Regioselective dehydrogenation of 3-keto-steroids to form conjugated enones using o-iodoxybenzoic acid and trifluoroacetic acid catalysis

Article abstract of DOI:10.1016/j.chemphyslip.2013.11.007

Mild and regioselective conversion of 3-keto-5α- and 3-keto-5β-steroids (trans A/B- and cis A/B-ring juncture, respectively) to the corresponding enones (Δ¹- and Δ⁴-3- ketones) by treatment with o-iodoxybenzoic acid (IBX) catalyzed by trifluoroacetic acid (TFA) in DMSO, is described. The IBX-mediated reaction involved dehydrogenation of the α- and β-hydrogen atoms of the 3-ketones to give the enones regioselectively in good isolated yields without concomitant formation of related dienones and trienones.

Full text of DOI:10.1016/j.chemphyslip.2013.11.007

Chemistry and Physics of Lipids 178 (2014) 45–51
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Regioselective dehydrogenation of 3-keto-steroids to form conjugated
enones using o-iodoxybenzoic acid and trifluoroacetic acid catalysis
Takashi Iida∗, Kaoru Omura, Ryou Sakiyama, Mitsuo Kodomari
Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Mild and regioselective conversion of 3-keto-5␣- and 3-keto-5␤-steroids (trans A/B- and cis A/B-ring
juncture, respectively) to the corresponding enones (ꢀ1- and ꢀ4-3-ketones) by treatment with o-
iodoxybenzoic acid (IBX) catalyzed by trifluoroacetic acid (TFA) in DMSO, is described. The IBX-mediated
reaction involved dehydrogenation of the ␣- and ␤-hydrogen atoms of the 3-ketones to give the enones
regioselectively in good isolated yields without concomitant formation of related dienones and trienones.
Received 17 October 2013
Received in revised form
19 November 2013
Accepted 21 November 2013
Available online 2 December 2013
© 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords:
o-Iodoxybenzoic acid (IBX)
Trifluoroacetic acid (TFA)
Nuclear magnetic resonance (NMR)
High-resolution electrospray ionization
mass spectrometry (HR-ESI-MS)
High-resolution atmospheric pressure
chemical ionization mass spectrometry
(HR-APCI-MS)
Thin-layer chromatography (TLC)
1. Introduction
␣-bromo-3-keto derivatives, which are obtainable from the cor-
responding 3-ketones by an ␣-substitution reaction with bromine
The biological and synthetic significance of ␣,␤-unsaturated
3-keto-steroids (ꢀ1- and ꢀ4-3-keto-steroids; enones) has gener-
ated much interest in their chemical synthesis for some decades.
All the practical syntheses start from abundantly available 3-
hydroxy-steroids and conventionally involve stepwise oxidation at
the 3-hydroxyl group and subsequent ␣,␤-dehydrogenation of the
resulting 3-ketones or dehydrohalogenation of ␣-haloketo inter-
mediates obtainable from the saturated 3-ketones.
Simultaneous oxidation of a hydroxyl group and migration of
a double bond in 3-hydroxy-ꢀ5-steroids is known as the Oppe-
nauner oxidation (Rasmusson and Arth, 1972), which proceeds
by an aluminum alkoxide [Al(OR)3]-catalyzed hydrogen exchange
with receptor carbonyl compounds. The Oppenauner oxidation
has found considerable application in steroid chemistry, since
it is a convenient method for converting naturally occurring
3-hydroxy-ꢀ5-steroids (e.g., cholesterol) directly into their ꢀ4-3-
keto-counterparts.
(Beard, 1972; Iida et al., 1991; Zhang and Qiu, 2006). When 3-
oxo-4␨-bromo-5␤-steroids are treated with a base (e.g., CaCO3) in
boiling N,N-dimethylformamide (DMF) or 2,4,6-trimethylpyridine
(␥-collidine), they induce the introduction of a carbon-carbon dou-
ble bond to form the ꢀ4-3-ketones. The procedure generally offers a
more satisfactory approach for introduction of carbon-carbon dou-
ble bonds than the halogenation–dehydrohalogenation sequence. A
quinone type of acceptor such as 2,3,5,6-tetrachlorobenzoquinone
(chloranil) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
also enjoys wide use in an alternative method for ␣,␤-
dehydrogenation of steroidal ketones (Beard, 1972). For instance,
DDQ in boiling dioxane converts 3-keto-5␣-steroids into the ꢀ1-,
ꢀ
1,4- and ꢀ1,4,6-3-keto-steroids (Turner and Ringold, 1967; Kirk
et al., 1990).
Selenium-based reagents have also been used in the prepara-
tion of steroidal enones. Probably the best known of this class
of reagents is selenium dioxide (SeO2) (Haines, 1985). However,
more recently, a iodoxybenzene-benzeneselenic anhydride (BSA)
system has found increasing application (Barton et al., 1980). Thus,
SeO2-induced dehydrogenation in t-butanol has been useful for
the conversion of 3-keto-5␣-steroids into the ꢀ1-, ꢀ4- and/or
Traditionally, a more practical method for the preparation
of steroidal enones is dehydrobromination of the appropriate
∗
ꢀ
1,4-3-ketones (Jerussi and Speyer, 1966). On the other hand, an
oxidation-dehydrogenation reaction in which 3-hydroxy-steroids
Corresponding author. Tel.: +81 3 3329 1151; fax: +81 3 3303 9899.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0009-3084/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemphyslip.2013.11.007

46
T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
2. Experimental
2.1. Materials and reagents
5␤-Cholestan-3␤-ol (coprostanol) was purchased from Ster-
aloids, Inc. (Newport, RI, USA) and it was oxidized with Jones
reagent to give 5␤-cholestan-3-one (1). 7␣,12␣-Diacetoxy-5␤-
cholestan-3-one (2), 5␣-cholestan-3-one (3) and 4,4-dimethyl-5␣-
cholestan-3-one (4), methyl 3-oxo-5␤-cholan-24-oate (5), methyl
3-oxo-7␣-formyloxy-5␤-cholan-24-oate (6), methyl 3-oxo-7␣-
acetoxy-5␤-cholan-24-oate (7), methyl 3-oxo-7␣,12␣-diacetoxy-
5␤-cholan-24-oate (8), methyl 3-oxo-5␣-cholan-24-oate (9),
methyl, 3␤-cathyloxy-4-oxo-7␣-acetoxy-5␤-cholan-24-oate (14),
and methyl 6-oxo-5␤-cholan-24-oate (15), methyl 3␣,12␣-
diacetoxy-7-oxo-5␤-cholan-24-oate (16), and methyl 12-oxo-5␤-
cholan-24-oate (17) were from collections in our laboratory.
5␤-Pregnan-3␣-ol and 5␣-pregnan-3␤-ol, which were available
from Steraloids were treated with pyridinium chlorochromate
(PCC) to afford 5␤-pregnan-3-one (10) and 5␣-pregnan-3-one (11),
respectively. 5␤-Androstane-3,17-dione (12) was available from
Steraloids. 17␤-Hydroxy-5␣-androstan-3-one was purchased from
Tokyo Chemical Industry Co. Ltd. and converted to 5␣-androstane-
3,17-dione (13) by PCC. All substrates were dried by azeotropic
distillation prior to use. Other chemicals and solvents were of ana-
lytical reagent grade.
2.2. Instruments
All melting points (mp) were determined on a micro hot-stage
apparatus and are uncorrected. NMR spectra were recorded at 23 ◦C
in CDCl3 by using a JEOL ECA-500 instrument (500.2 MHz for 1H and
125.8 MHz 13C). The 13C distortionless enhancement by polariza-
tion transfer (DEPT; 135◦, 90◦, and 45◦) spectra were also measured
to determine the exact 13C signal multiplicity and to differenti-
ate between CH3, CH2, CH, and C based on their environments.
High-resolution mass spectrometry (MS) using an electrospray ion-
ization (HR-ESI) or an atmospheric pressure chemical ionization
(HR-APCI) were carried out using a JEOL AccuTOF JMS-T100LC liq-
uid chromatography-mass spectrometer equipped with an ESI or
an APCI source and coupled to an Agilent 1200 series binary pump
(Agilent Technologies Inc., Santa Clara, CA, USA) operated in the
positive ion mode. The HR-ESI-MS and HR-APCI-MS of compounds
from 18 to 31 were performed in the flow injection mode, using
methanol as the mobile phase at a flow rate of 0.2 and 1.0 ␮L/min,
respectively. The ionization conditions were as follows: needle
voltage, 2000 kV; ion guide peak voltage, 2 and 2.5 kV (HR-ESI-
MS and HR-APCI-MS, respectively); ion source temperature, 80 ◦C;
orifice voltage, 75 and 85 V; mass range, 0–1000; nebulizing gas,
N2 gas. Normal-phase TLC was performed on pre-coated silica gel
plates (0.25 mm layer thickness; E.Merck, Darmstadt, Germany)
using a mixture of hexane-EtOAc (9:1–5:5, v/v) as the developing
solvent.
Fig. 1. Dehydrogenation process of 3-keto-5␤- and 3-keto-5␣-steroids to the con-
jugated enones, dienones and trienones.
were treated with iodoxybenzene catalyzed by BSA in boiling
toluene resulted in conversion to the ꢀ1,4-3-ketones (Iida et al.,
1988a).
These dehydrogenation reagents are, however, very sensitive
to experimental conditions, are somewhat unselective, and fre-
quently, may give rise to undesirable side reactions. In particular,
over-dehydrogenations of enones are frequently encountered in
these reactions to afford the dienones (ꢀ1,4- or ꢀ4,6-3-ketones)
and/or the trienones (ꢀ1,4,6-3-ketones). Thus, the previously
reported procedures usually provide a mixture of enones, dienones,
and trienones (Fig. 1). The available reactions therefore lead to fairly
low yields of the desired enones, even though they have been car-
ried out under strictly controlled experimental conditions.
Recently, Nicolaou et al. (2002) introduced a new reagent
system, consisting of o-iodoxybenzoic acid (IBX) and a catalytic
amount of trifluoroacetic acid (TFA), which converts certain 3-
keto-5␣-steroids into the ꢀ1-3-ketones. On the other hand, Li
and Tochtrop (2011) and we (Ogawa et al., 2013) have reported
that some 3-keto-5␤-steroids with the same reagent system are
exclusively transformed into the ꢀ4-3-ketones. The intriguing
results prompted us to examine in detail the IBX-mediated reac-
tion to other keto-steroids differing in the carbon length of the
side-chain at C-17, stereochemistry of the A/B-ring juncture (A/B
cis, 5␤-H or A/B trans, 5␣-H), and the distribution of the num-
ber, position and configuration of substituents in the steroid
nucleus. In this report we have explored the usefulness and lim-
itations of dehydrogenation with IBX-TFA system on a variety of
structurally different C27, C24, C21 and C19 3-keto-5␣- and 3-keto-
5␤-steroids.
2.3. Chemical synthesis of the conjugated enones from
keto-steroids
2.3.1. Preparation of IBX
IBX was prepared from 2-iodobenzoic acid and OXONE®
(monopersulfate compound, 2KHSO5/KHSO4/K2SO4) by the
procedure reported by Frigerio et al. (1999). Thus, to
a
magnetically stirred solution of 2-iodobenzoic acid (1.0 g,
4 mmol) in water (13 ml), OXONE® (monopersulfate compound,
2KHSO5/KHSO4/K2SO4; 3.62 g, 5.8 mmol) was added, and the
resulting suspension was stirred at 70 ◦C for 3.5 h and then 5 ◦C for
1.5 h. The precipitated semi-crystalline solid was filtered, washed
with water and acetone, and dried under reduced pressure at

T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
47
Table 1
13C NMR of ꢀ1- and ꢀ4-3-keto-steroids.
Carbon
18
35.6
19
35.4
20
21
22
35.6
23
35.4
24
35.4
25
35.4
26
27
35.7
28
29
35.7
30
31
38.2
1
158.6
127.3
200.2
41.0
44.3
31.3
27.6
35.6
49.9
38.9
21.2
39.8
42.7
56.2
24.1
28.2
56.3
12.1
12.9
35.7
18.6
36.1
23.8
39.5
28.0
22.5
22.8
157.3
125.8
205.3
44.6
51.7
32.2
20.9
35.5
52.5
39.2
21.7
39.7
42.6
56.2
24.1
28.2
56.5
12.1
16.4
35.8
18.6
36.1
23.8
39.5
28.0
22.6
22.8
158.6
127.4
200.3
41.0
44.3
31.3
27.6
35.6
49.9
39.0
21.2
39.7
42.7
55.8
24.1
28.1
56.3
12.2
13.0
35.4
18.2
30.9
31.0
174.7
158.7
127.3
200.3
41.0
44.4
31.4
27.7
35.7
50.4
39.1
21.0
23.1
42.3
53.0
24.4
28.1
55.8
12.7
13.0
37.9
13.3
157.8
127.6
199.9
40.9
44.3
27.3
30.1
35.2
51.3
39.0
20.5
31.4
47.8
50.1
21.7
35.8
220.6
13.0
13.9
2
3
4
5
6
7
8
9
34.0
199.5
123.7
171.6
32.9
32.0
35.7
53.8
38.6
21.0
39.6
42.4
55.9
24.1
28.1
56.1
11.9
17.4
35.7
18.6
36.1
23.8
39.5
28.0
22.5
22.8
33.8
198.7
126.5
166.0
37.4
70.7
41.0
38.3
37.7
25.5
74.9
44.8
43.1
22.9
27.2
47.7
12.2
16.9
35.0
17.8
35.9
23.8
39.4
27.9
22.5
22.8
21.1
21.2
170.4
170.5
33.9
199.5
123.7
171.5
32.8
31.9
35.5
53.7
38.5
20.9
39.5
42.4
55.7
24.1
28.0
55.8
11.9
17.3
35.3
18.2
30.9
31.0
174.6
33.9
198.7
126.5
165.9
37.4
71.1
38.3
46.2
38.2
20.8
39.0
42.5
50.0
23.4
27.8
55.6
11.7
17.1
35.2
18.2
30.8
30.9
174.6
34.0
199.0
126.3
166.8
37.4
71.1
38.3
46.3
38.3
20.8
39.0
42.5
50.2
23.5
27.9
55.6
11.7
17.0
35.2
18.2
30.9
30.9
174.6
33.8
198.6
126.5
165.8
37.3
70.6
41.0
38.3
37.7
25.5
74.8
44.9
43.2
22.8
27.0
47.3
12.2
16.9
34.6
17.5
30.7
30.8
174.5
34.0
199.7
123.8
171.7
33.0
32.1
35.7
52.9
38.7
20.8
23.1
42.0
54.2
24.5
28.1
55.4
12.5
13.3
37.7
17.4
33.9
199.3
124.2
170.3
32.6
30.8
35.2
53.8
38.6
20.3
31.3
47.5
50.9
21.8
35.7
220.4
13.7
17.4
21.1
21.4
23.9
58.9
212.7
46.8
37.9
54.3
41.8
20.4
39.5
43.0
56.8
25.2
27.9
55.8
12.0
13.0
35.3
18.2
30.9
31.1
174.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OCOCH3
21.1
21.1
21.1
170.3
170.4
OCOCH3
170.5
CH3-4␣
CH3-4␤
OCHO
27.0
21.9
160.4
51.5
COOCH3
51.4
51.5
51.5
51.5
51.5
1H NMR (500 MHz, CDCl3): ı = 0.71 (s, 3H, 18-H3), 0.86 (d, 3H,
J = 6.8 Hz, 26-H3), 0.87 (d, 3H, J = 6.8 Hz, 27-H3), 0.91 (d, 3H, J = 6.3 Hz,
21-H3), 1.18 (s, 3H, 19-H3), 5.72 (s, 1H, 4-H). HR-APCI-MS, calcd. for
C27H45O [M+H]+, 385.3470; found 385.3435.
room temperature for 16 h to afford IBX, which was directly used
without further purification.
2.3.2. General dehydrogenation procedure with IBX-TFA
Each of the reaction substrates 1–17 was subjected to IBX-TFA
reactions by two different procedures (A and B) as described below.
2.3.4. 7˛,12˛-Diacetoxy-cholest-4-en-3-one (19)
Obtained from 7␣,12␣-diacetoxy-5␤-cholestan-3-one (2) with
IBX (2 equiv.) at 40 ◦C for 36 h. Recrystallized from methanol-water:
yield, 66% (20%); mp, 145–148 ◦C. 1H NMR (500 MHz, CDCl3):
ı = 0.79 (s, 3H, 18-H3), 0.81 (d, 3H, J = 6.3 Hz, 21-H3), 0.86 (d, 6H,
J = 6.3 Hz, 26-H3 and 27-H3), 1.19 (s, 3H, 19-H3), 2.06, 2.08 (each
s, 3H, OCOCH3), 5.04 (brs, 1H, 7␤-H), 5.12 (brs, 1H, 12␤-H), 5.70
(s, 1H, 4-H). HR-ESI-MS, calcd. for C31H48NaO5 [M+Na]+, 523.3399;
found 523.3385.
(A) To a magnetically stirred solution of a keto-steroid [0.03 mmol
in 10 ml dry DMSO or 4, 12 or 13 in 10 ml fluorobenzene-DMSO
(2:1, v/v)] was added freshly prepared IBX (2–6 equiv.) and TFA
(3 drops). The mixture was stirred at 40–70 ◦C for 3–48 h; the
reaction was monitored by TLC. After cooling the mixture at
room temperature, the reaction product was extracted with
EtOAc. The combined extract was washed with saturated brine,
dried over MgSO4, and evaporated under reduced pressure. The
residue was poured onto a column of neutral silica gel (3–15 g).
Elution with EtOAc–hexane (1–9∼5–5, v/v) gave the major
reaction products which were recrystallized from appropri-
ate solvents. Physical and spectroscopic properties and yields
of the individual isolated compounds (18–30) were described
below. The 13C NMR data for respective isolated compounds
were compiled in Table 1.
2.3.5. 5˛-Cholest-1-en-3-one (20)
Obtained from 5␣-cholestan-3-one (3) with IBX (5 equiv.) at
65 ◦C for 3 h. Recrystallized from methanol: 65% (53%); mp,
121–125 ◦C [lit. 97–99 ◦C from ethanol (Horiuchi et al., 2004)]. 1H
NMR (500 MHz, CDCl3): ı = 0.68 (s, 3H, 18-H3), 0.86 (d, 3H, J = 6.9 Hz,
26-H3), 0.87 (d, 3H, J = 6.3 Hz, 27-H3), 0.90 (d, 3H, J = 6.9 Hz, 21-
H3), 1.01 (s, 3H, 19-H3), 5.84 (d, 1H, J = 9.8 Hz, 2-H), 7.14 (d, 1H,
J = 10 Hz, 1-H). HR-APCI-MS, calcd. for C27H45O [M+H]+, 385.3470;
found 385.3486.
(B) Standard reaction conditions: all of the IBX-TFA reactions
were carried out for the substrates 1–17 under the identical
experimental conditions: IBX, 2 equiv.: TFA, 3 drops; reaction
temperature, 65 ◦C; reaction time, 24 h. Values in the parenthe-
ses in each reaction described below exhibit isolated yields.
2.3.6. 4,4-Dimethyl-5˛-cholest-1-en-3-one (21)
Obtained from 4,4-dimethyl-5␣-cholestan-3-one (4) with IBX
(5 equiv.) at 65 ◦C for 14 h. Recrystallized from methanol: yield, 95%
(70%); mp, 88–89 ◦C [lit. 89–90 ◦C from ethanol (Mori et al., 1982)].
1H NMR (500 MHz, CDCl3): ı = 0.69 (s, 3H, 18-H3), 0.86 (d, 3H,
J = 6.3 Hz, 26-H3), 0.87 (d, 3H, J = 6.3 Hz, 27-H3), 0.91 (d, 3H, J = 6.3 Hz,
21-H3), 1.07 (s, 3H, 4␤-H3), 1.09 (s, 3H, 4␣-H3), 1.13 (s, 3H, 19-H3),
2.3.3. Cholest-4-en-3-one (18)
Obtained from 5␤-cholestan-3-one (1) with IBX (5 equiv.) at
70 ◦C for 4 h. Recrystallized from methanol: yield, 45% (38%); mp,
81–82 ◦C [lit. 78–80 ◦C from methanol (Lee and Biellmann, 1986)].

48
T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
5.87 (d, 1H, J = 10 Hz, 2-H), 7.10 (d, 1H, J = 10 Hz, 1-H). HR-APCI-MS,
2.3.14. Androst-4-ene-3,17-dione (29)
calcd. for C29H49O [M+H]+, 413.3783; found 413.3783.
Obtained from 5␤-androstane-3,17-dione (12) with IBX
(5 equiv.) at 60 ◦C for 40 h. Recrystallized from methanol: yield, 54%
(6%); mp, 158–161 ◦C [lit. 173–174 ◦C from acetone/CH2Cl2 (Peart
et al., 2011)]. 1H NMR (500 MHz, CDCl3): ı = 0.92 (s, 3H, 18-H3), 1.22
(s, 3H, 19-H3), 5.76 (s, 1H, 4-H). HR-ESI-MS, calcd. for C19H26NaO2
[M+Na]+, 309.1831; found 309.1806.
2.3.7. Methyl 3-oxo-chol-4-en-24-oate (22)
Obtained from methyl 3-oxo-5␤-cholan-24-oate (5) with IBX
(5 equiv.) at 65 ◦C for 10 h. Recrystallized from methanol: yield, 60%
(25%); mp, 130–133 ◦C [lit. 124–126 ◦C from aq. acetone (Ogawa
et al., 2004)]. 1H NMR (500 MHz, CDCl3): ı = 0.71 (s, 3H, 18-H3), 0.92
(d, 3H, J = 6.3 Hz, 21-H3), 1.18 (s, 3H, 19-H3), 3.66 (s, 3H, COOCH3),
5.72 (s, 1H, 4-H). HR-ESI-MS, calcd. for C25H39O3 [M+H]+, 387.2899;
found 387.2934.
2.3.15. 5˛-Androst-1-ene-3,17-dione (30)
Obtained from 5␣-androstane-3,17-dione (13) with IBX
(5 equiv.) at 60 ◦C for 40 h. Recrystallized from methanol: yield, 63%
(5%); mp, 101–104 ◦C [lit. 139.5–140.5 ◦C from ethanol (Zhang and
Qiu, 2006)]. 1H NMR (500 MHz, CDCl3): ı = 0.91 (s, 3H, 18-H3), 1.04
(s, 3H, 19-H3), 5.88 (d, 1H, J = 10 Hz, 2-H), 7.13 (d, 1H, J = 10 Hz, 1-H).
HR-ESI-MS, calcd. for C19H27O2 [M+H]+, 287.2011; found 287.1978.
2.3.8. Methyl 3-oxo-7˛-formyloxy-chol-4-en-24-oate (23)
Obtained from methyl 3-oxo-7␣-formyloxy-5␤-cholan-24-oate
(6) with IBX (2 equiv.) at 40 ◦C for 48 h. Recrystallized from
methanol: yield, 45% (30%); mp, 130–132 ◦C. 1H NMR (500 MHz,
CDCl3): ı = 0.72 (s, 3H, 18-H3), 0.93 (d, 3H, J = 6.3 Hz, 21-H3), 1.22 (s,
3H, 19-H3), 3.66 (s, 3H, COOCH3), 5.16 (brs, 1H, 7␤-H), 5.70 (s, 1H, 4-
H), 8.05 (s, 1H, OCHO). HR-ESI-MS, calcd. for C26H38NaO3 [M+Na]+,
453.2617; found 453.2578.
2.3.16. Methyl 6-oxo-5˛-cholan-24-oate (31)
Obtained from methyl 6-oxo-5␤-cholan-24-oate (15) with IBX
(2 equiv.)at 65 ◦Cfor 24 h. Recrystallizedfrom methanol:yield, 75%;
mp, 97–99 ◦C [lit. 85–87 ◦C from aq. methanol (Iida et al., 1993)].
1H NMR (500 MHz, CDCl3): ı = 0.66 (s, 3H, 18-H3), 0.73 (s, 3H, 19-
H3), 0.93 (d, 3H, J = 6.3 Hz, 21-H3), 3.66 (s, 3H, COOCH3). HR-ESI-MS,
calcd. for C25H40NaO3 [M+Na]+, 411.2875; found 411.2868.
2.3.9. Methyl 3-oxo-7˛-acetoxy-chol-4-en-24-oate (24)
Obtained from methyl 3-oxo-7␣-acetoxy-5␤-cholan-24-oate
(7) with IBX (2 equiv.) at 40 ◦C for 48 h. Recrystallized from
methanol: yield, 49% (40%); mp, 218–220 ◦C [lit. 207–208 ◦C from
aq. methanol (Ogawa et al., 2004)]. 1H NMR (500 MHz, CDCl3):
ı = 0.71 (s, 3H, 18-H3), 0.93 (d, 3H, J = 6.3 Hz, 21-H3), 1.20 (s, 3H,
19-H3), 2.02 (s, 3H, OCOCH3), 3.66 (s, 3H, COOCH3), 5.00 (brs, 1H,
7␤-H), 5.69 (s, 1H, 4-H). HR-ESI-MS, calcd. for C27H40NaO5 [M+Na]+,
467.2773; found 467.2732.
3. Results and discussion
Conversion of steroidal 3-ketones to the corresponding enones
(ꢀ1- and ꢀ4-3-ketones), key intermediates in steroid biosynthe-
sis and a key transformation for chemical synthesis of bioactive
steroids, has been improved significantly by the use of various
reagents. However, as noted in the introduction, the reaction still
has several drawbacks, especially when performed with 3-ketones
having other functional groups that are sensitive to the experimen-
tal conditions. The reaction products are often complex mixtures
of the enones, accompanied by the dienones and trienone result-
ing from over-dehydrogenation, from which the desired enones
are isolated with difficulty in low yields (Fig. 1). The recent suc-
cessful use of a IBX-TFA system for mild elimination of the ␣-
and ␤-hydrogens of carbonyl compounds prompted us to exam-
ine the usefulness and limitation of the reagent (Nicolaou et al.,
2002; Li and Tochtrop, 2011). The satisfactory results obtained by
our modified method (Ogawa et al., 2013) led us to investigate its
applicability to a wide variety of structurally different 3-keto-5␣-
and 3-keto-5␤-steroids (Fig. 2).
Since commercially available IBX (45 wt.%) contains an appre-
ciable amount of benzoic acid and isophthalic acid as a stabilizer,
freshly prepared IBX free from those additives was prepared
prior to the reaction. Our preliminary experiments also revealed
that when 3-keto-steroids (7 and 8) in DMSO are treated
with IBX (0.15 mmol equiv.) alone, the dehydrogenation dose not
proceed at all. In fact, IBX oxidative transformation of cholic acid
(3␣,7␣,12␣-trihydroxy-5␤-cholan-24-oic acid) and its 7␤-epimer
in the absence of TFA have been reported to yield the 7- and 12-
keto derivatives, respectively (Dangate et al., 2011). However, by
adding a catalytic amount of TFA (3 drops), the reaction proceeded
smoothly to give the desired enones (24 and 25) (Fig. 3). In addition,
isolated yields of the respective enones (18–30) from structurally
different 3-ketones (1–13) differed greatly under identical exper-
imental conditions (procedure B; see Section 2), depending upon
the presence or absence of substituents in the steroid nucleus, car-
bon length of the side chain at C-17, and the stereochemistry of the
A/B-ring juncture. In contrast, procedure A showed the most suit-
able experimental conditions for all of the substrates. These results
suggest that the yields of the desired enones by IBX-mediated dehy-
drogenation are influenced significantly by the reaction conditions
of temperature and time as well as the molar ratio of substrate to
2.3.10. Methyl 3-oxo-7˛,12˛-diacetoxy-chol-4-en-24-oate (25)
Obtained from methyl 3-oxo-7␣,12␣-diacetoxy-5␤-cholan-24-
oate (8) with IBX (2 equiv.) at 40 ◦C for 48 h. Recrystallized from
methanol: yield, 66% (45%); mp, 196–198 ◦C [lit. 186–189 ◦C from
aq. methanol (Ogawa et al., 2004)]. 1H NMR (500 MHz, CDCl3):
ı = 0.79 (s, 3H, 18-H3), 0.82 (d, 3H, J = 6.3 Hz, 21-H3), 1.19 (s, 3H,
19-H3), 2.06, 2.08 (each s, 3H, OCOCH3), 3.66 (s, 3H, COOCH3), 5.04
(brs, 1H, 7␤-H), 5.10 (brs, 1H, 12␤-H), 5.70 (s, 1H, 4-H). HR-ESI-MS,
calcd. for C29H42NaO7 [M+Na]+, 525.2828; found 525.2796.
2.3.11. Methyl 3-oxo-5˛-chol-1-en-24-oate (26)
Obtained from methyl 3-oxo-5␣-cholan-24-oate (9) with IBX
(5 equiv.) at 70 ◦C for 15 h. Recrystallized from methanol: yield, 68%
(35%); mp, 137–139 ◦C. 1H NMR (500 MHz, CDCl3): ı = 0.70 (s, 3H,
18-H3), 0.92 (d, 3H, J = 6.9 Hz, 21-H3), 1.00 (s, 3H, 19-H3), 3.66 (s,
3H, COOCH3), 5.85 (d, 1H, J = 10 Hz, 2-H), 7.13 (d, 1H, J = 10 Hz, 1-H).
HR-ESI-MS, calcd. for C25H39O3 [M+H]+, 387.2899; found 387.2942.
2.3.12. Pregn-4-en-3-one (27)
Obtained from 5␤-pregnan-3-one (10) with IBX (5 equiv.) at
65 ◦C for 20 h. Recrystallized from methanol: yield, 42% (30%); mp,
81–84 ◦C [lit. 102–103 ◦C from methanol (Lichtfouse and Albrecht,
1994)]. 1H NMR (500 MHz, CDCl3): ı = 0.62 (s, 3H, 18-H3), 0.88 (t,
3H, J = 7.5 Hz, 21-H3), 1.19 (s, 3H, 19-H3), 5.72 (s, 1H, 4-H). HR-APCI-
MS, calcd. for C21H33O [M+H]+, 301.2531; found 301.2501.
2.3.13. 5˛-Pregn-1-en-3-one (28)
Obtained from 5␣-pregnan-3-one (11) with IBX (6 equiv.) at
70 ◦C for 17 h. Recrystallized from methanol: yield, 91% (47%); mp,
94–96 ◦C [lit. 95 ◦C from aq. methanol (Longevialle, 1969)]. 1H NMR
(500 MHz, CDCl3): ı = 0.60 (s, 3H, 18-H3), 0.88 (t, 3H, J = 7.3 Hz, 21-
H3), 1.02 (s, 3H, 19-H3), 5.85 (d, 1H, J = 10 Hz, 2-H), 7.16 (d, 1H,
J = 10 Hz, 1-H). HR-APCI-MS, calcd. for C21H33O [M+H]+, 301.2531;
found 301.2516.

T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
49
Fig. 2. Chemical structures of keto-steroids examined.
In the 1H NMR spectrum of cholest-4-en-3-one (18), a charac-
teristic 1H signal arising from 4-H at 5.72 ppm as broad singlet (brs),
while the 13C signals at C-3, C-4 and C-5 occurred at a lower-field
of 199.5, 123.7 and 171.6 ppm. Differentiation between the quater-
nary C and the tertiary CH was made using the DEPT technique. On
the other hand, the 1H NMR spectrum of 5␣-cholest-1-en-3-one
(20) exhibited the specific 1H signals at 5.84 (2-H) and 7.14 ppm
(1-H) as doublets (d) and the 13C signals due to C-1 (158.6 ppm),
C-2 (127.3 ppm) and C-3 (200.2 ppm). Essentially identical charac-
teristics were detected in the 1H and 13C NMR spectra of the other
ꢀ4- and ꢀ1-3-keto-steroids (Table 1).
Based on the proposal made by Nicolaou et al. (2002), a possible
mechanism for the IBX-mediated dehydrogenation of 3-keto-5␤-
steroids was illustrated in Fig. 4. In short, the reaction is initiated
by single electron transfer (SET)-based pathway from the substrate
to IBX to form a radical cation which reacts further to give the final
product of the ꢀ4-3-ketone. Therefore, the abstraction reaction
of 3-keto-5␣- and 3-keto-5␤-steroids draws out the ␣-hydrogen
(H attached at C-2 or at C-4) of the carbonyl group at C-3 to give
that of IBX. In each reaction, the products were easily isolated by
a short chromatography on silica gel eluting with hexane–EtOAc
mixtures. The isolated yields of 18–30 were 45–95% for the proce-
dure A and 5–70% for the procedure B.
The 3-keto-steroids (1, 2, 5–8, 10 and 12) with 5␤-H ori-
entation (cis A/B-ring juncture), on treatment with IBX-TFA in
DMSO, afforded the corresponding ꢀ4-3-ketones (18, 19, 22–25,
27 and 29) regioselectively. For solubility reasons, a mixture of
fluorobenzene-DMSO was employed as a solvent for the reaction of
the substrates 4, 12 and 13. In some cases, a trace amount (evalu-
ated less than 5%) of the ꢀ1,4-3-ketones was also formed, indicating
a limited amount of over-dehydrogenation. In contrast, 3-keto-5␣-
steroids (trans A/B-ring juncture) were found to be the convenient
starting materials for ꢀ1 introduction. Thus ꢀ1-3-ketones (20, 21,
26, 28 and 30) were obtained exclusively the 3-keto-5␣-steroids
(3, 4, 9, 11 and 13) under the identical experimental conditions.
These findings suggest that in the 5␣-series enolization toward C-2
is favored, while in the 5␤-series enolization toward C-4 predomi-
nates (Beard, 1972).

50
T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
Fig. 3. Chemical structures of the reaction products by IBX-TFA.
Fig. 4. Possible dehydrogenation mechanism of the ␣- and ␤-hydrogens of 3-keto-5␤-steroids with IBX.

T. Iida et al. / Chemistry and Physics of Lipids 178 (2014) 45–51
51
only the corresponding ꢀ1- and ꢀ4-3-keto-steroids, respectively.
Alternatively, the presence of the ␣-hydrogen atom adjacent to
a carbonyl group is essential for the formation of enones and a
small amount of 1,4-dienones. Supporting evidence for the pro-
posed mechanism is that no 4,6-dienones and/or 1,4,6-trienones as
by-products were formed at all, differing from the results of DDQ-
(Turner and Ringold, 1967) and SeO2- (Jerussi and Speyer, 1966)
mediated hydrogenation reactions.
For the purpose of comparison of the reactivity, 4-, 6-, 7- and
12-keto-5␤-steroids (14–17) were also subjected to IBX-TFA reac-
tion. Interestingly, dehydrogenation of 14, 16 and 17 did not occur
at all, suggesting high site-selectivity of the reaction. Meanwhile,
methyl 6-oxo-5␤-cholan-24-oate (15), on treatment with IBX-TFA,
was found to our astonishment to undergo complete inversion at
the A/B-ring juncture to give the corresponding 5␣-isomer (31).
Attempted reaction of authentic 31 with IBX-TFA did not proceed
at al. The observation that allomerization of the A/B-ring juncture
from the less unstable cis 5␤-H form (15) to the more stable trans
5␣-H (31) is encountered in the IBX-mediated reaction may be
ascribed to the Lewis acid character of the TFA catalyst, via the
keto-enol tatomerism (Iida et al., 1988b).
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anhydride. Journal of Lipid Research 29, 1097–1101.
Iida, T., Momose, T., Tamura, T., Matsumoto, T., Chang, F.C., Goto, J., Nambara, T.,
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Kirk, D.N., Smith, C.Z., Honour, J.W., 1990. Synthesis of [11,11,12,12-
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methylated at position 2,3 or 6: molecular fossils of yet unknown biological
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In conclusion, the IBX-TFA procedure described herein repre-
sents a mild and simple method for regioselective dehydrogenation
of 3-keto-5␣- and 3-keto-5␤-steroids to the corresponding ꢀ1-
and ꢀ4-3-ketones, respectively, without concomitant formation of
related dienones and trienones.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scien-
tific Research© from the Japan Society for Promotion of Science (to
T.I. 21550091) for 2012-2-14 and the Supported Program for the
Strategic Research Foundation at Private Universities subsidized
MEXT 2009 (S0901022) for 2009–2013. We thank Dr. Alan F. Hof-
mann, University of California, San Diego, for his help in manuscript
preparation.
a
direct lactonization at C-20. Organic and Biomolecular Chemistry 2,
1013–1018.
Ogawa, S., Zhou, B., Kimoto, Y., Omura, K., Kobayashi, A., Higashi, T., Mitamura, K.,
Ikegawa, S., Hagey, L.R., Hofmann, A.F., Iida, T., 2013. An efficient synthesis of
7␣,12␣-dihydroxy-4-cholesten-3-one and its biological precursor 7␣-hydroxy-
4-cholesten-3-one: key intermediates in bile acid biosynthesis. Steroids 78,
927–937.
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