First synthesis of 7α- and 7β-amino-DHEA, dehydroepiandrosterone (DHEA) analogues and preliminary evaluation of their cytotoxicity on Leydig cells and TM4 Sertoli cells

Article abstract of DOI:10.1016/j.bmc.2007.02.036

Efficient syntheses of new DHEA analogues, and their apoptotic and necrotic effects on Leydig cells and TM4 Sertoli cells are described. The key step in the synthetic strategy of 7-amino-DHEA derivatives involves a bromination on C-7 position to give an epimeric mixture of bromides which were substituted by azides and reduced to give 7α- and 7β-amino-3β-hydroxyandrost-5-en-17-ones. No cytotoxic effect induced by apoptosis mechanism was observed on Leydig and TM4 Sertoli cells by treatment with these amino-DHEA analogues. A necrotic effect was induced only in TM4 Sertoli cells. The best activity was obtained with 7α,β-amino-androst-5-en-3β-ol and 7β-amino-3β-hydroxy-androst-5-en-17-one.

Full text of DOI:10.1016/j.bmc.2007.02.036

Bioorganic & Medicinal Chemistry 15 (2007) 3152–3160
First synthesis of 7a- and 7b-amino-DHEA,
dehydroepiandrosterone (DHEA) analogues and preliminary
evaluation of their cytotoxicity on Leydig cells and TM4 Sertoli cells
a
b
b
Marc-Antoine Bazin, Carine Travert, Serge Carreau,
a
a,
*
Sylvain Rault and La ¨ı la El Kihel
a
Centre d’Etudes et de Recherche sur le M e´ dicament de Normandie, UFR des Sciences Pharmaceutiques,
rue Vaub e´ nard, 14032 Caen cedex, France
Institut de Biologie Fondamentale et Appliqu e´ e (IBFA), Universit e´ de Caen Basse-Normandie, Caen cedex, France
5
b
Received 15 December 2006; revised 13 February 2007; accepted 20 February 2007
Available online 22 February 2007
Abstract—Efficient syntheses of new DHEA analogues, and their apoptotic and necrotic effects on Leydig cells and TM4 Sertoli cells
are described. The key step in the synthetic strategy of 7-amino-DHEA derivatives involves a bromination on C-7 position to give an
epimeric mixture of bromides which were substituted by azides and reduced to give 7a- and 7b-amino-3b-hydroxyandrost-5-en-17-
ones. No cytotoxic effect induced by apoptosis mechanism was observed on Leydig and TM4 Sertoli cells by treatment with these
amino-DHEA analogues. A necrotic effect was induced only in TM4 Sertoli cells. The best activity was obtained with 7a,b-amino-
androst-5-en-3b-ol and 7b-amino-3b-hydroxy-androst-5-en-17-one.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
or androgenic. The direct effect of DHEA is to act as a
weak androgen and that more pronounced androgenic-
ity/estrogenicity are secondary effects due to metabolic
conversion into testosterone/estradiol and derivatives.
Controlling estrogenic and androgenic steroid concen-
trations by inhibiting steroidogenic enzymes is a good
way to prevent and potentially cure breast and prostate
Dehydroepiandrosterone (3b-hydroxy-androst-5-en-17-
one or DHEA) is produced in the adrenals and brain.
It is the most abundant steroid in human plasma. Plas-
ma concentrations of this steroid reach maximum levels
during adolescence and decline with age. Studies have
shown that DHEA exerts physiological effects that are
supposed to play a beneficial role in diseases/disorders
1
2
3
1
6
hormone-dependent cancers.
4
5
6
as cancers, diabetes, atherosclerosis, cardiovascular
7
These previous studies showed clearly the essential role
of DHEA in various biological mechanisms. However,
the broad effect of DHEA itself limits its therapeutic
use. To circumvent the side effects of DHEA, one of
the promising ways is to develop DHEA derivatives
with a greater specificity. With this goal, we are
researching on DHEA analogues having targeted prop-
erties including cytotoxicity towards testicular cells and
we present herein the first promising results obtained
with new amino-DHEA derivatives.
8
9
disease, depression, immune function, acquired im-
12
mune deficiency syndrome (AIDS), aging, obesity,
1
0
11
1
3
14
memory, and Alzheimer’s disease. Beneficial effect of
additional DHEA treatment in the elderly population is
currently under study and seems to prevent these
1
5
disorders.
On the other hand, DHEA is an intermediate in the pro-
duction of both androgens and estrogens in the periph-
eral tissues. Effects of DHEA could be either estrogenic
It can be noticed that few DHEA analogues have been
synthesized to our knowledge. 7-Oxygenated DHEA
including DHEA metabolites have been mainly devel-
oped and their activities have been widely studied.
However, DHEA and its androgenous metabolites (7-
Keywords: DHEA; Aminosteroids; DHEA analogues; Hormones;
Cytotoxicity; TM4 cells.
1
7–19
*
Corresponding author. Tel.: +33 (0) 2 31 93 41 69; fax: +33 (0) 2 31
3 11 88; e-mail: laila.elkihel@unicaen.fr
9
oxo-DHEA, 7a- and 7b-hydroxyDHEA) have side
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.036

M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
3153
O
17
3
7
HO
DHEA
O
O
HO
NH2
HO
NH2
HO
NH2
I
II
III (7α/β : 85/15)
Scheme 1. DHEA and DHEA analogues.
2
0–24
effects that may limit their use in humans.
monohydroxylated derivatives of DHEA (4a, 5a, 7a,
b, 11b, 16a) and androst-5-ene-3b,17b-diol have been
Several
androst-5-en-7a,b-ol (3b) could be formed when anhy-
drous conditions were not respected (Scheme 3). The
structure and 7a/7b bromides ratio were established by
7
1
synthesized and evaluated on the induction of the
H NMR spectra. Due to the relative instability of the
thermogenic enzymes. Only 7-oxygenated derivatives
5
bromide product, the crude product was used in next step
without further purification. 7-Bromo derivatives 3
substituted by sodium azide gave 7a- and 7b-azido-and-
rost-5-ene (compounds 4 and 5) easily separable by col-
umn chromatography on silica gel. The stereochemistry
2
induced thermogenic enzymes. In this pathway, and
in our studies related to the synthesis of amino and
2
6–29
polyaminosterols,
7
we now report the synthesis of
-aminosteroids as new DHEA analogues (Scheme 1).
1
A first preliminary study consists to evaluate their
biological activities on purified Leydig cells and TM4
Sertoli cell lines.
of each azido epimer was established by H NMR spectra.
However, direct azidation on allylic position was at-
tempted using trimethylsilyl azide and lead(IV) acetate
3
2
in Zbiral conditions (Scheme 4). In this case, 7a,b-azi-
dosteroid mixture was not separable in classical chroma-
tography conditions.
2
. Results and discussion
2
.1. Chemistry
a- and 7b-amino-3b-hydroxy-androst-5-en-17-ones (I
The reduction of azides to afford primary amines is an
important synthetic operation. A number of reagents
described in the literature have been used for this reduc-
7
and II) were prepared from dehydroepiandrosterone.
As depicted in Scheme 2, the hydroxyl group of DHEA
was acetylated with acetic anhydride in pyridine to give
the acetate 1 (95%) and ketone functionality was pro-
tected using ethylene glycol and p-toluene sulfonic acid
3
3
tive process including Staudinger reaction, borohy-
3
4
35
drides, samarium diiodide and lithium aluminium
3
6
hydride.
But most of these reagents have some
disadvantages in relation to their general applicability.
7-Aminosteroids have been obtained in low yields (0–
25%). On the other hand, unfortunately, reduction of
azides 4 and 5 using recent methods such as either Sm/
(
(
PTSA) in refluxing toluene to give compound 2
80%). The exact catalytic quantities (0.5%) of PTSA
are necessary to obtain a good yield of ketal. Allylic bro-
mination was accomplished with 1,3-dibromo-5,5-dim-
ethylhydantoin (dibromantin) and sodium bicarbonate
in n-hexane at 60 ꢁC to afford 7a/b bromides. The liter-
ature reported the bromination in situ of cholesteryl
3
7
38
NiCl Æ6H O or Fe/AlCl in aqueous ethanol failed
2
2
3
to yield the desired product. Moreover, catalytic hydro-
genation could not be used because it has been limited
for being applied to unsaturated compounds. We at-
tempted a reduction of azides 4 and 5 using a high excess
of lithium aluminium hydride in presence of nickel(II)
chloride hexahydrate. Amines 6 and 7 were, respectively,
successfully obtained but with serious purification prob-
lems. These problems have been known in the literature
for purification of aminosteroids such as aminobrassi-
3
b-acetate with dibromantin followed by dehydrobro-
30
mination to give cholesta-4,6-dien-3b-ol. This syn-
thetic way has been applied to a variety of cholesterol
derivatives relevant to the synthesis of the human
3
1
metabolites of vitamin D3. In our case, we have
described the intermediate 7-bromosteroid because its
formation in the reaction must be confirmed. In
addition, this intermediate could be transformed by
dehydrobromination to undesired 3b-acetoxy-17,17-
ethylenedioxy-androst-5,7-diene (3a) observed in a small
amount (10–15%) and confirmed by NMR and MS
spectra. Moreover, 3b-acetoxy-17,17-ethylenedioxy-
3
9
nosteroids. Finally, the ketal was removed with
hydrochloric acid in tetrahydrofuran to give aminoster-
oids I and II.
As outlined in Scheme 5, 7a,b-amino-3b-hydroxy-
androst-5-enes (III) were prepared in the same way.

3154
M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
O
O
O
O
O
i
ii
iii
DHEA
AcO
AcO
AcO
Br
1
2
3
O
O
O
O
O
O
O
v
vi
AcO
AcO
N3
HO
HO
NH2
O
HO
HO
NH2
4
5
6
I
iv
O
3
O
v
vi
N3
NH2
NH2
7
II
Scheme 2. Synthesis of 7a- and 7b-amino-3b-hydroxy-androst-5-en-17-one (I and II). Reagents and conditions: (i) Ac
ethylene glycol, PTSA, toluene, reflux, 8 h, 80%; (iii) dibromantin, NaHCO , DMF, rt, 18 h, 45% (35% 7a and
O, THF, reflux, 45 min, 39–70%; (vi) 5% aqueous HCl, THF, rt, 18–24 h, 92–95%.
2
O, pyridine, rt, 12 h, 95%; (ii)
3
, n-hexane, 60 ꢁC, 45 min; (iv) NaN
3
6
5% 7b); (v) LiAlH
4
, NiCl
2
Æ6H
2
O
O
O
O
O
O
O
O
+
+
AcO
AcO
Br
AcO
AcO
OH
2
3
3a
3b
Scheme 3. Allylic bromination of 3b-acetoxy-17,17-ethylenedioxy-androst-5-ene with formation of secondary compounds.
O
O
Dehydroepiandrosterone was deoxygenated in Wolff–
Kishner modified conditions following the procedure
4
0
described by Huang-Minlon in a good yield (9). The
hydroxyl group was acetylated and allylic bromination
was accomplished by dibromantin/sodium bicarbonate.
The epimeric allylic bromides 11 were substituted by so-
dium azide to give 7a,b-azido-androst-5-ene epimers
(12) inseparable in this case in classical chromatography
conditions. The aminosteroids mixture III was obtained
after reduction of azide and ester.
AcO
AcO
N3
1
8 (7α/β : 70/30)
Scheme 4. Azidation in Zbiral conditions. Reagents and conditions:
(AcO)
4
Pb, Me SiN
3
3
, CH
2
Cl
2
, rt, 3 h, 70%.
i
ii
iii
DHEA
HO
AcO
AcO
Br
9
10
11
iv
v
1
1
AcO
N3
2 (7α/β : 85/15)
HO
NH2
1
III (7α/β : 85/15)
Scheme 5. Synthesis of 7a,b-amino-3b-hydroxy-androst-5-enes (III). Reagents and conditions: (i) hydrazine hydrate, KOH, ethylene glycol, reflux,
2
4 h, 74%; (ii) Ac
2
O, pyridine, rt, 12 h, 88%; (iii) dibromantin, NaHCO
Æ6H O, THF, reflux, 45 min, 31%.
3
3
, n-hexane, 60 ꢁC, 45 min; (iv) NaN , DMF, rt, 18 h, 75% (85% 7a and 15%
7b); (v) LiAlH , NiCl
4
2
2

M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
3155
Table 1. Cell viability percentage after 24 h treatment with either DHEA or DHEA analogues (DAPI test for determination of cell apoptosis
percentage)
Cell viability % (mean ± SD)
Control
I
II
III
DHEA
35 lM
95 ± 5
90 ± 3
Dose
Leydig cells
TM4 Sertoli
Untreated (0 lM)
95 ± 5
90 ± 3
35 lM
92 ± 3
87 ± 5
70 lM
93 ± 4
86 ± 4
35 lM
92 ± 3
82 ± 3
70 lM
89 ± 5
79 ± 3*
35 lM
92 ± 3
88 ± 3
70 lM
94 ± 4
77 ± 1*
70 lM
95 ± 5
90 ± 3
*p < 0.05, compared to untreated cells.
Table 2. Cell viability percentage after 24 h treatment with either DHEA or DHEA analogues (Trypan blue test for determination of cell necrosis
percentage)
Cell viability % (mean ± SD)
Control
I
II
III
DHEA
70 lM
Dose
Leydig cells
TM4 Sertoli
Untreated
95 ± 5
92 ± 6
35 lM
85 ± 5
88 ± 8
70 lM
78 ± 6
18 ± 3***
35 lM
93 ± 1
64 ± 3**
70 lM
85 ± 4
0
35 lM
82 ± 2
43 ± 6**
70 lM
45 ± 6
0
35 lM
86 ± 6
92 ± 6
*
*
*
76 ± 4
92 ± 6
*p < 0.05, **p < 0.01 and ***p < 0.001, compared to untreated cells.
2
.2. Biology
These results indicate that viability of purified Leydig
cells was not affected via apoptosis or necrosis pro-
cess after 24 h treatment with DHEA analogues I
and II. But, 35 and 70 lM of DHEA analogues II
and III significantly increased the percentage of
necrotic cells.
DHEA analogues (I, II and III) were tested on two types
of testicular cells (rat Leydig cells and mouse TM4
immortalized Sertoli cell line). Leydig cells were isolated
from testis of mature Sprague–Dawley rats and charac-
terized as previously described. TM4 Sertoli cell line
derived from testis of immature BALB/c mice provides
an ideal system for the in vitro analysis of function
and responsiveness to biochemical and hormonal
4
1
No TM4 cells’ death induced by apoptosis after treat-
ment with 35 lM of DHEA analogues was observed.
With increasing dose of DHEA analogue I (70 lM),
no significant effect was observed. But with 70 lM of
DHEA analogues II and III, the percentage of apoptotic
cells was increased. In contrast, DHEA analogues in-
duced more death by necrosis. When cells were exposed
to 35 lM, II and III appeared to induce necrosis death
on TM4 Sertoli cells. Increasing concentration to
70 lM, a high and significant necrosis was obtained by
III or by II exposures, less by I.
4
2
factors.
The cytotoxicity was studied via two separated pro-
cesses: apoptosis or necrosis.
To assess drug-induced apoptotic cell death, apoptosis
was measured by morphological and quantitative evalu-
ation of DNA stained with 4 ,6-diamidino-2-phenylin-
dole (DAPI) by fluorescence microscopy.
0
Finally, these results were then compared to those of
DHEA. DHEA did not appear to induce either apopto-
sis or necrosis of Leydig or Sertoli cells (except for
necrosis induced by a dose of 70 lM in Leydig cells).
Necrosis process was determined using Trypan blue
exclusion assays.
To evaluate viability of cells treated or not by DHEA or
DHEA analogues, the percentage of viable and non-via-
ble cells was determined (Tables 1 and 2). Cells were
incubated for 24 h with or without DHEA and DHEA
analogues at two different concentrations (35 and
3. Conclusion
7-Amino-DHEA derivatives constitute new analogues
7
0 lM). Preliminary tests showed that lower doses did
of DHEA. 7a- and 7b-amino-3b-hydroxy-androst-5-
en-17-one and 7a,b-amino-3b-hydroxy-androst-5-enes
were successfully prepared as new analogues of DHEA.
The key step in the synthetic strategy involves an allylic
bromination to give an epimeric mixture of 7a,b-bro-
mosteroids which were substituted by an azide function.
not affect cell viability. Viability and total number of
cells were determined, and the percentage of cell viabil-
ity was calculated by the formula (Fig. 1).
3b-Acetoxy-7a,b-azido-17,17-ethylenedioxy-androst-5-
enes were easily separated by classical chromatography.
In contrast, the intermediate epimer mixture 3b-acetoxy-
7a,b-azido-androst-5-ene and 3b-acetoxy-7a,b-azido-
androst-5-en-17-one was inseparable in the same
number of viable cells
cell viability =
x 100
total number of control cells
Figure 1.
conditions. It seems that the presence of ketal was

3156
M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
1
probably essential for the success of azide epimers’ sep-
aration. The azides were then reduced to amines.
1740 (C@O ester), 1725 (C@O ketone). H NMR
(400 MHz, CDCl , 25 ꢁC): d = 0.89 (s, 3H, 18-Me),
.05 (s, 3H, 19-Me), 2.04 (s, 3H, 3-CH COO), 4.60 (m,
3
3
1
1H, 3-H), 5.41 (d, J
3
13
In this preliminary study, no cytotoxic effect induced by
apoptosis or necrosis mechanism was observed on puri-
fied Leydig cells by treatment with DHEA analogues
(except with the highest dose of III that induced necrosis
in Leydig cells).
= 5.0 Hz, 1H, 6-H) ppm.
-7b
C
6
NMR (100 MHz, CDCl , 25 ꢁC): d = 13.5 (C-18), 19.3
(C-19), 20.2, 21.3 (3-OCOCH ), 21.8, 27.6, 30.7, 31.3,
31.4, 35.7, 36.6, 36.8, 38.0, 47.4, 50.0, 51.6, 73.6 (C-3),
121.8 (C-6), 139.8 (C-5), 170.4 (3-OCOCH ), 220.9 (C-
3
3
3
1
7) ppm. MS (30 eV, EI): m/z (%) = 270 (100)
+
DHEA analogues’ exposure to TM4 cells induced necro-
sis but no apoptosis (except with the highest dose of II
and III that induced apoptosis). A significant effect
was obtained by III and II, less by I. These results
showed that 7b-epimer (II) has a more cytotoxic effect
than 7a-epimer (I) at a concentration value of 70 lM.
It is clear that these results showed structure depen-
dent-cytotoxic effects on purified Leydig and immortal-
ized TM4 Sertoli cells via necrosis process. Further
investigation in other cell lines and the assessement of
androgenic and other effects will be developed.
[M ꢀAcOH], 255 (46), 121 (75). Anal. Calcd for
C H O (330.47): C, 76.33; H, 9.15. Found: C, 76.22;
30
2
1
3
H, 9.06.
4.1.3. 3b-Acetoxy-17,17-ethylenedioxy-androst-5-ene (2).
p-Toluene sulfonic acid (0.029 g, 0.15 mmol) and ethyl-
ene glycol (25 mL, 454 mmol) were added to a solution
of compound 1 (10 g, 30 mmol) in toluene (120 mL) un-
der argon atmosphere. The solution was refluxed for 8 h
with azeotropic distillation of water. After cooling, the
reaction mixture was made alkaline with saturated aque-
ous NaHCO3 and extracted with ethyl acetate (4·
3
0 mL). The organic layer was washed with brine, dried
4
. Experimental
over anhydrous sodium sulfate and evaporated. Recrys-
tallization from a methanol/ethanol mixture (1:1) affor-
4
4
4
4
.1. Chemistry
ded 2 as a white powder (9.1 g, 80%). Mp 140 ꢁC (lit.
40–141 ꢁC); R = 0.38 (cyclohexane/ethyl acetate,
1
f
ꢀ
1
1
.1.1. General remarks. All solvents were distilled and
80:20). IR (KBr): m(cm ): 1732 (C@O ester).
H
dried prior to use. Reagents and materials were obtained
from commercial suppliers and were used without fur-
ther purification. The reactions were monitored by
TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm
thick). The spots were detected by spraying sulfuric
acid/ethanol (2:8) on TLC and heating. Column chro-
matography was carried out using silica gel 60 (0.063–
NMR (400 MHz, CDCl , 25 ꢁC): d = 0.86 (s, 3H, 18-
3
Me), 1.03 (s, 3H, 19-Me), 2.04 (s, 3H, 3-CH COO),
3.86–3.94 (m, 4H, –O(CH ) O–), 4.61 (m, 1H, 3-H),
3
2
2
3
5.38 (d, J6-7b = 4.9 Hz, 1H, 6-H) ppm. C NMR
13
(100 MHz, CDCl , 25 ꢁC): d = 14.2 (C-18), 19.3 (C-19),
3
20.4, 21.4 (3-OCOCH ), 22.7, 27.7, 30.5, 31.2, 32.0,
3
35.1, 36.5, 36.9, 38.0, 45.6, 49.8, 50.5, 64.5 and 65.1
(–O(CH ) O–), 73.8 (C-3), 119.4 (C-17), 122.4 (C-6),
139.5 (C-5), 170.5 (3-OCOCH ) ppm. MS (30 eV, EI):
0
.2 mm) (Merck). Melting points were determined on a
2
2
Kofler block and are uncorrected. IR spectra were
recorded on a Perkin-Elmer 1600 FT-IR spectrometer.
Specific rotation was measured in methanol with a
Perkin-Elmer 353 polarimeter. EI mass spectra were
recorded on a Jeol-GCmate (GC–MS system) spectrom-
eter with ionisation energy from 30 to 40 eV. H NMR
and C NMR spectra were recorded using CDCl or
CD OD, respectively, at 400 MHz (Jeol Lambda 400
3
3
+
m/z (%) = 314 (51) [M ꢀAcOH], 252 (95), 226 (59), 99
(100). Anal. Calcd for C H O (374.53): C, 73.76; H,
2
3
35
4
9.15. Found: C, 73.72; H, 9.53.
1
4.1.4. 3b-Acetoxy-7a,b-bromo-17,17-ethylenedioxy-and-
rost-5-ene (3). Sodium bicarbonate (6.1 g, 72 mmol) was
added to a solution of compound 2 (5 g, 13 mmol) and
1,3-dibromo-5,5-dimethylhydantoin (2.30 g, 8 mmol) in
n-hexane (50 mL). The mixture was refluxed for 40 min
under argon atmosphere. The solution was cooled, fil-
tered and evaporated. The crude product was used in next
1
3
3
spectrometer) and at 100 MHz. Chemical shifts are
reported relative to TMS; J values are given in Hertz.
1
3
1
C NMR spectra are H-decoupled. Elemental analyses
were performed at the ‘Institut de Recherche en Chimie
Organique Fine’ (Rouen).
1
step without purification. IR and H NMR spectra
have been carried out for characterization. IR (KBr)
ꢀ
1
4
.1.2. 3b-Acetoxy-androst-5-en-17-one (1). Acetic anhy-
m(cm ): 2971–2832 (C–H alkane), 1729 (C@O ester),
1
dride (26 mL, 277 mmol) was added dropwise to a solu-
tion of dehydroepiandrosterone (10 g, 35 mmol) in
pyridine (25 mL). The solution was stirred at room tem-
perature and under argon for 12 h. Ice water (20 mL)
was poured into the mixture. The white precipitate
formed was dissolved in CH Cl (200 mL). The organic
560 (C–Br). H NMR (CDCl , 400 MHz, 25 ꢁC),
d = 0.90 (s, 3H, 18-Me), 1.06 (s, 3H, 19-Me), 2.05 (s, 3H,
3
CH COO–), 3.82–3.97 (m, 4H, –O(CH ) O–), 4.60 (m,
3
2 2
3
1H, 3-H), 4.66 (dd, J7a-8 = 8.0 Hz, J7a-6 = 2.1 Hz,
3
3
0.4H, 7a-H of b epimer), 4.69 (dd, J7b-8 = 5.0 Hz,
7b-6
J6-7a = 2.1 Hz, 0.4H, 6-H of b epimer), 5.77 (d,
J6-7b = 5.2 Hz, 0.6H, 6-H of a epimer).
J
= 5.0 Hz, 0.6H, 7b-H of a epimer), 5.41(d,
2
2
3
layer was washed with 1 M HCl (3· 20 mL), 5% NaH-
3
CO (1· 30 mL), brine (1· 30 mL) and water (1·
3
3
0 mL), dried over anhydrous sodium sulfate and evap-
orated under reduced pressure. The crude product was
4.1.5. 3b-Acetoxy-7a-azido-17,17-ethylenedioxy-androst-
5-ene (4) and 3b-acetoxy-7b-azido-17,17-ethylenedioxy-
androst-5-ene (5). The crude product 3 was dissolved in
DMF (15 mL) and sodium azide (8.7 g, 133 mmol) was
recrystallized from acetone to give 1 (10.9 g, 95%) as a
4
3
white powder. Mp 172 ꢁC (lit. 169–171 ꢁC); R = 0.28
f
ꢀ
1
(
cyclohexane/ethyl acetate, 80:20). IR (KBr): m(cm ):

M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
3157
added to the mixture that was stirred for 18 h at room
temperature under argon atmosphere. The reaction mix-
ture was poured into water (50 mL) and extracted with
diethyl ether (4· 50 mL). The combined organic layers
were dried over anhydrous sodium sulfate and evapo-
rated. The crude product was purified by column chro-
matography (silica gel, cyclohexane/ethyl acetate, 98:2)
to give two separated epimers as white amorphous solids
(400 MHz, CDCl , 25 ꢁC): d = 0.88 (s, 3H, 18-Me),
3
3
1.01 (s, 3H, 19-Me), 3.09 (ddd,
J
= 4.8 Hz,
b-8
7
3
5
J7b-6 = 4.8 Hz, J7b-4 = 2.0 Hz, 1H, 7b-H), 3.55 (m,
1H, 3-H), 3.84–3.96 (m, 4H, –O(CH ) O–), 5.54 (dd,
2
2
3
4
J6-7b = 5.1 Hz, J6-4 = 1.5 Hz, 1H, 6-H) ppm.
13
C
NMR (100 MHz, CD OD, 25 ꢁC): d = 14.5 (C-18),
3
19.2 (C-19), 21.2, 23.7, 31.3, 32.1, 35.0, 38.3, 38.4,
38.6, 42.9, 43.0, 45.1, 46.8, 47.7, 65.6 and 66.2
(–O(CH ) O–), 72.1 (C-3), 120.4 (C-17), 125.6 (C-6),
7
a (16%) and 7b (29%).
2
2
145.5 (C-5) ppm. MS (30 eV, EI): m/z (%) = 357 (2)
[M ], 235 (5), 100 (84), 85 (100). HRMS-EI (20 eV):
+
4
.1.5.1. Compound 4. R = 0.35 (cyclohexane/ethyl
f
+
m/z [M ] Calcd for C H NO : 347.2460. Found:
347.2464.
acetate, 80:20). [a] ꢀ86ꢁ (c 0.1 in methanol). IR
D
21 33
3
ꢀ1
1
(
KBr): m(cm ): 2098 (N ), 1726 (C@O ester).
H
3
NMR (400 MHz, CDCl , 25 ꢁC): d = 0.85 (s, 3H, 18-
3
Me), 1.04 (s, 3H, 19-Me), 2.05 (s, 3H, 3-CH COO),
4.1.7. 7b-Amino-17,17-ethylenedioxy-androst-5-en-3b-ol
(7). Azide 5 (0.8 g, 1.93 mmol) was reduced as described
for compound 6 to yield 7 (0.47 g, 70%) as beige crystals.
3
3
4
.60 (m, 1H, 7b-H), 3.85–3.95 (m, 4H, –O(CH ) O–),
2 2
3
.67 (m, 1H, 3-H), 5.37 (d, J
ppm. C NMR (100 MHz, CDCl , 25 ꢁC): d = 14.3
C-18), 19.2 (C-19), 20.5, 21.4 (3-OCOCH ), 24.1, 27.6,
9.9, 35.2, 36.4, 36.6, 37.0, 37.6, 46.2, 48.5, 49.8, 63.9,
4.5 and 65.3 (–O(CH ) O–), 73.1 (C-3), 118.8 (C-17),
21.2 (C-6), 145.2 (C-5), 170.5 (3-OCOCH ) ppm. MS
3
= 4.8 Hz, 1H, 6-H)
-7b
6
1
3
Mp 176 ꢁC; R = 0.20 (dichloromethane/methanol, 90:10
3
f
(
then 80:20). [a] +189ꢁ (c 0.1 in methanol). IR (KBr):
3
D
ꢀ
1
1
2
6
1
m(cm ): 3386 and 3310 (O–H and N–H). H NMR
(400 MHz, CDCl , 25 ꢁC): d = 0.84 (s, 3H, 18-Me),
2
2
3
3
0.98 (s, 3H, 19-Me), 3.09 (d, J
H), 3.47 (m, 1H, 3-H), 3.80–3.92 (m, 4H, –O(CH ) O–),
= 8.0 Hz, 1H, 7a-
7a-8
+
Å
(
30 eV, EI): m/z (%) = 373 (15) [M ꢀN ], 314 (37),
3
55 (51), 226 (27), 99 (100). HRMS-EI (20 eV):
2 2
1
3
2
m/z [M ] Calcd for C H N O : 415.2471. Found:
5.11 (s, 1H, 6-H) ppm. C NMR (100 MHz, CDCl3,
25 ꢁC): d = 14.3 (C-18), 19.3 (C-19), 20.5, 25.2, 30.1,
31.5, 35.2, 36.1, 37.0, 41.8, 42.0, 46.3, 49.0, 50.2, 53.8,
+
2
3
33
3
4
4
15.2455.
64.4 and 65.1 (–O(CH ) O–), 70.9 (C-3), 118.6 (C-17),
126.4 (C-6), 142.1 (C-5) ppm. MS (40 eV, EI): m/z
2 2
4
.1.5.2. Compound 5. R = 0.32 (cyclohexane/ethyl
f
+
(%) = 357 (14) [M ], 315 (11), 235 (24), 99 (100).
acetate, 80:20). [a] +86ꢁ (c 0.1 in methanol). IR
D
ꢀ1
1
+
HRMS-EI (20 eV): m/z [M ] Calcd for C H NO :
347.2460. Found: 347.2449.
(
KBr): m(cm ): 2100 (N ), 1732 (C@O ester).
H
3
21 33
3
NMR (400 MHz, CDCl , 25 ꢁC): d = 0.89 (s, 3H, 18-
3
Me), 1.07 (s, 3H, 19-Me), 2.05 (s, 3H, 3-CH COO),
3
3
4
.40 (m, 1H, 7a-H), 3.83–3.96 (m, 4H, –O(CH ) O–),
4.1.8. 7a-Amino-3b-hydroxy-androst-5-en-17-one (I). Five
percent aqueous HCl solution (5 mL) was added to a solu-
tion of compound 6 (0.05 g, 0.13 mmol) in THF (5 mL).
The mixture was stirred for 24 h at room temperature.
The solution was cooled to 0 ꢁC and treated with 10%
2
2
1
.61 (m, 1H, 3-H), 5.35 (s, 1H, 6-H) ppm. C NMR
3
(
2
100 MHz, CDCl , 25 ꢁC): d = 14.3 (C-18), 19.2 (C-19),
3
0.5, 21.3 (3-OCOCH ), 24.1, 27.6, 30.0, 35.2, 36.5,
3
3
(
6.6, 37.0, 37.6, 46.2, 48.4, 49.8, 63.8, 64.5 and 65.2
–O(CH ) O–), 73.1 (C-3), 118.8 (C-17), 121.2 (C-6),
NaHCO (10 mL) and extracted with chloroform (4·
2
2
3
1
45.2 (C-5), 170.4 (3-OCOCH ) ppm. MS (30 eV, EI):
3
10 mL). The organic layer was washed with brine, dried
over anhydrous sodium sulfate and evaporated. The
crude product was purified by chromatography (silica
gel, dichloromethane/methanol/triethylamine, 80:18:2)
to afford 0.048 g (95%) of I as an amorphous solid.
+
Å
+
Å
m/z (%) = 373 (15) [M ꢀN ], 372 (30) [M ꢀCH CO ],
3
14 (24), 255 (100), 240 (63), 99 (86). ESI-MS:
3
3
m/z = 438.2 [M+Na] .
+
4
(
.1.6. 7a-Amino-17,17-ethylenedioxy-androst-5-en-3b-ol
6). Azide 4 (0.40 g, 0.96 mmol) and nickel(II) chloride
R = 0.21 (dichloromethane/methanol, 80:20). [a]D
f
ꢀ
1
ꢀ193ꢁ (c 0.1 in methanol). IR (KBr): v(cm ): 3380 and
1
hexahydrate (0.23 g, 0.96 mmol) were dissolved in
anhydrous THF (40 mL) under argon atmosphere. The
solution was stirred at room temperature for 30 min.
Lithium aluminium hydride (0.37 g, 9.6 mmol) was
then added to the solution and the mixture was refluxed
for 6 h, cooled and quenched by dropwise addition of
water (20 mL). The product was diluted with 2 M
aqueous sodium hydroxide solution (100 mL) and
extracted with dichloromethane (4· 50 mL). The
combined dichloromethane extracts were washed with
water (2· 30 mL), dried over calcium chloride and
magnesium sulfate and filtered. Removal of the solvent
in vacuo and purification by chromatography (silica
gel, dichloromethane/methanol, 90:10 then 80:20)
afforded a pure product 6 (0.13 g, 39%) as a beige solid.
3154 (O–H and N–H), 1738 (C@O ketone). H NMR
(400 MHz, CDCl , 25 ꢁC): d = 0.89 (s, 3H, 18-Me), 1.02
3
(s, 3H, 19-Me), 3.19 (s, 1H, 7b-H), 3.55 (m, 1H, 3-H),
5.54 (d, J6-7b = 5.4 Hz, 1H, 6-H) ppm. C NMR
3
13
(100 MHz, CDCl , 25 ꢁC): d = 13.3 (C-18), 18.6 (C-19),
3
20.0, 22.1, 31.1, 31.4, 35.6, 36.6, 37.2, 37.5, 42.0, 42.1,
45.4, 46.0, 47.1, 71.3 (C-3), 125.3 (C-6), 142.8 (C-5),
220.4 (C-17) ppm. MS (30 eV, EI): m/z (%) = 303 (57)
+
[M ], 286 (57), 150 (80), 83 (100). HRMS-EI (20 eV): m/
z [M ] Calcd for C H NO : 303.2198. Found: 303.2185.
+
1
9
29
2
4.1.9. 7b-Amino-3b-hydroxy-androst-5-en-17-one (II).
Compound 7 (0.35 g, 1 mmol) was deprotected in the
same manner as 6 to yield product II (0.28 g, 92%) as
an amorphous solid. R = 0.20 (dichloromethane/metha-
f
Mp > 260 ꢁC; R = 0.22 (dichloromethane/methanol,
nol, 80:20). [a] +191ꢁ (c 0.1 in methanol). IR (KBr):
f
D
ꢀ
1
9
0:10). [a] ꢀ193ꢁ (c 0.1 in methanol). IR (KBr):
D
m(cm ): 3385 and 3256 (O–H and N–H), 1742 (C@O
ꢀ1
1
1
m(cm ): 3386 and 3295 (O–H and N–H). H NMR
ketone). H NMR (400 MHz, CDCl , 25 ꢁC): d = 0.84
3

3158
M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
ꢀ
1
1
(
3
(
2
4
2
s, 3H, 18-Me), 1.00 (s, 3H, 19-Me), 3.20 (m, 1H, 7a-H),
70:30). IR (KBr): m(cm ): 1732 (C@O ester). H NMR
1
.46 (m, 1H, 3-H), 5.16 (s, 1H, 6-H) ppm. C NMR
3
(400 MHz, CDCl , 25 ꢁC): d = 0.72 (s, 3H, 18-Me), 1.03
(s, 3H, 19-Me), 2.04 (s, 3H, 3-CH COO), 4.60 (m, 1H, 3-
3
100 MHz, CDCl , 25 ꢁC): d = 13.6 (C-18), 19.1 (C-19),
3
3
3
H), 5.38 (d, J
13
0.5, 20.9, 24.0, 29.6, 31.2, 31.2, 35.8, 36.3, 36.9, 41.6,
8.0, 51.3, 60.4, 70.8 (C-3), 125.3 (C-5), 142.8 (C-6),
20.4 (C-17) ppm. MS (30 eV, EI): m/z (%) = 303 (17)
= 4.9 Hz, 1H, 6-H) ppm. C NMR
-7b
6
(100 MHz, CDCl , 25 ꢁC): d = 17.2 (C-18), 19.3 (C-19),
3
20.5, 21.1 (3-OCOCH ), 21.4, 25.6, 27.8, 32.1, 32.2,
36.7, 37.1, 38.1, 38.6, 40.2, 40.6, 50.3, 54.8, 73.9 (C-3),
122.6 (C-6), 139.6 (C-5), 170.5 (3-OCOCH ) ppm. MS
3
+
[M ], 270 (31), 99 (85), 83 (100). HRMS-EI (20 eV):
m/z [M ] Calcd for C H NO : 303.2198. Found:
3
+
1
9
29
2
3
+
(30 eV, EI): m/z (%) = 316 (1) [M ], 256 (100)
03.2213.
+
[
M ꢀAcOH], 241 (16), 148 (23), 135 (21). Anal. Calcd
4
.1.10. 3b-Acetoxy-7a,b-azido-androst-5-en-17-one (8).
for C H O (316.49): C, 79.70; H, 10.19. Found: C,
21 32 2
Trimethylsilyl azide (8.0 mL, 60.5 mmol) was added
dropwise to a solution of 1 (2 g, 6.1 mmol) and lead(IV)
acetate (5.37 g, 12.1 mmol) in methylene chloride
79.98; H, 10.33.
4.1.13. 3b-Acetoxy-7a,b-azido-androst-5-ene (12). Com-
pound 12 was obtained from compound 10 (0.5 g,
1.6 mmol) via 11 as described for the preparation of
compounds 4 and 5 to yield (0.42 g, 75%) azide epimers
mixture (7a/b: 15/85 ratio, respectively) inseparable by
chromatography (silica gel, cyclohexane/ethyl acetate,
(
20 mL). The mixture was stirred for 2 h at room tem-
perature. The solution was diluted with water and the
lead(II) azide precipitate was removed by filtration and
decomposed with sodium nitrite/dilute hydrochloric
acid. The organic layer was dried over sodium sulfate
and evaporated. The crude product was purified by
chromatography (cyclohexane/ethyl acetate, 8:2) to give
95:5) as a colourless oil. R = 0.60 (cyclohexane/ethyl
f
ꢀ
1
acetate, 70:30). IR (KBr): m(cm ): 2094 (N ), 1735
3
1
1
.6 g (70% of epimeric mixture: a epimer 70% and b epi-
mer 30%) of 8 as a white amorphous solid. IR: m: 2095
(C@O ester). H NMR (400 MHz, CDCl , 25 ꢁC):
3
d = 0.71 (s, 3H, 18-Me), 1.08 (s, 3H, 19-Me), 2.04 (s,
1
3
3H, 3-CH COO), 3.37 (d, J
(
(
N ), 1738 (C@O ester), 1725 (C@O ketone). H NMR
= 9.0 Hz, 0.85H, 7a-
3
3
7a-8
400 MHz, CDCl , 25 ꢁC): d = 0.86 (s, 3H, 18-Me),
.02 (s, 3H, 19-Me), 2.16 (s, 3H, 3-CH COO), 3.59 (d,
J7a-8 = 9.4 Hz, 0.3H, 7a-H of azide b epimer), 3.73
H), 3.61 (m, 0.15H, 7b-H), 4.61 (m, 1H, 3-H), 5.36
(s, 0.85H, 6-H of azide b epimer), 5.57 (d,
J6-7b = 5.1 Hz, 0.15H, 6-H of azide a epimer) ppm.
3
3
1
3
3
13
C
(
5
m, 0.7H, 7b-H of azide a epimer), 4.65 (m, 1H, 3-H),
NMR (100 MHz, CDCl , 25 ꢁC): d = 16.9, 17.3, 18.5,
3
3
.35 (s, 0.3H, 6-H of azide b epimer), 5.55 (dd, J
.1 Hz and J = 1.4 Hz, 0.7H, 6-H of azide a epimer)
=
19.2 (C-18 and C-19), 20.5, 20.8, 21.1 and 21.3
(CH COO), 25.3, 25.9, 26.8, 27.1, 27.4, 27.6, 30.1,
30.4, 32.8, 33.2, 36.6, 36.7, 37.0, 37.2, 37.5, 37.6, 37.9,
6
-7b
3
5
ppm.
6
-4
3
3
64.2, 73.1 and 73.6 (C-3), 119.1 and 121.3 (C-6), 145.2
8.3, 40.4, 41.1, 48.6 and 48.8 (C-7), 53.9, 58.5, 64.1,
4
(
.1.11. Androst-5-en-3b-ol (9). Hydrazine hydrate
2.16 mL, 69 mmol) was added to a solution of dehydro-
epiandrosterone (2 g, 7 mmol) in ethylene glycol
20 mL) and heated to 100 ꢁC for 2 h. Potassium
and 147.3 (C-5), 170.3 and 170.4 (3-CH COO) ppm.
3
+
MS (30 eV, EI): m/z (%) = 269 (9) [M ꢀAcOH, –N ],
2
+
Å
(
255 (56) [M ꢀAcOH, –N ], 186 (7), 159 (18), 133
3
(36), 108 (85), 95 (67), 82 (100). ESI-MS: m/z = 480.4
hydroxide (4.2 g, 75 mmol) was added to the stirred
solution and stirring was continued with no external
heating over 24 h. The solution was cooled to 0 ꢁC.
The precipitate was filtered and dissolved in chloroform
+
[M+Na] .
4.1.14. 7a,b-Amino-androst-5-en-3b-ol (III). Epimeric
azide mixture 12 (0.38 g, 1.1 mmol) was reduced in the
same manner as compound 6 to yield III (0.12 g, 31%)
after purification by chromatography (silica gel, dichlo-
romethane/methanol, 90:10) as an amorphous solid.
(
(
30 mL). The organic phase was washed with 1 M HCl
3· 15 mL), 10% NaHCO (1· 15 mL), brine and dried
3
over calcium chloride and magnesium sulfate. The solu-
tion was evaporated and the crude product was purified
by chromatography (silica gel, cyclohexane/ethyl ace-
tate, 80:20) to afford compound 9 as a white solid
R = 0.19 (dichloromethane/methanol, 90:10). IR
f
1
(KBr): m: 3362 and 3280 (O–H and N–H). H NMR
4
5
(
(
1.39 g, 74%). Mp 136 ꢁC (lit. 133–134 ꢁC); R = 0.23
(400 MHz, CDCl , 25 ꢁC): d = 0.73 (s, 3H, 18-Me),
f
3
ꢀ
1
3
cyclohexane/ethyl acetate, 70:30). IR (KBr): m(cm ):
1.03 (s, 3H, 19-Me), 3.01 (ddd,
J
= 7.8 Hz,
a-8
7
1
3
3
5
J7a-6 = 2.1 Hz, J
J7b-8 = 5.1 Hz, J
3
246 (O–H). H NMR (400 MHz, CDCl , 25 ꢁC):
< 1 Hz, 0.55H, 7a-H), 3.10 (ddd,
7a-4
5
7b-6
3
3
d = 0.72 (s, 3H, 18-Me), 1.02 (s, 3H, 19-Me), 3.53 (m,
1
= 5.0 Hz, J
= 1.5 Hz, 0.45H,
7b-4
3
H, 3-H), 5.36 (d, J
13
3
= 6.6 Hz, 1H, 6-H) ppm.
C
7b-H), 3.53 (m, 1H, 3-H), 5.18 (dd, J6-7a = 2.1 Hz,
J6-4 < 1 Hz, 0.55H, 6-H of amine b epimer), 5.52 (dd,
6
-7b
4
NMR (100 MHz, CDCl , 25 ꢁC): d = 17.2 (C-18), 19.4
3
3
4
(
C-19), 20.5, 21.1, 25.6, 31.7, 32.1, 32.1, 36.6, 37.3,
J6-7b = 5.0 Hz, J < 1 Hz, 0.45H, 6-H of amine a epi-
6-4
13
3
6
8.7, 40.3, 40.6, 42.3, 50.4, 54.8, 71.8 (C-3), 121.7 (C-
), 140.7 (C-5) ppm. MS (30 eV, EI): m/z (%) = 274
mer) ppm.
C NMR (100 MHz, CDCl , 25 ꢁC):
3
d = 17.0, 17.4, 18.7, 19.4 (C-18 and C-19), 20.5, 20.8,
25.9, 27.1, 27.6, 27.9, 30.1, 30.6, 31.4, 31.7, 36.3, 37.2,
37.4, 37.5, 38.2, 38.7, 39.7, 40.1, 40.5, 41.4, 42.0, 42.1,
42.2, 42.5, 47.4 and 48.1 (C-7), 54.1, 54.4, 71.3 and
+
(100) [M ], 256 (63), 241 (74), 189 (64), 163 (65). Anal.
Calcd for C H O (274.45): C, 83.15; H, 11.02. Found:
C, 83.37; H, 11.16.
1
9
30
7
1.4 (C-3), 125.9 and 126.9 (C-6), 141.9 and 143.1 (C-
5) ppm. MS (30 eV, EI): m/z (%) = 289 (48) [M ], 272
+
4
.1.12. 3b-Acetoxy-androst-5-ene (10). Using the same
method described for compound 1, compound 9 (1.3 g,
.7 mmol) was converted to 10 (1.14 g, 88%) as a white
powder. Mp 98 ꢁC; R = 0.64 (cyclohexane/ethyl acetate,
+
(27) [M ꢀNH ], 177 (100), 136 (38). HRMS-EI
3
+
(20 eV): m/z [M ] Calcd for C H NO: 289.2406.
Found: 289.2402.
4
1
9
31
f

M.-A. Bazin et al. / Bioorg. Med. Chem. 15 (2007) 3152–3160
3159
4
4
.2. Biology
4.2.6. Statistical analysis. Data are presented as
means ± SD of triplicate determinations performed in
three different experiments. Student’s t-test was used to
compare the effects of DHEA or DHEA analogues on
viability of Leydig cells or TM4 Sertoli cell line. Signif-
.2.1. Animals. Seventy-day-old male Sprague–Dawley
rats, obtained from our own colony, were maintained
on a 12 h light/dark cycle at 20–22 ꢁC. Food and water
were provided ad libitum.
*
**
icance was assumed at p < 0.05 ( ) p < 0.01 ( ) or
**
*
p < 0.001 ( ).
4
.2.2. Isolation and purification of Leydig cells. Rats were
killed and the testes removed, decapsuled and placed in
DMEM/Ham F12 medium (1:1; v/v). The crude intersti-
tial cells were isolated following a collagenase/dispase
Acknowledgment
Dr. R e´ mi Legay is gratefully acknowledged for his assis-
tance in measurement of mass spectra.
(
0.05%) treatment in the presence of soybean trypsin
inhibitor (STI) (0.005%) and DNase (0.001%). The
interstitial cells were separated from the seminiferous
tubular pellet by filtration through a nylon mesh. The
Leydig cells were purified on discontinuous (20–80%)
References and notes
4
1
Percoll gradients as described previously. Leydig cell
fractions were collected, washed with medium and their
purity was analyzed by histochemical staining for the
specific 3b-hydroxysteroid dehydrogenase (3b-HSD po-
1
2
3
. Endoh, A.; Kristiansen, S. B.; Casson, P. R.; Buster, J. E.;
Hornsby, P. J. J. Clin. Endocrinol. Metab. 1996, 81, 3558.
. Dehennin, L.; Ferry, M.; Lafarge, P.; P e´ r e` s, G.; Lafarge,
J. P. Steroids 1998, 63, 80.
4
6
sitive cells). Leydig cells’ viability was evaluated by
Trypan blue test. Purity and cell viability were, respec-
tively, around 90% and 95%.
. Baulieu, E. E. J. Clin. Endocrinol. Metab. 1996, 81, 3147.
4. Parish, E. J.; Li, S. R.; Rao, Z. G. Lipids 2000, 35, 271.
5. Kawano, H.; Yasue, H.; Kitagawa, A.; Hiral, N.; Yoshi-
ada, T.; Soejima, H.; Miyamoto, S.; Nakano, M.; Ogu-
awa, H. J. Clin. Endocrinol. Metab. 2003, 88, 3190.
6. Fukui, M.; Kitagawa, Y.; Nakamura, N.; Kadono, M.;
Yoshida, M.; Hirata, C.; Wada, K.; Hasengawa, G.;
Yoshikawa, T. Atherosclerosis 2005, 181, 339.
4
.2.3. Treatment of Leydig cells. Cells were incubated
5
for 24 h at a density of 3 · 10 cells/mL of medium under
an atmosphere of 95% air-5% CO . DHEA and DHEA
2
analogues were dissolved in ethanol before being added
to cell culture media. Then, the Leydig cells were treated
during 24 h with 0, 35 and 70 lM of DHEA or DHEA
analogues (0.007% ethanol in culture media).
7
. Feldman, H. A.; Johannes, C. B.; Araujo, A. B.; Mohr, B.
A.; Longcope, C.; McKinlay, J. B. Am. J. Epidemiol. 2001,
1
53, 79.
8
9
. Dean, C. E. Ann. Pharmacother. 2000, 34, 1419.
. Morales, A. J.; Nolan, J. J.; Nelson, J. C.; Yen, S. S. C.
J. Clin. Endocrinol. Metab. 1994, 78, 1360.
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3
1
(
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