Transformations of 3-Hydroxy Steroids with Lewis and Anhydrous Protic Acids: The Case of Pregn-4-en-3β,17α,20β-Triol

Article abstract of DOI:10.1111/j.1747-0285.2011.01147.x

The acid-catalyzed dehydration is one of the most important processes, which transforms 3-hydroxy steroids into their corresponding unsaturated derivatives. This reaction is of great importance because it can produce molecules that play a key role in the understanding of the natural metabolism of steroids. Sterol dehydration is generally performed with aqueous acidic systems, and the treatment often affords low yields of the desired compounds and/or complex mixtures of by-products. In this paper, we report the results obtained from the study of the structural and stereochemical effects of the acid-induced reaction of pregn-4-en-3β,17α,20β-triol in anhydrous systems. In particular, the treatment of this trihydroxy steroid model with Lewis acids leads to the corresponding Δ^3,5-steradiene as the only product and in very high yields. With Lewis acids, no modifications of the 1,2-diol function on the D-ring are observed, even when the reactions are performed at high temperatures. Protic acid catalysis in non-aqueous organic solvents causes the formation of an epimeric mixture of the corresponding Δ^3,5-steradiene derivatives by a partial stereochemical inversion of the asymmetric C-17. The reactivity of the 17α,20β-diolic residue is also evaluated by exposing pregn-4-en-3β,17α,20β-triol and the corresponding Δ^3,5-steradiene to the prolonged action of anhydrous protic acid systems under thermal conditions. Pregn-4-en-3β,17α,20β-triol generates only pregn-3,5-dien-17α,20β-diol by treatment with Lewis acids. Pregn-4-en-3β,17α,20β-triol is converted into a 3:2 mixture of pregn-3,5-dien-17α,20β-diol and its 17β epimer upon anhydrous protic acid catalysis

Full text of DOI:10.1111/j.1747-0285.2011.01147.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01147.x
Chem Biol Drug Des 2011; 78: 269–276
Research Article
Transformations of 3-Hydroxy Steroids with
Lewis and Anhydrous Protic Acids: The Case of
Pregn-4-en-3b,17a,20b-Triol
biological functions of animal life, for example, in the peripheral
biosynthesis of active estrogens and androgens starting from adre-
nal precursors (1). They are involved in the mechanism of glucu-
ronidation (2,3), and it is well known that 3-hydroxy steroids are
considered to be important synthetic and naturally occurring pre-
cursors for the regioselective preparation of cardenolides, gly-
cosylated conjugates of neuroactive steroids proposed as
pharmaceutical agents (4,5). In recent years, the widespread appli-
cations of 3-hydroxy steroids have motivated an extensive study of
the chemical and biochemical molecular transformations of this
kind of compounds. Biotransformation of this kind of compound
strictly depends on the activity of carbonyl reducing enzymes ⁄
hydroxy steroids dehydrogenases (6). The metabolic chain of
3-hydroxy steroids is also regulated by acid-catalyzed processes,
which are able to produce profound modifications of the molecular
structure, by generating D3,5-steradiene derivatives. These com-
pounds can further evolve to more complex steroid derivatives
through the intervention of carbocation species, to give stable
intermediates of primary importance in the understanding of the
mechanisms that regulate many chemical and biochemical reac-
tions of steroids. A series of acid-catalyzed rearrangements are
implicated in the enzymatic reactions involved in the biosynthesis
of animal hormones and plant sterols (7,8). These kinds of trans-
formations have also been used in many chemical reactions car-
ried out in the laboratory, with the aim of synthesizing suitable
molecular probes useful in the investigation of the pathways
related to the metabolic processes of steroid transformation that
take place in nature (9–11). Reactions assisted by aqueous acidic
systems often lead to steroidal backbone rearrangement products
(12–14). For example, the acid treatment of 3-hydroxy-D4-steroids
affords the corresponding D3,5-steradiene derivatives, probably via
an allylic carbocation; dehydration of 3-hydroxy-D4-steroids has
been accomplished by treatment with 70% aqueous acetic acid at
100 ꢀC to yield the corresponding D3,5-steradienes (15). The con-
version of 3-hydroxy-D4-steroids (or 3-acetoxy-D4-steroids) into
Rosaria De Marco, Antonella Leggio,
Angelo Liguori*, Francesca Perri and
Carlo Siciliano
Dipartimento di Scienze Farmaceutiche, Universitꢀ della Calabria,
Edificio Polifunzionale, I-87036 Arcavacata di Rende, Cosenza, Italy
*Corresponding author: Angelo Liguori, A.Liguori@unical.it
The acid-catalyzed dehydration is one of the most
important processes, which transforms 3-hydroxy
steroids into their corresponding unsaturated
derivatives. This reaction is of great importance
because it can produce molecules that play a key
role in the understanding of the natural metabo-
lism of steroids. Sterol dehydration is generally
performed with aqueous acidic systems, and the
treatment often affords low yields of the desired
compounds and ⁄ or complex mixtures of by-prod-
ucts. In this paper, we report the results obtained
from the study of the structural and stereochemi-
cal effects of the acid-induced reaction of pregn-
4-en-3b,17a,20b-triol in anhydrous systems. In par-
ticular, the treatment of this trihydroxy steroid
model with Lewis acids leads to the corresponding
D3,5-steradiene as the only product and in very
high yields. With Lewis acids, no modifications of
the 1,2-diol function on the D-ring are observed,
even when the reactions are performed at high
temperatures. Protic acid catalysis in non-aqueous
organic solvents causes the formation of an epi-
meric mixture of the corresponding D3,5-steradiene
derivatives by a partial stereochemical inversion
of the asymmetric C-17. The reactivity of the
17a,20b-diolic residue is also evaluated by expos-
ing pregn-4-en-3b,17a,20b-triol and the correspond-
ing D3,5-steradiene to the prolonged action of
anhydrous protic acid systems under thermal
conditions.
D
3,5-steradiene-related structures has been used as a smart tool
Key words: acid-catalyzed rearrangement, carbocations, Lewis
acids, pregn-4-en-3b,17a,20b-triol, sterol dehydration, D3,5-steradienes,
17,20-diols, 3-hydroxy steroids
in determining gestagenic hormones by spectrophotometry (16) and
by liquid chromatography ⁄ mass spectrometry (17). The role of
D
3,5-steradiene derivatives is essential in a number of applica-
tions, such as the analysis of human urine of patients with can-
cer(18–20), chemical and physical laboratory experiments for the
study of the biotic chemistry of steroids (21–23), the use of conju-
gated steroids as molecular markers for food control (24,25), pro-
duction of aromatic derivatives by gas-phase pyrolysis of plant
sterols (26), and the use as ideal precursors in the synthesis of
Received 2 August 2010, revised 4 May 2011 and accepted for
publication 13 May 2011
3-Hydroxy steroids and polyhydroxy-related systems display a piv-
otal role in the metabolic pathways of keto steroids. These com-
pounds are hormones which play vital roles in a series of
269

De Marco et al.
polyhydroxy steroid derivatives of biological and pharmaceutical
importance (27). Thus, the clean and easy synthesis in large
amounts and in excellent yields and the knowledge of the behav-
ior of 3,5-steradienes obtained from naturally occurring or syn-
thetic 3-hydroxy steroids may offer the basis for the preparation
of modified analogues that could be useful as standards in instru-
mental analyses of human matrices and other materials of biologi-
cal importance. All the reported conversions of 3-hydroxy steroids
into the corresponding D3,5-steradiene derivatives usually require
harsh experimental conditions characterized by the use of concen-
trated aqueous acid solutions and high temperatures. Moreover,
these processes induce skeletal modifications of steroids and pro-
duce the desired conjugated steradiene systems in low to moder-
ate yields together with consistent amounts of unknown
compounds.
Methods and Materials
General methods
All solvents were purified and dried by standard procedures and
distilled prior to use. Commercially available reagents were pur-
chased from Aldrich Chemical Co. and were used without further
1
purification. H NMR spectra were recorded at 300 MHz on a Bru-
ker Avance 300 spectrometer. 13C NMR spectra and Distorsionless
Enhancement by Polarization Transfer (DEPT) experiments were
recorded at 75.5 MHz on the same instrument. All NMR spectra
were recorded at 25 ꢀC, using standard pulse sequence programs
from the Bruker BioSpin firm. DMSO-d6 or CDCl3 was used as sol-
vent. Chemical shift values (d) are expressed in ppm relative to the
residual proton of DMSO-d6 fixed at 2.50 ppm (central line of the
1
quintet) for H NMR spectra and relative to the DMSO-d6 resonance
fixed at 39.7 ppm (central line of the septet) for 13C NMR spectra.
All coupling constants (J) are reported in hertz (Hz). The experiment
for the NMR study of the reaction of compound 1 with camphor-
sulfonic acid (CSA) as the catalyst was performed in CDCl3 at
)10 ꢀC. In this case, chemical shift values are expressed in ppm
relative to the residual proton of the solvent at 7.23 ppm for 1H
NMR spectra. In case of signal overlapping, resonances were cor-
rectly assigned by proton homodecoupling experiments. GC ⁄ MS
Another class of acid-induced processes in which the hydroxyl
groups of steroids are involved is the rearrangement of the D-ring
in 17,20-dihydroxy steroids. Steroid structures having a 17,20-diol
functionality are compounds of extraordinary importance secreted
by the follicle and involved in the biological process of the oocyte
maturation in the reproductive cycle of living organisms (28–30).
Similar diols are also recognized in the disorders of neurosteroid
synthesis implicated in grave human pathological abnormalities and
malformations of brain, heart, and gastrointestinal tract (31). The
17,20-diol moiety shows particular reactivity. The pinacol–pinaco-
lone rearrangement of steroids with a 17,20-dihydroxy group, for
example, proceeds by the initial protonation of one of the two OH
groups followed by migration of hydrogen or an alkyl residue
(32,33). The second hydroxyl group assists this rearrangement
through the formation of a carbon–oxygen p-bond. Protonation of
the hydroxyl group at C-17 determines a shift of the proton from C-
20, while the possible formation of a carbocation at C-20 depends
on the type of substituent (34) and can drive the D-homoannulation
of the steroid precursor (35). D-homoannulated products can also be
obtained by the acid treatment of 17b,20-dihydroxy-23-oxosteroids
(36). Unfortunately, many of these reactions take place in the pres-
ence of concentrated aqueous acid solutions and at high tempera-
tures, affording complex mixtures of products that often contain
variable amounts of unidentifiable species. Thus, a more profound
study of the transformation of natural or synthetic steroids contain-
ing a 3-OH group together with a 17,20-diol residue under different
and milder acidic conditions could be of particular interest. More-
over, as polyhydroxy steroids often feature characteristics that are
able to determine structural modifications via competitive chemical
pathways regulating the formation of 3,5-steradiene simple and ⁄ or
homoannulated derivatives, it is reasonable to exploit the reactivity
of 3,17,20-trihydroxy systems, such as pregn-4-en-3b,17a,20b-triol,
to obtain more insights into the formation of some derivatives that
could find applications in chemical biology and in drug design as
molecular probes to better understand the role of structurally simi-
lar intermediates in the metabolic pathways of 17,20-diol-based
potential drugs. Finally, the investigation of the reactivity of pregn-
4-en-3b,17a,20-triol under acidic conditions could be of great inter-
est in the synthesis of new polyhydroxy steradienes, which can be
recognized as metabolites in Smith–Lemli–Opitz syndrome (37,38),
and in the development of new pharmaceuticals active against mel-
anoma cells (39).
analyses were performed by
a HP-5MS (30 m · 0.25 mm,
PhMesiloxane capillary column). The mass detector was operated in
the electron impact ionization mode (EIMS) with an electron energy
of 70 eV. The injection port was heated to 250 ꢀC. The oven tem-
perature was initially set at 100 ꢀC with a hold of 2 min and
ramped to 280 ꢀC at 14 ꢀC ⁄ min with a hold of 10 min. Methane
gas at a pressure of ca. 2 Torr was used as the CI reagent gas.
Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. Elemental analysis was performed on a Per-
kin–Elmer Elemental Analyzer. Reaction mixtures were monitored by
thin layer chromatography (TLC) using Merck silica gel 60-F254 pre-
coated glass plates. Kieselgel 60H without gypsum purchased from
Merck was used for short-column flash chromatography (SCFC). All
reactions were performed using flame-dried glassware and under
an inert atmosphere (dry N2).
Treatment of pregn-4-en-3b,17a,20b-triol (1)
with aluminum trichloride
Procedure A
Pregn-4-en-3b,17a,20b-triol (1; 1.0 g, 3.0 mmol) was dissolved in
dry 1,4-dioxane (50 mL), and aluminum trichloride (1.2 g, 9.0 mmol)
was added to the resulting solution. After stirring for 20 min at
room temperature, TLC analysis of the reaction mixture (chloro-
form ⁄ methanol, 20:1) showed complete conversion of the precursor
into the corresponding D3,5-steradiene 2. The organic solvent was
removed under vacuum, and the residue was treated with distilled
water (30 mL), then extracted with ethyl acetate (3 · 30 mL). The
combined organic layers were washed once with brine (30 mL),
dried over Na2SO4, filtered, and evaporated to dryness under vac-
uum. The crude solid residue was purified by flash column chroma-
tography (chloroform ⁄ methanol 20:1 v ⁄ v) to give 0.88 g (92%) of 2
as pure product.
270
Chem Biol Drug Des 2011; 78: 269–276

3-Hydroxy Steroids with Lewis and Anhydrous Protic Acids
Procedure B
0.99 (d, J = 6.2 Hz, 3H, 21-CH3), 0.86 (s, 3H, 19-CH3), 0.73 (s, 3H, 18-
Pregn-4-en-3b,17a,20b-triol (1; 1.0 g, 3.0 mmol) was dissolved in dry
1,4-dioxane (120 mL), and aluminum trichloride (1.2 g, 9.0 mmol) was
added. The resulting solution was refluxed under stirring until TLC
analysis (chloroform ⁄ methanol, 20:1) showed the complete consump-
tion of the precursor. The organic solvent was removed under vac-
uum, and the residue was treated with distilled water (30 mL), then
extracted with ethyl acetate (3 · 50 mL). The combined organic lay-
ers were washed once with brine (30 mL), dried over Na2SO4, fil-
tered, and evaporated to dryness under vacuum. The crude solid
residue was purified by flash column chromatography (chloro-
form ⁄ methanol, 20:1) to afford 0.8 g (84%) of 2 as pure product.
Compound 2 obtained by this procedure showed physicochemical
properties, and NMR spectra identical to those observed for a sam-
ple of the same compound prepared by applying the procedure A.
CH3); 13C NMR (DEPT, 75 MHz, DMSO-d6) d 14.6 (CH3), 16.8 (CH3),
18.9 (CH3), 21.0 (CH2), 23.8 (CH2), 31.0 (CH2), 31.6 (CH2), 32.0 (CH2),
33.8 (CH2), 36.2 (CH), 37.0 (C), 37.9 (CH2), 46.9 (C), 49.5 (CH), 54.7
(CH), 69.5 (CH), 84.6 (C), 117.2 (CH), 122.9 (CH), 123.3 (CH), 145.8 (C).
Treatment of pregn-4-en-3b,17a,20b-triol (1)
with titanium tetrachloride
A 1 M dichloromethane solution of titanium tetrachloride (5.4 mL,
5.4 mmol) was added to a suspension of pregn-4-en-3b,17a,20b-
triol (1; 0.6 g, 1.8 mmol) in dry dichloromethane (40 mL). The mix-
ture was maintained under magnetic stirring for 20 min at room
temperature, until TLC analysis of the reaction mixture (chloro-
form ⁄ methanol 20:1 v ⁄ v) showed complete consumption of the pre-
cursor. The organic solvent was removed under vacuum, and the
residue was treated with distilled water (25 mL), then extracted
with ethyl acetate (3 · 25 mL). The combined organic layers were
washed once with brine (25 mL), dried over Na2SO4, filtered, and
evaporated to dryness under vacuum. The crude solid residue was
purified by flash column chromatography (chloroform ⁄ methanol 20:1
v ⁄ v) to afford 0.52 g (89%) of 2 as pure compound. 1H and 13C
NMR spectra recorded for a sample of the pure product obtained
after chromatography showed spectroscopic characteristics equal to
those obtained for the same compound prepared by the procedure
A, as previously described.
Pregn-3,5-dien-17a,20b-diol (2)
White solid. M.p. 139)141 ꢀC; Rf 0.37 (chloroform ⁄ methanol 20:1
1
v ⁄ v). H NMR (300 MHz, DMSO-d6,): d 5.89 (d, J = 9.7 Hz, 1H, 4-
H), 5.57 (m, 1H, 6-H), 5.35 (m, 1H, 3-H), 4.04 (d, J = 6.8 Hz, 1H, 20-
OH), 3.74 (m, 1H, 20-H), 3.42 (s, 1H, 17-OH), 1.98)2.21 (m, 3H),
1.03)1.81 (m, 14H), 1.01 (d, J = 6.2 Hz, 3H, 21-CH3), 0.89 (s, 3H,
19-CH3), 0.75 (s, 3H, 18-CH3); 13C NMR (DEPT, 75 MHz, DMSO-d6) d
14.7 (CH3), 18.7 (CH3), 18.9 (CH3), 20.3 (CH2), 22.7 (CH2), 23.7 (CH2),
31.6 (CH), 31.7 (CH2), 31.8 (CH2), 33.4 (CH2), 33.8 (CH2), 34.7 (C),
46.9 (C), 47.6 (CH), 50.4 (CH), 69.5 (CH), 84.7 (C), 123.1 (CH), 124.7
(CH), 129.1 (CH), 141.0 (C). GC ⁄ MS (EI) m ⁄ z (%): 316 (43) [M+], 298
(7), 271 (43), 253 (100), 239 (3), 226 (11), 213 (63), 197 (13), 173 (8),
159 (12), 157 (17), 145 (27), 143 (24), 131 (15), 105 (34), 91 (36), 81
(38), 67 (14). Anal. Calcd. for C21H32O2: C, 79.70; H, 10.19. Found: C
79.42; H 10.22.
Treatment of pregn-4-en-3b,17a,20b-triol (1)
with CSA in toluene
A solution of 1 (120 mg, 0.36 mmol) and catalytic amounts of CSA
(8.4 mg, 0.04 mmol, 10% mol) in dry toluene were refluxed in a
Dean–Stark apparatus for 5 h. After this time, the solvent was dis-
tilled under reduced pressure and the residue was dissolved in
ethyl acetate (10 mL) and then washed once with a 5% NaHCO3
aqueous solution (10 mL). The organic layer was washed once with
brine (10 mL), dried over Na2SO4, filtered, and evaporated to dry-
ness under vacuum. The crude material was fractionated by short-
column flash chromatography (chloroform ⁄ methanol 20:1 v ⁄ v). Purifi-
cation gave 103 mg (90%) of a mixture of the epimeric dienes 2
and 3, which was subjected to the next thermal treatment, and
very small amounts (3 mg) of a second fraction (Rf = 0.88) contain-
Treatment of pregn-4-en-3b,17a,20b-triol (1)
with CSA
To a solution of pregn-4-en-3b,17a,20b-triol (1; 1.0 g, 3.0 mmol) in
dry 1,4-dioxane (50 mL), CSA (0.08 g, 0.32 mmol) was added. The
resulting mixture was refluxed under stirring for 2 h. After this
time, TLC analysis (chloroform ⁄ methanol, 20:1) showed complete
consumption of the precursor. The organic solvent was removed
under vacuum, and the residue was treated with distilled water
(30 mL), then extracted with ethyl acetate (3 · 30 mL). The com-
bined organic layers were washed once with brine (30 mL), dried
over Na2SO4, filtered, and evaporated to dryness under vacuum.
The crude solid residue was purified by chromatography (ethyl ace-
tate ⁄ petroleum ether, 50:50) to give 0.85 g (89% total yield) of a
1
ing 4, which was directly analyzed by H- and 13C NMR and by GC-
MS. The mixture of epimers 2 and 3 showed NMR data equal to
those observed for the same pair of epimers obtained by the treat-
ment of 1 with CSA in 1,4-dioxane. For compound 4, the following
main physicochemical data were determined: 1H NMR (300 MHz,
CDCl3): d 5.92 (d, J = 9.6 Hz, 1 H, 4-H), 5.60 (m, 1 H, 6-H), 5.38 (m,
1 H, 3-H), 2.48 (m, 1 H, 17-H), 2.18 (m, 3 H, 21-CH3). 13C NMR
(75 MHz, DMSO-d6, 25 ꢀC): d 209.1, 141.0, 128.8, 125.3, 122.3.
GC ⁄ MS (CI): m ⁄ z (%) 339 (8) [M+C3H5]+, 327 (12) [M+C2H5]+, 299
(100) [M+H]+, 281 (73) [(M-H2O)+H]+, 255 (33) [(M-COCH3)+H]+.
1
mixture of the two epimers 2 and 3. H NMR analysis performed
on a sample of the product obtained after chromatography indicated
an approximately 3:2 ratio of the C-17 a ⁄ b epimers. H, 13C, and
1
DEPT analyses of the same mixture allow also the complete assign-
ment of resonances for the epimer 3.
Pregn-3,5-dien-17 b,20b-diol (3)
Treatment of pregn-4-en-3b,17a,20b-triol (1)
with CSA in toluene
A mixture of epimers 2 and 3 (250 mg) recovered by chromatogra-
phy from the thermal treatment of 1 as previously described, and
1H NMR (300 MHz, DMSO-d6,): d 5.75 (m, 1H, 4-H), 5.60 (m, 1H, 6-
H), 5.46 (m, 1H, 3-H), 4.07 (d, J = 6.8 Hz, 1H, 20-OH), 3.73 (m, 1H,
20-H), 3.40 (s, 1H, 17-OH), 2.01)2.28 (m, 3H), 1.01)1.81 (m, 14H),
Chem Biol Drug Des 2011; 78: 269–276
271

De Marco et al.
1
catalytic amounts of CSA (20 mg, 10% mol) in dry toluene were
refluxed in a Dean–Stark apparatus for 5 h. After a work-up similar
to that described in the previous section, the starting material was
recovered in a quite quantitative yield. GC ⁄ MS analysis of the reac-
tion mixture showed also the presence of traces of compound 4.
one-dimensional H NMR spectroscopy demonstrated that the func-
tionalities of the C-17 side chain of the starting material 1
remained unaltered. Treatment of 1 with AlCl3 determined only the
loss of water from the allylic site of the A-ring with the consequent
formation of the related 3,5-steradiene derivative. The driving force
for this rapid dehydration was supposed to be the increased stabil-
ity of the 3,5-steradiene structure, determined by the formation of
the conjugated double bond system, with respect to the other pos-
sible isomers that could be produced from competitive pathways
involving the transposition of the allylic carbocation. This charged
species is most likely generated by the initial coordination of the
aluminum core of the Lewis species to the OH function at the C-3.
It is important to remark here that no evidence of D-ring rearrange-
ments was detected by NMR spectroscopy. In particular, DEPT tech-
niques were used to confirm the carbon backbone of 2 and to
exploit the possible presence in the molecular structure of D-homo-
annulated moieties. The DEPT 13C analysis confirmed a five-mem-
bered structure for the D-ring, showing that no homoannulation of
the D-ring of compound 1 occurred under the experimental condi-
tions adopted for the Lewis acid treatment. Therefore, we investi-
gated whether it would be possible to induce the formation of D-
homoannulated products using experimental conditions already
reported in the literature for the Lewis acid-assisted D-ring rear-
rangement of 17-hydroxy-20-keto steroids (41). Following the previ-
ous protocol, one molar equivalent of pregn-4-en-3b,17a,20b-triol
(1) was dissolved in dry 1,4-dioxane and treated with three equiva-
lents of aluminum trichloride under reflux until the complete con-
sumption of 1. Hydrolytic work-up followed by column
chromatography of the crude mixture afforded exclusively the prod-
uct 2 in a 84% yield. Furthermore, to verify the reactivity of the
1,2-diol system under the acid conditions used, one molar equiva-
lent of the D3,5-steradiene 2 was dissolved in dry 1,4-dioxane and
treated with a molar excess (five equivalents) of aluminum trichlo-
ride, under reflux for 3 h. Surprisingly, compound 2 was not reac-
tive and recovered unchanged in a quantitative yield.
Results and Discussion
The effects of non-aqueous acid media in the competitive structural
rearrangements possible for polyhydroxylated steroids were
exploited using pregn-4-en-3b,17a,20b-triol (1), chosen as an ideal
model of trihydroxy steroid characterized by a molecular backbone
including a 4-en-3-ol allylic system and a 17,20-diol function. Com-
pound 1 was considered to be capable of generating highly reactive
charged intermediates involving both the A- and D-rings, and an
optimal target in exploiting the transformations of the three differ-
ent hydroxyl functions present in the structure. Initially, we investi-
gated any possible competitive and ⁄ or simultaneous conversion of
the subskeletons of the A- and D-rings under milder experimental
conditions than those already reported, performing all reactions in
non-aqueous organic solvents and using Lewis acids or anhydrous
protic acids. Pregn-4-en-3b,17a,20b-triol (1) was obtained by the
stereospecific reduction in 17a-hydroxyprogesterone with NaBH4 in
methanol, according to a known synthetic route (40).
The reaction of 1 and 3 molar equivalents of aluminum trichloride
was analyzed (Scheme 1). The process was carried out in dry 1,4-
dioxane at room temperature and required reaction times not
exceeding 20 min for the total consumption of the starting material.
After a simple work-up, the crude residue was recovered and puri-
fied by column chromatography to obtain only one product in an
excellent yield (92%) based on the initial amount of 1. The com-
pound thus obtained was recognized as pregn-3,5-dien-17a,20b-diol
(2), and the expected molecular structure was unambiguously con-
1
firmed by one-dimensional H and 13C NMR spectroscopy, with the
aid of extensive 1H homodecoupling to establish the correlations
between the most important proton spin systems of the molecule.
The reaction performed using aluminum trichloride did not lead to
epimerization at C-17 or modifications of the 17,20-diol system on
the D-ring. The different reaction courses observed in this case
could be the result of the formation of a very stable coordination
complex between the oxygen atoms of the two adjacent hydroxyl
groups and the metallic core of the Lewis species. To confirm this
hypothesis, we performed another experiment using TiCl4. It is
known that the Lewis acid TiCl4 easily generates cyclic stable
adducts involving oxygen and nitrogen atoms (42). Treatment of one
molar equivalent of 1 with three equivalents of TiCl4, in dry DCM
at room temperature for 20 min, gave 2 exclusively in a 89% yield
and with complete retention of the configuration at C-17. Proton
NMR analysis confirmed that no epimeric mixtures were formed.
The absence of any epimerization could reasonably be considered
as a direct proof of the stability of the supposed cyclic intermediate
created by the coordination between the hydroxyl groups at C-17
and C-20 of 1 with the Lewis species. This assumption was further
verified by performing the same reaction under reflux for longer
periods (3–5 h). Under these new conditions, no modifications of
the 17,20-diol functionality on the D-ring were observed, as con-
firmed by proton NMR spectroscopy of a sample obtained from the
hydrolytic work-up of the crude reaction mixture.
1
Clean H and 13C NMR spectra recorded in DMSO-d6 at 25 ꢀC were
obtained for 2, showing the typical signals attributable to the con-
1
jugated 3,5-diene system. In particular, the H spectrum was char-
acterized by a well-defined doublet at 5.89 ppm, and two multiplets
were centered at 5.56 and 5.34 ppm. These signals were indicative
for the resonances of the 4-H, 6-H, and 3-H protons, respectively.
The multiplet at 3.75 ppm was attributed to the 20-H, and the pro-
tons of the C-17 and C-20 hydroxyl groups featured one singlet at
3.40 ppm and one doublet at 4.03 ppm, respectively. The assign-
ment of the signals attributable to the hydroxyl protons was
confirmed by isotopic exchange with D2O. The data collected by
Scheme 1: Treatment of pregn-4-en-3b,17a,20b-triol (1) with
AlCl3.
272
Chem Biol Drug Des 2011; 78: 269–276

3-Hydroxy Steroids with Lewis and Anhydrous Protic Acids
Successively, we extended our study to the reactivity of 1 toward
A
protic acids (Scheme 2). The trihydroxy steroid 1 was then allowed
to react in the presence of catalytic amounts of anhydrous CSA in
dry 1,4-dioxane. The acid was used in a 10% molar ratio with
respect to 1, and the reaction mixture was maintained under reflux
for 2 h. Under these conditions, we observed the formation of a
mixture of the two epimers 2 and 3, recovered in a 89% total yield
B
1
after column chromatography. The H NMR spectra recorded on a
dilute sample of the crude reaction product clearly indicated the
presence of the two C-17 epimers 2 and 3 in an approximately 3:2
ratio. In fact, the one-dimensional 1H NMR spectrum recorded in
DMSO-d6 at 25 ꢀC revealed, for the obtained mixture of epimers,
two sets of well-distinguishable resonances attributable to the con-
jugated 3,5-diene system, the hydroxyl group at C-17, and the C-18,
C-19, and C-20 methyl protons in both of the two epimers, together
a series of overlapping signals generated by the protons of the
other rings of both molecules.
Scheme 3: Camphorsulfonic acid catalysis: formation of the
3,5-steradiene frame of 2 and 3 from 1 (A) and epimerization of 2
to give 3 (B). The partial chemical structures IV and VII are,
respectively, used to indicate the stereochemistry of 2 and 3.
On the basis of the comparison with the spectroscopic data
obtained for the pure epimer 2, the major isomer formed under the
reaction conditions described earlier was recognized as the 17a epi-
mer. The last experiment confirmed that organic protic acid cataly-
sis converts the allylic alcoholic subskeleton of the A-ring into the
corresponding 3,5-diene moiety by dehydration but causes epimer-
ization at the C-17 stereocenter.
the ring opening of the charged epoxide generates 2 and 3 and
did not activate the other reaction channels already recognized in
case of steroid systems similar to 1 when they are treated with
concentrated aqueous acid solutions to produce mixtures of uniden-
tified products (32).
With regard to the stereochemical aspects that accompany the rear-
rangement of 1 to yield 2 and 3 when the trihydroxy precursor is
treated under protic conditions, the formation of the two epimeric
3,5-dienes could reasonably be interpreted by postulating the devel-
opment of a highly reactive carbocation of type V (Scheme 3B) at
the C-17 atom of the steroid structure during the protic acid treat-
ment. Furthermore, it could be supposed that this charged species
is stabilized as a protonated epoxide (VI), the formation of which
involves both the two C-17 and C-20 atoms. Thus, it seems that
The reactivity of 1 with CSA as the catalyst was also tested by an
NMR experiment, recording proton spectra in CDCl3 at )10 ꢀC at
different times. A one-dimensional 1H spectrum was initially
obtained for the precursor 1. The spectral window between 3.50
and 5.60 ppm clearly showed resonances at 5.28, 4.20, and
4.03 ppm attributable to the 4-H, 3-H, and 20-H protons, respec-
tively. Catalytic amounts of CSA (10% molar ratio) were then added
to the same sample, and a new set of two-one-dimensional proton
spectra was recorded at )10 ꢀC after 5 and 10 min. The spectrum
recorded after 10 min showed the complete consumption of 1. In
particular, the signal at 4.20 ppm attributable to the 3-H proton dis-
appeared, and three new signals were visible at 5.93, 5.60, and
5.38 ppm for the 4-H, 3-H, and 6-H protons, respectively. These res-
onances were attributed to the 3,5-diene moiety, as confirmed by
comparing the spectral data with those obtained for a sample of 2
prepared by reacting 1 with catalytic amounts of CSA in dry 1,4-
dioxane. The spectrum recorded after 10 min also showed the pres-
ence of two multiplets centered at 5.50 and 5.74 ppm, together a
signal that partially overlaps the multiplet at 5.60 ppm. All these
resonances were assigned to the protons of the 3,5-diene system
of the second epimer 3, according to the spectral data obtained for
the mixture of epimers 2 and 3 prepared by treatment with CSA
in 1,4-dioxane. Finally, no discernable resonances attributable to
D-homoannulated compounds were observed in the 1H spectra
recorded.
To conclude our investigation, we performed a study of the possible
transformations of the C-17 and C-21 hydroxy groups in the D-ring
of 1 by testing the structure resistance to the protic conditions
adopted in the treatment with CSA. To this aim, we exploited the
Scheme 2: Treatment of pregn-4-en-3b,17a,20b-triol (1) with
camphorsulfonic acid.
Chem Biol Drug Des 2011; 78: 269–276
273

De Marco et al.
reactivity of the triolic precursor 1 by subjecting it to a stronger
thermal treatment in the presence of catalytic amounts of CSA in
refluxing toluene for more prolonged times. Reaction was performed
in a Dean–Stark apparatus, and the reaction mixture was warmed
for 5 h. Fractionation by column chromatography of the crude mate-
rial obtained from the thermal treatment allowed the recovery of a
predominant fraction containing a mixture of both the epimeric 3,5-
steradienes 2 and 3 in a 90% yield. Another fraction, characterized
by a Rf value higher than that of the pair of dienes 2 and 3, was
recovered in a 3% yield. Owing to its small amount, this second
fraction was directly analyzed by NMR and GC ⁄ MS under chemical
ionization conditions, without further treatments. The spectral win-
conditions adopted for 1 as previously described. Although GC ⁄ MS
analysis under CI conditions of the reaction mixture at the end of
the treatment showed the presence of traces of 4, the starting
material was recovered in a quite quantitative yield. Last results
confirmed the exceptional stability of the 17,20-diol moiety to the
prolonged action of protic acids in anhydrous environments.
Conclusions
The aqueous acidic treatment of polyhydroxy steroids often pro-
duces complex mixtures of species that present strong modifications
of the precursor ring systems. As demonstrated in the literature,
steroids containing hydroxyl groups in different parts of their skele-
ton can give rise to a series of homoannulated and ⁄ or unsaturated
derivatives when treated with acids. However, the resulting mix-
tures are often characterized by the presence of unknown side
products generated via the formation of highly reactive and not iso-
lable cationic intermediates. The reaction of polyhydroxy steroids
with Lewis acids or anhydrous organic acids presents different fea-
tures. In particular, and conversely to the data reported in the liter-
ature for the aqueous acid treatments of pregn-4-en-3b,17a,20b-
triol, the reactions of the same trihydroxy steroid with AlCl3 or TiCl4
influenced only the allylic system of the A-ring of the precursor,
affording the related D3,5-steradiene. Moreover, for the studied
reaction, there was no evidence of D-ring rearrangement or C-17
side-chain modification. The product can also be manufactured in
safety on the gram scale. The organic acid CSA produced only the
epimerization of the C-17 atom, with no further modifications of the
3,5-diene system. The stability of the 1,2-diol system under all the
acid conditions applied was verified by performing the same treat-
ments under reflux. In case of Lewis acid-induced reactions, the
1
dow between 5.0 and 6.2 ppm of the H NMR spectrum registered
in CDCl3 at 25 ꢀC showed a series of signals at 5.92, 5.60, and
5.38 attributable to the typical resonances of the 4-H, 6-H, and 3-H
protons, respectively. These values confirmed the spin system of
the 3,5-diene skeleton to be present in the molecular structure of
the principal component of the minor fraction. The same spectrum
showed an interesting singlet resonating at 2.18 ppm probably
diagnostic for the methyl protons of a ketone moiety at the C-17
center and a not well-defined multiplet centered at 2.48 ppm rea-
sonably attributable to the 17-H proton. This hypothetic attribution
was supported also by the signals displayed in the spectral window
between 110 and 220 ppm of the 13C NMR spectrum recorded in
CDCl3 at the same temperature. In particular, resonances at 141.0,
128.8, 125.3, and 122.3 were attributed to the carbon skeleton of
the conjugated system of the 3,5-steradiene, while the presence of
a peak at 209.1 ppm suggested a 20-keto structure such as 4
(Scheme 3). The strong overlapping of multiplets from hydrogen
atoms in the methylene groups in the low-frequency window of the
1H spectrum made very difficult and complicated the total assign-
ments of other resonances for the structure of the plausible 20-keto
steroid derivative 4. The GC ⁄ MS analysis of the smaller fraction
recovered by column chromatography showed a prevalent chromato-
graphic peak. The MS spectrum obtained under chemical ionization
conditions was characterized by the appearance of a set of three
peaks, the pseudo-molecular ion [M+H]+ of m ⁄ z 299, followed by
the ions of m ⁄ z 281 and 255 corresponding to the [(M-H2O)+H]+
and [(M-43)+H]+ species, respectively. In the CI-MS spectrum also
appeared two peaks related to the ion species of m ⁄ z 327 and
339, the clusters [M+C2H5]+ and [M+C3H5]+, respectively. Therefore,
NMR and CI-MS analysis seemed to be in agreement with the 20-
keto structure hypothesized for compound 4, which is probably
formed through an intermediate having the structure of type IX
after the acid-induced dehydration of the 17,20-diol function as
depicted in VIII (Scheme 4).
D
3,5-steradiene precursor was recovered totally unchanged, while
CSA and high temperatures were responsible only for the epimer-
ization occurring at the C-17. In light of the data collected in this
study, it is possible to assume that the most likely reason for the
different courses observed for the Lewis acid treatments with
respect to the reaction performed with the anhydrous protic acid
CSA lies in the formation of stable complexes involving the two
adjacent hydroxy oxygen atoms and the metal core of the Lewis
acid. This kind of coordination should block the elimination of water
from the dihydroxylated D-ring side chain, avoiding the expected D-
homoannulation. Alternatively, when the anhydrous organic acid
CSA is used, the protonation of one of the two hydroxyl groups
determines the loss of water and the concomitant formation of a
stable tertiary carbocation at C-17, which is responsible for the
observed epimerization. Moreover, the data obtained from both the
thermal treatments of 1 and of a mixture of 2 and 3 under the
same conditions demonstrated that the functional side chains in the
C-17 of the D-ring are extraordinary stable to the prolonged expo-
sure to anhydrous protic acids.
For completeness, the mixture of epimers 2 and 3 was subjected
to a thermal treatment performed under the same experimental
Acknowledgments
This work was supported by grants from Ministero dell'Istruzione,
dell'Universitꢀ e della Ricerca (MIUR), Italy.
Scheme 4: Structure of compound 4 and the plausible enol
intermediate involved in its formation.
274
Chem Biol Drug Des 2011; 78: 269–276

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16. Gçrçg S., Csizꢃr ꢄ. (1971) Analysis of steroids. XVI. Spectropho-
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