higher
polarity than DHCA, but their amounts were much too low to
allow identification.
Results from chemical reductions were very different
(Scheme 2). As previously reported by Fantin et al. [19],
NaBH4 in MeOH afforded a mixture of 7␣,12␣-dihydroxy-3-
keto-5␤-cholan-24-oic acid (4a) and 7␣,12␤-dihydroxy-3-keto-
5␤-cholan-24-oic acid (4b). Using ethanol as solvent we found
lesser yields of these compounds while the major product was
cholic acid (5), as observed when working in CHCl3.
The reduction of DHCA with HCOO− NH4+and catalytic
Pd/C under MW irradiation [21] rapidly converted it to cholic
Scheme 2 – Chemical reductions of DHCA.
Table 1 – Chemical reduction of DHCA (1) in different conditions
Entry
Reducing agent and conditions
Time (h)
Yield (%)
2
4a
4b
5
I
II
III
IV
V
NaBH4, MeOH, Stirring rt
NaBH4, EtOH, Stirring rt
NaBH4, CHCl3, Stirring rt
Pd/C, MeOH, Parr reactor, 105 ◦C
HCOO− NH4+, CH2Cl2/prop. glycol, MW 50 W rfx
Raney Nickel, H2O, US 70 W 30 ◦C
10
10
10
15
1.5
2
–
–
–
12a
–
80
70
23
19
–
–
–
20
15
–
–
–
–
42
61
–
85
–
VI
–
a
In mixture with 3 (11%).

steroids 7 1 ( 2 0 0 6 ) 469–475
473
acid. Identification of products was done by comparison with
published data. Yields and reaction times are listed in Table 1.
It is remarkable that hydrogenation of DHCA with Ni/Raney in
water proved highly regio- and stereoselective affording 2 in
high yield.
Although DHCA is a starting material for the synthesis of
5␤-cholanic acid derivatives, no 1H NMR data for this com-
pound exist in the literature with the exception of a single
reference for carbon-13 assignments in CDCl3 [25]. Thus, we
proceeded to study the proton and carbon NMR spectra in two
solvents (CDCl3 and, more conveniently, pyridine-d5); obvi-
ously methanol could not be used because of the reactivity
of DHCA.
As regards the 7- and 3-C O carbon atoms, we noticed that
whereas the signal at higher frequency (ı 209.12 ppm) showed
correlations to protons belonging to a CH2 and a CH group,
the signal at ı 208.86 ppm was connected to two CH2 groups.
On this basis the resonances could be attributed to the car-
bonyl groups at positions 7 and 3, respectively. This attribution
was confirmed by the connectivity between the 7-C O and H-
8, easily recognized from its correlation with the previously
assigned C-11.
Once enabled to ascertain the regiochemistry of the reduc-
tion reaction, we turned to its stereoselectivity. To this end,
NOE experiments were carried out irradiating the new 3-, or
7- or 12-H (easily recognizable in the 1H NMR spectrum); they
allowed the distinction between ␣ and ␤ epimers of a monore-
duced compound by preventive identification of H-5 (for 3-OH)
or H-8 (for 7- or 12-OH).
For example, if H-8 has been previously identified in com-
pound 1, H-5 can be unambiguosly assigned from the gHMBC
experiment, as C-5 (CH unit from DEPT spectrum) will show
correlations (see Table 2) to both 19-Me and 6-CH2, thus
allowing the attribution. The same holds for the monore-
duced compounds 2 and 3. On these grounds we found that
reduction of DHCA sodium salt with T. hirsuta afforded 7,12-
diketolithocholic acid (2) whose stereochemistry (3␣-OH) was
confirmed by NOE experiments carried out in CDCl3, CD3OD
In order to assign all the 1H and 13C resonances we carried
out a series of 1D and 2D NMR experiments runs including
DEPT, NOESY, H–H COSY, gHSQC and gHMBC (Table 2).
For our immediate aim, the structural determination of
compounds arising from the reduction of DHCA, a sound C
O
attribution in the latter was mandatory. Thus, we started from
the HMBC spectrum of 1 attributing to the 12-CO the qua-
ternary carbon resonating at ı 211.98 owing to its correlation
peak (3J coupling) with a methyl group (ı 1.02 ppm, singlet) that
must be assigned to the 18-Me; the same carbon showed cor-
relations to both protons at position 11 (ı 2.82 and 2.22 ppm,
11-CH2 at ı 38.92 from HSQC).
Table 2 – 1H and 13C NMR spectral data and HMBC most significant correlations for DHCA (1) (400 MHz)
Carbon DEPT
1H NMRa (ı) 13C NMR (ı) HMBCb
Observed NOEsc
Pyridine-d5
CDCl3
Pyridine-d5
CDCl3
35.26d
1
CH2
1.80␣; 1.43␤
2.00␣; 1.65␤
35.26
2
19,1␤, 11␣, 2;
1␣, 19, 5, 2
2
3
4
5
6
CH2
C
CH2
CH
CH2
2.28 and 2.09
–
2.38 and 2.26
2.18
2.93␤ (dd, J = 13.0, 5.9);
1.98␣ (dd, J = 13.0, 2.4)
2.29
36.67
208.86
43.11
46.88
45.22
36.46d
209.08
42.76
46.83d
44.95
2, 4
6␤
19, 6
2.23 and 2.16
2.37
2.96␤ (dd, J = 13.1,
6.2); 2.07␣
19.6␣, 5
7
8
9
10
11
C
–
209.12
49.05
45.40
36.22
38.92
208.68
48.97d
45.54
35.99
38.61
6, 8
CH
CH
C
3.02 (pt, J = 11.7)
2.57
–
2.93 (pt, J = 12.5)
2.39
6␣, 11␣, 9
19, 1␤, 11, 8
6␣, 9, 8
9
18, 19, 11␤
18, 19, 8, 11␣
CH2
2.82␤ (pt, J = 12.2); 2.22␣
2.88␤ (pt, J = 12.7);
2.18␣
12
13
14
15
16
17
18
19
20
21
22
23
24
C
C
–
–
1.96
2.45 and 1.32
1.95 and 1.35
2.20
1.02
1.29
211.98
57.16
52.06
25.38
27.97
46.28
11.88
21.60
36.02
19.19
31.40
32.17
176.34
211.96
56.88
51.73d
25.11
27.59
45.62d
11.84
21.89
35.46
18.58
30.18d
31.11d
179.22
18, 11
18, 14, 17
18, 8
CH
CH2
CH2
CH
CH3
CH3
CH
CH3
CH2
CH2
C
1.88
2.34 and 1.29
2.08 and 1.38
2.08
1.11
1.43
14, 8
18, 21
14, 17
9
21, 23
22
21, 23
22
22, 23
8, 11␤
1.46
1.34
1.08 (d, J = 6.8)
2.13 and 1.59
2.64 and 2.57
–
0.89 (d, J = 6.6)
1.90 and 1.46
2.48 and 2.34
a
1H directly attached to 13C as determined from HSQC experiment.
1H–13C long range correlation (HMBC) corresponding to two- or three-bond connectivities.
Through-space interactions.
b
c
d
Revised assignment with respect to literature data [25].

474
steroids 7 1 ( 2 0 0 6 ) 469–475
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Financial support from Italian MIUR (COFIN 2004; prot.
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