Synthesis of 7α-hydroxy-dehydroepiandrosterone and 7β-hydroxy-dehydroepiandrosterone

Article abstract of DOI:10.1016/j.steroids.2005.07.006

The fermentation of dehydroepiandrosterone synthesized from the starting material diosgenin using Mucor racemosus produced 7α-hydroxy- dehydroepiandrosterone and 7β-hydroxy-dehydroepiandrosterone. The bioactivity of the microbial metabolites is also discussed. The species M. racemosus was isolated by screening among stains from soil samples collected from various parts of China.

Full text of DOI:10.1016/j.steroids.2005.07.006

Steroids 70 (2005) 970–973
Synthesis of 7␣-hydroxy-dehydroepiandrosterone and
7␤-hydroxy-dehydroepiandrosterone
a
a,∗
b
a
a
Heping Li , Hong-Min Liu , Wenzhong Ge , Lihua Huang , Lihong Shan
a
Department of Chemistry, Zhengzhou University, Zhengzhou 450052, PR China
b
Art and Science school, Heilongjiang August First Land Reclamation University, PR China
Received 16 February 2005; received in revised form 30 June 2005; accepted 12 July 2005
Available online 6 September 2005
Abstract
The fermentation of dehydroepiandrosterone synthesized from the starting material diosgenin using Mucor racemosus produced 7␣-
hydroxy-dehydroepiandrosterone and 7␤-hydroxy-dehydroepiandrosterone. The bioactivity of the microbial metabolites is also discussed.
The species M. racemosus was isolated by screening among stains from soil samples collected from various parts of China.
©
2005 Elsevier Inc. All rights reserved.
Keywords: Microbial transformation; DHEA; Hydroxylation; Mucor racemosus; 7-Hydroxy
1
. Indroduction
debate about the stereoconfiguration of the active metabo-
lite. In peripheral tissues, 7-OH-DHEA has been found to
up-regulate immunity and to counteract immunosuppres-
sion [22–24], Moreover, in both Alzheimer’s patients and
a control group, the conversion of DHEA to ∆5-androstene-
3␤,17␤-diol and to 7␣-OH-DHEA occurred in the frontal
cortex, hippocampus, amygdala, cerebellum. The forma-
tion of these metabolites within distinct brain regions neg-
atively correlated with the density of ␤-amyloid deposits
[24].
The microbiological hydroxylation of steroids has been
described in terms of a triangular relationship between
two binding sites and the site of hydroxylation [1,2]. The
oxidoreduction of steroids governs the biological activity
and metabolic fate. 7-Oxygenated steroids are widespread
in mammals, birds, fish, and plants [3]. The major 3␤-
hydroxysteroids, including dehydroepiandrosterone (DHEA,
4
), pregnenolone, cholesterol and androstane-3␤,17␤-diol
are efficiently 7␣-hydroxylated in diverse tissues including
the brain[4–11]. In recent years, The significant bioactivity
of DHEA and 7-OH-DHEA has been noted. The metabolism
of DHEA is also of some interest. DHEA and its metabo-
lites has been shown to promote the immune response in
experimental animals [12–17]. The major metabolic path-
way for DHEA in extra-hepatic tissues is via 7-hydroxylation
With the aim of synthesizing 7-hydroxy-DHEA, we first
synthesized 4 from diosgenin (1) which is a readily available
commercal product. Our next objective was to screen micro-
organisms capable of producing 7-hydroxy steroids. In our
search for various physiologically important steroid deriva-
tives by microbial transformations, we were able to isolat a
common strain of Mucor racemosus (A.C.C.C. 0401) from
soil using 4 as the substrate.
[18–20]. Some scientists have observed that 7␣-OH-DHEA
is more active than DHEA in preventing hypoxic cell death of
neurons in vitro. Therefore, 7-oxygenation seems to be asso-
ciatedwiththeactivationofDHEA[21]. Thereisstillongoing
In this study, M. racemosus was used to convert 4 into
7␣-hydroxy-dehydroepiandrosterone (5) and 7␤-hydroxy-
dehydroepiandrosterone (6). The metabolites were charact-
ernized by various spectroscopic methods. It was reported
that membrane-bound M. racemosus lipases could be
entrapped in cryogel beads obtained from polyvinyl alcohol
∗
Corresponding author. Tel.: +86 371 7767200; fax: +86 371 7427200.
E-mail address: liuhm@public.zz.ha.cn (H.-M. Liu).
0
039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.07.006

H. Li et al. / Steroids 70 (2005) 970–973
971
(PVA) by a freezing–thawing method in two-phase system,
2.3. 3β-Acetoxy-5,16-pregnadien-20-oxime (3)
and due to the porosity of PVA-cryogels, high molecular-
weight substances can penetrate beads of the biocatalyst.
The biocatalyst could be applied for various hydrolysis and
synthesis reactions [25]. But we believe this to be the first
reported instance of the microbial hydroxylation of steroids
using M. racemosus. Moreover, we produced 5 and 6 from the
readily available material diosgenin, and with a considerable
yield.
A mixture of 2 (2.5 g, 6.7 mmol), hydroxylamine
hydrochloric acid (0.85 g, 12.2 mmol), pyridine (3 ml) and
95% ethanol (13 ml) was refluxed for 30 min, the progress
of the reaction was monitored by TLC (acetone/ether (bp
◦
60–90 C) (3:7). Then the mixture was cooled with ice,
washed with hot water, dried under a vacuum. A white solid
◦
was obtained, 2.27 g; yield 87%. mp 227–229 C (literature
◦
2
1
28–230 C [27]), IR (KBr) ν: 3476, 2939, 1721, 1373, 1269,
−
1 1
034 cm . H NMR (see Table 1).
2
. Experimental
2.4. Dehydroepiandrosterone(4)
2
.1. Instrumental methods
A mixture of 3 (0.70 g, 1.9 mmol) and unhydrous pyri-
◦
Melting points were determined on a XT5 melting point
dine (70 ml) was first stirred for 2 h at 10–15 C, then for
an additional 2 h at room temperature. The mixture was then
poured into ice water and put into a refrigerator for one night.
The next day, the mixture was extracted with ethyl acetate
apparatusandareuncorrected. Infraredspectrawererecorded
using KBr discs on a Bruker Vectror-22 spectrometer. Mass
spectra were obtained on an Esquire3000 mass spectrometer
by electrospray ionization. Optical rotations were measured
(
3 × 20 ml). The organic layer was evaporated under reduced
◦
in 1-dm cells at 20 C on a Perkin-Elmer 341 automatic
pressure to obtain a solid which was dissolved in methanol
and 2.5% potassium hydroxide (30 ml) and refluxed. The
mixture was then extracted with ethyl acetate (3 × 20 ml),
and the organic layer was evaporated under reduced pressure
to obtain product 4, which was recrystallized with methanol
1
13
spectropolarimeter in methanol solutions. The H and
C
nuclear magnetic resonance spectra were obtained using a
Bruker Avance DPX-400 spectrometer at 400 and 100 MHz,
respectively, with tetramethylsilane as internal standard in
◦
DMSO-d ,and chemical shifts are given as δ values. Coupling
6
to afford 0.476 g; yield: 51.8%. mp 150.6–151.7 C (litera-
◦
constants (J) are given in hertz (Hz). Thin layer chromatog-
raphy (TLC) was performed on a 0.25 mm thick layer of
silica gel G (Qingdao Marine Chemical Factory, China).
ture 150–152 C). IR (KBr) ν: 3462, 2935, 2854, 1732, 1460,
−
1 1
1
371, 1298, 1219, 1136, 1061, 839, 802, 592 cm . H and
1
3
C NMR data (see Tables 1 and 2).
Chromatography was performed with petroleum ether (bp
◦
6
0–90 C)/acetone (7:3) or chloroform/methanol (8:1) and
2
7
.5. 7α-Hydroxy-dehydroepiandrosterone(5) and
β-hydroxy –dehydroepiandrosterone(6)
visualized by spraying the plates with 50% sulfuric acid solu-
◦
tion and heating in an oven at 100 C for 3 min until the colors
developed.
Ten 500-ml Erlenmeyer flasks, each containing 100 ml
sterilized (peptone dextrose agar) broth, were inoculated with
2
.2. 3β-Acetoxy-5,16-pregnadien-20-one (2)
Table 2
Compound 2 was synthesized as described in the liter-
13
C NMR (CDCl3, 400 MHz) data of compounds 4, 5 and 6
ature [26] with a few variations noted in the Results and
Carbon
4
5
6
◦
◦
discussion. mp 172–174 C. [α]D = −30 (c, 0.11, CH3OH).
1
2
3
4
5
6
7
8
9
37.2
31.5
71.4
42.2
35.8
31.1
71.2
41.9
36.8
31.2
71.3
41.6
IR (KBr) ν: 2944, 2905, 2865, 1730, 1661, 1583, 1438,
−
370, 1248, 1307 cm . H NMR (DMSO-d , 400 MHz) see
6
1 1
1
13
Table 1. C NMR (DMSO-d , 400 MHz) δ: 196.3 (C20),
6
141.3
120.8
31.5
31.5
50.3
36.7
20.4
30.8
47.5
51.8
21.8
35.8
221.3
13.2
19.4
146.6
123.6
64.3
37.2
42.6
37.2
20.1
31.3
47.1
44.9
21.9
37.0
220.0
13.3
18.3
143.7
125.5
72.9
40.5
48.2
36.7
20.4
31.5
47.8
51.2
24.6
36.0
221.2
13.6
19.2
1
1
69.8 (CH3COO), 154.4 (C17), 145.2 (C16), 140.1 (C5),
21.9 (C6), 73.3 (C3), 56.0 (C14), 50.0 (C9), 45.6 (C13)
+
ppm. MS (ESI) m/z [M + Na] 379.
1
1
0
1
Table 1
1
12
H NMR (CDCl3, 400 MHz) data of compounds 2, 3, 4, 5 and 6
1
1
3
4
3-H
6-H
7-H
18-H
19-H
2
3
4
5
6
4.45, m
4.61, m
3.54, m
3.58, m
3.57, m
5.36, d J = 4.4
5.38
5.40, d J = 5.20
5.64, d J = 2.0
5.32
0.85, s
0.63, s
0.90, s
0.89, s
0.90, s
1.01, s
1.02, s
1.04, s
1.02, s
1.08, s
15
16
17
18
19
3.97
3.95, d J = 8.0

9
72
H. Li et al. / Steroids 70 (2005) 970–973
◦
◦
Scheme 1. Reagents and conditions: (a) (CH3CO)2O, NH4Cl, Py, 140 C; (b) CrO3, CH3CO2H, ClCH2CH2Cl, 0 C; (c) AcON␣·3H2O ꢀ; (d) NH2OH·HCl,
Py, C2H5OH, reflux; (e) POCl3, Py, benzene, condensed HCl; (f) CH3OH, KOH.
freshly obtained spores from agar slope cultures and incu-
bated for 2 days at 27 C in a rotary shaker (150 rpm). DHEA
toluene was used as a solvent instead of benzene because
of its lower toxicity. Finally methanol and 2.5% potas-
sium hydroxide was used in the hydrolysis step, which pro-
duced fairly quickly (Scheme 1.). The target compounds
7␣-hydroxy-dehydroepiandrosterone (5) and 7␤-hydroxy-
dehydroepiandrosterone (6) were obtained via microbiolog-
ical hydroxylation. The steroid-converting fungus was iso-
lated by screening among 96 stains from samples collected
◦
(1.5 g) was dissolved in 20 ml acetone, and 2 ml of this solu-
tion was added to each 500-ml Erlenmeyer flask. Incubation
was continued for 4 days under the same conditions. The
fermentation media were extracted exhaustively with ethyl
acetate (5 × 200 ml) and filtered to separate the broth from the
mycelium. The extract was evaporated under reduced pres-
sure. The transformation products were separated by silica
gel column chromatography by using CHCl3/CH3OH (8:1)
as eluent. Two metabolites, 5 (613 mg)and 6 (276 mg), were
◦
from various parts of China. The strain was grown at 27 C on
◦
a peptone dextrose agar slope, stored at 4 C, and freshly sub-
cultured before the transformation experiment. The selected
strain was identified as the species M. racemosus by China
General Microbiological Culture Collection Center.
◦
purified. Compound 5: yield 40.9%. mp 179.4∼181.7 C
◦
(
literature 181.5 ∼ 183.5 C [28]). IR (KBr) ν: 3364, 3301,
2
1
930, 2855, 1730, 1660, 1629, 1460, 1380, 1297, 1132,
Two metabolites, 5, 6, and the unconverted substrate 4
were purified and identified by melting points and spectral
−
1
1
13
056 cm . H and C NMR data (see Tables 1 and 2). MS
m/z 326.7, [M + Na] , 342.7 [M+K] . Analysis calculated
+
+
1
13
data (IR, MS, H NMR, C NMR) (Scheme 1). The mass
for C19H28O3: C, 74.96%; H, 9.27% O, 15.77%. Found: C,
spectraofmetabolites5and6showedthequasi-molecularion
[M + Na] at m/z 326.7, which suggested that they incorpo-
+
7
4.83%; H, 9.34%; O, 15.70%.
◦
Compound 6, yield 18.0%. mp 216.2∼217.6 C (liter-
rate one oxygen atom into the substrate. In the NMR spectra
for compound 5, the signal at δ 3.58 was assigned to H-3,
which was similar to that of compound 6. In the HMQC
spectra for compound 5, the signal at δ 5.64 correlated to
C-6 and at 3.97 correlated to C-7,which were assigned to
H-6 and H-7 respectively. Similarly, in compound 6, there
were corresponding signals at 5.32 and at 3.95, which were
also assigned respectively to H-6 and H-7. Regarding the
H-H COSY spectra for compound 5, the resonance of H-
7(δ = 3.97) correlated with H-6 (δ = 5.64) and H-8(δ = 1.69),
while for compound 6, the resonance of H-7(δ = 3.95) only
correlated with H-8(δ = 1.83). These correlated peaks show
that the hydroxyl group attached at C-7 of compound 5 has
an was axial orientation, while the hydroxyl group attached
at C-7 of compound 6 has an equatorial orientation.
◦
ature 215 ∼ 216 C [28]). IR (KBr) ν: 3431, 2933, 1730,
−
1
1
657,1621, 1461, 1375, 1247, 1112, 1057, 1029 cm
.
1
13
H and C NMR data (see Tables 1 and 2). MS m/z,
+
+
3
26.7 [M + Na] , 342.7 [M + K] . Analysis calculated for
C19H28O3: C, 74.96%; H, 9.27% O, 15.77% found C,
7
4.78%; H, 9.16%; O, 15.67%.
3
. Results and discussion
3
␤-Acetoxy-5, 16-pregnadien-20-one (2) was prepared
according to the literature [25] from diosgenin. However, we
obtained better results by keeping the reaction at a higher
temperature after the oxidant (CrO3/CH3CO2H) was added,
and by cooling the product after it was treated with hot
water rather than by treating it with cold water directly.
With 2 as the precursor of 3␤-acetoxy-5,16-pregnadien-20-
oxime (3) according to the literature [26], we produced
Acknowledgements
3
. Next, dehydroepiandrosterone (4) was prepared from 3
We are grateful to NNSF of PRC for financially supporting
of this research.
using Bechmanns’ reaction and hydrolysis. In this step,

H. Li et al. / Steroids 70 (2005) 970–973
973
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