Synthesis of 7β-hydroxy-epiandrosterone

Article abstract of DOI:10.1016/j.steroids.2010.08.003

The synthesis of 7β-hydroxy-epiandrosterone (6) possessing strong anti-inflammatory properties was achieved starting from 3β-acetoxy-17,17- (ethylenedioxy)-5-androsten (1). This approach involved as a main step an allylic oxidation of the C-7 followed by two reduction reactions of the double bond and of the carbonyl group. This stereoselective synthesis in 5 steps gave 7β-hydroxy-epiandrosterone in 63% overall yield.

Full text of DOI:10.1016/j.steroids.2010.08.003

Steroids 76 (2011) 28–30
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 7␤-hydroxy-epiandrosterone
a
a
a,∗
Christophe Ricco , Gilbert Revial , Clotilde Ferroud
,
b
b
Olivier Hennebert , Robert Morfin
Laboratoire de Transformations Chimiques et Pharmaceutiques, UMR7084, Conservatoire National des Arts et Métiers,
rue Conté, 75003 Paris, France
a
2
b
Laboratoire de Biologie EA3199, Conservatoire National des Arts et Métiers, 2 rue Conté, 75003 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2010
Received in revised form 30 July 2010
Accepted 6 August 2010
Available online 19 August 2010
The synthesis of 7␤-hydroxy-epiandrosterone (6) possessing strong anti-inflammatory properties was
achieved starting from 3␤-acetoxy-17,17-(ethylenedioxy)-5-androsten (1). This approach involved as a
main step an allylic oxidation of the C-7 followed by two reduction reactions of the double bond and of
the carbonyl group. This stereoselective synthesis in 5 steps gave 7␤-hydroxy-epiandrosterone in 63%
overall yield.
©
2010 Elsevier Inc. All rights reserved.
Keywords:
Stereoselective synthesis
7
7
␤-Hydroxy-epiandrosterone
␤-Hydroxy-EpiA
Anti-inflammatory compound
1
. Introduction
was then necessary to develop a chemical synthesis with better
yields and producing gram quantities of the compound needed for
clinical and pharmaceutical investigations. In this paper, we report
a very efficient 5-step stereoselective synthesis of 7␤-hydroxy-
EpiA (6) (Scheme 1).
The 7␤-hydroxy-epiandrosterone (7␤-hydroxy-EpiA), steroidal
compound naturally present in humans and animals, is generated
by two enzymatic steps from epiandrosterone (EpiA). Initially, oxi-
dation of EpiA by CYP-7B1 gives 7␣-hydroxy-EpiA followed by
the 11␤-hydroxysteroid dehydrogenase type 1-catalyzed inter-
conversion of the secondary alcohol to produce 7␤-hydroxy-EpiA
2. Experimental
2.1. General remarks
[
1,2]. Strong anti-inflammatory properties of 7␤-hydroxy-EpiA
have been reported in vivo in rats [3] as well as in vitro in
human peripheral blood monocytes cultured in the presence of
TNF-␣ (tumor necrosis factor alpha) [4]. Very low concentrations
of compound were sufficient to reduce inflammation. Milligram
quantities of 7␤-Hydroxy-EpiA were first produced, according to
the published procedure [5] using bioconversion of EpiA by Rhizo-
pus nigricans. The best yields were 48% but the process could not
be increased to gram-scale production because of contaminating
side products (6␤-hydroxy-EpiA, 7␣-hydroxy-EpiA) and the large
volumes of solvents needed in extractions. Nevertheless, the quan-
tities produced were enough for reference use in characterization
of 7␤-hydroxy-EpiA as a native metabolite of epiandrosterone in
human tissue preparations [6]. Use of the two recombinant human
enzymes for the preparation of 7␤-hydroxy-EpiA was considered
impractical because of project cost in research and equipment. It
The reactions were monitored by thin-layer chromatography
TLC) on aluminium plates (0.20 mm) precoated with fluores-
(
cent silica gel using EtOAc/cyclohexane as eluent. The reaction
components were visualized under UV light and then dipped
in iodine vapor. Silica gel (40–63 ␮m) was used for flash
chromatography separations and EtOAc/cyclohexane was the
eluent. Gas chromatography–mass spectroscopy (GC–MS) was per-
formed with an Agilent 6890N (equipped with a 12 m × 0.20 mm
dimethylpolysiloxane capillary column) linked to a Model 5973N
(
ionisation energy 70 eV). Melting points were determined on a
Kofler block. Optical rotations were measured on a Perkin-Elmer
−
1
model 241. Infrared spectra (wavenumbers in cm ) were recorded
on Nicolet apparatus model Avatar 320. 1H and C NMR spectra
13
were recorded in CDCl , respectively, at 400 MHz (Bruker 400) and
3
1
00 MHz. Chemical shifts were reported in ppm relative to TMS.
Reagents and materials were obtained from commercial suppliers
and were used without further purification. 3␤-Acetoxy-17,17-
(
ethylenedioxy)-5-androsten (1) was prepared according to the
∗ Corresponding author.
E-mail address: clotilde.ferroud@cnam.fr (C. Ferroud).
literature [7].
0
039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.08.003

C. Ricco et al. / Steroids 76 (2011) 28–30
29
Scheme 1. Reaction conditions: (i) CrO3-3,5-dimethylpyrazole, CH2Cl2, 4 h, −20 ◦C; (ii) H2, 10%Pd/C, EtOAc, 1 h, RT; (iii) NaBH4, CeCl3·7H2O, MeOH, 2 h, 0 ◦C; (iv) TsOH, H2O,
Me2CO, 12 h, RT; (v) K2CO3, MeOH, 1.5 h, RT.
2.2. 3ˇ-Acetoxy-17,17-(ethylenedioxy)-5-androsten-7-one (2)
(CH), 50.01 (CH), 54.79 (CH), 64.47 (CH ), 65.20 (CH ), 72.69 (CH),
2 2
1
18.6 (C), 170.5 (C), 210.6 (C).
◦
To 8.11 g (81 mmol) of chromium trioxide, suspended at −20 C
in dry CH Cl , 7.80 g (81 mmol) of 3,5-dimethylpyrazole [8] was
2
2
2
3
.4.
quickly added. After 30 min, a solution of 2.02 g (5.40 mmol) of
␤-acetoxy-17,17-(ethylenedioxy)-5-androsten (1) in dry 20 mL
of CH Cl was added dropwise and the mixture was stirred at
ˇ-Acetoxy-17,17-(ethylenedioxy)-7ˇ-hydroxy-5˛-androstane
3
(
4)
2
2
◦
20 C for 4 h. The reaction medium was diluted with a mixture
−
To 900 mg (2.30 mmol) of 3␤-acetoxy-17,17-(ethylenedioxy)-
-oxo-5␣-androstane (3), dissolved in 15 mL of MeOH, 944 mg
2.5 mmol) of cerium (III) chloride heptahydrate was added. At
of toluene–ethyl acetate (7:3) and filtered through a short column
of silica gel layered with celite. The column was washed with the
same solvent mixture, and the solvents were evaporated under
reduced pressure. The residue was purified by flash chromatog-
raphy (30% EtOAc in cyclohexane) to give 1.65 g (79% yield) of 2 as
white crystals.
7
(
0
◦
C, 131 mg (3.5 mmol) of NaBH was added portionwise. After 2 h
4
the reaction was quenched with 2 mL of acetone and the mixture
was stirred for 30 min. The solvent was removed under reduced
pressure and the residue was taken up with 20 mL of water and
extracted with EtOAc. The combined organic layers, dried over
Compound 2: Rf = 0.45 (30% EtOAc in cyclohexane); mp
◦
◦
23
1
(
2
78–180 C (EtOAc/cyclohexane) (lit. [8] 176–177 C); [␣]D −135
anhydrous MgSO , were evaporated under reduced pressure. The
c 2.55, CHCl ); EIMS m/z 328 (M, 60, 7%), 313 (11), 268 (1), 256 (9),
4
3
−
1
1
crude resulting product was then purified by flash chromatography
41 (5), 229 (20), 213 (2), 99 (100); IR (KBr) 1727, 1677 cm
; H
(
50% EtOAc in cyclohexane) to give 801 mg (89% yield) of 4 as white
crystals.
Compound 4: R = 0.5 (50% EtOAc in cyclohexane); mp 131 C
NMR 0.83 (s, 3H), 1.18 (s, 3H), 2.02 (s, 3H), 2.20 (t, 1H, J = 11.4 Hz),
2
.45 (dd, 1H, J = 11.6, 2.0 Hz), 2.52 (ddd, 1H, J = 13.9, 5.11, 2.0 Hz),
.85 (m, 4H), 4.68 (tt, 1H, J = 11.5, 4.6 Hz), 5.67 (d, 1H, J = 1.7 Hz);
C NMR 14.41 (CH ), 17.34 (CH ), 20.64 (CH ), 21.25 (CH ), 25.06
◦
3
f
23
13
(EtOAc/cyclohexane); [␣]
−5.5 (c 1.47, CHCl ); EIMS m/z 392
D
3
3
3
2
3
+
(
M , 1%), 374 (0.2), 332 (0.4), 312 (0.3), 291 (15), 292 (3), 99 (100),
(
(
(
(
CH ), 27.32 (CH ), 29.60 (CH ), 34.13 (CH ), 36.00 (CH ), 37.77
2 2 2 2 2
−
1 1
1
3
1
4
2
3
00 (19); IR (KBr) 3489, 1731 cm ; H NMR 0.69 (ddd, J = 11.6, 11.6,
.9 Hz, 1H), 0.80 (s, 3H), 0.81 (s, 3H), 0.92 (ddd, J = 13.7, 13.6, 3.9 Hz,
H), 1.97 (s, 3H), 3.36 (ddd, J = 10.4, 10.2, 5.2 Hz, 1H), 3.76–3.91 (m,
H), 4.62 (tt, J = 11.3, 4.9 Hz, 1H); 13C NMR 12.32 (CH ), 14.61 (CH ),
CH ), 38.29 (C), 44.37 (CH), 45.36 (CH), 46.14 (C), 49.76 (CH), 64.46
2
CH ), 65.21 (CH ), 72.11 (CH), 118.6 (C), 126.6 (CH), 164.2 (C), 170.3
2
2
C), 201.3 (C).
3
3
0.74 (CH ), 21.42 (CH ), 25.50 (CH ), 27.38 (CH ), 30.28 (CH ),
2
3
2
2
2
2
.3. 3ˇ-Acetoxy-17,17-(ethylenedioxy)-7-oxo-5˛-androstane (3)
3.55 (CH ), 34.28 (CH ), 34.97 (C), 36.65 (CH ), 38.25 (CH ), 41.83
2
2
2
2
(
6
CH), 43.43 (CH), 46.59 (C), 49.68 (CH), 52.14 (CH), 64.51 (CH ),
5.21 (CH ), 73.32 (CH), 75.11 (CH), 118.6 (C), 170.7 (C).
2
2
1
.13 g (2.9 mmol) of 3␤-acetoxy-17,17-(ethylenedioxy)-5-
androsten-7-one (2) and 510 mg of 10% Pd/C in 50 mL of EtOAc
were stirred under an atmospheric pressure of hydrogen at RT for
1
h. After filtration and evaporation of the solvent, the resulting
2.5. 3ˇ-Acetoxy-7ˇ-hydroxy-5˛-androstan-17-one (5)
crude product was purified by flash chromatography (20–30%
EtOAc in cyclohexane) to give 1.10 g (97% yield) of 3 as white
To 460 mg (1.2 mmol) of 3␤-acetoxy-17,17-(ethylenedioxy)-
7␤-hydroxy-5␣-androstane (4) in 20 mL of acetone, 30 mg
(1.7 mmol) of water and 44 mg (0.26 mmol) of p-toluenesulfonic
acid were added. The mixture was stirred overnight at room tem-
perature. The solvent was removed under reduced pressure and the
residue was taken up with EtOAc. The organic solution was washed
(NaHCO , water and brine), dried (MgSO ) and evaporated under
reduced pressure. The resulting product was purified by flash chro-
matography (50% EtOAc in cyclohexane) to give 400 mg (98% yield)
of 5 as white crystals.
crystals.
◦
Compound 3: R = 0.45 (30% EtOAc in cyclohexane); mp 185 C
f
2
3
(
(
EtOAc/cyclohexane); [␣]D −54.8 (c 1.24, CHCl ); EIMS m/z 390
3
+
M , 2%), 262 (0.1), 347 (0.1), 330 (0.3), 318 (0.7), 300 (0.2), 99 (100);
−
1 1
IR (KBr) 1730, 1701 cm ; H NMR 0.83 (s, 3H), 1.09 (s, 3H), 2.02 (s,
3
1
2
3
H), 2.24–2.36 (m, 3H), 3.80–3.95 (m, 4H), 4.68 (tt, J = 11.2, 5.1 Hz,
3
4
13
H); C NMR 11.70 (CH ), 14.47 (CH ), 21.26 (CH ), 21.33 (CH ),
3
3
2
3
3.80 (CH ), 27.09 (CH ), 29.63 (CH ), 33.80 (CH ), 34.06 (CH ),
2
2
2
2
2
5.87 (C), 35.89 (CH ), 43.36 (CH), 45.65 (CH ), 45.70 (C), 45.99
2
2

3
0
C. Ricco et al. / Steroids 76 (2011) 28–30
◦
+
Compound 5: R = 0.4 (50% EtOAc in cyclohexane); mp 162 C
pound: 7,17-dioxo-13␣-androst-5-en-3␤-yl actetate [10]. Thus,
the crystallized ketone 3 was obtained in almost quantitative yield.
The trans A/B stereochemistry was confirmed by the multiplicity in
f
20
(
1
1
EtOAc/cyclohexane); [␣]D +98.1 (c 2.0, CHCl ); EIMS m/z 348 (M ,
3
6%), 330 (3), 315 (4), 299 (8), 288 (12), 273 (37), 255 (13), 220 (82),
60 (58), 145 (100), 132 (56), 119 (50), 107 (65), 93 (87), 79 (81), 67
H NMR 0.76 (ddd,
J = 11.6, 11.6, 3.9 Hz, 1H), 0.87 (s, 3H), 0.88 (s, 3H), 0.99 (ddd, J = 13.6,
1
the H NMR of 3␣-hydrogen (ı 4.68, tt, J = 11.2, 5.1 Hz).
−
1 1
(
62), 55 (60); IR (KBr) 3605, 1735, 1725 cm
;
The next step, reduction of the carbonyl group to the equatorial
secondary alcohol, was crucial regarding the stereochemistry of C-
7. We thought the conditions of the Luche reduction described with
the enones could be interesting in our case and we tested it with the
cyclic and rigid ketone 3. In these conditions the reduction of ketone
3 was achieved in a highly stereoselective manner in 89% yield,
using NaBH /CeCl [11] in MeOH medium to give 4 as a single com-
1
1
(
(
6.5, 3.7 Hz, 1H), 2.02 (s, 3H), 2.07 (dd, J = 19.5, 9.8 Hz, 1H), 2.24 (m,
H), 2.43 (dd, J = 19.5, 8.6 Hz, 1H), 3.48 (td, J = 10.5, 5.2 Hz, 1H), 4.67
tt, J = 11.4, 5.0 Hz, 1H); 13C NMR 12.34 (CH ), 14.05 (CH ), 20.67
3 3
CH ), 21.43 (CH ), 24.92 (CH ), 27.31 (CH ), 31.46 (CH ), 33.51
CH ), 35.09 (C), 36.04 (CH ), 36.53 (CH ), 38.58 (CH ), 41.83 (CH),
2 2 2 2
2 3 2 2 2
(
4
3
4
1
3
2.74 (CH), 48.28 (C), 50.95 (CH), 52.37 (CH), 73.19 (CH), 74.50 (CH),
pound [12]. Again, the multiplicity of the 7␣-hydrogen resonance
(ı 3.35, ddd, J = 10.6, 10.4, 5.3 Hz) confirmed its axial position.
The ketal 4 was easily hydrolyzed in acidic acetone/water
medium to give the carbonyl compound 5 in quantitative yield,
and finally the desired 7␤-hydroxy-EpiA (6) was obtained in 94%
yield through basic hydrolysis of the 3␤-acetate (K CO , MeOH).
+
70.7 (C), 221.5 (C); HRMS-ESI m/z [M+Na ] calcd for C21H32O Na:
4
71.21928. found: 371.21966.
2.6. 7ˇ-Hydroxy-epiandrosterone (6)
2
3
To 603 mg (1.7 mmol) of 3␤-acetoxy-7␤-hydroxy-5␣-
In conclusion, we reported a stereoselective synthesis of 7␤-
hydroxy-EpiA (6), a very strongly anti-inflammatory compound in
63% overall yield starting from 3␤-acetoxy-17,17-(ethylenedioxy)-
5-androsten (1). It should be noticed that this synthesis was
conducted in a reproducible manner at scales ranging from 0.2 to
1 g of final compound.
androstan-17-one (5) in 20 mL of MeOH, 2.3 mL of K CO3 solution
2
(
1.5 M in water, 3.5 mmol) was added, and the solution was stirred
at RT for 1.5 h. After evaporation of the solvent under reduced
pressure, the resulting crude product was purified by flash chro-
matography (5% MeOH in EtOAc) to yield 496 mg (94%) of 6 as
white crystals.
◦
Compound 6: R = 0.4 (5% MeOH in EtOAc); mp 241 C (EtOAc);
Appendix A. Supplementary data
f
␣]D23 +41.0 (c 1.53, CHCl ); EIMS m/z 306 (M , 26%), 291 (5), 273
+
[
3
(
(
3
0
15), 255 (7), 249 (4), 215 (17), 178 (100), 160 (12), 145 (36), 133
21), 120 (36), 107 (35), 93 (42), 79 (35), 67 (30), 55 (28); IR (KBr)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2010.08.003.
−1 1
457, 1739 cm
.73 (m, 1H), 2.08 (m, 1H), 2.18 (dd, J = 19.3, 8.6 Hz, 1H), 3.00 (d,
; H NMR 0.51 (m, 1H), 0.63 (s, 3H), 0.66 (s, 3H),
References
13
J = 5.5 Hz, 1H), 3.19 (m, 1H), 3.27 (m, 2H); C NMR 12.31 (CH ),
1
3
3
[
[
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3.94 (CH ), 20.55 (CH ), 24.76 (CH ), 31.19 (CH ), 31.43 (CH ),
3 2 2 2 2
4.95 (C), 35.96 (CH ), 36.74 (CH ), 37.55 (CH ), 38.82 (CH ), 41.95
2
2
2
2
(
CH), 42.44 (CH), 48.11 (C), 51.02 (CH), 52.41 (CH), 70.10 (CH),
+
7
3
3.91 (CH), 221.8 (C); HRMS-ESI m/z [M+Na ] calcd for C19H30O Na:
11␤-hydroxysteroid dehydrogenase type 1.
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The synthesis of the target compound 7␤-hydroxy-EpiA (6)
starting from the known 3␤-acetoxy-17,17-(ethylenedioxy)-5-
androsten (1) [7] involved, as a main step, an allylic oxidation of
the C-7 followed by two reduction steps of the double bond and the
carbonyl group. Although an approach to the titled compound was
already reported [9], we decided to develop a stereoselective and
more efficient synthesis in order to obtain only the required isomer
[
[
[
6] Morfin R, Di Stefano S, Charles JF, Floch HH. Precursors for 6␤- and
7␣-hydroxylations of 5␣-androstane-3␤,17␤-diol by human normal and
7
␤-hydroxy-EpiA. Indeed, in the published synthesis, the authors
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oxidation led to the corresponding enone in a 60% yield. Then the
two reduction steps were successively performed “one pot” under
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Med Chem 2007;15:3152–60. In addition to the reported spectral data, the
2
6
optical rotation of the starting compound 1 was measured: [␣]D −82.7 (c 2.3,
CHCl3).
the Birch reaction conditions (Na, liquid NH ). The authors obtained
3
[
[
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after work-up the 65/35 mixture of 7␤- and 7␣-hydroxy-EpiA in a
0% overall yield.
1
Our synthesis started by the allylic oxidation of 1 under mild
conditions to avoid ketal hydrolysis. According to the literature
8], this oxidation was carried out with CrO /3,5-DMP in CH Cl
[
10] Hanson JR, Hunter AC, Roquier S. The preparation of some 13␣-androstanes.
[
Collect Czech Chem Commun 1998;63:1646–54.
3
2
2
◦
[11] Luche JL, Rodriguezhahn L, Crabbe P. Reduction of natural enones in presence
of cerium trichloride. J Chem Soc Chem Commun 1978;14:601–2.
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rated steroidal ketones was published Stastna E, Cerny I, Pouzar V, Chodounska
H. Stereoselectivity of sodium borohydride reduction of saturated steroidal
ketones utilizing conditions of Luche reduction. Steroids 2010;75:721–5.
at −20 C to lead to enone 2 in 79% yield. Under these conditions,
no trace of 17-carbonyl compound was detected.
[
The first reduction step involved the stereoselective catalytic
hydrogenation of the enone 2 to ketone 3 over palladium on
charcoal in EtOAc, conditions already described with a similar com-