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3.62 (1H, t, J=8.5 Hz, 17α-H), 3.91 (1H, dt, J=5.1 Hz, J=10.5 Hz,
11β-H).
8.23; found. C, 71.59; H, 8.21%. IR νmax (cm−1): 3370, 1719, 1654, 1
H
NMR (CDCl3) 1.29 (3H, s, 19-H), 1.35 (3H, s, 18-H), 3.84 (1H, dt,
J=4.5 Hz, J=10.1 Hz, 11β-H), 5.73 (1H, s, 4-H).
3β,11α-Dihydroxy-17a-oxa-D-homo-5α-androstan-17-one (9):
mp. 257–258 ° C (Me2CO, needles) (lit. 260° [14]); C19H30O4: calcd.
C, 70.77; H, 9.38; found. C, 70.70; H, 9.35%. IR νmax (cm−1): 3443,
1719, 1H NMR (CDCl3) 0.90 (3H, s, 19-H), 1.28 (3H, s, 18-H), 3.59 (1H,
m, 3α-H), 3.75 (1H, dt, J=4.8 Hz, J=11.4 Hz, 11β-H).
3.1.6. Transformation of progesterone (6)
After 24 h of transformation of 100 mg of progesterone (6),
the isolates were (% mol): the unreacted substrate (9 mg, 9%), 4 mg
(4%) of 11α-hydroxyprogesterone (16), 14 mg (13%) of 6β,11α-
dihydroxyprogesterone (17), 19 mg (20%) of 11α-hydroxytestoster-
one (14), and 19 mg (20%) of 11α,17β-dihydroxy-5β-androstan-3-
one (18).
11α-Hydroxyprogesterone (16): mp. 169 °C (MeOH, needles) (lit.
165–168° [31]); C21H30O3: calcd. C, 76.33; H, 9.15; found. C, 76.27; H,
9.12%. IR νmax (cm−1): 3454, 1695, 1656, 1620, 1H NMR (CDCl3) 0.70
(3H, s, 18-H), 1.32 (3H, s, 19-H), 2.14 (3H, s, 21-H), 4.05 (1H, dt,
J=4.8 Hz, J=10.5 Hz, 11β-H), 5.74 (1H, s, 4-H).
3.1.2. Transformation of DHEA (2)
After 48 h transformation of 100 mg of DHEA (2), there were
isolated (% mol): 3β,11α,17β-trihydroxyandrost-5-ene (11) (48 mg,
45%) and 3β,11α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one
(12) (29 mg, 27%). After 3 h transformation of 100 mg of 2, the
additional isolates were 20 mg (20%) of androstenediol (3) and 7 mg
(7%) of 11α-hydroxy-DHEA (10).
Androstenediol (3) IR νmax (cm−1): 3423, 3210, 1651, 1H NMR
(CDCl3) 0.76 (3H, s, 18-H), 1.02 (3H, s, 19-H), 3.54 (1H, m, 3α-H), 3.64
(1H, t, J=8.5 Hz, 17α-H), 5.35 (1H, d, J=5.1 Hz, 6-H).
6β,11α-Dihydroxyprogesterone (17): mp. 246–247 ° C (MeOH,
needles) (lit. 244–246° [31]); C21H30O4: calcd. C, 72.80; H, 8.73; found.
1
11α-Hydroxy-DHEA (10): mp. 210–212 ° C (Me2CO, needles) (lit.
210° [29]); C19H28O3: calcd. C, 74.96; H, 9.27; found. C, 74.99; H, 9.25%.
IR νmax (cm−1): 3446, 1739, 1654, 1H NMR (CDCl3) 0.90 (3H, s, 18-H),
1.18 (3H, s, 19-H), 3.53 (1H, m, 3α-H), 4.09 (1H, dt, J=5.1 Hz,
J=11.1 Hz, 11β-H), 5.43 (1H, d, J=5.2 Hz, 6-H).
3β,11α,17β-Trihydroxyandrost-5-ene (11): mp. 261–265 ° C
(MeOH, plates) (lit. 265–267° [30]); C19H30O3: calcd. C, 74.47; H,
9.87; found. C, 74.50; H, 9.84%. IR νmax (cm−1): 3398, 3222, 1640, 1H
NMR (CD3OD) 0.81 (3H, s, 18-H), 1.21 (3H, s, 19-H), 3.42 (1H, m, 3α-
H), 3.65 (1H, t, J=8.5 Hz, 17α-H), 4.06 (1H, dt, J=4.8 Hz, J=11.0 Hz,
11β-H), 5.44 (1H, d, J=4.9 Hz, 6-H).
C, 72.15; H, 8.71%. IR νmax (cm−1): 3444, 3378, 1696, 1658, H NMR
(pyr-d5) 0.74 (3H, s, 18-H), 1.81 (3H, s, 19-H), 2.05 (3H, s, 21-H), 4.35
(1H, dt, J=4.5 Hz, J=10.2 Hz, 11β-H), 4.55 (1H, br s, 6α-H), 6.08
(1 H, s, 4-H).
11α,17β-Dihydroxy-5β-androstan-3-one (18): mp. 212–216 °C
(MeOH, prisms); C19H30O3: calcd. C, 74.47; H, 9.87; found. C, 74.20; H,
9.84%. IR νmax (cm−1): 3478, 1709, 1H NMR (CDCl3) 0.77 (3H, s, 18-H),
1.14 (3H, s, 19-H), 3.70 (1H, t, J=8.5 Hz, 17α-H), 4.00 (1H, m, 11β-H).
CD (c=1.1×10−3 mol/l in methanol) Δε269−0.70.
3.2. Structural identification of metabolites
3β,11α-Dihydroxy-17a-oxa-D-homo-androst-5-en-17-one (12):
mp. 234–236 ° C (MeOH, needles colorless). C19H28O4: calcd. C,
71.22; H, 8.81; found. C, 71.15; H, 8.78%. IR νmax (cm−1): 3440, 1716,
1622, 1H NMR (CD3OD) 1.20 (3H, s, 19-H), 1.38 (3H, s, 18-H), 3.45 (1H,
m, 3α-H), 3.88 (1H, dt, J=4.8 Hz, J=11.0 Hz, 11β-H), 5.45 (1H, d,
J=5.6 Hz, 6-H).
Incubation of epiandrosterone (1) resulted in isolation of three
metabolites. The first product had a resonance at δH 3.56 ppm (m),
confirming that the 3β-OH group was maintained. This product also
had new resonances at δC 69.2 ppm and δH 3.95 ppm (dt, J=5.1 Hz,
J=10.5 Hz), which suggested hydroxylation at an equatorial position
of the steroid molecule. In comparison to the spectrum of substrate,
the C-19 methyl signal of the product demonstrated significant
downfield shift indicating that hydroxylation had occurred at the
11α-position. This was further supported by the 13C NMR of 7 which
showed downfield shifts for β-carbons C-9 (Δ 5.4 ppm) and C-12
(Δ 10.9 ppm) that were consistent with reference shifts values [32].
All these results confirmed that the product was 3β,11α-dihydroxy-
5α-androstan-17-one (7). The spectroscopic data of this compound
correspond to those described in literature [29]. The 1H NMR spectrum
of 8 possessed a resonance at δH 3.53 ppm (m), confirming that the
3β-hydroxy-5α-androstan skeleton had been retained. In comparison
to the spectrum of 1, it contained two new signals at δH 3.62 ppm (t,
J=8.5 Hz) and δH 3.91 ppm (dt, J=5.1 Hz, J=10.5 Hz) thus suggest-
ing the presence of two hydroxyl groups. The upfield shift of the C-18
methyl group (Δ 0.14 ppm) in 1H NMR and the loss of the non-
protonated resonance signal at δC 224.1 ppm, coupled with the
appearance of a new methine carbon signal at δC 82.1 ppm in the 13C
NMR spectrum, confirmed that the C-17 ketone had been reduced to a
C-17β alcohol. Further evidence of the reduction was provided by
upfield shifts of C-13 (Δ 4.6 ppm) and C-16 (Δ 5.9 ppm) signals of this
metabolite in comparison to the spectrum of substrate 1. The site and
the stereochemistry of the additional hydroxyl group were estab-
lished from changes (β-deshielding effects) in the 13C NMR spectra
and the characteristic shape and multiplicity of the CH(OH) resonance
in the 1H NMR. All these results fully confirmed the structure of
metabolite 8 as 3β,11α,17β-trihydroxy-5α-androstane. Comparison
of 1H NMR spectrum of the product 9 to that of epiandrosterone
(1) revealed a new signal at δH 3.75 (dt, J=4.8 Hz, J=11.4 Hz),
indicating monohydroxylation. The characteristic shape and multiplic-
ity of this signal suggested that the hydroxyl group was introduced at
11α-position. It was confirmed by the long-range correlation of the
3.1.3. Transformation of androstenediol (3)
After 4 days of transformation of 100 mg of androstenediol (3), the
following compounds were isolated (% mol): the unreacted substrate
(9 mg, 9%), 3β,11α,17β-trihydroxyandrost-5-ene (11) (22 mg, 21%)
and 3β,11α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one (12)
(63 mg, 58%).
3.1.4. Transformation of pregnenolone (4)
Transformation of pregnonolone (4) with the fungus for 6 days
resulted in no observable transformation with full recovery of the
starting material. It was identified by comparison of the NMR data
with that of an authentic sample.
3.1.5. Transformation of androstenedione (AD) (5)
After 48 h of transformation of 100 mg of androstenedione (5), the
isolates were (% mol) 8 mg (8%) of 11α-hydroxy-AD (13), 42 mg
(40%) of 11α-hydroxytestosterone (14), and 27 mg (25%) of 11α-
hydroxy-17a-oxa-D-homo-androst-4-en-3,17-dione (15).
11α-Hydroxy-AD (13): mp. 196 ° C (MeOH, cubes) (lit. 196–197°
[20]); C19H26O3: calcd. C, 75.46; H, 8.67; found. C, 75.37; H, 8.64%.
IR νmax (cm−1): 3412, 1740, 1654, 1621, 1H NMR (CDCl3) 0.96 (3H, s,
18-H), 1.36 (3H, s, 19-H), 4.09 (1H, dt, J=5.1 Hz, J=10.1 Hz, 11β-H),
5.77 (1H, s, 4-H).
11α-Hydroxytestosterone (14): mp. 221 ° C (MeOH, cubes) (lit.
218–219° [20]); C19H28O3: calcd. C, 74.96; H, 9.27; found. C, 74.85; H,
9.25%. IR νmax (cm−1): 3420, 1660, 1618, 1H NMR (CDCl3) 0.80 (3H, s,
18-H), 1.30 (3H, s, 19-H), 3.66 (1H, t, J=8.5 Hz, 17α-H), 4.02 (1H, dt,
J=5.0 Hz, J=10.1 Hz, 11β-H), 5.71 (1H, s, 4-H).
11α-Hydroxy-17a-oxa-D-homo-androst-4-en-3,17-dione (15):
mp. 102–103 °C (chloroform, plates); C19H26O4: calcd. C, 71.67; H,