4
N. Milecka-Tronina et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
a
-Hydroxy-5
a
-androstan-3,17-dione (15): mp 160–163 °C
and
(Fig. 1). The appearance of a new methine carbon signal at dC
74.78 ppm, in combination with downfield shifts for C-6 (
10.34 ppm), C-8 ( 7.87 ppm) and -carbon upfield shift for C-5
2.91 ppm), C-9 ( 2.01 ppm) and C-14 ( 0.4 ppm) further
a-oriented H-9 (dH 0.74 ppm) and H-14 (dH 1.43 ppm)
(Me2CO, needles) (lit. 158–160 °C33); C19H28O3: calcd C, 75.10; H,
9.32; found. C, 74.80; H, 9.21%. IR
m
max(cmÀ1): 3393, 1740, 1716,
D
1H NMR (CDCl3) 0.86 (3H, s, 18-H), 1.01 (3H, s, 19-H), 3.99 (1H,
D
c
m, 7b-H).
(
D
D
D
7b-Hydroxy-5
(Me2CO, needles); C19H28O3: calcd C, 75.10; H, 9.32; found. C,
74.80; H, 9.21%. IR
max(cmÀ1): 3340, 1738, 1714, 1H NMR (CDCl3)
0.91 (3H, s, 18-H), 1.06 (3H, s, 19-H), 3.51 (1H, m, 7 -H).
a
-androstan-3,17-dione (16): mp 157–162 °C
determine the product as 7b-hydroxy-EpiA (12). The spectral
data are in agreement with the literature.25 The 1H NMR spec-
trum of 15 shows a new downfield signal for the oxygen-bearing
methine proton at dH 3.99 ppm. The signal shape and position
suggest hydroxylation at an axial C-7 position of the steroid mol-
ecule, in agreement with the literature data.33,36 The shift of C-18
m
a
9a-Hydroxy-5a-androstan-3,17-dione (17): mp 211–215 °C
(Me2CO, needles) (lit. 212–214 °C33); C19H28O3: calcd C, 75.10; H,
9.30; found. C, 74.80; H, 9.20%. IR mmax (cmÀ1): 3340, 1741, 1714,
1H NMR (CDCl3), 0.88 (3H, s, 18-H), 1.14 (3H, s, 19-H).
methyl signal (
in comparison to the spectrum of substrate 4 correspond to the
shifts accompanying introduction of the 7
-hydroxyl group.35
D 0.01 ppm) and C-19 methyl signal (D 0.02 ppm)
11
a
-Hydroxy-5
a
-androstan-3,17-dione (18): mp 190–193 °C
a
(Me2CO, needles) (lit. 191–193 °C34); C19H28O3: calcd C, 75.10; H,
This assignment of position of hydroxyl group was further sup-
ported by the HMBC correlations of H-7 (dH 3.99 ppm) with C-5
(dC 39.03 ppm) and C-14 (dC 45.68 ppm) (Fig. 1). The signal at
9.30; found. C, 74.80; H, 9.20%. IR m
max(cmÀ1): 3336, 1739, 1716,
1H NMR (CDCl3) 0.88 (3H, s, 18-H), 1.13 (3H, s, 19-H), 4.00 (1H,
m, 11b-H).
dC 66.2 ppm and downfield shifts for C-6 (
4.09 ppm), and -gauche upfield shifts for C-5 (
9 ( 8.48 ppm) and C-14 ( 5.53 ppm) constitute confirmation
of the C-7-hydroxylation. The -stereochemistry at C-7 OH group
D
8.22 ppm), C-8 (
D
c
D
7.56 ppm), C-
D
D
3.2. Structural identification of metabolites
a
was supported on the basis of NOESY correlation between the
geminal H-7 (dH 3.99 ppm) and H-8 (dH 1.40 ppm) (Fig. 1). Finally,
that metabolite was identified as 7a-hydroxy-5a-androstan-
Metabolism of DHEA (1), androstenediol (2) and EpiA (3) by A.
coerulea AM93 yielded known metabolites identified as 7
xy-DHEA (5), 7b-hydroxy-DHEA (6), 7-oxo-DHEA (7), 3b,7
trihydroxyandrost-5-ene (8), 3b,7b,17b-trihydroxyandrost-5-ene
(9), 3b,17b-dihydroxyandrost-5-en-7-one (10), 7 -hydroxy-EpiA
-hydroxy-EpiA (14) by com-
a
-hydro-
a,17b-
3,17-dione (15). The spectral data are in agreement with the lit-
erature.33 The structure of 16 was determined as a result of com-
parison of its spectroscopic data with that of the starting material
4. The appearance of a new signal at dH 3.51 ppm suggests mono-
hydroxylation, while the signal shape indicates an equatorial
configuration of the introduced hydroxyl group. This observation
is supported by the appearance of a new methine carbon signal at
dC 74.24 ppm which, in combination with the downfield shift for
a
(11), 9a-hydroxy-EpiA (13) and 11a
parison of their spectral data with the literature values23 and on
the basis of identity of their Rt from GC and Rf from TLC with stan-
dards available in our laboratory.
The metabolite 12 has a resonance at dH 3.58 ppm (m), con-
firming that the 3b-OH group is maintained; a broad multiplet
at dH 3.46 ppm suggests hydroxylation at an equatorial position
of the steroid molecule. The shape and position of this signal
and downfield shift of the C-18 and C-19 methyl signals by
0.04 ppm and 0.03 ppm respectively, with respect to the sub-
strate 3, correspond to the literature data of the steroidal 7b-
alcohols.35 The stereochemistry of the newly introduced hydroxyl
group was inferred from NOESY correlation of H-7 (dH 3.46 ppm)
the C-6 (
D 10.17 ppm) and C-8 (D 7.73 ppm) is an important con-
firmation of 7-hydroxylation. The stereochemistry of the newly
introduced OH group at C-7 was deduced as b (equatorial) on
the basis of NOESY correlation between H-7 (dH 3.51 ppm) and
H-5 (dH 1.64 ppm), H-9 (dH 0.82 ppm), and H-14 (dH 1.41 ppm).
Additionally, changes in signal shifts of the methyl groups at C-
18 and C-19 correspond to those accompanying introduction of
the 7b-hydroxyl groups into the substrate.35 In the 1H NMR spec-
trum of 17, having visibly higher polarity on TLC than 4, the
CHOH signal was absent and the 19-methyl group underwent a
significant downfield shift (
D 11 ppm) suggesting the presence
of a non-protonated carbon, possibly due to functionalization at
C-9. A new resonance at dC 75.57 ppm, the downfield shifts of
signals C-10 (
upfield shifts of C-1
8.93 ppm), C-12 (
D
4.56 ppm) and C-11 (
6.9 ppm), C-5
4.33 ppm) and C-14 (
D 7.73 ppm) and c-gauche
(
D
(
D
8.13 ppm), C-7
(
D
D
D
7.02 ppm) signals
of this metabolite in comparison to the spectrum of substrate 4,
confirmed hydroxylation at the 9 position. Additional support
a
was the HMBC spectrum showing correlation of C-9 with H-12
(dH 1.52 ppm). The spectral data of 17 are in agreement with
the literature.35,36 NMR spectra of 18 have new resonances at
dH 4.00 ppm (m) and dC 68.62 ppm, indicating monohydroxyla-
tion. In comparison to the substrate, the C-19 methyl signal of
the product demonstrated significant downfield shift
0.1 ppm) suggesting that hydroxylation had occurred at the
11
-position. This was further supported by the 13C NMR of 18
which showed downfield shifts for b-carbons C-9 ( 6.08 ppm)
and C-12 ( 11.53 ppm) and the HMBC spectrum which showed
(D
a
D
D
long-range couplings between H-11 (dH 4.00 ppm) and C-9
(dC 59.94 ppm), C-12 (dC 42.99 ppm), and C-13 (dC 47.92 ppm).
The stereochemistry of the newly introduced hydroxyl group
was further supported by NOESY spectrum which showed corre-
lation of 11b-H with the proton signals of C-18 (dC 14.55 ppm)
and C-19 (dC 11.75 ppm) methyl groups (Fig. 1). The spectral data
Figure 1. Crucial NOESY and HMBC correlations of 7b-hydroxy-EpiA (12), 7
hydroxy-5 -androstan-3,17-dione (15), and 11 -hydroxy-5 -androstan-3,17-
dione (18).
a-
a
a
a