Ergosteroids II: Biologically active metabolites and synthetic derivatives of dehydroepiandrosterone

Article abstract of DOI:10.1016/S0039-128X(97)00159-1

An improved procedure for the synthesis of 3β-hydroxyandrost-5-ene- 7,17-dione, a natural metabolite of dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained in steroids that are reduced to the corresponding 17β-hydroxy derivative, or hydroxylated at 7α or 7β, and is considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did. Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5 double bond, were inactive.

Full text of DOI:10.1016/S0039-128X(97)00159-1

Ergosteroids II: Biologically active
metabolites and synthetic derivatives
of dehydroepiandrosterone
Henry Lardy, Nancy Kneer, Yong Wei, Bruce Partridge, and Padma Marwah
Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin, USA
An improved procedure for the synthesis of 3␤-hydroxyandrost-5-ene-7,17-dione, a natural metabolite of
dehydroepiandrosterone (DHEA) is described. The synthesis and magnetic resonance spectra of several other
related steroids are presented. Feeding dehydroepiandrosterone to rats induces enhanced formation of several
liver enzymes among which are mitochondrial sn-glycerol 3-phosphate dehydrogenase (GPDH) and cytosolic
malic enzyme. The induction of these two enzymes, that complete a thermogenic system in rat liver, was used as
an assay to search for derivatives of DHEA that might be more active than the parent steroid. Activity is retained
in steroids that are reduced to the corresponding 17␤-hydroxy derivative, or hydroxylated at 7␣ or 7␤, and is
considerably enhanced when the 17-hydroxy or 17-carbonyl steroid is converted to the 7-oxo derivative. Several
derivatives of DHEA did not induce the thermogenic enzymes whereas the corresponding 7-oxo compounds did.
Both short and long chain acyl esters of DHEA and of 7-oxo-DHEA are active inducers of the liver enzymes when
fed to rats. 7-Oxo-DHEA-3-sulfate is as active as 7-oxo-DHEA or its 3-acetyl ester, whereas DHEA-3-sulfate is
much less active than DHEA. Among many steroids tested, those possessing a carbonyl group at position 3, a
methyl group at 7, a hydroxyl group at positions 1, 2, 4, 11, or 19, or a saturated B ring, with or without a 4-5
double bond, were inactive. (Steroids 63:158–165, 1998) © 1998 by Elsevier Science Inc.
Keywords: 7-oxo-dehydroepiandrosterone; DHEA; ergosteroids; acyl esters of 7-oxo-DHEA
possible clinical utility of DHEA is limited by its low
activity and by its conversion to sex hormones. It can be
used by women in only small doses and for limited periods
of time, for reasonable doses lead to greatly elevated blood
testosterone and dihydrotestosterone concentrations.14 In fe-
male rats DHEA administration leads to excess estrogen and
androgen synthesis and the production of polycystic ova-
ries.15,16
To search for possible metabolites of DHEA that might
have greater biological activity, greater specificity, and less
propensity to form sex hormones, we initiated a program of
synthesizing steroids that are derivable from DHEA. The
activity of synthesized compounds was monitored by mea-
suring the induction of thermogenic enzymes in rats.17,18
Hepatic mitochondrial sn-glycerol-3-phosphate dehydroge-
nase and cytosolic malic enzyme function to bypass electron
transport from mitochondrial NADH to ubiquinone. The
result is a decreased efficiency of coupling phosphorylation
to the oxidations of the tricarboxylic acid cycle,17,19,20
greater heat production, and decreased efficiency of food
utilization for growth, fat synthesis or work output.21
There are several cytochrome P450 enzymes that hy-
droxylate steroids at specific positions, and products of such
reactions on DHEA have been isolated from tissues, blood
and urine of normal and tumor-bearing humans and other
mammals.22–28 Hydroxylations of DHEA in tissue homog-
Introduction
Dehydroepiandrosterone (3␤-hydroxyandrost-5-en-17-one;
hereafter DHEA), the most abundant steroid in human
blood, is an intermediate in the biosynthesis of testosterone
and estrogens but also exerts several physiological effects
independent of the sex hormones.1 It is an anti-obesity agent
for genetically obese2 and normal3 animals but does not
affect food intake; it is probably not effective in humans.4 It
decreases blood cholesterol concentration in several spe-
cies,5–7 lessens the severity of diabetes in genetically pre-
disposed mice,8 enhances the immune system,9,10 sup-
presses tumor development,11 and improves memory in
aged mice.12
It is unlikely that these widely different metabolic re-
sponses are elicited by a single steroid molecule for which
no receptor has been found in liver.13 Another reason to
doubt that DHEA, per se, is responsible is that most of these
effects can be observed only when large amounts (0.4–
0.6% of the diet) of this steroid are administered. The
This work was supported by Humanetics Corp., St. Louis Park, Minnesota.
Address reprint requests to Henry Lardy, University of Wisconsin at
Madison, Institute for Enzyme Research, 1710 University Avenue, Madi-
son, WI 53705-4098.
Received October 1, 1997; accepted November 21, 1997.
Steroids 63:158–165, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(97)00159-1

Biologically active derivatives of DHEA: Lardy et al.
Sigma, St. Louis and Research Plus, Bayonne, New Jersey. Some
steroids, other reagents and anhydrous solvents were from Aldrich,
Milwaukee, Wisconsin. Many of the compounds tested were pre-
pared by procedures in the literature. We record here the synthesis
of only new compounds and improved procedures for some known
structures. To save space trivial names are used for well known
compounds.
The progress of chemical reactions was monitored by TLC.
Melting points (°C) were determined in open capillaries in an
electrically heated and stirred Thiele-type bath. They are uncor-
rected. Nuclear magnetic resonance spectra were acquired with
Bruker 200, 300, and 500 MHz spectrometers. Tetramethyl silane
(TMS) in CDCl3 was used as internal standard. Chemical shifts are
reported on the ␦ scale with peak multiplicities: d, doublet; dd,
double doublet; m, multiplet; q, quartet; s, singlet; and t, triplet.
Magnetic resonance data are included for some known compounds
where such data were not previously reported.
enates and cell fractions have been reported from many
laboratories.29–37 7␣-Hydroxy-DHEA23,26,27 and 7-oxo-
DHEA38–41 are known to occur naturally, mainly as
esters. Both 7␣- and 7␤-hydroxylated DHEA are pro-
duced23,29–33,36 and converted to 7-oxo-DHEA42–46 by
cell fractions.
After testing some commercially available steroids18,47
we prepared several monohydroxylated derivatives of
DHEA [4␣, 5␣, 7␣, 7␤, 11␤, 16␣, 19] and of 5-androstene-
3␤, 17␤-diol [7␣, 19]. Only the 7-hydroxy derivatives were
active. Because 7-hydroxy-DHEA was fully as active as
DHEA48 and 7-oxo-DHEA was more active49 we suggested
that these compounds might be the first members of a
metabolic sequence to more active hormone(s). This hy-
pothesis is consistent with the finding that agents that in-
duce the formation of cytochrome P450 2A1, a steroid 7␣-
hydroxylase,37,50 enhance the response of rats to DHEA.18
None of these 7-substituted structures is convertible to
compounds with androgenic or estrogenic activity and they
are therefore potentially useful medications for women
whereas DHEA is not because, in large doses, it causes
masculinization. Because of the ability of these
7-oxygenated steroids to induce thermogenic enzymes that
provide a pathway for heat production,17,20 we have named
them ergosteroids.49
Syntheses
3␤,7␣-Dihydroxyandrost-5-en-17-one (2)
This compound was initially synthesized according to the method
of Starka.55 However, the low yields and lack of generality of that
method prompted a search for another route to the 7-hydroxy
steroids. In the method described below DHEA acetate is subjected
to allylic bromination by a procedure developed by Confalone et
al.56 to prepare 7␣ derivatives of cholesterol. The mixture of 7␣-
and ␤-bromo DHEA acetate that is formed may be isomerized to
predominantly the 7␣-bromo compound and then subjected to a
stereospecific oxidative debromination in the presence of acetic
acid and silver acetate.
Ten g of DHEA acetate (1; 0.03 mol) and 13.6 g of NaHCO3
(0.016 mol) were stirred with 1 L hexane (b.p. 69–71°C) and
heated to reflux under N2. Dibromantin (1,3-dibromo-5,5-
dimethylhydantoin (6.11 g; 0.021 mol) was added and the heating
continued for 30 min. The mixture was then cooled to room
temperature and filtered. The residue was extracted with 50 mL of
CH2Cl2 and the combined organic phases were rotary evaporated
to near dryness at less than 35°C. The creamy white product is
unstable to storage and should be used immediately.
Experimental
The ability of steroids to induce the formation of liver cytosolic
malic enzyme and mitochondrial sn-glycerol-3-phosphate dehy-
drogenase in rats was used as a criterion of DHEA-like activity.
Steroids to be assayed were finely pulverized and ground with a
small amount of Purina rat chow, then mixed with the requisite
amount of chow to give the desired steroid concentration. Records
of daily food consumption were kept to ensure that dietary restric-
tion was not influencing the response of the malic enzyme.51 In
some cases the steroid was dissolved in olive oil and injected
intraperitoneally.
Male Sprague–Dawley rats (140–160 g) were fed diets con-
taining steroids for 6 days. On Day 7 (at about 200 g), animals
were sacrificed, and the large left lobe of the liver was excised and
placed in an ice-cold medium containing 250 mM mannitol, 70
mM sucrose, and 3 mM Hepes at pH 7.4. The liver samples were
weighed, washed, homogenized and mitochondrial and cytosolic
fractions were separated.52 Mitochondrial glycerol-3-phosphate
dehydrogenase was assayed by the procedure of Wernette et al.53
and cytosolic malic enzyme by that of Hsu and Lardy.54 Enzyme
activities in control rats are expressed as nmol/(min times mg
protein). The activities in rats fed steroids are reported as a percent
of the activity found in control rats treated similarly but without
steroid supplementation. We wish to emphasize that because of
variation in the enzyme activity of both control and treated groups
from one experiment to another the reported activities are not
highly precise—they serve mainly as a guide for further syntheses.
Compounds are considered inactive unless they increased the
enzyme activity at least 50% above that of the livers of control rats.
Maximum increases obtained are about five-fold for glycerophos-
phate dehydrogenase and ten-fold for malic enzyme. To improve
sensitivity, assays were designed with amounts of steroids that
gave about half maximal responses; the variability between exper-
iments was found to be relatively greater under these conditions
than when maximally effective doses were given.
The 7␣- and 7␤-bromo derivatives were dissolved in 80 mL
CH2Cl2, and 8 g anhydrous LiBr in 320 mL ice-cold acetone was
added. The mixture was shielded from light and stirred on ice for
3 h during which time it was converted predominantly to the
7␣-bromo enantiomer. Silver acetate (26 g) suspended in 320 mL
CH2Cl2 and 80 mL of glacial acetic acid was stirred for 20 min at
room temperature and then poured into the solution of 7-bromo-
DHEA acetate. The mixture darkened as it was stirred for 30 min.
The mixture was filtered and the solids were rinsed with CH2Cl2.
The combined filtrate was extracted 3 times with 1 L H2O, residual
acid was removed with 5% NaHCO3, extracted twice with water
and rotary evaporated to dryness. The ester was dissolved in 500
mL methanol and heated to reflux under N2 with stirring; 250 mL
of 5% Na2CO3 were added and refluxing continued for 45 min.
The methanol was evaporated and the solution carefully neutral-
ized with acetic acid. Extraction into CH2Cl2 was followed by
evaporation and azeotropic drying with absolute ethanol and twice
with acetone. The solid 7␣-hydroxy DHEA was dissolved in a
minimum amount of warm acetone and hexane was added to
incipient turbidity. Crystals form at room temperature (m.p. 187–
189°C) and a second crop may be obtained from the filtrate by
storing at lower temperature. Depending on the scale of the reac-
tion the yield is from 50 to 80% based on DHEA acetate. The
recrystallized compound melted at 192–193° (lit.29 182–183°C). In
the event that crystallization does not occur the solution may be
evaporated, dried as above and the product purified by chroma-
Dehydroepiandrosterone and some other steroids were pur-
chased from Steraloids, Wilton, New Hampshire; some were from
Steroids, 1998, vol. 63, March 159

Papers
Scheme 1
tography on silica gel (petroleum ether:AcOEt 2.5:1, 1.5:1, then
1:1). An alternative route to either 3␤-acetoxy-7␣—or 3␤-
acetoxy-7␤-hydroxyandrost-5-en-17-one has been reported re-
cently from this laboratory.57
The mother liquor, when chromatographed on silica gel
(eluent–ethyl acetate/hexane 1:5) yielded another 5.0 g of pure
3␤-acetoxyandrost-5-ene-7,17-dione (combined yield 72%). U.V.:
max 235 nM, E ϭ 13,700 (c ϭ 0.032 g/L, ethanol). Infra red:
1
1741, 1724, 1669, 1240 cm. H NMR (500 MHz, CDCl3): ␦ 5.78
(1H, s, 6-H), 4.74 (1H, m, 3␣-H), 2.08 (3-H, s, CH3-acetate), 1.24
(s, 3H, 19-CH3), 0.90 (s, 3H, 18-CH3). 13C NMR (200 MHz,
CDCl3): ␦ 221 (C-17), 200 (C-7), 170 (CAO, acetate), 164.5
(C-5), 126.3 (C-6), 71.8 (C-3), and 50, 47.7, 45.8, 44.3, 38.4, 37.8,
35.9, 35.4, 30.7, 27.2, 24.0, 21.0, 20.5, 17.3, 13.6.
3␤-Acetoxyandrost-5-ene-7,17-dione (3)
The synthesis of the 7-oxo-DHEA acetate used in the bioassays
described in this paper was based on a patent by Forischer et al.58
All starting material should be thoroughly purified and free of any
acidic or basic impurities; if not, reaction time increases and the
yield of product decreases.
3␤-Hydroxyandrost-5-ene-7,17-dione (4)
In a two-necked flask N-hydroxyphthalimide (10.86 g, 0.066
mol) was taken up in a mixture of acetone and ethyl acetate (1:1,
200 mL). 3␤-Acetoxyandrost-5-en-17-one (1; 22.0 g, 0.066 mol)
and azobis (cyclohexane-carbonitrile, 1.1 g) were added in suc-
cession. Subsequently, a weak stream of compressed air was
passed into the mixture which was allowed to reflux for 9 h. After
completion of the reaction (as monitored by TLC: ethyl acetate:
hexane 1:1.5) the mixture was evaporated to dryness and the
resultant mass taken up in 150 mL of toluene. Stirring at 50°C for
30 min followed by cooling at room temperature gave a white
crystalline precipitate of N-hydroxyphthalimide which was filtered
off, washed with two 10 mL portions of toluene, and dried under
suction. Thereby, 10 g (90%) of N-hydroxyphthalimide was re-
covered and may be reused.
The organic layer was washed thoroughly with saturated so-
dium bicarbonate solution until the aqueous layer was colorless,
then with water. Toluene is distilled off under suction and the
residue was heated to dissolve in 130 mL of methanol. On cooling
the 7-oxo compound crystallized. It was collected on a filter,
washed with cold methanol and dried under vacuum. 3␤-
Acetoxyandrost-5-ene-7,17-dione (3) was thus obtained as an off
white colored compound (13.2 g, 56.7% yield). It was further
purified by recrystallization from acetone-hexane, which gave
11.5 g of white crystalline product (50% yield), with a melting
point of 185–187°C.
3␤-Hydroxyandrost-5-ene-7,17-dione (4) was prepared from the
acetate (3) by saponification with Na2CO3 in methanol M.p. 236–
239°C. 1H NMR (200 MHz, CDCl3): ␦ 5.74 (d, J ϭ 1.47 Hz, 6-H),
3.69 (m, 3␣-H), 1.23 (s, 19-CH3), 0.90 (s, 18-CH3). 13C NMR (200
MHz, CDCl3): ␦ 219.8 (C-17), 200.7 (C-7), 166.2 (C-5), 125.7
(C-6), 70.2 (C-3), 50.3, 47.8, 45.9, 44.4, 41.9, 38.4, 36.4, 35.5,
31.14, 30.8, 24.1, 20.6, 17.4, 13.71.
Acyl esters of 3␤-hydroxyandrost-5-ene-7,17-dione
and related steroids (5–13)
Propionyl, butyryl, and long-chain fatty acyl esters of DHEA have
been described in the literature and some are items of commerce.
Isobutyryl DHEA and propionyl, butyryl, and isobutyryl esters of
7-oxo-DHEA were prepared by reacting the parent steroid with a
10% molar excess of the corresponding acid anhydride in anhy-
drous pyridine; acid chlorides were used for preparing the long
chain esters. When acylation was completed the mixture was
poured into ice-water, carefully neutralized with HCl, collected by
filtration, washed with water, dried and crystallized from ethanol
or acetone-hexane. A second crop was usually obtained by storing
the filtrate at 4°C. Some esters of 7-oxo-DHEA were also prepared
by oxidizing the corresponding ester of DHEA.
160 Steroids, 1998, vol. 63, March

Biologically active derivatives of DHEA: Lardy et al.
3␤-isoButyroxyandrost-5-en-17-one (5). M.p. 187–188°C. 1H
NMR (200 MHz, CDCl3): ␦ 5.4 (1H, d, J ϭ 5.0 Hz, 6-H), 4.59
(1H, m, 3␣-H), 1.18 (3H, s, CH3), 1.14 (3H, s, CH3), 1.05 (3H, s,
19-CH3), 0.89 (3H, s, 18-CH3).
Table 1 Enzyme Induction by Esters of DHEA and 7-Oxo-DHEA
GPDH ME
No.
Concentration of
% of
1
Compound
in diet
rats control
3␤-Butyroxyandrost-5-ene-7,17-dione (6). M.p. 219–220°C. H
NMR (200 MHz, CDCl3): ␦ 5.76 (1H, d, J ϭ 1.4 Hz, 6-H), 4.75
(1H, m, 3␣-H), 1.24 (3H, s, 19-CH3), 0.96 (3H, t, J ϭ 7.4 Hz,
CH3), 0.90 (3H, s, 18-CH3).
DHEA
0.05
5
232 295
DHEA-acetate
0.05
13 210 276
13 238 378
7-Oxo-DHEA-acetate
DHEA-propionate
7-Oxo-DHEA-propionate
DHEA
DHEA-acetate
DHEA-butyrate
7-Oxo-DHEA-butyrate
7-Oxo-DHEA-butyrate
DHEA-isobutyrate
7-Oxo-DHEA-isobutyrate
DHEA-acetate
DHEA-laurate
7-Oxo-DHEA-laurate
DHEA-palmitate
7-Oxo-DHEA-palmitate
7-Oxo-DHEA-palmitate
7-Oxo-DHEA-acetate
DHEA-stearate
0.052
0.052
0.054
0.05
6
6
3
5
2
3
4
6
6
6
6
3
5
2
5
2
2
2
3
2
2
240 292
250 379
202 311
263 351
175 196
119 196
291 459
97 125
208 285
152 261
159 228
158 221
194 235
146 154
238 292
209 396
202 205
151 228
305 356
120 143
134 165
3␤-isoButyroxyandrost-5-ene-7,17-dione (7). M.p. 185–186°C.
1H NMR (300 MHz, CDCl3): ␦ 5.78 (1H, s, 6-H), 4.74 (1H, m,
3␣-H), 1.22 (3H, s, 19-CH3), 1.18 (6H, s, (CH3)2), 0.9 (3H, s,
18-CH3).
0.057
0.062
0.013
0.065
0.062
0.064
0.05
3␤-Dodecanoyloxyandrost-5-ene-7,17-dione (8). M.p. 90–91°C.
1H NMR (300 MHz, CDCl3): ␦ 5.77 (1H, s, 6H), 4.73 (1H, m,
3␣-H), 1.24 (s, 19-CH3), 0.9 (s, 18-CH3).
0.08
3␤-Hexadecanoyloxyandrost-5-ene-7,17-dione (9). M.p. 87–88°C.
1H NMR (200 MHz, CDCl3): ␦ 5.76 (1H, s, 6H), 4.73 (1H, m, 3␣-H),
1.2 (s, 19-CH3), 0.9 (s, 18-CH3).
0.08
0.08
0.05
0.09
0.02
3␤-Octadecanoyloxyandrost-5-ene-7,17-dione (10). M.p. 87–88°C.
1H NMR (200 MHz, CDCl3): ␦ 5.76 (1H, s, J ϭ 1.4 Hz, 6H), 4.74
(1H, m, 3␣-H), 0.90 (3H, s, 18-CH3).
0.096
0.036
0.067
0.035
0.036
7-Oxo-DHEA-stearate
7-Oxo-DHEA-sulfate
3␤-Acetoxy-A-17␤-stearate
3␤-Acetoxy-17␤-stearoyl-A-7-
one
3␤-Hemisuccinoyloxyandrost-5-ene-7,17-dione (11). M.p. 265–
265.5°C. 1H NMR (200 MHz, CDCl3): ␦ 5.76 (1H, s, J ϭ 1.6 Hz,
6-H), 4.76 (1H, m, 3␣-H), 1.24 (3H, s, 19-CH3), 0.9 (3H, s,
18-CH3).
DHEA-acetate
0.05
0.06
0.05
0.012
0.03
0.06
8
4
2
2
2
6
220 346
205 353
268 429
172 177
206 213
333 488
DHEA-hemisuccinate
7-Oxo-DHEA-acetate
7-Oxo-DHEA-hemisuccinate
7-Oxo-DHEA-hemisuccinate
7-Oxo-DHEA-hemisuccinate
3␤-Acetoxyandrost-5-en-17␤-octadecanoate (13). To 0.85 g
of 3␤-acetoxyandrost-5-en-17␤-ol (12) in 10 mL dry pyridine,
1 g of stearoyl chloride was added at room temperature. Ninety
percent of the starting steroid had reacted within 30 min and
none could be detected the following day when the reaction
mixture was worked up as described above. The crystalline
product, 1.394 g (91%) melted at 82–83°C. 1H NMR (200 MHz,
CDCl3): ␦ 5.37 (1H, s, J ϭ 5.4 Hz, 6H), 4.6 (2H, m, 3␣-H ϩ
17␣-H), 2.04 (3H, s, COCH3), 1.03 (3H, s, 19-CH3), 0.80 (3H,
s, 18-CH3).
In each assay, enzyme activities of rats fed the new esters for six
days were compared with rats fed DHEA or DHEA-acetate and
with non-supplemented controls. All acyl groups are at the 3␤
position unless designated at 17␤. GPDH, sn glycerol
3-phosphate dehydrogenase; ME, malic enzyme. In this table -A-
is an abbreviation for androst-5-ene.
3␤-Acetoxy-17␤-octadecanoyloxyandrost-5-en-7-
one (14)
M.p. 87–90°C. 1H NMR (200 MHz, CDCl3): ␦ 5.71 (1H, s, J ϭ 1.0
Hz, 6-H), 4.71 (1H, m, 3␣-H), 4.64 (1H, m, 17␣-H), 2.05 (3H, s,
COCH3), 1.22 (3H, s, 19-CH3), 0.81 (3H, s, 18-CH3).
pyridine was added to cleave the osmate ester. After stirring for
20 min the mixture was extracted repeatedly with dichlorometh-
ane (100, 50, 50, 25 mL) which was washed with saline and
water, and dried over MgSO4. When concentrated under re-
duced pressure the product crystallized, was collected by fil-
tration and dried in vacuo. Yield 930 mg. M.p. 238–240°C. The
mother liquor yielded 191 mg (m.p. 237–238°C) for an overall
1
yield of 88%. H NMR (200 MHz, CDCl3): ␦ 5.05 (m, 3␣-H),
3␤,5␣,6␣-Trihydroxyandrostan-17-one (15)59
4.12 (d, J ϭ 1.00 Hz, 6␤-H), 2.03 (s, OCOCH3), 1.32 (s,
19-CH3), 0.88 (s, 18-CH3). 13C NMR (300 MHz, CDCl3): ␦
219.6 (C-17), 209.3 (C-7), 170.5 (CAO acetate), 81.3 (C-5),
77.1, 70.0 (C-3,6), 47.1, 46.7, 44.3 (C-methines), 35.5, 34.9,
30.7, 30.6, 26.5, 22.6, 21.0 (C-methylenes), 21.3, 16.0, 13.8
(methyls).
M.p. 239–242°C. 1H NMR (200 MHz, CDCl3): ␦ 4.05 (m, 3␣-H),
3.73, 3.67 (dd, J ϭ 5.4, 11.2 Hz, 6␤-H), 0.96 (s, 19-CH3), 0.84
(18-CH3). 13C NMR (300 MHz, CDCl3): ␦ 210 (C-17), 70.2, 67.1
(C-3,6), 50.9, 44.4, 33.3 (C-methines), 38.4, 35.8, 33.8, 31.5, 31.1,
30.6, 21.7, 20.4 (C-methylenes), 15.5, 13.8 (C-methyls).
3␤-Acetoxy-5␣,6␣-dihydroxyandrostane-7,17-dione
(16)
3␤-17␤-Diacetoxy-5␣,6␣-dihydroxyandrostan-7-one
(17)
A solution of 1.15 g of 3␤-acetoxyandrost-5-ene-7,17-dione (3) in
10 mL of pyridine was treated with 0.98 g of OsO4 in 15 mL of
pyridine and stirred for 2.5 h at room temperature. A solution of
1.8 g of sodium bisulfite in 30 mL of water and 20 mL of
This compound, prepared as for the diketone above, melted at
1
159–160°C when recrystallized. H NMR (200 MHz, CDCl3): ␦
5.06 (m, 3␣-H), 4.63 (t, J ϭ 8.3 Hz, 17␣-H), 4.08 (d, J ϭ 2.9 Hz,
Steroids, 1998, vol. 63, March 161

Papers
Table 2 Steroids that Induce the Synthesis of Mitochondrial Glycerophosphate Dehydrogenase and Cytosolic Malic Enzyme in Rats’
Livers
GDPH
ME
Steroid
Concentration in diet
No. of rats
% of control
3␤-ol-A-17-one (DHEA)
0.01
8
3
135
68
4
3
42
9
6
5
11
6
9
124
107
0.025
157
178
0.05
0.1
0.2
253 Ϯ 56
295 Ϯ 68
285
327 Ϯ 76
428 Ϯ 86
645
3␤-acetoxy-A-17-one
0.023
148
285
0.057
241 Ϯ 55
285 Ϯ 64
168 Ϯ 17
290 Ϯ 32
341 Ϯ 88
366
329 Ϯ 75
455 Ϯ 105
244 Ϯ 61
499 Ϯ 47
507 Ϯ 71
704
0.1
3␤-ol-A-7,17-dione (7-oxo-DHEA)
0.0105
0.026
0.052
0.105
3␤-acetoxy-A-7,17-dione
3␤,7␣-diol-A-17-one
0.02–0.03
215 Ϯ 66
304 Ϯ 107
270 Ϯ 59
308
341 Ϯ 85
392 Ϯ 40
463 Ϯ 113
374
0.052–0.06
19
15
2
0.1–0.12
0.033
0.05
2
292
423
3␤-acetoxy-7␤-ol-A-17-one
A-3␤,17␤-diol
0.05
3
219
339
0.05
4
234
261
0.2
3
275
494a
3␤,17␤-diol-A-7-one
0.01
8
216
219
0.05
5
378
416
0.1
6
231
652
3␤-acetoxy-A-17␤-ol
3␤-acetoxy-17␤-ol-A-7-one
3␤,17␤-diacetoxy-A
3␤,17␤-diacetoxy-A-7-one
3␤,16␣-diol-A-17-one
A-3␤,7␣,17␤-triol
3␤,16␣,17␣-triacetoxy-A
3␤,16␣,17␣-triacetoxy-A-7-one
3␤,16␣,17␣-triol-A-7-one
3␤,16␣-diacetoxy-7␣-ol-A-17-one
3␤,16␤-diacetoxy-7␤-ol-A-17-one
3␤,16␤-diacetoxy-7␣-ol-A-17-one
3␤-propionoxy-7␣-F-A-17-one
3␤-acetoxy-7␣-F-A-17-one
3␤-acetoxy-7␤-F-A-17-one
3␤-acetoxy-7␣-F-A-17-one
3␤-acetoxy-7␤-F-A-17-one
0.058
2
222
338
0.13
3
232
452
0.065
8
114
131
0.068
9
290
274
0.05
0.1
5
99
135
2
227
611
0.075
3
145
107
0.078
3
198
164
0.056
3
102
227
0.05
2
246
270
0.05
2
228
507
0.05
5
242
340
30 mg/day times 3
30 mg/day times 3
30 mg/day times 3
20 mg/day times 3
20 mg/day times 3
3
213
164
3
157
290
3
167
258
3
185
270
3
224
231
In this table, -A- is an abbreviation for androst-5-en. Enzyme activity in the livers of rats fed the stock diet without supplementation is
termed 100%.
aData from Su and Lardy (18).
6␤-H), 3.83 (d, OH), 2.04, 2.02 (2s, 3,17-acetate), 1.31 (s, 19-
CH3), 0.80 (s, 18-CH3). 13C NMR (300 MHz, CDCl3): ␦ 209.5
(C-7), 171.05, 170.48 (CAO, acetates), 81.27 (C-5), 81.78, 77.02,
70.16, (C-3,6,17), 47.27, 46.86, 43.48 (C-8,9,14), 35.74, 34.81,
30.7, 27.48, 26.46, 24.3, 21.22 (C-1,2,4,11,12,15,16), 21.31, 21.1
(acetate-CH3), 15.96, 12.08 (C-18,19).
Scheme 2
162 Steroids, 1998, vol. 63, March

Biologically active derivatives of DHEA: Lardy et al.
Table 3 Compounds that Do Not Induce Mitochondrial Glycerophosphate Dehydrogenase or Cytosolic Malic Enzyme in Rats’ Livers
Androst-4-ene-3␤,17␤-diol; androst-4-ene-3,17-dione; androst-4-ene-3,11,17-trione (0.052); 4-hydroxyandrost-4-ene-3,17-dione
(0.015); 11␤-hydroxyandrost-4-ene-3,17-dione (0.05); 19-hydroxyandrost-4-ene-3,17-dione (0.024); 7␣-hydroxytestosterone;
17␣-ethynyltestosterone; 17-hydroxyandrosta-4,6-dien-3-one; 3␤-acetoxy-5␣-ol-androst-6-en-17-one (0.05).
Androst-5-en-17-one; androst-5-ene-7,17-dione; 3␤-hydroxy-7-methylandrost-5-en-17-one; 4␤-hydroxy-DHEA; 16␣-hydroxy-DHEA;
16␣-bromo-DHEA; 19-ol-DHEA; androst-5-ene-3␤,7␣,19-triol; 1␣,3␤-diacetoxyandrost-5-ene-7,17-dione; 2␣,3␤-diacetoxyandrost-6-
en-17-one; androst-5-ene-3␤,16␣-diol; 3␤,17␤-dihydroxyandrost-5-en-11-one (0.053); 3␤-acetoxy-5␣-hydroxyandrost-6-en-17-one;
androst-5-ene-3␤,19,17␤-triol; 2␤,3␤,17␤-triacetoxyandrost-5-en-7-one; 3␤,7␣-dihydroxy-16␤-acetoxyandrost-5-en-17-one (0.05);
3␤-hydroxyandrost-5-ene-17-carboxylic acid; 3␤-acetoxyandrosta-1,5-diene-7,17-dione; androsta-3,5-diene-7,17-dione (0.05);
17␤-hydroxyandrosta-3,5-dien-7-one; 3␤-hydroxyandrosta-5,7-dien-17-one; androsta-5,16-dien-3␤-ol, and its 3-acetate.
11␤-hydroxyandrosterone; 11-oxo-androsterone (0.04); 3␤-acetoxy-5␣-androstane-7␣,17␤-diol (0.01);
3␤-acetoxy-5␣,6␣-dihydroxyandrostane-7,17-dione; 3␤,5␣,6␣-trihydroxyandrostan-17-one (0.056);
3␤,17␤-diacetoxy-5␣,6␣-dihydroxyandrostan-7-one; 3␤,5␣,6␣,16␣,17␣-pentacetoxyandrostane;
2␣,3␣-dihydroxy-5␣-androstane-7,17-dione; 3␤,5␣,6␤-trihydroxyandrostan-17-one; 17␤-hydroxy-5␣-androstane-3,7-dione (0.027).
5␤-androstane-3␣,17␤-diol (0.067); 5␤-androstane-3␤,17␤-diol (0.077), and its 3-acetate (0.05); 3␤-hydroxy-5␤-androstan-17-one
(0.077) and its 3-acetate (0.077); 5␤-androstane-3,17-dione (0.077); 5␤-androstane-3␣,7␣,17␤-triol (0.043); 7␣,17␤-dihydroxy-5␤-
androstan-3-one-(0.05); 3␣,7␣,16␣-trihydroxy-5␤-androstan-17-one (0.08), and its 16-acetate (0.05); 7␣-hydroxy-16␣-acetoxy-5␤-
androstane-3,17-dione (0.05).
7-hydroxyestrone (0.01); androsta-1,4,6-triene-3,7-dione; 17␣-ethyl-17␤-ol-19-nor androst-4-en-3-one; 3␣,6␣-dihydroxy-5␤-pregnan-
20-one (0.05); 3␣-hydroxy-5␤-pregnan-20-one (0.05); 16␣-hydroxyprogesterone (0.06); pregnenolone; 7␣-hydroxypregnenolone;
3␤-acetoxypregnene-7,20-dione (0.06); 3␤,17␣-diacetoxypregnen-7,20-dione.
These steroids were fed at 0.1 or 0.2% of the diet unless a lower concentration is indicated. For comparative purposes, some additional
inactive compounds are reported in Tables 1 and 2.
over silica gel. The white solid was crystallized from MeOH/Et2O
3␤,16␣-diacetoxyandrost-5-en-17-one (18)
Four g of 3␤,16␣-dihydroxyandrost-5-en-17-one60 in 24 mL Ac2O
to yield triol 20 (180 mg, 56%. M.p. Ͼ 230°C. 1H NMR (300
MHz, DMSO) ␦ 5.42 (d, 1H, J ϭ 2 Hz, 6-H), 5.30 (br s, 1H, OH,
and 16 mL pyridine was stirred for 12 h at room temperature. After
D2O exch.), 4.68 (br s, 1H, OH, D2O exch.), 4.26 (d, 1H, J ϭ 2 Hz,
removal of solvents in vacuo, the residue was purified over silica
OH, D2O exch.), 3.64 (s, 1 H, 17-H), 3.60 (br s, 1H, 7-H), 3.32 (m,
gel (CHCl3: Me2CO ϭ 60:1) to yield compound 18 (3 g, 67%) as
3-H), 0.92 (s, CH3), 0.61 (s, CH3).
a white solid which was crystallized from AcOEt/pet ether. M.p.
1
165–167°C. HMR (300 MHz, CDCl3) ␦ 5.20 (m, 2 H, 6-H and
16-H), 4.60 (m, 1 H, 3-H), 2.09 (s, OCOCH3), 2.02 (s, OCOCH3),
1.06 (s, CH3), 1.01 (s, CH3).
Results and discussion
Many therapeutically useful steroids are administered as esters
and the widespread distribution of esterase activity61 in mam-
malian tissues ensures the cellular availability of the free ste-
roid. Several aliphatic esters of DHEA and of its 7-oxo analog
were synthesized to ascertain their possible utility in therapy
and to provide standards for metabolic studies; their activity as
inducers of rat liver glycerol-3-phosphate dehydrogenase and
malic enzyme is shown in Table 1. Unexpected selective
hydrolyses appear to be involved in the availability of some of
these steroids. Among the monoesters prepared and tested all
were active except the isobutyryl derivative of DHEA; how-
ever, isobutyryl-7-oxo-DHEA was fully active. Other exam-
ples of inactive esters matched by active 7-oxo derivatives are
presented in Table 2 and are discussed below. The sulfate ester
of 7-oxo-DHEA, which is found in humans,40 was approxi-
mately as active as equimolar amounts of DHEA-acetate
whereas DHEA sulfate was poorly active orally and especially
when injected intraperitoneally.18
Long chain fatty acyl derivatives of steroids occur natu-
rally62–66 and some may have special functions.64 The weak
enzyme induction by 17␤-stearoyl derivatives of 3␤-
acetoxy-androstenediol and the corresponding 7-oxo com-
pound indicates that these esters are not readily hydrolyzed
by tissue esterases. Hochberg et al. have observed that fatty
acyl esters of testosterone64 and estrogens65 also have pro-
longed half lives in rats.
3␤,16␣-diacetoxy-7-␣-hydroxyandrost-5-en-17-one
(19) and 3␤,7␣,16␣-triacetoxyandrost-5-en-17-one
(20)
Compound 18 (3 g) was brominated, treated with LiBr and sub-
sequently with AgOAc as in the procedure for compound 2 except
that toluene was the solvent to which the LiBr solution was added.
Thirty min after the addition of AgOAc, solids were removed by
filtration and washed; the filtrate was concentrated and 300 mL
H2O was added to the residue. The pH was brought to neutral with
NaHCO3 and the solution was extracted with AcOEt (150 mL
times 5). The combined organic solution was washed with brine,
dried and concentrated to dryness. The crude product was purified
over silica gel (AcOEt:pet ether ϭ 1:3, 1:2, and then 1:1) to yield
1.5 g of diacetate 19 (48%) and 700 mg of triacetate 20 (20%).
Both compounds were crystallized from Et2O.
Compound 19. M.p. 155–158°C. 1H NMR (300 MHz, CDCl3)
␦ 5.64 (d, 1H, J ϭ 4 Hz, 6-H), 5.44 (d, 1H, J ϭ 5 Hz, 16-H), 4.63
(m, 1H, 3-H), 3.92 (m, 1H, 7-H), 2.15 (s, OCOCH3), 2.04 (s,
OCOCH3), 1.02 (s, CH3), 0.97 (s, CH3).
Compound 20. M.p. 170–172°C. 1H NMR (300 MHz, CDCl3)
␦ 5.61 (d, 1H, J ϭ 2 Hz, 6-H), 5.40 (d, 1H, J ϭ 4 Hz, 16-H), 5.02
(dd, 1H, J ϭ 2 Hz, 7-H), 4.70 (m, 1H, 3-H), 2.10 (s, OCOCH3),
2.02 (s, OCOCH3), 1.02 (s, CH3), 0.98 (s, CH3).
3␤,7␣,17␤-Trihydroxyandrost-5-en-16-one (21)
The finding that oxygenated derivatives of dehydroepi-
androsterone occur in the urine of normal subjects and of
patients with adrenal tumors22,23 led Okada et al.23 to pos-
Diacetate 19 (400 mg) was stirred with 342 mg K2CO3 in 25 mL
MeOH at room temperature for 2 h. After removal of insoluble salt
the filtrate was concentrated in vacuo and the residue was purified
Steroids, 1998, vol. 63, March 163

Papers
tulate that the parent C19 ketosteroid might be converted to
other active hormones as well as to testosterone and estro-
gens. Mammalian tissue preparations hydroxylate DHEA to
produce 7␣, 7␤, and 16␣-hydroxy derivatives and 7-oxo-
DHEA.29–37 Such oxygenated analogs of DHEA had appar-
ently not been tested for biological activity in animals until
recently.18,47–49 The fact that there is no known receptor for
DHEA and that relatively massive amounts (ca. 0.5% of the
diet; or ca. 500 mg/kg body wt. per day) are required to
elicit its antiobesity effect in mice and rats indicates that
DHEA is not an active hormone but only a precursor of an
active anti-obesity factor. We felt that some of the known
metabolites of DHEA might be intermediates in the biolog-
ical conversion of DHEA to more active hormones that have
yet to be discovered. We therefore tested these known
metabolites and synthesized additional compounds that
might be produced from DHEA in mammalian tissues or
that might shed light on structure/activity relations.
A comparison of the structures of compounds that induce
the formation of liver mitochondrial sn-glycerol-3-phosphate
dehydrogenase and cytosolic malic enzyme in rats (Table 2)
with those that do not (Table 3) is informative. The conversion
of precursors of DHEA to active steroids in the brain and
testes of rats apparently does not occur rapidly enough to
provide DHEA to the liver for feeding pregnenolone, 7␣-
hydroxypregnenolone, or 7-oxo-17␣-hydroxypregnenolone
had no influence on the activity of these enzymes in liver.
Introduction of hydroxyl groups into DHEA or 7-oxo-
DHEA at position 1, 2, 4, 11, or 19 abolished the activity as
did saturating the B ring or moving the double bond from
5-6 to 4-5. Introducing a second double bond at 1-2 or 7-8
abolished activity as did oxidizing the 3␤-hydroxyl group to
form a ketone. The 16␣-hydroxy derivative of DHEA is
produced in normal and tumor bearing humans. It did not
induce the formation of glycerol-3-phosphate dehydroge-
nase and had only a minor influence on malic enzyme
(Table 2); likewise the 16␣-hydroxy derivative of andro-
stenediol assayed as the triacetate was inactive but the
corresponding 7-oxo derivatives of both were active. The
diacetyl ester of androstenediol was inactive but 7-oxo-
androstenediol diacetate was as effective as free andro-
stenediol (Table 2).
responds so readily to administered DHEA, androstene diol
and their 7-oxygenated derivatives. Because pregnenolone
and 17␣-hydroxy pregnenolone did not enhance the liver
enzymes, it does not seem likely that small amounts of
DHEA produced in brain and testes or ovaries are rapidly
converted to an active steroid to which liver is responsive.
It seems likely that the 7-hydroxy derivatives of DHEA,
and 7-oxo-DHEA are on an enzyme-catalyzed pathway to
one or more active hormones that are the ultimate effectors
of the several metabolic responses to DHEA.
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