Preparation of 3β-acetoxy-17a-oxo-androst-5-ene-7,17-dione, a biologically active impurity isolated from the production of 3β-acetoxyandrost-5-ene-7,17-dione

Article abstract of DOI:10.1081/SCC-200051712

An impurity isolated during the kilo-lab preparation of 3β-acetoxyandrost-5-ene-7,17-dione was identified as 3β-acetoxy-17a- oxo-androst-5-ene-7,17-dione. This novel D-ring secosteroid was postulated to be a Baeyer-Villiger oxidation by-product of the preparation reaction. A genuine sample was prepared by the selective peracid-mediated Baeyer-Villiger reaction on 3β-acetoxyandrost-5-ene-7,17-dione (7-oxo-DHEA acetate), confirming the novel structure.

Full text of DOI:10.1081/SCC-200051712

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Preparation of
3β‐Acetoxy‐17a‐Oxo‐Androst‐5‐Ene‐7,17‐Dione,
a Biologically Active Impurity
Isolated from the Production of
3β‐Acetoxyandrost‐5‐Ene‐7,17‐Dione
Lisa D. Coutts a , Russell J. DeOrazio a & Harold Meckler a
a Albany Molecular Research Inc., Chemical Development and Medicinal
Chemistry Departments , Albany, New York, USA
Published online: 21 Aug 2006.
To cite this article: Lisa D. Coutts , Russell J. DeOrazio & Harold Meckler (2005) Preparation of
3β‐Acetoxy‐17a‐Oxo‐Androst‐5‐Ene‐7,17‐Dione, a Biologically Active Impurity Isolated from the Production
of 3β‐Acetoxyandrost‐5‐Ene‐7,17‐Dione, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 35:7, 979-985, DOI: 10.1081/SCC-200051712
To link to this article: http://dx.doi.org/10.1081/SCC-200051712
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Synthetic Communicationsw, 35: 979–985, 2005
Copyright # Taylor & Francis, Inc.
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1081/SCC-200051712
Preparation of 3b-Acetoxy-17a-Oxo-
Androst-5-Ene-7,17-Dione, a Biologically
Active Impurity Isolated from the Production
of 3b-Acetoxyandrost-5-Ene-7,17-Dione
Lisa D. Coutts, Russell J. DeOrazio, and Harold Meckler
Albany Molecular Research Inc., Chemical Development and Medicinal
Chemistry Departments, Albany, New York, USA
Abstract: An impurity isolated during the kilo-lab preparation of 3b-acetoxyandrost-
5-ene-7,17-dione was identified as 3b-acetoxy-17a-oxo-androst-5-ene-7,17-dione.
This novel D-ring secosteroid was postulated to be a Baeyer–Villiger oxidation by-
product of the preparation reaction. A genuine sample was prepared by the selective
peracid-mediated Baeyer–Villiger reaction on 3b-acetoxyandrost-5-ene-7,17-dione
(7-oxo-DHEA acetate), confirming the novel structure.
Keywords: 3b-Acetoxyandrost-5-ene-7,17-dione, allylic oxidation, Baeyer–Villiger
oxidation, lactone, 7-oxo-DHEA acetate
INTRODUCTION
The conversion of 3b-acetoxyandrost-5-ene-17-one [dehydroepiandrosterone
(DHEA) acetate], 2, to 3b-acetoxyandrost-5-ene-7,17-dione [7-oxo-DHEA
acetate†], 3, has been reported as early as 1949 in the chemical literature.[1]
Most of these preparations utilize a transition-metal-catalyzed allylic
Received in the USA December 9, 2004
Address correspondence to Harold Meckler, Albany Molecular Research Inc., P.O.
Box 15098, 30 Corporate Circle, Albany, NY 12212, USA. E-mail: hmeckler@
albmolecular.com
†Trade name: 7-KetoTM, developed and distributed by Humanetics Corporation,
Chanhassen, MN 55317.

980
L. D. Coutts et al.
oxidation protocol. Since the early 1990s, there have been several reports of a
free-radical-mediated process for the oxidation of acidic or allylic alkyl
groups,[2–4] which avoids the downstream problems posed by the transition-
metal-mediated oxidations.
While developing this oxidation method for the commercial production of
3, we observed a minor by-product in the crude product, which was slightly
more polar than 3 by TLC and HPLC analysis. The level of this impurity
varied 3–7% by area under the curve HPLC analysis. After recrystallization
from methanol, this impurity was routinely reduced to ,1%, but it was
never totally removed from the purified 3. The recrystallization mother
liquors were found to be enriched with this impurity to the level of ꢀ20%
by HPLC analysis.
The recrystallization mother liquor concentrates were purified by silica-
gel column chromatography (eluted with 3:2 hexanes/ethyl acetate) to
provide a sample of this impurity as a white powder. Preliminary mass
spectral analysis indicated that the impurity had an M þ 16
(Mþ þ H ¼ 361), in comparison to the molecular weight for
3
(Mþ þ H ¼ 345). The combustion analysis was correct for the molecular
formula of C21H28O5 (3 þ one oxygen atom). There were no major changes
observed in the proton NMR spectrum of this impurity as compared to 3.
Scheme 1.

3b-Acetoxy-17a-Oxo-Androst-5-Ene-7,17-Dione
981
There was a significant difference in the carbon NMR spectrum with the dis-
appearance of the D-ring ketone carbonyl carbon at d 219 ppm and the appear-
ance of a lactone carbonyl carbon at d 171 ppm. The initial biological activity
of this impurity has been reported by Lardy’s group,[5] but their report does not
describe where or how the sample was obtained.
In the presence of a peracid, the Baeyer–Villiger oxidation is a syntheti-
cally useful process for the conversion of cyclic and acyclic ketones into
lactones and esters, respectively. The regioselectivity and stereospecificity
of this reaction have been reviewed.[6–8] There are reports of the rearrange-
ment of a steroid’s D-ring ketone, but none of them contain an additional
carbonyl or a,b-unsaturated carbonyl group in the B-ring. To selectively
rearrange the D-ring ketone in the presence of the a,b-unsaturated ketone in
the B-ring, without any further rearrangements or epoxidation, would be a
worthwhile synthetic transformation.
To confirm that the impurity formed during the conversion of 2 to 3 was
indeed a Baeyer–Villiger oxidation product, we directly synthesized
compound 4 (Scheme 1). The synthesis of lactone 4 was accomplished
using the method of Hassall,[6] which involved oxidizing a compound
similar to 3 with toluenesulfonic acid and peracetic acid in acetic acid.
Following an aqueous workup and purification by column chromatography,
this product was confirmed to be more polar than 3 by TLC and HPLC
analyses and coeluted with the impurity isolated from the free-radical-
mediated conversion of 2 to 3. Solid 4 prepared by this new route was charac-
terized by 1H NMR, 13C NMR, IR (KBr), DI MS, TLC, HPLC, melting point,
and elemental analysis and found to be identical with the impurity isolated
from the production process.
In summary, the careful investigations of the oxidation to prepare 3b-
acetoxyandrost-5-ene-7,17-dione (7-oxo-DHEA acetate), 3, under free
radical conditions, resulted in the identification and characterization of the
novel lactone 4. A sample of 4 was prepared directly from 3 by utilizing a
mild variation of the Baeyer–Villiger oxidation, thus completing the
structure proof.
EXPERIMENTAL
The reactions were performed under a dry nitrogen atmosphere. Reagents and
solvents were obtained from commercial sources and used as received. Proton
magnetic resonance spectra were obtained on a Bruker AMX-500 MHz NMR,
using either tetramethylsilane or chloroform as an internal reference. Carbon
magnetic resonance spectra were obtained on a Bruker AMX-300 at 75 MHz
using CDCl3 as the solvent. Infrared spectra were obtained as KBr pellets on a
Perkin Elmer Spectrum 1000 Infrared Spectrophotometer. Mass spectrum
analysis was performed on a Shimadzu QP-5000 GC/MS (CI mass

982
L. D. Coutts et al.
spectrometry). Melting points were obtained on a Thomas Hoover Capillary
Melting Point Apparatus. Elemental analysis was obtained from Quantitative
Technologies, Inc. HPLC data were obtained on a Perkin Elmer chromato-
gram equipped with a Phenomenex Hypersil 5 C18 (150 ꢀ 4.6 mm) column
at a detector-wavelength setting of 205 nm.
Preparation of 3b-Acetoxyandrost-5-Ene-17-One (DHEA Acetate) (2)
To a vigorously stirred slurry of 3b-hydroxyandrost-5-ene-17-one (1, 4995 g,
17.3 mol) in glacial acetic acid (15.2 L) was added acetic anhydride (3.3 L),
followed by sodium acetate (1700 g, 20.7 mol, 1.2 equiv). The resulting
slurry was heated to 658C for 18 h; TLC analysis (silica-gel plate eluted
with a 1:1.5 ethyl acetate/hexanes mixture) indicated that the starting
material had been consumed. The reaction mixture was cooled to 508C and
water (36 L) was added over 15–20 min. The resulting slurry was cooled to
ambient temperature and stirred for 4 h. The solids were collected by
vacuum filtration, washed with water (2 ꢀ 10 L), and air-dried for 12 h. The
solids were dissolved in ethanol (44 L) and heated to 708C. The solution
was clarified through a steam-heated line filter and allowed to cool to
ambient temperature with moderate agitation. The product crystallized at
about 508C. The solid product was collected by vacuum filtration, washed
with ethanol (2 ꢀ 4 L), and air-dried for 4 h. The solids were transferred to
glass-drying trays and vacuum-dried at 608C for 24 h to afford 2 as a white
solid (4322 g, 75%). A second crop was isolated by concentrating the
combined mother and wash liquors to about 10 L and allowing the slurry to
age for 24 h. These solids were vacuum-dried at 608C to afford additional 2
as a white solid (1084 g, 19%), making the combined yield of 2 94% (5406 g).
1
Mp 172–1758C. H NMR (500 MHz, CDCl3) d ppm ¼ 5.40 (d, 1H),
4.68–4.55 (m, 1H), 2.52–2.40 (m, 1H), 2.40–2.30 (m, 3H), 2.18–2.05
(m, 2H), 2.05 (s, 3H), 1.99–1.75 (m, 3H), 1.70–1.10 (m, 7H), 1.05 (s, 3H),
0.95 ppm (3H, s). DI MS (m/z): 331 [M þ H]þ. Anal. calcd. for C21H30O3:
C, 69.98; H, 7.83. Found: C, 69.77; H, 7.70.
Preparation of 3b-Acetoxyandrost-5-ene-7,17-dione (3)
A stirred mixture of N-hydroxyphthalimide (1996 g, 12.2 mol) and 2-butanone
(26 L) was heated to 808C and then 3b-acetoxyandrost-5-ene-17-one (DHEA
acetate) (2, 4000 g, 12.1 mol) was added in one portion. After the solids
dissolved, the solution was allowed to return to 808C; 2,20-azobisisobutyroni-
trile (AIBN, 450 g, 2.7 mol) was added in one portion. Oxygen was bubbled
into the solution for 2 h, and the reaction was analyzed by TLC (silica-gel
plate eluted with a 1:1.5 ethyl acetate/hexanes mixture) to ensure the

3b-Acetoxy-17a-Oxo-Androst-5-Ene-7,17-Dione
983
oxidation of 2 had begun. The reaction was continued for an additional 6 h and
then tested for completion by TLC. An additional portion of AIBN (100 g) was
added. The reaction continued for 12 h and was tested for completion by TLC.
The oxygen flow to the stirred reaction mixture was replaced with nitrogen
and the heat was turned off. Once cooled, most of the 2-butanone (22 L)
was removed by vacuum concentration and replaced with methylene
chloride (13 L). The resulting slurry was stirred at ambient temperature for
24 h and vacuum-clarified through a table-top funnel lined with celite filter
aid. The reaction flask and filter cake (recovered N-hydroxyphthalimide)
were rinsed with methylene chloride (5 L) and this rinse was added to the
clarified reaction mixture.
To this stirred solution was added pyridine (1.2 L) followed by acetic
anhydride (0.6 L); the mixture was stirred for 3 h. The reaction mixture was
concentrated under reduced pressure to dryness and the residue was redis-
solved in methyl t-butyl ether (25 L). The resulting solution was washed
with 2 N hydrochloric acid (2 ꢀ 8 L), water (2 ꢀ 4 L), saturated aqueous
sodium bicarbonate solution (4 ꢀ 8 L), water (2 ꢀ 4 L), and saturated
sodium chloride solution (2 ꢀ 4 L). The organic solution was concentrated
under reduced pressure to about 7 L volume and cooled to 08C for 4 h while
the product crystallized. The solids were collected by vacuum filtration,
rinsed with cold methyl t-butyl ether, and vacuum-dried for 24 h to afford
1850 g of crude 3 as a white solid.
A stirred suspension of crude 3 (1800 g) in methanol (25 L) was heated to
reflux until the solids dissolved. This stirred solution was treated with a
mixture of bentonite clay (200 g of Clarion 470) and decolorizing carbon
(200 g of Darco G-60) and was clarified through a celite-coated filter. The
flask and filter cake were washed with hot methanol (4 ꢀ 1 L). The filtrate
was allowed to cool to ambient temperature and was stirred for 18 h. The solid
was collected by vacuum filtration, rinsed with cold methanol, and dried under
reduced pressure at 608C for 24 h to afford a 23% yield (951.6 g) of purified 3.
The combined mother/wash liquors were concentrated under reduced pressure
to about 10 L and the remaining solution was allowed to cool for 12 h. The
resulting solids were collected and dried under reduced pressure at 608C for
24 h to afford an additional 6% yield (251 g) of purified 3. The combined yield
of recrystallized 3 was 29% of a white solid.
1
Mp 186–1928C. H NMR (500 MHz, CDCl3) d ppm ¼ 5.75 (s, 1H),
4.75–4.65 (m, 1H), 2.88–2.75 (m, 1H), 2.65–2.40 (m, 3H), 2.15 (dd, 1H,
J ¼ 12, 12 Hz), 2.05 (3H, s), 2.04–1.95 (dd, 1H, J ¼ 12, 11 Hz), 1.88–1.55
(m, 7H), 1.35–1.20 (m, 6H), and 0.90 ppm (s, 3H). 13C NMR (CDCl3):
220.2, 200.8, 170.3, 164.5, 126.6, 82.1, 72.1, 50.1, 47.9, 45.9, 44.5, 38.6,
37.9, 36.1, 35.7, 30.8, 27.5, 24.3, 21.3, 20.7, 17.5, and 16.8 ppm; IR (KBr):
2954, 1741, 1673, 1629, 1457, 1379, 1298, 1235, 1180, 1031, 903, 871,
607, and 533 cm21. DI MS (m/z) 345 [M þ H]þ. Anal. calcd. for
C21H28O4: C, 73.23; H, 8.19. Found: C, 73.33; H, 8.07.

984
L. D. Coutts et al.
Preparation of 3b-Acetoxy-17a-Oxo-Androst-5-Ene-7,17-Dione (4)
To a vigorously stirred solution of 3b-acetoxyandrost-5-ene-7,17-dione (7-
oxo-DHEA acetate) (3, 35.0 g, 0.10 mol) in acetic acid (700 mL) was added
p-toluenesulfonic acid monohydrate (p-TsOH, 0.70 g), followed by commer-
cial peracetic acid solution (32 wt% in glacial acetic acid, 49.43 g, 0.65 mol,
6.5 equiv); the mixture was stirred for 18 h at ambient temperature. The
solution was slowly diluted with water (2.5 L) and the slurry was stirred for
15 min at ambient temperature. The resulting solids were collected by
filtration, washed with water (3 ꢀ 50 mL), and then dried at ambient tempera-
ture under reduced pressure to remove residual solvents. The white solid was
further purified by column chromatography (silica-gel column eluted using a
gradient system: 1:4:5 ethyl acetate/hexanes/methylene chloride to 2:3:5
ethyl acetate/hexanes/methylene chloride to 3:2:5 ethyl acetate/hexanes/
methylene chloride). Recrystallization of the solid isolated from the
combined fractions of purified 4, from a methanol/diisopropyl ether
mixture provided 4 (21.9 g, 67%) as a white solid.
Mp 267–2718C. 1H NMR (500 MHz, CDCl3) d ppm ¼ 5.75 (s, 1H), 4.71
(m, 1H), 2.84 (m, 1H), 2.72–2.56 (m, 3H), 2.47 (dd, 1H, J ¼ 12, 2 Hz), 2.16
(dd, 1H, J ¼ 12, 11 Hz), 2.05 (3H, s), 1.99–1.63 (7H, m), 1.49–1.46 (3H, m),
1.32 (3H, s), 1.29 (dd, 1H, J ¼ 11, 4 Hz), 1.21 ppm (3H, s). 13C NMR
(CDCl3): 199.4, 171.3, 170.1, 163.5, 126.2, 82.1, 71.9, 48.8, 46.3, 40.3,
38.8, 37.8, 37.5, 35.8, 28.9, 27.1, 21.5, 21.2, 20.7, and 16.8. IR (KBr):
2968, 1731, 1669, 1461, 1386, 1243, 1090, 1029, and 965 cm21. DI MS
(m/z): 361 [M þ H]þ. Anal. calcd. for C21H28O5: C, 69.98; H, 7.83. Found:
C, 69.77; H, 7.70. Rf 0.43 (silica-gel plate eluted with 5:3:2 methylene
chloride/ethyl acetate/hexanes).
ACKNOWLEDGMENT
Albany Molecular Research, Inc. thanks Henry Lardy for an authentic sample
of 4 and for his exciting and enlightening discussion during this project. We
also thank Humanetics Corporation, Chanhassen, Minnesota, for their per-
mission to publish this article. The authors thank Sandy Birk for her assistance
in the preparation of this paper.
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