Microbial hydroxylation of some steroids by Aspergillus wentii MRC 200316

Article abstract of DOI:10.1135/cccc2010112

Biotransformations of epiandrosterone (1), dehydroepiandrosterone (2) and pregnenolone (3) by Aspergillus wentii MRC 200316 for 5 days have been reported. Incubation of epiandrosterone (1) afforded 11α-hydroxy-5α- androstane-3,17-dione (4) and 3β,11α-dihydroxy-5α-androstan-17- one (5). Incubation of dehydroepiandrosterone (2) afforded 3β,7β- dihydroxyandrost-5-en-17-one (6) and 3β,7α-dihydroxyandrost-5-en-17- one (7). Incubation of pregnenolone (3) afforded only 11α-hydroxypregn-4- ene-3,20-dione (8).

Full text of DOI:10.1135/cccc2010112

Hydroxylation of Some Steroids by Aspergillus wentii
1273
MICROBIAL HYDROXYLATION OF SOME STEROIDS BY
Aspergillus wentii MRC 200316
Kudret YILDIRIM
Chemistry Department, Sakarya University, 54187 Sakarya, Turkey;
e-mail: kudrety@sakarya.edu.tr
Received September 12, 2010
Accepted October 19, 2010
Published online November 26, 2010
Biotransformations of epiandrosterone (1), dehydroepiandrosterone (2) and pregnenolone (3)
by Aspergillus wentii MRC 200316 for 5 days have been reported. Incubation of epi-
androsterone (1) afforded 11α-hydroxy-5α-androstane-3,17-dione (4) and 3β,11α-dihydroxy-
5
α-androstan-17-one (5). Incubation of dehydroepiandrosterone (2) afforded 3β,7β-di-
hydroxyandrost-5-en-17-one (6) and 3β,7α-dihydroxyandrost-5-en-17-one (7). Incubation of
pregnenolone (3) afforded only 11α-hydroxypregn-4-ene-3,20-dione (8).
Keywords: Steroids; Biotransformation; Hydroxylation; Aspergillus wentii.
Microbial biotransformation of steroids has found worldwide application
for the preparation of more valuable and functionalized compounds such
1
as steroid drugs and hormones due to its high regio- and stereoselectivity .
For example, a number of investigations involving microbial biotransform-
ations of a wide range of steroidal substrates have been reported in recent
years1 . There are still enormous efforts to increase the efficiency of micro-
bial steroid biotransformations and to find new useful microorganisms and
–3
1
reactions . Some of these steroid biotransformations have recently been car-
ried out using Penicillium digitatum4 and Aspergillus terreus . Penicillium
digitatum and Aspergillus terreus showed some low 5α-reductase and Baeyer–
Villiger monooxygenase activities, respectively.
5
Aspergillus wentii Wehmer is a toxigenic, ubiquitous, soil-inhabiting fun-
6
,7
gus usually found on decayed vegetation and moist grains . It produces
8
6
9
several toxic metabolites such as aflatoxins , emodin and ochratoxin A as
1
0
well as other secondary metabolites like kojic acid , 1-amino-2-nitrocyclo-
pentanecarboxylic acid , hexadecylcitraconic acid , 3-nitropropionic
1
1
12
1
3
14
acid , and wentilactone A and B
.
As far as biotransformations by A. wentii are concerned, the only steroid
1
5
incubations with this fungus have been reported recently . In the men-
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281
©
2010 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2010112

1
274
Yildirim:
tioned incubations, A. wentii MRC 200316 has hydroxylated testosterone
and progesterone in high yields. In this work, the biotransformations of
epiandrosterone (1), dehydroepiandrosterone (2) and pregnenolone (3) by
A. wentii MRC 200316 for 5 days were investigated. A. wentii also showed
some high hydroxylase activities on these steroids.
RESULTS AND DISCUSSION
Incubation of epiandrosterone (1) with A. wentii for 5 days afforded two
metabolites (Scheme 1). The first metabolite was identified as 11α-hydroxy-
5
α-androstane-3,17-dione (4). The metabolite had characteristic reso-
1
6,17
nances
at δ 3.98 ppm (1 H, dt, J = 5.0 and 10.0 Hz) and δ 68.66 ppm,
H
C
which were consistent with the hydroxylation of 11α-equatorial position.
The 13C NMR spectrum of the metabolite demonstrated β-carbon down-
field shifts for C-9 (∆ 5.47 ppm) and C-12 (∆ 11.36 ppm), and a γ-carbon
upfield shift for C-8 (∆ 0.92 ppm), all of which consistent with 11α-
1
7
hydroxylation . The metabolite lacked the 3α-H resonance of 1 at δ 3.53
H
(
1 H, tt, J = 5.0 and 12.0 Hz) and had a new resonance at δC 212.72 ppm,
suggesting that the 3β-hydroxy group was oxidized to a 3-keto group.
SCHEME 1
Biotransformation of steroidal substrates by A. wentii MRC 200316
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

Hydroxylation of Some Steroids by Aspergillus wentii
1275
The second metabolite was identified as 3β,11α-dihydroxy-5α-androstan-
1
5
7-one (5). The metabolite had new resonances at δ 3.88 ppm (1 H, dt, J =
H
.0 and 10.0 Hz) and δC 68.43 ppm, which were in accordance with the
1
3
presence of an 11α-hydroxyl group. The C NMR spectrum of the metabo-
lite showed downfield shifts for C-9 (∆ 6.02 ppm) and C-12 (∆ 11.34 ppm),
and a γ-carbon upfield shift for C-8 (∆ 0.84 ppm), which were further in ac-
1
7
cordance with 11α-hydroxylation . The metabolite had the 3α-H reso-
nance of 1 at δ 3.50 ppm (1 H, tt, J = 5.0 and 10.0 Hz), suggesting that the
H
3
β-hydroxy group was maintained.
During the time course experiment for epiandrosterone (1), the
1
9-methyl resonance of 1 shifted from 0.84 to 0.92 ppm and the compari-
1
son of the methyl group integrations in the H NMR spectra indicated that
7
1
1
5% of the substrate was converted into 3β,11α-dihydroxy-5α-androstan-
7-one (5) and the hydroxylation had been completed by 72 h. At 96 h, the
9-methyl resonance of 1 shifted from 0.84 to 0.89 and 0.92 ppm, and the
1
comparison of the methyl group integrations in the H NMR spectra indi-
cated that almost 30% of 3β,11α-dihydroxy-5α-androstan-17-one (5) was
converted into 11α-dihydroxy-5α-androstane-3,17-dione (4). Further com-
1
parison of the methyl group integrations in the H NMR spectra suggested
that no more reactions took place after 96 h. Time course experiment re-
sults indicated that epiandrosterone (1) was first converted into 3β,11α-
dihydroxy-5α-androstan-17-one (5) by a 11α-hydroxylase and some of this
diol was then converted into 11α-hydroxy-5α-androstan-3,17-dione (4) via
an oxidation at C-3 (Scheme 2).
Incubation of dehydroepiandrosterone (2) with A. wentii for 5 days af-
forded two metabolites (Scheme 1). The first metabolite was identified as
1
3
β,7β-dihydroxyandrost-5-en-17-one (6). The H NMR spectrum of 6 had
a new resonance at δH 3.95 ppm (1 H, dt, J = 3.0 and 8.0 Hz) and experi-
enced an upfield shift for the 6-H resonance (∆ 0.03 ppm), which was con-
sistent with the presence of a 7β-hydroxy group. Its 13C NMR spectrum, in
comparison with 2, showed downfield shifts for C-6 (∆ 4.54 ppm) and C-8
(
∆ 8.93 ppm), whereas it showed a γ-gauche upfield shift for C-9 (∆ 2.01 ppm),
1
8
which are comparable with literature values . The metabolite had a reso-
nance at δH 3.55 ppm (1 H, tt, J = 5.0 and 10.0 Hz), indicating that the
3
β-hydroxy group retained.
The second metabolite was identified as 3β,7α-dihydroxyandrost-5-en-
1
1
7-one (7). The H NMR spectrum of 7 had a new resonance at δ 3.96 ppm
H
(
1 H, bs, W = 12.0 Hz) and experienced a downfield shift for the 6-H reso-
h
nance (∆ 0.28 ppm), which was in accordance with a 7α-hydroxy group. Its
1
3
C NMR spectrum, in comparison with 2, showed downfield shifts for C-6
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

1
276
Yildirim:
(
∆ 2.40 ppm) and C-8 (∆ 5.80 ppm), whereas it showed a γ-gauche upfield
1
8
shift for C-9 (∆ 8.53 ppm), which are comparable with literature values .
The resonance at δ 3.56 ppm (1 H, m, W = 24.0 Hz) was still present in
the product H NMR spectrum, indicating that the 3β-hydroxy group main-
tained.
H
h
1
During the time course experiment, the 19-methyl signal of dehydro-
epiandrosterone (2) moved from 1.01 to 1.08 and 1.03 ppm, respectively,
1
and the comparison of the methyl group integrations in the H NMR spec-
trum indicated that 75% of dehydroepiandrosterone (2) had been con-
verted into 3β,7β-dihydroxyandrost-5-en-17-one (6) and 3β,7α-dihydroxy-
androst-5-en-17-one (7), and the hydroxylation had been completed by
1
7
2 h. Further comparison of the methyl group integrations in the H NMR
spectra suggested that no more reactions were observed after that time. Ac-
cording to time course experiment results for dehydroepiandrosterone (2),
A. wentii showed both 7α- and 7β-hydroxylase activities (Scheme 2). This
fungus had a major 7α-hydroxylase activity and a minor 7β-hydroxylase ac-
tivity on dehydroepiandrosterone (2) as in the incubations of the same sub-
strate with Rhizomucor tauricus19 and Mucor racemosus .
20
SCHEME 2
Metabolism of steroidal substrates by A. wentii MRC 200316
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

Hydroxylation of Some Steroids by Aspergillus wentii
1277
Incubation of pregnenolone (3) with A. wentii for 5 days afforded only
1
1
1α-hydroxypregn-4-ene-3,20-dione (8) (Scheme 1). The H NMR spectrum
of the metabolite demonstrated a significant downfield shift (∆ 0.30 ppm)
for the 19-methyl group. The metabolite had characteristic resonances1
6,17
at δH 3.98 ppm (1 H, dt, J = 5.0 and 10.0 Hz) and δ 68.63 ppm, suggesting
C
1
that an 11α-hydroxylation had taken place. The H NMR spectrum of the
metabolite lacked the resonance of starting material at δ 3.45 ppm (1 H, tt,
H
J = 5.0 and 12.0 Hz, 3-H), and the double bond signal of 3 at δH 5.32 ppm
(
(
1 H, d, J = 5.0 Hz, 6-H) had an important downfield shift to 5.69 ppm
∆ 0.37 ppm), suggesting that the 5-ene-3β-hydroxy moiety of 3 was con-
verted into the 4-ene-3-keto moiety.
During the time course experiment, the 19-methyl signal of pregnen-
2
1
olone (3) shifted from 0.98 to 1.17 ppm and the comparison of the meth-
1
yl group integrations in the H NMR spectrum indicated that 80% of preg-
nenolone (3) was converted into 3β,11α-dihydroxypregn-5-en-20-one (9)
and hydroxylation had been completed by 72 h. At 96 h, the presence of
the 19-methyl signal of pregnenolone (3) shifted from 0.98 to 1.28 ppm,
and the lack of the 19-methyl resonance of 3β,11α-dihydroxypregn-5-en-
2
5
3
0-one (9) at δH 1.17 ppm indicated that all of 3β,11α-dihydroxypregn-
-en-20-one (9) had been converted into 11α-hydroxypregn-4-ene-
,20-dione (8). Further comparison of the methyl group integrations in the
1
H NMR spectra suggested that no more reactions took place after 96 h.
Time course experiment results demonstrated that A. wentii first had an
1
1α-hydroxylase activity on pregnenolone (3) and this afforded 3β,11α-
dihydroxypregn-5-en-20-one (9). All of this diol was then converted into
1
2
1α-hydroxypregn-4-ene-3,20-dione (8) by an isomerase enzyme (Scheme
). This type of isomerization of a few 11α-hydroxylated metabolites was
also observed during the incubations of pregnenolone (3) with Botrytis
cinerea22 and Cunninghamella elegans .
23
This study has demonstrated that A. wentii MRC 200316 is capable to
hydroxylate a range of steroids including epiandrosterone (1), dehydro-
epiandrosterone (2) and pregnenolone (3). A. wentii showed high 11α-
hydroxylase activities on epiandrosterone (1) and pregnenolone (3) as in
1
3
the incubation of progesterone . A range of fungi including Rhizopus
2
4
25
26
nigricans , Aspergillus ochraceus and Cephalosporium aphidicola are capa-
ble to functionalize at the 11α position which is influenced by the presence
of a C-17 side chain. Interestingly, the C-17 side chain had no influence on
hydroxylation at this position by A. wentii.
Uniquely in this study, dehydroepiandrosterone (2) was hydroxylated at
5
C-7 by A. wentii. This suggested that the introduction of a ∆ -double bond
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

1
278
Yildirim:
completely altered the results from the incubation of epiandrosterone (1)
by A. wentii leading to only non-stereospesific allylic hydroxylation at C-7
in place of the attack at C-11α. Its hydroxylase activity at the axial proton
was higher. Apart from a minor hydroxylation at C-14α, A. wentii also
hydroxylated testosterone mainly at the allylic, 6β-axial position, although
it did not hydroxylate pregnenolone (3) and progesterone at any allylic po-
1
5
5
4
sition . This suggested that the presence of a ∆ - or ∆ -double bond and
the lack of a side chain at C-17 resulted in mainly allylic hydroxylations by
A. wentii.
In conclusion, it has been shown that A. wentii has the ability to
hydroxylate steroidal substrates and it is a good 11α-hydroxylator for
epiandrosterone (1) and pregnenolone (3). It has also been shown that the
presence of a double bond in ring A or B and the lack of a side chain at
C-17 resulted in allylic hydroxylations by A. wentii. Our work on steroid
biotransformation by A. wentii and some other fungi is in progress.
EXPERIMENTAL
Epiandrosterone, dehydroepiandrosterone and pregnenolone were purchased from Fluka
(
Istanbul, Turkey). Aspergillus wentii MRC 200316 was obtained from TUBITAK (Marmara
Research Center, Food Science and Technology Research Institute, Culture Collection Unit,
Kocaeli, Turkey). Stock cultures were maintained at 4 °C on PDA slopes. Solvents were of
analytical grade and were purchased from Merck (Istanbul, Turkey). Potato dextrose agar
and agar for PDA slopes and ingredients for liquid medium were also purchased from Merck
(
Istanbul, Turkey).
The steroids were separated by column chromatography on silica gel 60 (Merck 107734)
with increasing concentrations of ethyl acetate in hexane as eluent. TLC was carried out
with 0.2 mm thick Merck Kieselgel 60 F254 TLC plates using ethyl acetate/hexane (1:1, v/v)
as eluent. TLC plates were dipped into an anisaldehyde–H SO reagent and heated to 120 °C
2
4
–
1
for 3 min in order to visualize the spots. Infrared spectra (wavenumbers in cm ) were re-
corded using a Shimadzu IR Prestige-21 apparatus. Optical rotation mesurements were car-
ried out on a WXG-4 polarimeter. Elemental analysis was performed using a Thermo
1
Finnigan Flash EA 1112 elemental analyser. H NMR spectra were recorded in deuterio-
chloroform with tetramethylsilane as an internal standard with a Varian Mercury 300 spec-
1
3
trometer. C NMR spectra were recorded in deuteriochloroform at 75 MHz with a Varian
Mercury 300 spectrometer. Chemical shifts are given in ppm (δ-scale), coupling constants (J)
and width multiplets (W) in Hz. Melting points were determined by an Electrothermal
IA 9200 melting point apparatus and are uncorrected. Biotransformation experiments were
run with control flasks containing non-inoculated sterile medium and one of the substrates.
After 5 days of incubation, all controls were also harvested and analysed by TLC. No metab-
olites were detected in controls.
2
7
Time course experiments were conducted in order to determine the metabolic pathway.
Conditions were identical to those in main biotransformation experiments except that each
individual steroidal substrate (300 mg), dissolved in DMF (6 ml), was evenly distributed be-
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

Hydroxylation of Some Steroids by Aspergillus wentii
1279
tween 6 flasks (each containing 100 ml of medium). One flask was harvested and extracted
after 8 h. Then one flask was harvested and extracted every 24 h. TLC analysis was per-
formed immediately on the isolated mixture. Following 6 h under high vacuum, the product
1
H NMR spectra were determined in CDCl3 to confirm the steroidal nature of the extracts.
Biotransformation of Epiandrosterone (1) by A. wentii
Spores freshly obtained from PDA slopes were transferred aseptically into 10 Erlenmeyer
flasks of 250 ml, containing 100 ml of a sterile synthetic medium28 (1.5% sucrose, 1.5%
glucose, 0.5% polypeptone, 0.05% MgSO ·7H O, 0.05% KCl, 0.1% K HPO and 0.001%
4
2
2
4
FeSO ·7H O, pH 7.2) in a biological safety cabinet. After cultivation at 27 °C for 2 days on a
4
2
rotary shaker (150 rpm), epiandrosterone (1; 500 mg, 1.72 mmol) dissolved in 10 ml of DMF
was evenly distributed aseptically among the flasks. The biotransformation of the substrate
was carried out in 10 flasks for 5 days under the same conditions. The fungal mycelium was
separated from the broth by filtration under the vacuum and the mycelium was rinsed with
ethyl acetate (500 ml). The broth was saturated with NaCl and then extracted 3 × each with
1
l of ethyl acetate. The organic extract was dried over anhydrous sodium sulfate and the
solvent was evaporated in vacuo to give a brown gum (735 mg), which was then chroma-
tographed on a silica gel. Elution with 30% ethyl acetate in hexane afforded the unreacted
1
13
starting material (60 mg, 12%), which was identified by comparison of its H and C NMR
spectra with those of an authentic material.
Elution with 50% ethyl acetate in hexane afforded 11α-hydroxy-5α-androstane-3,17-dione
(
4; 95 mg, 18%), which was crystallized from petroleum ether as needles, m.p. 194–195 °C,
20
29
20
[
α] +63, c 0.1, CHCl3 (lit. gives m.p. 194–195 °C, [α]D +66, c 1, CHCl ). For C19H28O3
D
3
(
(
3
304.41) calculated: 74.96% C, 9.27% H; found: 74.35% C, 9.73% H. IR: 3335 (O–H), 1719
C=O), 1735 (C=O). 1H NMR (300 MHz, CDCl ): 0.89 s, 3 H (H-18); 1.14 s, 3 H (H-19);
3
1
3
.98 dt, 1 H, J(11β,12β) = 5.0, J(11β,9) = J(11β,12α) = 10.0 (H-11). C NMR (75 MHz,
CDCl ): 219.82, 212.72, 68.66, 59.72, 50.09, 47.96, 47.21, 44.96, 42.75, 39.83, 38.21, 37.30,
3
3
5.79, 34.01, 30.21, 29.02, 21.75, 14.50, 11.73.
Elution with 60% ethyl acetate in hexane afforded 3β,11α-dihydroxy-5α-androstan-17-one
2
0
(
5; 285 mg, 54%), which was crystallized from acetone as cubes, m.p. 104–106 °C, [α] +48,
D
2
9
20
c 0.1, CHCl3 (lit. gives m.p. 93–103 °C, [α]D +53, c 1, CHCl ). For C19H30O3 (306.43) calcu-
lated: 74.47% C, 9.87% H; found: 74.50% C, 9.65% H. IR: 3580 (O–H), 1740 (C=O). H NMR
3
1
(
1
300 MHz, CDCl ): 0.83 s, 3 H (H-19); 0.92 s, 3 H (H-18); 3.50 tt, 1 H, J(3,2) = 5.0, J(3,4) =
0.0 (H-3); 3.88 dt, 1 H, J(11β,12β) = 5.0, J(11β,9) = J(11β,12α) = 10.0 (H-11). C NMR
3
1
3
(
75 MHz, CDCl ): 219.90, 70.65, 68.43, 60.27, 50.19, 47.94, 44.97, 42.73, 38.42, 38.35,
3
3
7.25, 35.81, 34.09, 31.55, 30.65, 28.80, 21.72, 14.44, 12.64.
Biotransformation of Dehydroepiandrosterone (2) by A. wentii
Under similar conditions, the incubation of dehydroepiandrosterone (2; 500 mg, 1.73 mmol)
with A. wentii afforded a brown gum (746 mg), which was then chromatographed on a silica
gel. Elution with 30% ethyl acetate in hexane afforded the unreacted starting material
50 mg, 10%), which was identified by comparison of its 1H and C NMR spectra with
13
(
those of an authentic material.
Elution with 80% ethyl acetate in hexane afforded 3β,7β-dihydroxyandrost-5-en-17-one
(
6; 63 mg, 12%), which was crystallized from ethyl acetate–petroleum ether as needles, m.p.
Collect. Czech. Chem. Commun. 2010, Vol. 75, No. 12, pp. 1273–1281

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Yildirim:
08–209 °C, [α]D +67, c 0.1, CHCl3 (lit.30 gives m.p. 213–215 °C, [α]D +63, c 1.3, CHCl ). For
2
0
20
2
3
C19H28O3 (304.41) calculated: 74.96% C, 9.27% H; found: 74.75% C, 9.03% H. IR: 3237
1
(
(
O–H), 1736 (C=O), 1665 (C=C). H NMR (300 MHz, CDCl ): 0.90 s, 3 H (H-18); 1.08 s, 3 H
3
H-19); 3.50 tt, 1 H, J(3,2) = 5.0, J(3,4) = 10.0 (H-3); 3.95 dt, 1 H, J(7α,8) = 8.0, J(7α,6) =
J(7α,4β) = 3.0 (H-7); 5.32 bs (H-6). C NMR (75 MHz, CDCl ): 221.33, 143.64, 125.44,
1
3
3
7
2
2.79, 71.19, 51.12, 48.16, 47.75, 41.57, 40.38, 36.82, 36.61, 35.96, 31.40, 31.16, 24.16,
0.34, 19.14, 13.55.
Elution with pure ethyl acetate in hexane afforded 3β,7α-dihydroxyandrost-5-en-17-one
(
7; 269 mg, 51%), which was crystallized from ethyl acetate–petroleum ether as needles,
20
30
20
m.p. 174–175 °C, [α]D –73, c 0.1, CHCl3 (lit. gives m.p. 178–180 °C, [α]D –80, c 0.14,
CH OH). For C19H28O3 (304.41) calculated: 74.96% C, 9.27% H; found: 74.81% C, 9.10% H.
3
1
IR: 3347 (O–H), 3163 (O-H), 1734 (C=O), 1622 (C=C). H NMR (300 MHz, CDCl ): 0.89 s,
3
3
5
6
1
H (H-18); 1.03 s, 3 H (H-19); 3.56 m, 1 H, W = 24 (H-3); 3.96 bs, 1 H, Wh = 12 (H-7);
h
.63 d, 1 H, J(6,7β) = 5.0 (H-6). 1 C NMR (75 MHz, CDCl ): 221.89, 145.97, 123.30, 70.75,
3
3
3.89, 47.01, 44.69, 42.24, 41.64, 37.25, 36.98, 36.73, 35.65, 30.84 (2 × C), 21.67, 19.84,
8.07, 13.08.
Biotransformation of Pregnenolone (3) by A. wentii
Under similar conditions, the incubation of pregnenolone (3; 500 mg, 1.58 mmol) with
A. wentii afforded a brown gum (753 mg), which was then chromatographed on a silica gel.
Elution with 30% ethyl acetate in hexane afforded the unreacted starting material (60 mg,
1
13
1
2%), which was identified by comparison of its H and C NMR spectra with those of an
authentic material.
Elution with 50% ethyl acetate in hexane afforded 11α-hydroxypregn-4-ene-3,20-dione
(
[
(
(
8; 407 mg, 78%), which was crystallized from ethyl acetate as needles, m.p. 163–164 °C,
20
31
20
α] +165, c 0.1, CHCl3 (lit. gives m.p. 165–167 °C, [α]D +169, CHCl ). For C21H30O3
D
3
330.45) calculated: 76.32% C, 9.15% H; found: 76.21% C, 9.08% H. IR: 3380 (O–H), 1654
C=O), 1610 (C=C). 1H NMR (300 MHz, CDCl ): 0.65 s, 3 H (H-18); 1.28 s, 3 H (H-19);
3
2
1
5
1
.10 s, 3 H (H-21); 3.98 dt, 1 H, J(11β,12β) = 5.0, J(11β,9) = J(11β,12α) = 10.0 (H-11); 5.69 s,
1
3
H (H-4). C NMR (75 MHz, CDCl ): 209.02, 200.49, 171.40, 124.34, 68.63, 63.02, 58.77,
3
5.21, 50.18, 44.04, 39.86, 37.32, 34.82, 34.06, 33.51, 31.43, 31.27, 24.12, 22.83, 18.18,
4.38.
The author would like to thank F. Sonmez and H. Genc for running the 1H and 13C NMR spectra.
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