Spironolactone-related inhibitors of type II 17β-hydroxysteroid dehydrogenase: Chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities

Article abstract of DOI:10.1016/S0968-0896(98)00260-0

The family of 17β-hydroxysteroid dehydrogenases (17β-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E₂), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17β-HSD enzymes, type II catalyzes the oxidation of E₂ into estrone (E₁), T into androstenedione, DHT into androstanedione, and 20α-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17β-HSD. In this context, we have efficiently synthesized a series of 7α-thioalkyl and 7α-thioaryl derivatives of spironolactone that inhibit type II 17β-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-γ-lactone and a bulky side-chain at the 7α-position. It was found that a para-substituted benzylthio group at the 7α-position enhances the inhibitory potency of spironolactone derivatives on type II 17β-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC₅₀ value of 0.5μM against type II 17β-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC₅₀ value of 0.7μM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17β-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00260-0

Bioorganic & Medicinal Chemistry 7 (1999) 1013±1023
Spironolactone-related Inhibitors of Type II 17ꢀ-Hydroxysteroid
Dehydrogenase: Chemical Synthesis, Receptor Binding Anities,
and Proliferative/Antiproliferative Activities
Martin R. Tremblay, Van Luu-The, Gilles Leblanc, Patricia Noel, Esther Breton,
Fernand Labrie and Donald Poirier*
Medicinal Chemistry Division, Laboratory of Molecular Endocrinology, Laval University Medical Research Center,
2705 Laurier Boulevard, Quebec G1V 4G2, Canada
Received 23 June 1998; accepted 2 October 1998
AbstractÐThe family of 17b-hydroxysteroid dehydrogenases (17b-HSDs) catalyzes the formation and inactivation of testosterone
(T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target
tissues. Among the ®ve known 17b-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione,
DHT into androstanedione, and 20a-dihydroprogesterone into progesterone. Speci®c inhibitors are thus an interesting means to
study the regulation and to probe the structure of type II 17b-HSD. In this context, we have eciently synthesized a series of 7a-
thioalkyl and 7a-thioaryl derivatives of spironolactone that inhibit type II 17b-HSD. These new C19-steroidal inhibitors possess
two important pharmacophores, namely 17-spiro-g-lactone and a bulky side-chain at the 7a-position. It was found that a para-
substituted benzylthio group at the 7a-position enhances the inhibitory potency of spironolactone derivatives on type II 17b-HSD.
In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 mM against type II 17b-HSD, whereas the
compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 mM. The latter
inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any
anity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative e€ect on
androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These ®ndings contribute to a better knowledge of the
structure of type II 17b-HSD and o€er an interesting tool to study the regulation of this enzyme in several biological systems.
# 1999 Elsevier Science Ltd. All rights reserved.
Introduction
mainly focused on the ®rst two types. Type I is the best
known 17b-HSD: its role and its structure have been
elucidated. It has a high substrate anity for estrone
(E1), a C18 steroid, and its preferred reaction is reduc-
tion using NADPH as cofactor. Clearly, the cytosolic
type I 17b-HSD is involved in estradiol (E2) biosynth-
esis from several E2-producing tissues, including nor-
mal and neoplastic breast.15,16 The 3D structures of the
type I enzyme alone as well as the complexed enzyme
Over the last decade, the involvement of steroid hor-
mones in the development and proliferation of hormone-
sensitive tumors (e.g. breast and prostate) has been
clearly demonstrated.1,2 Steroid receptors and steroido-
genic enzymes are thus attractive targets for drugs
designed to treat hormone-sensitive diseases.3,4 Among
the steroidogenic enzymes, the 17b-hydroxysteroid dehy-
drogenase (17b-HSD) family exerts an important role in
the regulation of active hormone levels in extraglandular
tissues (Fig. 1).5,6 These peripheral tissues contribute to a
large proportion of steroid hormone formation from the
adrenal precursor dehydroepiandrosterone (DHEA) and
its conjugated sulfate (DHEAS).7
with E2 and cofactor NAD+ were reported.17±19
A
consensus sequence Tyr-X-X-X-Lys was clearly identi-
®ed as a key feature of the active site. During the past
few years, ®rst and second generation inhibitors against
type I 17b-HSD have been reported by our group and
others.20±25
Although ®ve types of 17b-HSD cDNAs and genes have
The presence of a microsomal enzyme, named type II
17b-HSD, was ®rst revealed by Blomquist and co-
workers.26 After isolation and characterization, it was
shown that type II 17b-HSD is a protein of 387 amino
acids containing an amino-terminal signal-anchor motif
and a carboxy-terminal endoplasmic reticulum retention
been cloned from human tissues,6,8±14 our group has
Key words: Steroid derivatives; inhibitors; dehydrogenases; steroid-
ogenesis; hormones.
*Corresponding author. Tel.: (418) 654-2296; fax: (418) 654-2761.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(98)00260-0

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M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
Figure 1. Peripheral biosynthesis of active estrogens and androgens from the adrenal precursor dehydroepiandrosterone (DHEA).
motif. The type II cDNA sequence showed only 20%
homology with type I 17b-HSD.11 Although the 3D
structure of cytosolic type I 17b-HSD has been eluci-
dated, the structure of microsomal type II remains
unknown. Therefore, synthetic inhibitors should help to
probe the active site as well as allosteric sites of type II
17b-HSD, and could possibly clarify the role of type II
17b-HSD, which is not fully understood. Type II 17b-
HSD catalyzes the interconversion of testosterone (T)
and androstenedione (Á4-dione), dihydrotestosterone
(DHT) and androstanedione as well as E2 and E1.11,27
This isoenzyme also catalyzes the interconversion of
20a-dihydroprogesterone and progesterone to a lower
extent. Experiments with cellular models revealed that
the type II prefers an oxidative process using NAD+ as
cofactor.27 The in vitro kinetic data showed that the
substrate binding anity is between 0.2 and 0.4 mM for
all of the reduced substrates reported above.11 Type II
17b-HSD is expressed mainly in the placenta, liver,
small intestine, endometrium, kidney, pancreas, and
colon.12 It has also been reported that benign and
malignant prostate tissue as well as human meningioma
tumors possess signi®cant levels of type II 17b-HSD
activity.28,29 These data suggest that type II 17b-HSD
inactivates the reduced steroidal hormone by oxidation
in peripheral tissues. In classical endocrine tissues such
as endometrium and placenta, it has been hypothesized
that the type II isoenzyme together with the type I may
control the intracellular ratio of active C18 and C19
steroid hormones during pregnancy.30,31 Moreover, the
20a-HSD activity may provide high progesterone levels
during pregnancy. Our speci®c inhibitors of both type I
and type II 17b-HSDs have been used to further inves-
tigate this ®ne regulation and to clarify the role of type
II 17b-HSD.27,32 Thus, inhibitors of type II 17b-HSD
are useful tools to elucidate the role of this enzyme in
particular biological systems and they are also helpful
for improving our knowledge of its 3-D structure. In
this view, we are interested in developing various kinds
of steroidal inhibitors of type II 17b-HSD.
Recently, we reported a spiro-g-lactone derivative of E2
(compound 1) as the ®rst C18-steroidal inhibitor of type
II 17b-HSD (Fig. 2).33 Concurrently, we also observed
that spironolactone (2) was the most potent inhibitor of
type II 17b-HSD among a series of tested C19-ster-
oids.34 They both possess the spiro-g-lactone that is now
established as an important pharmacophore for the
inhibition of type II 17b-HSD. Moreover, the 7a-thioa-
cetyl group was shown to enhance the inhibitory
Figure 2. Chemical structure of the reported C18-steroidal inhibitor of
type II 17b-HSD (1) and the lead compound, spironolactone (2). The
strategy for lead optimization is the substitution of position 7ꢁ.

M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
1015
potency of C19-steroid derivatives bearing a spiro-g-
lactone.34 Thus, we wanted to develop a potent C19-
steroid inhibitor of type II 17b-HSD to modify the
endocrine pro®le of our ®rst estrogen-like inhibitor
(compound 1) because the latter may have limited uses
due to its estrogenic activity. Therefore, position 7a of
spironolactone was substituted with various thioalkyl
and thioaryl side chains. Most importantly, a 2-(1-
piperidinyl)-ethoxybenzyl group was introduced at the
position 7a to assess the ability of type II 17b-HSD to
tolerate this pharmacophore. It was shown that closely
related chemical groups provide interesting properties
to both steroidal and non steroidal derivatives toward
estrogen receptors.35±38 Herein, we report the synthesis
and biological activities of novel spironolactone deriva-
tives that represent another class of type II 17b-HSD
inhibitors.
Subsequent reduction of the aldehydes using sodium
borohydride and thioacetylation according to the Mit-
sunobu procedure43 yielded compounds 15 and 16.
Reduction of the thioacetyl group by lithium aluminum
hydride gave the thiols 17 and 18. This synthetic
sequence allowed us to generate a gram-scale of both 17
and 18 in a short period of time. The eciency of this
sequence was necessary because of the special need of
the subsequent 1,6-conjugate addition (Scheme 1). In
fact, the yield of this addition was unsatisfactory
(<20%) when canrenone (3) was solubilized in 1,4-
dioxane with only 3±10 equivalents of thiol 17. How-
ever, target product 10 was obtained in a substantially
improved yield when the two components were mixed
neat with a catalytic amount of metallic sodium. After-
ward, the cleavage of the TBDMS group of compound
10 using TBAF at a low temperature ( 78 ꢀC) gave the
phenol 11. It is noteworthy that the ¯uoride ion is basic
enough to undergo the retro-Michael elimination when
the reaction is carried out at higher temperatures (0 ꢀC
and 25 ꢀC). Direct O-alkylation of 11 with 1-(2-chloro-
ethyl)-piperidine hydrochloride required drastic condi-
tions giving mainly the retro-Michael elimination. We
thus alternatively used with success the same procedure
as for the synthesis of 10 to produce compound 12 (vide
infra).
Chemistry
A simple and straightforward synthesis was used to
generate the 7a-analogues of spironolactone (Scheme 1).
First, the thioacetyl group of spironolactone (2) was
removed by a retro-Michael elimination to yield canre-
none (3). Then, a sodium-mediated 1,6-conjugate addi-
tion of the appropriate thioalkyl side chain was
performed following the conditions described by
Brueggemeier and co-workers.39 For the synthesis of
compounds 4±6 and 8±9, canrenone was simply solubi-
lized in commercially available thioalkyls or thioaryls
with metallic sodium (1 equiv). Thiobenzoyl 7 was
prepared by adding thiobenzoic acid to canrenone using
a standard procedure.40±42 Compounds 10±12 were
synthesized by convergent approaches using synthetic
thiols 17 and 18 (Scheme 2). The starting material
4-hydroxybenzaldehyde was protected as a t-butyl-
dimethylsilyl ether or O-alkylated with 1-(2-chloroethyl)-
piperidine hydrochloride according to standard pro-
cedures to yield compounds 13 and 14, respectively.
The synthesis of nine analogues of spironolactone was
achieved by a short-step and ecient sequence. The
1,6-conjugate addition proceeded smoothly without
a€ecting other functionalities on the key intermediate 3.
Indeed, the formation of the thioether bond is regio-
and stereoselective. The regiospeci®city could be attrib-
uted to the soft character of both the sulfur atom and
the carbon atom at position 7, which is the most elec-
tropositive carbon. The stereoselectivity is mainly due to
the steric hindrance provoked by 19-CH3 and 18-CH3
on the b face of the substrate. The assignment of the
position as well as the con®guration of the alkylthio
substituents was made using NMR spectroscopy.
Scheme 1. Reagents and conditions: (a) NaOMe, THF, rt (79%); (b) Na(s), commercial or synthetic thiols (neat), 60 ꢀC (49±93%); (c) TBAF, THF,
78 ꢀC (88%).

1016
M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
Scheme 2. Reagents and conditions: (a) TBDMS±Cl, imidazole, DMF, rt (83%); (b) K2CO3, ClCH2CH2N(CH2)5, DMF; (c) NaBH4, MeOH, 0 ꢀC;
(d) DEAD, PPh3, CH3COSH, THF, 0 ꢀC (63±82%, two steps); (e) LiAlH4, THF, 0 ꢀC (43±76%).
Heteronuclear shift correlation (HSC) experiments
made on compound 10 allowed us to clearly identify 7-H
signal. Afterward, the 8b-H and 6b-H signals were
localized by combination of HSC and nuclear Over-
hauser e€ect (nOe) experiments. The absence of axial±
axial coupling constant (Jꢁ10 Hz) between all these
protons was easily revealed by J-resolve experiments
and con®rmed that the proton at position 7 is equatorial
(b). Moreover, all spectroscopic data (1H and 13C
NMR) were in agreements with studies reported by
Brueggemeier and co-workers.39
further investigated the inhibitory e€ect provoked by
the thiobenzyl group. Thus, we synthesized the phenolic
derivative 11 and its substituted analogue 12. As can be
seen in Table 2, the phenolic derivative 11 is better than
its substituted analogue 12, which is more potent than
the lead compound 9. It is interesting to note that com-
pounds 11 and 12 displayed inhibitory potency very
similar to our previously reported C18-steroid inhibitor
1. Moreover, compound 12 did not show any activity
against types I, III, and V of 17b-HSD when tested at
3 mM.
We decided to test these type II 17b-HSD inhibitors on
the human P450 aromatase because the 7a-derivatives
of Á4-dione are well known as inhibitors of this
enzyme.39,44,45 Results shown in Table 3 reveal that only
compounds 8 and 11 weakly inhibited P450 aromatase
activity compared with the established inhibitor 4-OH-
androstenedione,46 while other newly synthesized com-
pounds were totally inactive toward this enzyme.
Biological Results
In a previous study on the reductive activity of type II
17b-HSD, we observed that spironolactone (2), bearing
a 7a-thioacetyl group on a Á4-dione nucleus, was a
more potent inhibitor than the analogue without a 7a-
substituent.34 We aimed to further characterize the
importance of the 7a-substituent on this C19-steroid
nucleus in relation to the inhibition of type II 17b-HSD.
We ®rst changed the thioacetyl group for thioalkyls (4±
6), thiobenzoyl (7), thioaryl (8), and thiobenzyl (9)
groups (Scheme 1). To avoid contamination by aroma-
tase, which is also expressed in microsomes, we chose a
system that is free of endogenous aromatase activity.
Transformed human embryonic kidney (293) cells were
transfected with an expression vector encoding type II
17b-HSD. Assays were performed using subcellular
fractions by a previously reported method.27
Afterward, we were interested in studying the hormonal
pro®le of our type II 17b-HSD inhibitors. Thus, binding
anities on the steroid receptors were determined using
Table 1. Inhibitory e€ects of 7a-derivatives of spironolactone on the
activity of transfected type II 17b-HSD
A screening test was performed on our ®rst series of 7a-
derivatives of spironolactone (Table 1). The reductive
activity was initially tested to make the connection with
our previously published work that was reported before
the preferred activity of the enzyme was established.34
The ability of the compound to inhibit the reduction of
radiolabelled Á4-dione into testosterone was assessed. It
can be seen that neither compound 3 without substitu-
tion at position 7a nor compounds bearing a thioester
group (2 and 7) are very potent against type II 17b-
HSD. On the other hand, thioalkyl (4±6) and particu-
larly thioaryl compounds (8 and 9) displayed a more
potent inhibitory e€ect. As shown in Table 1, the 7a-
thiobenzyl analogue 9 exhibited the highest inhibitory
e€ect with an IC50 value of 0.42 mM, while other com-
pounds showed IC50 values close to micromolar. More-
over, analogue 9 had a better anity (threefold) than
the natural oxidized substrate Á4-dione (1.5 mM). We
No.
R
Inhibition of reductase activity
Type II 17b-HSD*
% at 0.1 mM % at 1.0 mM IC50 (mM)
2
3
4
5
6
7
8
9
SCOCH3
Á6-7
SCH2CH3
S(CH2)2CH3
S(CH2)5CH3
SCOPh
SPh
SCH2Ph
substrate
3
7
6
9
12
0
22
22
47
42
41
4
nd
nd
0.9‹0.3
1.1‹0.4
1.4‹0.3
nd
17
25
Ð
56
58
Ð
1.0‹0.4
0.42‹0.07
1.5‹0.4
Á4-dione
*For the reduction of [3H]-Á4-dione (5 nM) to [3H]-T catalysed by
transfected type II 17b-HSD.

M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
1017
Table 2. Inhibitory e€ects of 7a-derivatives of spironolactone on the
activity of transfected type II 17b-HSD
Discussion
We have described an ecient synthesis of 7a-deriva-
tives of spironolactone. Target compounds 4±9 were
synthesized within two chemical steps from spir-
onolactone when the thiols introduced at position 7a
were commercially available. A convergent synthesis of
six chemical steps was performed to generate the two
other inhibitors (11 and 12). The ®rst series of inhibitors
(2 to 9) was tested against the reductive activity of type
II 17b-HSD (transformation of Á4-dione (5 nM) into
testosterone). We then established the 7a-thiobenzyl
analogue as an interesting lead compound. During
optimization of this lead compound, it was reported
that type II 17b-HSD prefers oxidation over reduction.27
Consequently, our newly synthesized spironolactone
derivatives (11 and 12) as well as the compounds used as
references (1, 2, and 9) were evaluated for the inhibition
of the oxidation of [14C]-testosterone into [14C]-Á4-
dione catalyzed by type II 17b-HSD. These compounds
that inhibit type II 17b-HSD with IC50 values between
0.5 and 1.0 mM have two pharmacophores: a 17-spiro-g-
lactone group and a 7a-thioaryl group. Our results from
previous reports33,34 together with the present paper
clearly demonstrate that the 17-spiro-g-lactone group is
a very important pharmacophore that provides to both
C18 and C19 steroids an inhibitory e€ect on type II
17b-HSD. In addition, we have shown that a thiobenzyl
group at position 7a can enhance the inhibitory potency
of spironolactone. More importantly, we have suitably
substituted the spironolactone with a 7a-(para-hydro-
xybenzyl sul®de) group to obtain a C19-steroid inhi-
bitor (compound 11) that has similar inhibitory potency
to the ®rst reported C18-steroid inhibitor 1. Although
the combination e€ect of these two pharmacophores is
signi®cant (twofold), it is too weak to conclude that the
7a-substitution leads to a speci®c and strong binding to
type II 17b-HSD. However, these results have demon-
strated that a bulky pharmacophore such as a 2-(1-
piperidinyl)-ethoxybenzylthio group (compound 12) is
well tolerated by the type II enzyme. On the other hand,
it is known that the type I isoform does not allow strong
binding with E2 bearing a bulky side chain at position
7a.25,47 Although type I 17b-HSD has a strong pre-
ference for C18 over C19 steroids, we reported that the
spiro-g-lactone derivative of E2 (compound 1) does not
inhibit this enzyme.27 The spiro-g-lactone group thus
provides selectivity toward the type I isoenzyme even in
the C18-steroid series. Consequently, it is logical that
the newly synthesized compound 12 with the spiro-g-
lactone group placed on a 7a-substituted C19-steroid
nucleus is selective for type II over type I 17b-HSD.
However, the reasons why compound 12 does not inhi-
bit type III or type V 17b-HSD are less clear because no
SAR studies concerning these enzymes are reported in
the literature so far.
No.
R
Inhibition of oxidative activity*
Type II 17b-HSD IC50 (mM)
2
9
11
SCOCH3
SCH2Ph
SCH2Ph(4-OH)
1.1‹0.4
1.0‹0.2
0.53‹0.03
0.7‹0.3
0.7‹0.1
12 SCH2Ph(4-OCH2CH2N(CH2)5)
C18 inhibitor
1
*For the oxidation of [14C]-T (0.1 mM) into [14C]-Á4-dione catalysed
by transfected type II 17b-HSD.
standard conditions. Percentages of binding for two
concentrations of the tested compound are shown in
Table 4. The ®rstly reported inhibitor 1 eciently
bound to the estrogen receptor, while none of the newly
synthesized inhibitors had any anity for this receptor.
However, several 7a-derivatives of spironolactone
bound to progestin or glucocorticoid receptors and, to a
lesser extent, to androgen receptor. Among the tested
compounds, only 12 was devoid of any anity for all of
the tested steroid receptors. This compound did not
display signi®cant proliferative/antiproliferative activity
on estrogen-sensitive ZR-75-1 cells and showed weak
antiproliferative e€ect on androgen-sensitive Shionogi
cells (Fig. 3).
Table 3. Inhibitory e€ects of 7a-derivatives of spironolactone on the
activity of transfected P450 aromatase
No.
R
Inhibition of P450 aromatase*
% at 0.3 mM
% at 3.0 mM
2
3
4
5
6
7
8
9
11
SCOCH3
Á6-7
SCH2CH3
S(CH2)2CH3
S(CH2)5CH3
SCOPh
SPh
SCH2Ph
SCH2Ph(4-OH)
12
20
17
14
16
12
8
14
38
12
88
13
Ð
Ð
22
17
11
40
Ð
52
14
93
In addition to the inhibitory potency for type II 17b-HSD,
we have evaluated several other biological parameters
that are related to this type of steroidal compound.
According to previous reports from Brueggemeier and
co-workers,39,44,45 the para-substituted thiobenzyl groups
at position 7a also provoke inhibition of P450 aromatase
12 SCH2Ph(4-OCH2CH2N(CH2)5)
4-OH-androstenedione**
Ð
*For the aromatization of [14C]-Á4-dione to [14C]-E1 catalyzed by
transfected P450 aromatase.
**Standard inhibitor of P450 aromatase.

1018
M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
Table 4. Percentages of binding anity of compounds 2±12 on four steroid receptors
No.
Androgen receptor
Progestin receptor
Glucocorticoid receptor
Estrogen receptor
10 nM
1 mM
10nM
1 mM
10 nM
1 mM
10 nM
1 mM
1*
2
3
4
5
6
7
8
9
11
12
DHT
R5050
DEX
E2
0‹1
5‹2
1‹1
0‹4
0‹2
5‹3
5‹1
1‹3
5‹1
0‹3
0‹2
70‹1
1‹4
0‹1
0‹2
29‹1
53‹1
8‹2
28‹1
41‹3
12‹3
53‹2
35‹1
2‹2
9‹2
0‹2
100‹1
28‹2
2‹1
15‹2
0‹2
0‹2
0‹1
0‹2
7‹4
3‹2
0‹2
0‹2
0‹2
0‹1
3‹2
65‹2
0‹3
6‹3
90‹1
23‹5
26‹2
29‹2
59‹2
87‹1
62‹2
30‹1
42‹1
31‹4
0‹2
0‹1
2‹4
0‹3
3‹3
0‹3
6‹3
0‹3
0‹2
1‹2
0‹3
4‹2
2‹2
9‹2
66‹2
5‹2
74‹1
17‹3
1‹4
59‹2
53‹3
57‹3
10‹3
3‹3
33‹1
0‹1
0‹5
0‹3
1‹1
0‹2
0‹2
0‹1
0‹1
0‹1
0‹2
2‹2
5‹2
0‹3
75‹1
98‹1
2‹2
0‹2
0‹1
0‹2
0‹2
0‹2
0‹2
0‹1
0‹1
0‹2
4‹1
5‹2
0‹1
100‹1
12‹3
0‹4
4‹2
40‹2
99‹2
1‹2
6‹2
85‹2
99‹1
12‹2
34‹1
25‹2
*Concentrations are 100 nM and 10 mM instead of 10 nM and 1 mM. Abbreviations of steroids used as standard: DHT: dihydrotestosterone; R5050:
synthetic progestin; DEX: dexamethasone; E2: estradiol.
if they are linked to a Á4-dione nucleus. In our series,
we found that only the phenolic derivative 11 displays a
weak activity on P450 aromatase. The inhibitory
potency is clearly abolished when the phenol is alky-
lated or when the 7a position was substituted with a
thioalkyl instead of a thiobenzyl group.
We also evaluated the steroid receptor binding anity
of our series of inhibitors. We found that our synthetic
spironolactone derivatives with 7a-thioalkyl and 7a-
thioaryl bound to glucocorticoid, progestin, and andro-
gen receptors. The spironolactone template should pro-
vide the anity for the androgen receptor because
spirolactone itself eciently binds to this receptor. The
anity for glucocorticoid and progestin receptors dis-
played by some of our inhibitors may be attributable to
the 7a-substitution. It has been identi®ed that sub-
stituents, particularly a 4-dimethylamino phenyl group,
at C11b or C7a of an appropriate steroidal template
leads to progesterone antagonists and some of them
retain anity for the glucocorticoid receptor.48±50 How-
ever, the compound 12, which has a 2-(1-piperidinyl)-
ethoxybenzylthio group, was totally devoid of such
anity for all tested steroid receptors (AR, PR, GR, ER).
Finally, we conclude that compound 12 is a new potent
selective inhibitor of type II 17b-HSD that does not
bind to the estrogen receptor as well as the three other
tested receptors. This interesting hormonal pro®le
makes it suitable for further studies to establish the
regulation of steroid hormone biosynthesis by 17b-
HSDs in several biological systems. It can also be used
to study the structure of type II 17b-HSD.
Experimental
General method for chemical synthesis
Figure 3. (A) E€ect of compound 12 on the growth of androgen-sen-
sitive Shionogi mouse mammary carcinoma cells in absence and in
Chemical reagents were purchased from the Aldrich
presence of dihydrotestosterone (DHT). (B) E€ect of compound 12 on
Chemical Company (Milwaukee, WI) and spir-
onolactone was purchased from Steraloids Inc. (Wilton,
the growth of estrogen-sensitive human breast cancer ZR-75-1 cells in
absence and in presence of estradiol (E2).

M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
1019
NH). Solvents were obtained from BDH Chemicals
(Montreal, Canada) or Fischer Chemicals (Montreal,
Canada). Tetrahydrofuran (THF) used in anhydrous
conditions was distilled from sodium benzophenone
ketyl; other commercially available dry solvents were
stored under argon. Glassware used in anhydrous con-
ditions was baked for 1 h at 80 ꢀC, assembled hot and
®lled with argon before use. Standard inert-atmosphere
techniques were used for solvents transfers by syringe.
Flash-column chromatographies were performed using
230±400 mesh ASTM silica gel 60 (E. Merck, Darm-
stadt, GE). Infrared spectra (IR) were recorded on a
Perkin±Elmer series 1600 FT-IR spectrometer and
are reported in cm 1. High-resolution mass spectra
(HRMS) obtained from fast atom bombardment (FAB)
were provided by the Centre Regional de Spectrometrie
de Masse (Universite de Montreal, Canada). The NMR
35.49 (C-16), 36.05 (C-10), 37.80 (C-8), 46.44 (C-13),
47.05 (C-9), 50.46 (C-14), 95.31 (C-17), 124.00 (C-4),
128.36 (C-6), 139.34 (C-7), 162.98 (C-5), 176.48 (C-22),
199.30 (C-3); HRMS-FAB calcd for C22H29O3 (MH+),
341.2117. Found, 341.2111.
Synthesis of 7ꢁ-thioalkyl and 7ꢁ-thioaryl derivatives of
spironolactone (4±6 and 8±9) (general procedure). Can-
renone (3) was dissolved in the appropriated thiol (15±
20 equiv) under argon and the mixture was gently
heated at 45 ꢀC. Then, sodium metal (1 equiv) was
added to the solution which was allowed to stir at 60 ꢀC
for 45 min to 24 h. Afterward, saturated aqueous NH4Cl
was added until the neutrality was obtained and the
crude product was extracted with diethyl ether (4±6) or
CH2Cl2 (8±9). The combined organic layers were dried
over MgSO4 and concentrated in vacuo. Puri®cation of
the crude compounds was performed by column chro-
matography (4: hexane/EtOAc, 1/1; 5: hexane/EtOAc,
6/4; 6: hexane/EtOAc, 4/6; 8: hexane/EtOAc, 1/1; 9:
hexane/EtOAc, 1/1).
1
spectra were recorded at 300 MHz for H and 75 MHz
for 13C on a Bruker AC/F300 spectrometer. The
chemical shifts (d in ppm) were referenced to CHCl3 (d
1
7.26 ppm and 77.00 ppm for H and 13C, respectively).
The structures shown below are given to help clarify the
assignment of carbon and related proton signals.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(ethylthia)-21, 17-carbolact-
one (4). White solid (59% yield); IR n (®lm) 1775
(CˆO, lactone), 1667 (CˆO, conjugated ketone), 1616
Synthesis of spironolactone derivatives 3±12 (Scheme 1)
1
(CˆC); H NMR (CDCl3) d 0.97 (s, 3H, 18-CH3), 1.20
3-Oxo-17ꢁ-pregna-4, 6-diene 21, 17-carbolactone (canre-
none, 3). To a solution of spironolactone (2) (2.0 g,
4.8 mmol) in dry THF (150 mL) was added sodium
methoxide (1.6 g, 28 mmol) at room temperature under
argon atmosphere. The mixture was allowed to stir 18 h.
Then, water was added and THF was removed in
vacuo. The aqueous phase was acidi®ed to pH 2 and
extraction of the crude product was performed with
CH2Cl2. The combined organic layers were washed with
brine, dried over MgSO4 and concentrated under
reduced pressure. Puri®cation by column chromato-
graphy (CH2Cl2/EtOAc, 65/35) gave 1.2 g (72% yield)
of canrenone 3: White amorphous solid; IR n (KBr)
1770 (CˆO, lactone), 1660 (CˆO, conjugated ketone),
(s, 3H, 19-CH3), 1.22 (t, J=7.3 Hz, 3H, SCH2CH3),
3.06 (m, 1H, 7b-CH), 5.76 (s, 1H, 4-CH); 13C NMR d
(CDCl3) 14.47 (C-18), 14.68 (SCH2CH3), 17.94 (C-19),
20.48 (C-15), 22.23 (C-11), 24.68 (SCH2CH3), 29.23
(C-21), 31.18 (C-12 and C-21), 33.90 (C-2), 35.25 (C-16),
35.47 (C-1), 38.40 (C-6 and C-10), 39.87 (C-8), 44.92 (C-
7), 45.22 (C-14), 45.38 (C-13), 46.85 (C-9), 95.70 (C-17),
126.92 (C-4), 166.90 (C-5), 176.36 (C-22), 198.59 (C-3);
HRMS-FAB calcd for C24H35O3S (MH+), 403.2307.
Found, 403.2291.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(propylthia)-21, 17-carbo-
lactone (5). White solid (72% yield); IR n (®lm) 1770
(CˆO, lactone), 1672 (CˆO, conjugated ketone), 1620
1
1
1617 (CˆC); H NMR (CDCl3) d 1.03 (s, 3H, 18-CH3),
(CˆC); H NMR (CDCl3) d 0.98 (s, 3H, 18-CH3), 0.98
1.12 (s, 3H, 19-CH3), 5.68 (s, 1H, 4-CH), 6.07 (d,
J=10.7 Hz, 6-CH), 6.12 (dd, J1=10.7 Hz and J2=
2.2 Hz, 1H, 7-CH); 13C NMR (CDCl3) d 14.42 (C-18),
16.33 (C-19), 20.11 (C-15), 22.46 (C-11), 29.22 (C-21),
31.17 (C-20), 31.65 (C-12), 33.90 (C-2), 33.96 (C-1),
(t, J=7.3 Hz, 3H, SCH2CH2CH3), 1.21 (s, 3H, 19-
CH3), 3.03 (m, 1H, 7b-CH), 5.78 (s, 1H, 4-CH); 13C
NMR (CDCl3) d 13.55 (SCH2CH2CH3), 14.47 (C-18),
17.94 (C-19), 20.48 (C-15), 22.28 (C-11), 22.91 (CH2CH3),
29.23 (C-21), 31.18 (C-12 and C-20), 33.03 (SCH2), 33.90

1020
M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
(C-2), 35.26 (C-16), 35.46 (C-1), 38.39 (C-10), 38.54 (C-
6), 39.94 (C-8), 45.23 (C-7), 45.39 (C-13 and C-14),
46.81 (C-9), 95.71 (C-17), 126.91 (C-4), 166.97 (C-5),
176.68 (C-22), 198.64 (C-3); HRMS-FAB calcd for
C25H37O3S (MH+), 417.2463. Found, 417.2451.
29 mg (40% yield) of 7 as an amorphous white solid: IR
n (®lm) 1770 (CˆO, lactone), 1663 (CˆO, conjugated
ketone and thioester), 1616 (CˆC); 1H NMR (CDCl3) d
1.01 (s, 3H, 18-CH3), 1.26 (s, 3H, 19-CH3), 2.91 and
2.95 (2m, 1H), 4.25 (m, 1H, 7b-CH), 5.71 (s, 1H, 4-CH),
7.45 (m, 2H, 300-CH), 7.58 (m, 1H, 400-CH), 7.94 (d,
J=7.4 Hz, 2H, 200-CH); 13C NMR (CDCl3) d 14.57 (C-
18), 17.78 (C-19), 20.54 (C-15), 22.35 (C-11), 29.13 (C-
21), 31.05, 31.17, 33.90 (C-2), 35.15 (C-16), 35.63 (C-1),
38.54 (C-10), 39.11 (C-7), 40.07 (C-6), 45.01 (C-8), 45.46
(C-16), 46.07 (C-14), 49.68 (C-9), 95.51 (C-17), 126.99
(C-4), 127.35 (2C, C-300), 128.64 (C-400), 133.64 (2C, C-
200), 136.94 (C-100), 165.69 (C-5), 176.51 (C-22), 190.26
(SCˆO), 198.61 (C-3); HRMS-FAB calcd for C29H35
O4S (MH+), 479.2256. Found, 479.2248.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(hexylthia)-21, 17-carbo-
lactone (6). White solid (62% yield); IR n (®lm) 1771
(CˆO, lactone), 1670 (CˆO, conjugated ketone), 1618
(CˆC); 1H NMR (CDCl3) d 0.87 (t, J=6.9 Hz, 3H,
S(CH2)5CH3), 0.97 (s, 3H, 18-CH3), 1.20 (s, 3H, 19-
CH3), 3.03 (m, 1H, 7b-CH), 5.76 (s, 1H, 4-CH); 13C
NMR (CDCl3) d 14.01 (CH2CH3), 14.52 (C-18), 17.97
(C-19), 20.51 (C-15), 22.34 (C-11), 22.52 (CH2CH3),
28.63 (CH2), 29.26 (C-21), 29.56 (CH2), 31.02 (CH2),
31.23 (C-12 and C-20), 31.39 (SCH2), 33.93 (C-2), 35.31
(C-16), 35.49 (C-1), 38.43 (C-10), 38.55 (C-6), 39.98 (C-
8), 45.28 (C-7), 45.41 (C-13), 45.47 (C-14), 46.85 (C-9),
95.75 (C-17), 126.97 (C-4), 166.97 (C-5), 176.71 (C-22),
198.65 (C-3); HRMS-FAB calcd for C28H43O3S
(MH+), 459.2933. Found, 459.2952.
Synthesis of 7ꢁ-(40-O-alkylated-thiobenzyl) derivatives
of spironolactone (10 and 12) (general procedure). Can-
renone (3) was dissolved in the appropriated synthetic
thiols 17 or 18 (15±20 equiv) under argon and the mix-
ture was gently heated at 45 ꢀC. Then, sodium metal
(1 equiv) was added to the solution which was allowed
to stir for 45 min (10) or for 60 min (12). Afterward,
saturated aq NH4Cl was added to the cooled reaction
mixture until the neutrality was obtained and the crude
product was extracted with diethyl ether. The combined
organic layers were dried over MgSO4 and concentrated
in vacuo. Puri®cations of the crude compounds were
performed by column chromatography (hexane/EtOAc,
95/5 for 10; CH2Cl2/MeOH, 95/5 for 12).
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(phenylthia)-21, 17-carbolact-
one (8). White solid (59% yield); IR n (®lm) 1770
(CˆO, lactone), 1665 (CˆO, conjugated ketone), 1618
1
(CˆC); H NMR (CDCl3) d 0.99 (s, 3H, 18-CH3), 1.20
(s, 3H, 19-CH3), 3.46 (m, 1H, 7b-CH), 5.68 (s, 1H, 4-
CH), 7.28 (m, 3H, 400-CH and 200-CH), 7.38 (m, 2H, 300-
CH); 13C NMR (CDCl3) d 14.46 (C-18), 17.81 (C-19),
20.44 (C-15), 22.19 (C-11), 29.18 (C-21), 31.14 (C-12),
31.19 (C-20), 33.88 (C-2), 35.25 (C-16), 35.48 (C-1),
38.03 (C-6), 38.44 (C-10), 39.92 (C-8), 45.44 (C-13 and
C-14), 46.81 (C-9), 50.15 (C-7), 95.56 (C-17), 127.15 (C-
4), 127.62 (C-400), 129.14 (2C, C-300), 133.32 (2C, C-200),
133.94 (C-100), 166.41 (C-5), 176.56 (C-22), 198.60 (C-3);
HRMS-FAB calcd for C28H35O3S (MH+), 451.2307.
Found, 451.2296.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-[(4-t-butyldimethylsilyloxy)-
benzylthia]-21, 17-carbolactone (10). White amorphous
solid (91% yield): IR n (®lm) 1774 (CˆO, lactone), 1672
(CˆO, conjugated ketone), 1609 (CˆC); 1H NMR
(CDCl3) d 0.17 (s, 6H, Si(CH3)2), 0.92 (s, 3H, 18-CH3),
0.96 (s, 9H, SiC(CH3)3), 1.18 (s, 3H, 19-CH3), 2.84 (m,
1H, 7b-CH), 3.58 (qapp, ABX system, 2H, CH2Ph), 5.74
(s, 1H, 4-CH), 6.76 (d, J=8.6 Hz, 2H, 300-CH), 7.12 (d,
J=8.5 Hz, 2H, 200-CH); 13C NMR (CDCl3) d 4.47
(Si(CH3)2), 14.45 (C-18), 17.87 (C-19), 18.20 (SiC(CH3)3),
20.49 (C-15), 21.78 (C-11), 25.63 (SiC(CH3)3), 29.23 (C-
21), 31.18 (C-12 and C-20), 33.90 (C-2), 34.50 (SCH2),
35.27 (C-16), 35.46 (C-1), 38.14 (C-6), 38.39 (C-10),
39.73 (C-8), 44.35 (C-7), 45.15 (C-14), 45.28 (C-13),
47.32 (C-9), 95.70 (C-17), 120.07 (2C, C-300), 126.96 (C-
4), 129.92 (2C, C-200), 130.59 (C-100), 154.82 (C-400),
166.92 (C-5), 176.69 (C-22), 198.65 (C-3); HRMS-FAB
calcd for C35H51O3SSi (MH+), 595.3278. Found,
595.3262.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(benzylthia)-21, 17-carbo-
lactone (9). White solid (74% yield); IR n (®lm) 1769
(CˆO, lactone), 1665 (CˆO, conjugated ketone), 1618
1
(CˆC); H NMR (CDCl3) d 0.90 (s, 3H, 18-CH3), 1.16
(s, 3H, 19-CH3), 2.86 (m, 1H, 7b-CH), 3.63 (qapp, ABX
system, 2H, CH2Ph), 5.72 (s, 1H, 4-CH), 7.27 (m, 5H,
Ph); 13C NMR (CDCl3) d 14.37 (C-18), 17.82 (C-19),
20.43 (C-15), 21.75 (C-11), 29.15 (C-21), 31.11 (C-12
and C-20), 33.84 (C-2), 35.11 (SCH2), 35.21 (C-16),
35.40 (C-1), 38.13 (C-6), 38.34 (C-10), 39.72 (C-8), 44.72
(C-7), 45.07 (C-14), 45.25 (C-13), 47.19 (C-9), 95.61 (C-
17), 126.88 (C-4), 127.12 (C-400), 128.41 (2C, C-300),
128.81 (2C, C-200), 137.99 (C-100), 166.75 (C-5), 176.59
(C-22), 198.54 (C-3); HRMS-FAB calcd for C29H36O3S
(MH+), 465.2463. Found, 465.2479.
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-{4-[2-(1-piperidinyl)-ethoxy]-
benzylthia}-21, 17-carbolactone (12). O€-white solid
(82% yield): IR n (®lm) 1772 (CˆO, lactone) 1664
(CˆO, conjugated ketone), 1610 (CˆC); 1H NMR
(CDCl3) d 0.89 (s, 3H, 18-CH3), 1.15 (s, 3H, 19-CH3),
2.78 (t, J=5.9 Hz, 2H, 2000-CH2N), 2.79 (m, 1H, 7b-
CH), 3.57 (qapp, ABX system, 2H, CH2Ph), 4.08 (t,
J=5.9 Hz, 2H, 1000-CH2O), 5.70 (s, 1H, 4-CH), 6.80 (d,
J=8.4 Hz, 2H, 300-CH), 7.15 (d, J=8.5 Hz, 2H, 200-
CH); 13C NMR (CDCl3) d 14.34 (C-18), 17.78 (C-19),
20.38 (C-15), 21.75 (C-11), 23.82 (C-40000), 25.47 (2C, C-
30000;), 29.14 (C-21), 31.07 (C-12 and C-20), 33.80 (C-2),
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(benzoylthia)-21, 17-carbo-
lactone (7). According to the procedure described by
Cella and Tweit,40±42 canrenone (3) (52 mg, 0.15 mmol)
was dissolved in 1 mL of thiobenzoic acid. The mixture
was allowed to stir at 60 ꢀC for 3 h. Then, water was
added and the crude product was extracted with diethyl
ether. The organic layers were dried over MgSO4 and
concentrated under reduced pressure. Puri®cation by
silica gel chromatography (hexane/EtOAc, 4/6) gave

M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
1021
34.36 (SCH2), 35.15 (C-16), 35.33 (C-1), 38.03 (C-6),
38.27 (C-10), 39.61 (C-8), 44.44 (C-7), 45.01 (C-14),
45.19 (C-13), 47.16 (C-9), 54.83 (2C, 20000-CH2N), 57.62
(2000-CH2N), 65.58 (1000-CH2O), 95.65 (C-17), 114.44 (2C,
C-300), 126.77 (C-4), 129.81 (2C, C-200), 129.89 (C-100),
157.70 (C-400), 166.98 (C-5), 176.68 (C-22), 198.64 (C-3);
HRMS-FAB calcd for C36H50O4NS (MH+), 592.3461.
Found, 592.3471.
extracted three times with EtOAc. The combined
organic layers were washed with brine, dried over
MgSO4, and removed under reduced pressure. The
crude oil was puri®ed by chromatography on silica gel
(CHCl3/MeOH, 95/5) to give 512 mg (54% yield) of 14
as a yellow liquid: 1H NMR (CDCl3) d 1.43 (m, 2H, 4000-
CH2), 1.58 (m, 4H, 3000-CH2), 2.50 (m, 4H, 2000-CH2N),
2.78 (t, J=6.0 Hz, 2H, 200-CH2N), 4.17 (t, J=6.0 Hz,
2H, 100-CH2O), 6.98 (d, J=8.7 Hz, 2H, 30-CH), 7.80 (d,
J=8.3 Hz, 2H, 20-CH), 9.85 (s, 1H, CHO); 13C NMR
(CDCl3) d 24.01 (C-4000), 25.80 (2C, C-3000), 54.96 (2C,
2000-CH2N), 57.55 (200-CH2N), 66.31 (100-CH2O), 114.74
(2C, C-30), 129.80 (2C, C-20), 131.82 (C-40), 163.78 (C-
10), 190.64 (CHO).
3-Oxo-17ꢁ-pregna-4-ene-7ꢁ-(4-hydroxy-benzylthia)-21,
17-carbolactone (11). To a solution of 10 (237 mg,
0.40 mmol) in 30 mL of dry THF at 78 ꢀC was added
0.6 mL of tetrabutylammonium ¯uoride (1 M solution
in THF). The resulting mixture was allowed to stir
45 min at 78 ꢀC under argon. Then, water was added
and extraction of the crude product was performed
using CH2Cl2. The combined organic layers were
washed with brine, dried over MgSO4 and evaporated in
vacuo. Silica gel chromatography (CH2Cl2/acetone, 95/
5) gave 159 mg (88% yield) of 11 as a white amorphous
solid: IR n (®lm) 3325 (OH, phenol), 1762 (CˆO, lactone),
Synthesis of thioacetyl derivatives 15 and 16. To a solu-
tion of the aldehyde 13 (31.5 g, 130 mmol) or 14 (7.3 g,
27 mmol) in MeOH (240 mL or 60 mL, respectively) at
0 ꢀC was added sodium borohydride (7.6 g, 200 mmol or
1.6 g, 41 mmol) and the reaction was stirred at 0 ꢀC for
45 min (13) and for 3 h (14). Water was then added and
MeOH removed under reduced pressure. The crude
alcohol was extracted twice with diethyl ether and twice
with EtOAc. Organic solvents were dried over MgSO4
and removed in vacuo. The crude alcohols were used
without puri®cation for the next step. To an eciently
stirred solution of triphenylphosphine (52.5 g, 200 mmol
or 7.1 g, 27 mmol) in THF (200 mL or 50 mL) was
added diethyl azodicarboxylate (31.5 mL, 200 mmol or
4.3 mL, 27 mmol) at 0 ꢀC. The mixture was stirred for
30 min, after which a thick white precipitate was
obtained. Then, the crude alcohols dissolved in THF
and thiolacetic acid (21.4 mL, 300 mmol or 5.8 mL,
81 mmol) were added dropwise while the temperature
was maintained below 0 ꢀC. The reaction was stirred for
2 h at 0 ꢀC. Solvent was removed in vacuo. The residue
was puri®ed by silica gel chromatography (hexane/
EtOAc, 95/5 for 15; CH2Cl2/MeOH, 98/2 for 16).
1
1678 (CˆO, conjugated ketone), 1619 (CˆC); H NMR
(CDCl3) d 0.93 (s, 3H, 18-CH3), 1.18 (s, 3H, 19-CH3), 2.89
(m, 1H, 7b-CH), 3.60 (qapp, ABX system, 2H, CH2Ph),
5.72 (s, 1H, 4-CH),006.78 (d, J=8.6 Hz, 2H, 300-CH), 7.15
(d, J=8.3 Hz, 2H, 2 -CH); 13C NMR (CDCl3) d 14.46 (C-
18), 17.89 (C-19), 20.49 (C-15), 21.90 (C-11), 29.27 (C-21),
31.18 (C-12 and C-20), 33.84 (C-2), 34.72 (SCH2), 35.27
(C-16), 35.38 (C-1), 38.31 (C-10), 38.43 (C-6), 39.81 (C-8),
44.84 (C-7), 45.14 (C-14), 45.32 (C-13), 47.29 (C-9), 95.91
(C-17), 115.42 (2C, C-300), 126.81 (C-4), 129.55 (C-100),
130.06 (2C, C-200), 155.31 (C-400), 167.85 (C-5), 177.00 (C-
22), 199.33 (C-3); HRMS-FAB calcd for C29H37O3S
(MH+), 481.2413. Found, 481.2428.
Preparation of synthetic thiols 17 and 18 (Scheme 2)
4-(t-Butyldimethylsilyloxy)-benzaldehyde (13). To a solu-
tion of p-hydroxybenzaldehyde (20.0 g, 160 mmol) in
150 mL of dry DMF was added imidazole (55.7 g,
800 mmol) and TBDMS±Cl (48.2 g, 320 mmol) under
argon atmosphere. The resulting mixture was stirred for
4 h at room temperature. Then, water was added and
the crude compound was extracted twice with ether and
twice with CH2Cl2. Organic solvents were dried over
MgSO4 and removed under reduced pressure. The crude
oil was puri®ed by ®ltration on silica gel (hexane/
EtOAc, 95/5) to give 31.5 g (83% yield) of 13 as a col-
4-(t-Butyldimethylsilyloxy)-ꢁ-thioacetyl-toluene (15).
1
Colorless oil (63% yield); H NMR (CDCl3) d 0.18 (s,
6H, Si(CH3)2), 0.97 (s, 9H, SiC(CH3)3), 2.34 (s, 3H,
COCH3), 4.07 (s, 2H, CH2Ph), 6.75 (d, J=8.5 Hz, 2H,
30-CH), 7.13 (d, J=8.3 Hz, 2H, 20-CH); 13C NMR
(CDCl3) d 4.48 (Si(CH3)2), 18.11 (SiC(CH3)3), 25.60
(SiC(CH3)3), 30.25 and 32.94 (SCH2 and COCH3),
120.05 (2C, C-30), 129.81 (2C, C-20), 131.82 (C-10),
154.82 (C-40), 195.14 (COCH3).
1
orless oil: H NMR (CDCl3) d 0.23 (s, 6H, Si(CH3)2),
0.97 (s, 9H, SiC(CH3)3), 6.92 (d, J=8.6 Hz, 2H, 30-CH),
7.77 (d, J=8.6 Hz, 2H, 20-CH), 9.86 (s, 1H, CHO); 13C
NMR (CDCl3) d 4.44 (Si(CH3)2), 18.17 (SiC(CH3)3),
25.48 (SiC(CH3)3), 120.40 (2C, C-30), 130.32 (C-40),
131.91 (2C, C-20), 161.44 (C-10), 190.80 (CHO).
4-[2-(1-Piperidinyl)-ethoxy)]-ꢁ-thioacetyl-toluene (16).
1
White wax (89% yield); H NMR (CDCl3) d 1.46 (m,
2H, 4000-CH2), 1.66 (m, 4H, 3000-CH2), 2.32 (s, 3H,
COCH3), 2.63 (m, 4H, 2000-CH2N), 2.88 (t, J=5.8 Hz,
2H, 200-CH2N), 4.05 (s, 2H, SCH2Ph), 4.15 (t, J=
5.8 Hz, 2H, 100-CH2O), 6.80 (d, J=8.7 Hz, 2H, 30-CH),
7.17 (d, J=8.7 Hz, 2H, 20-CH); 13C NMR (CDCl3) d
23.36 (C-4000), 24.78 (2C, C-3000), 30.29 and 32.81 (SCH2
and COCH3), 54.51 (2C, 2000-CH2N), 57.18 (200-CH2N),
64.91 (100-CH2O), 114.56 (2C, C-30), 129.91 (2C, C-20),
131.88 (C-10), 154.47 (C-40), 195.27 (COCH3).
4-[2-(1-Piperidinyl)-ethoxy]-benzaldehyde (14). To a sol-
ution of p-hydroxybenzaldehyde (0.5 g, 4.1 mmol) in
500 mL of dry acetonitrile was added K2CO3 (8.49 g,
61.5 mmol) and 1-(2-chloroethyl)piperidine hydro-
chloride (7.53 g, 41.0 mmol) under argon atmosphere.
The resulting mixture was gently re¯uxed overnight.
Then, an aqueous solution of 5% HCl was added until a
neutral pH was obtained. The crude compound was
Synthesis of thiols 17 and 18. To a stirred solution of 15
or 16 (5 g, 17 mmol or 6.2 g, 21 mmol) in dry THF (200

1022
M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
or 120 mL) was added lithium aluminum hydride
(960 mg, 25 mmol or 1.2 g, 32 mmol) at 0 ꢀC. The reac-
tion was stirred for 3 h at 0 ꢀC. Then, EtOAc and water
were added. The pH was brought to 5 with an aqueous
solution of 10 % HCl and the resulting slurry was ®l-
tered. The crude product was extracted with EtOAc and
the combined organic layers were washed successively
with Rochelle's salt, brine, dried over MgSO4 and eva-
porated under reduced pressure. Puri®cations were per-
formed by silica gel chromatography (hexane/EtOAc,
98/2 for 17; CH2Cl2/acetone/MeOH, 80/15/5 for 18).
trans. of control % trans. of compound)/(% trans. of
control)]Â100. When several concentrations of an inhi-
bitor were used in the enzymatic assay, an inhibition
curve was plotted using the percentage of transforma-
tion versus the concentration of inhibitor. From this
inhibition curve, the IC50 value was calculated by a
computer using an unweighted iterative least-squares
method for 4-parameters logistic curve ®tting.
Inhibition of type II 17ꢀ-HSD (Table 2). The same
enzymatic source as reported above was used to catalyze
the oxidation of 0.1 mM of [14C]-testosterone to [14C]-Á4-
dione, using NAD+ as cofactor. The enzymatic assay was
performed exactly as described in our earlier report.27
4-(t-Butyldimethylsilyloxy)-benzyl mercaptan (17). Col-
1
orless liquid (76% yield); H NMR (CDCl3) d 0.19 (s,
6H, Si(CH3)2), 0.98 (s, 9H, SiC(CH3)3), 1.73 (t, J=
7.3 Hz, 1H, SH), 3.70 (d, J=7.3 Hz, 2H, CH2S), 6.78 (d,
J=8.5 Hz, 2H, 30-CH), 7.18 (d, J=8.5 Hz, 2H, 20-CH);
13C NMR (CDCl3) d 4.44 (Si(CH3)2), 18.23 (SiC(CH3)3),
25.66 (SiC(CH3)3), 28.43 (CH2SH), 120.16 (2C, C-30),
129.06 (2C, C-20), 133.30 (C-10), 155.00 (C-40).
Inhibition of P450 aromatase (Table 3). Aromatase
activity was obtained from 293 cells transfected with the
cDNAs encoding P450 aromatase and NADPH P450
reductase. The cells were sonicated in 50 mM sodium
phosphate bu€er (pH 7.4), containing 20% glycerol and
1 mM EDTA, and were used as the enzymatic pool
without further puri®cation (3.6 mg protein/tube). The
transformation of 0.1 mM of [14C]-Á4-dione to [14C]-
estrone was evaluated. The enzymatic reaction was car-
ried out at 37 ꢀC in 1 mL 50 mM sodium phosphate
bu€er (pH 7.4), containing 20% glycerol and 1 mM
EDTA, in the presence of the indicated concentrations
of inhibitor (0.3 mM and 3.0 mM in EtOH or EtOH/
DMSO (95/5)) for 1 h. After incubation, steroids were
extracted twice with diethyl ether and separated by TLC
(toluene/acetone, 4/1). Radioactivity signals associated
to [14C]-Á4-dione and [14C]-estrone were detected and
quanti®ed using a Phosphor Imager (Sunny Vale, CA).
Data were calculated as mentioned above.
4-[2-(1-Piperidinyl)-ethoxy]-benzyl mercaptan (18). Col-
1
orless liquid (43% yield); H NMR (CDCl3) d 1.47 (m,
2H, 4000-CH2), 1.63 (m, 4H, 3000-CH2), 1.73 (t, J=7.3 Hz,
1H, SH), 2.54 (m, 4H, 2000-CH2N), 2.80 (t, J=6.0 Hz,
2H, 200-CH2N), 3.70 (d, J=7.3 Hz, 2H, SCH2Ph), 4.11
(t, J=5.8 Hz, 2H, 100-CH2O), 6.85 (d, J=8.5 Hz, 2H, 30-
CH), 7.22 (d, J=8.3 2H, 20-CH); 13C NMR (CDCl3) d
24.07 (C-4000), 25.81 (2C, C-3000), 28.32 (CH2SH), 54.95
(2C, 2000-CH2N), 57.82 (200-CH2N), 65.85 (100-CH2O),
114.65 (2C, C-30), 129.03 (2C, C-20), 133.22 (C-10),
157.75 (C-40).
Biology
Inhibition of type II 17ꢀ-HSD (Table 1). Transfected
293 cells with cDNA encoding for type II 17b-HSD
were sonicated to liberate the crude enzyme that was
used as the enzymatic pool without further puri®cation.27
The enzymatic assay was performed as follows: a stock
solution was ®rst prepared containing the radiolabelled
substrate [3H]-Á4-dione (5 nM), NADH (1 mM) in a
phosphate bu€er (pH 7.4, 50 mM KH2PO4, EDTA
1 mM, 20% glycerol). For the assay, 890 mL of the stock
solution and 10 mL of a solution of inhibitor (EtOH or
EtOH/DMSO, 95/5) were added in a tube. The reaction
was started by adding 100 mL of a solution of crude
enzyme (0.4 mg protein/tube) prepared as above. The
mixture was incubated for 1 h at 37 ꢀC, and the reaction
was stopped by adding an excess of unlabeled Á4-dione
and T. Steroids were extracted with diethyl ether and
solvent was removed under reduced pressure. The resi-
due was dissolved in CH2Cl2, spotted on a silica gel
plate (TLC, 20Â20Â0.2 cm, Kieselgel 60 F254) and
eluted with CH2Cl2/EtOAc (9/1). Less polar Á4-dione
and more polar T were identi®ed on TLC as two rows
of visible spots under UV light. Each spot was cut from
the plate and stored in a vial with 1 mL EtOH and
10 mL of scintillating solution. Radioactivity was mea-
sured in a b-counter. The percent of transformation of
[3H]-Á4-dione into [3H]-T was calculated as follows: %
trans.=100 X {[3H]-T(cpm)/([3H]-Á4-dione(cpm)+[3H]
-T(cpm))}. Subsequently, percent of inhibition=[(%
Steroid receptor binding anity screening (Table 4). The
anity binding assays on estrogen and progestin recep-
tors from rat uterine were carried out under the stan-
dard procedure established in our laboratory.51 Assays
for androgen receptor from rat ventral prostate were
performed according to the procedure described by Luo
and co-workers.52 For binding assay on glucocorticoid
receptor from rat liver, a slightly modi®ed version of the
procedure described by Asselin and co-workers was
used.53 In this case, separation of bound and free ster-
oids was achieved with dextran-coated charcoal
adsorption instead of protamine sulfate precipitation.
Proliferative/antiproliferative activities (Fig. 3). The in
vitro evaluation of compound 12 on the proliferation
of estrogen-sensitive human breast cancer ZR-75-1 cells
as well as its ability to inhibit the 0.1 nM E2-induced
proliferation was performed according to the proce-
dure described by our laboratory.54 Assays on the
proliferation of Shionogi mammary carcinoma cells as
well as on the inhibition of 0.3 nM DHT-induced pro-
liferation were carried out using a previously described
method.52
Acknowledgements
The authors thank the Medical Research Council of
Canada (MRC) and Le Fonds de la recherche en sante

M. R. Tremblay et al. / Bioorg. Med. Chem. 7 (1999) 1013±1023
1023
du Quebec (FRSQ) for operating grants (D.P.). M.R.T
is holder of a NSERCC and FCAR scholarships. We
are grateful to Mr. Guy Reimnitz and Mr. Serge Auger
for enzymatic tests, Dr. Jacques Simard and Mrs. Diane
Michaud for providing biological evaluation on ZR-75-
1 and Shionogi cells as well as Dr. Patricia Dionne for
the judicious NMR experiments.
25. Tremblay, M. R.; Poirier, D. J. Steroid Biochem. Molec.
Biol. 1998, 66, 179.
26. Blomquist, C. H.; Lindemann, N. J.; Hakanson, E. Y.
Arch. Biochem. Biophys. 1985, 239, 206.
27. Luu-The, V.; Zhang, Y.; Poirier, D.; Labrie, F. J. Steroid
Biochem. Molec. Biol. 1995, 55, 581.
28. Elo, J. P.; Akinola, L. A.; Poutanen, M.; Vihko, P.; Kyl-
lonen, A. P.; Lukkarineen, O.; Vihko, R. Int. J. Cancer 1996,
66, 37.
29. Carsol, J.-L.; Martin, P.-M.; de Launoit, Y. Neuroendo-
crinology 1994, 60, 445.
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