Development of 3-substituted-androsterone derivatives as potent inhibitors of 17β-hydroxysteroid dehydrogenase type 3

Article abstract of DOI:10.1016/j.bmc.2011.06.003

17Beta-hydroxysteroid dehydrogenase type 3 (17β-HSD3) is a steroidogenic enzyme that catalyzes the transformation of 4-androstene-3,17- dione (Δ⁴-dione) into androgen testosterone (T). To provide effective inhibitors of androgen biosynthesis, we synthesized two different series (amines and carbamates) of 3β-substituted-androsterone derivatives and we tested their inhibitory activity on 17β-HSD3. From the results of our structure-activity relationship study, we identified a series of compounds producing a strong inhibition of 17β-HSD3 overexpressed in HEK-293 cells (homogenized cells). The most active compound when tested in intact HEK-293 transfected cells, namely (3α,5α)-3-{[trans-2,5-dimethyl-4-{[2- (trifluoromethyl)phenyl] sulfonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-17- one (15b), shows an IC₅₀ value of 6 nM, this compound is thus eight times more active than our reference compound D-5-2 (IC₅₀ = 51 nM). This new improved inhibitor did not stimulate the proliferation of androgen-sensitive Shionogi cells, suggesting a non-androgenic profile. Compound 15b is thus a good candidate for further in vivo studies on rodents.

Full text of DOI:10.1016/j.bmc.2011.06.003

Bioorganic & Medicinal Chemistry 19 (2011) 4652–4668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of 3-substituted-androsterone derivatives as potent inhibitors
of 17b-hydroxysteroid dehydrogenase type 3
René Maltais a, Michelle-Audrey Fournier a, Donald Poirier a,b,
⇑
a Laboratory of Medicinal Chemistry, Endocrinology and Genomic Unit, CHUQ (CHUL) – Research Center, Quebec, Canada
b Department of Molecular Medicine, Faculty of Medicine, Laval University, Quebec, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
17Beta-hydroxysteroid dehydrogenase type 3 (17b-HSD3) is a steroidogenic enzyme that catalyzes the
transformation of 4-androstene-3,17-dione (D
4-dione) into androgen testosterone (T). To provide effec-
Received 21 January 2011
Revised 30 May 2011
Accepted 2 June 2011
Available online 13 June 2011
tive inhibitors of androgen biosynthesis, we synthesized two different series (amines and carbamates) of
3b-substituted-androsterone derivatives and we tested their inhibitory activity on 17b-HSD3. From the
results of our structure–activity relationship study, we identified a series of compounds producing a
strong inhibition of 17b-HSD3 overexpressed in HEK-293 cells (homogenized cells). The most active com-
Keywords:
Steroid
Androsterone
Enzyme
Hydroxysteroid dehydrogenase
Cancer
pound when tested in intact HEK-293 transfected cells, namely (3a,5a)-3-{[trans-2,5-dimethyl-4-{[2-
(trifluoromethyl)phenyl] sulfonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-17-one (15b), shows an
IC50 value of 6 nM, this compound is thus eight times more active than our reference compound D-5-2
(IC50 = 51 nM). This new improved inhibitor did not stimulate the proliferation of androgen-sensitive
Shionogi cells, suggesting a non-androgenic profile. Compound 15b is thus a good candidate for further
in vivo studies on rodents.
Androgen
Hormone
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
to the production of approximately 60% of total active androgens
in men.16 The other part of active androgens (40%) would be di-
Prostate cancer is the most common cancer among men in the
United States with an estimate of 217,730 new diagnosed cases
in 2010 and with 32,050 associated deaths.1 Endocrine therapy
has been recognized as one of the most efficient treatments for
prostate cancer with a positive response in cancer regression in
nearly 80% of cases at first treatment.2 Different endocrine treat-
ments are now available to block either the testicular source of
androgens (medical or surgical castration) or the effect of the
androgens testosterone (T) and dihydrotestosterone (DHT) on the
androgen receptor (antiandrogens).3,4 Although they improve sur-
vival, these androgen deprivation treatments are linked to impor-
tant side effects and are rarely curative for advanced prostate
cancer.5–7 Thus, the development of new therapeutic options are
strongly needed to improve survival as well as the quality of life
for treated patients.8
rectly synthesized in the prostate from inactive adrenal precur-
sors dehydroepiandrosterone (DHEA) and
4-dione by the
action of 3b-hydroxysteroid dehydrogenases, 5 -reductases and
D
a
other 17b-HSDs such as type 5 or type 15.17,18 However, although
the 17b-HSD3 expression level is found to be very low in normal
prostate tissues, it has been reported that 17b-HSD3 is suspected
to play an important role in the conversion of adrenal steroids
into potential androgens in prostate cancer tissues.19,20 In fact,
the expression levels of the 17b-HSD3 mRNA in prostatic tissues
with malignancy is significantly higher (31 times) than those in
prostatic tissues without malignancy. Furthermore, Nelson’s
group recently showed that 17b-HSD3 is overexpressed (8 times)
in LuCaP 23 and LuCAP 35 cell lines, which were obtained from
metastatic tissues of patients resistant to castration therapy.21–
24
Importantly, despite a castrated level of T in the bloodstream,
17b-Hydroxysteroid dehydrogenase type 3 (17b-HSD3), also
named testicular 17b-hydroxysteroid dehydrogenase, is a steroi-
dogenic enzyme that catalyzes the reduction of non androgenic
the level of T within the metastatic tumors was found to be suf-
ficiently high to stimulate the proliferation of cancer cells. Thus,
the 17b-HSD3 enzyme represents a promising target for the treat-
ment of advanced prostate cancer by blocking the different
sources of active androgen T from testis (endocrine), adrenals
(endocrine) and intratumoral tissues (intracrine) (Fig. 1). Further-
more, the use of 17b-HSD3 inhibitors complementary to actual
endocrine therapy could increase the efficiency of antiandrogens,
LHRH agonists, and inhibitors of androgen biosynthesis enzymes
4-androstene-3,17-one (D
4-dione)9 to potent androgen T using
NADPH as cofactor.10–15 This enzyme is found principally in the
Leydig cells in the microtubule part of the testis and contributes
⇑
Corresponding author. Address: Laboratory of Medicinal Chemistry, Oncology
and Genomic Unit, CHUQ (CHUL)
– Research Center, 2705 Laurier Boulevard,
(5
a
-reductase and 17
a-lyase) by producing
a
synergistic
Quebec, Canada G1V 4G2. Tel: +1 418 654 2296; fax: +1 418 654 2761.
E-mail address: donald.poirier@crchul.ulaval.ca (D. Poirier).
effect.25,26
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.003

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4653
(Brain)
LH
(Adrenals)
(Bloodstream)
Δ4-dione
LH
17β-HSD3
Δ4-dione
17β-HSD3
Δ4-dione
T
T
17β-HSD5
17β-HSD15
5α-R
17β-HSD3
5α-R
Δ4-dione
T
DHT
DHT
AR
AR
(Testis)
(Metastases)
(Prostate tumors)
(Bloodstream)
Figure 1. Schematic view of the contribution of 17b-HSD3 in androgen testosterone (T) biosynthesis from the non androgenic 4-androstene-3,17-dione (
D
4-dione) in
different sources including the testis (endocrine), the adrenals (endocrine), and the intratumoral tumor tissues (intracrine). LH: Luteinizing hormone, 5
a-R: 5
a-reductase, AR:
androgen receptor, DHT: dihydrotestosterone, 17b-HSD5 and 17b-HSD15: types 5 and 15 of 17b-hydroxysteroid dehydrogenase.
(A)
2
(R ) ramified alkyl chain > linear alkyl chain
O > OH
O
and better inhibition for alkyl chain
O
between 1-4 carbons
CH > H
3
H
H
X
O
H
H
H
H
R2
N
R1
R
H
H
R
N
O
OH
H
O
H
R1
R2
H
H
3β chains > 3α chains
1
(R ) better inhibition for alkyl chain
between 5-8 carbons
(R) hydrophobic ester
chain well tolerated
OH > H and Oalkyl
O
O
O
(B)
O
N
H
N
N
O
N
O
O
O
N
O
HO
HO
N
HO
O
O
3
2
1
5
4
Series I
(tertiary amines)
O
O
Series II
(carbamates)
H
H
7a-j, 10a-j, 11a-j,
12a-e, 13a-d,
14a-c and 15a-c
H
H
R2
H
H
R1
N
O
N
R1
17a-j, 18, 21,
22 and 23a-g
O
H
OH
H
Figure 2. (A) Relative importance of different chemical groups (alkyl, alkylamide and carbamate derivatives) added at position 3b of androsterone steroid scaffold and their
effect on 17b-HSD3 inhibition as established from our previous SAR studies.27–29 (B) Representative first-generation inhibitors (compounds 1–5) and two new series of
second-generation inhibitors (compounds 7, 10–15, 17, 18, 22 and 23). See Tables 1–3 for the representation of all new synthesized and tested compounds.
Our previous structure–activity relationship (SAR) studies on
the inhibition of 17b-HSD3 have identified important criteria for
inhibitory activity (Fig. 2A).27–33 Despite their good efficiency in
homogenized HEK-293 cells overexpressing 17b-HSD3, these
inhibitors were however not optimal in different aspects. As an
example, first generation inhibitors of the 3b-alkyl-androsterone
series showed an androgenic profile on Shionogi (AR+) cells which
is obviously undesirable in the context of a treatment for an andro-
gen-sensitive disease. The inhibitory potency of these inhibitors
was also lower in intact than in homogenized HEK-293 cells over-
expressing 17b-HSD3, thus suggesting low cell permeability. To
provide 17b-HSD3 inhibitors with a non-androgenic profile and

4654
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
O
O
a
H
R1
H
H
(see Table 1
for R1 and R2)
N
R2
HO
O
H
6
7a-j
O
R
b
N
R1
c
N
HO
R
10 a-j (R = H)
11 a-j (R = CH )
(see Table 2
for R1)
O
3
O
R
R
d
N
N
R1
N
HO
HO
HN
(see Table 2
for R1)
O
R
12a-e (R = H)
13a-d (R = CH )
R
8 (R = H)
9 (R = CH3)
3
O
R
e
N
O
S
O
(see Table 2
for R1)
N
R1
HO
R
14a-c (R = H)
15a-c (R = CH )
3
Scheme 1. Reagents and conditions: (a) R1R2NH, ethanol, 60 °C; (b) piperazine or 2,5-dimethylpiperazine, ethanol, 60 °C; (c) R1CH2Br; TEA, DCM, rt; (d) R1COCl, TEA, DCM, rt;
(e) R1SO2Cl, TEA, DCM. The 2,5-dimethylpiperazine derivatives are in the trans configuration. The stereochemistry of carbons 5, 8, 9 and 14 is shown only for compound 6 but
is the same for all other steroids.
O
O
a
b
R
N
H
O
HO
16
6
O
O
c
R
N
O
N
O
17
O
O
18
R =
OCH3
(17c)
(17a)
(17b)
F3C
N
CF3
(17e)
CF3
CF3
(17d)
(17f)
CF3
CF3
(17h)
(17g)
(17i)
Scheme 2. Reagents and conditions: (a) R-NH2, ethanol, 60 °C; (b) triphosgene, DIPEA, DCM; (c) NaH, MeI, DMF (from 17b).
with more drug-like properties, we explored the enzyme tolerance
for new diversified substituents introduced at position 3 of the
androsterone (ADT) nucleus. As an extension of our previous work
illustrated with inhibitors 1–5, two series of ADT derivatives (ter-
tiary amines and carbamates) were designed, synthesized and
tested as new inhibitors of 17b-HSD3 (Fig. 2B).

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4655
19, which was treated with triphosgene to give the intermediate
carbamate 20. The TBDMS protecting group was then removed
using tetrabutylammonium fluoride (TBAF) in tetrahydrofuran
(THF) and the resulting primary alcohol 21 was transformed to
the corresponding bromide 22 using triphenylphosphine and car-
bon tetrabromide in DCM.
2. Results and discussion
2.1. Chemical synthesis
The ADT derivatives 7a–j were easily obtained in acceptable
yields (35–70%) from the opening in refluxing ethanol of the oxi-
rane 6 with a series of commercially available secondary amines
(Scheme 1). Similarly, the oxirane 6 was also opened with pipera-
zine or trans-2,5-dimethylpiperazine to give the intermediate
compounds 8 and 9. The free NH of 8 and 9 was next submitted
to a reaction with different kinds of building blocks (benzyl bro-
mide, acyl chloride and sulfonyl chloride) to obtain the corre-
sponding piperazine derivatives 10a–j and 11a–j (amines), 12a–e
and 13a–d (amides) and 14a–c and 15a–c (sulfonamides).
The carbamate derivatives 17a–i, 18, 21, 22 and 23a–g were
prepared following two different methods. In the first sequence
of reactions (Scheme 2), carbamates 17a–i and 18 were obtained
by reacting the appropriate secondary amines with the oxirane 6
in refluxing ethanol and cyclizing the resulting amino-alcohol
using triphosgene with diisopropylethylamine (DIPEA) in dichloro-
methane (DCM). The C16-dimethylation of 17b giving 18 was done
using sodium hydride as deprotonation base and an excess of MeI
as reagent of methylation. In the second sequence of reactions
(Scheme 3), the carbamates 23a–g were obtained by different N,
O and S-alkylation reactions of the key alkyl bromide intermediate
22, which was obtained from 6 in four steps. Briefly, the aminolysis
of oxirane 6 with aminoethanol-O-TBDMS gave the amino alcohol
2.2. Inhibition of 17b-HSD3 in homogenized cells
2.2.1. Tertiary amine derivatives
Our previous SAR study showed that a hydrophobic chain at po-
sition 3b of ADT was well tolerated by the 17b-HSD3.29 Unfortu-
nately, these compounds were found to stimulate the
proliferation of androgen-sensitive cells and to possess relatively
high hydrophobicity (high Log P value) as illustrated in Figure 2B
with phenylpropyl-ADT (1). We then turned our attention towards
the synthesis of tertiary alkylamine (series I) which seems more
promising considering their lower hydrophobic character as well
as their high potential for structural diversification. We first diver-
sified the ADT scaffold using various acyclic (7a–d) and cyclic (7e–
j) amines at position 3b and evaluated their potential as inhibitors
of 17b-HSD3 (Table 1). Thus the ability of compounds 7a–j to
inhibit the 17b-HSD3 transfected in human embryonic kidney
(HEK)-293 cells (homogenized) was done by measuring the
amount of labeled
D
T formed from natural labeled substrate
4-dione in the presence of NADPH as cofactor. The results were
expressed as the percent of inhibitory activity for
a given
O
O
b
a
TBDMSO
N
H
HO
O
19
6
O
O
c
d
HO
TBDMSO
N
O
N
O
O
20
O
21
O
O
e, f , g or h
R
Br
N
O
N
O
O
O
23
22
N
R =
O
S
N
O
O
(23a)
(23c)
(23b)
(23d)
O
N
N
N
(23f)
(23e)
(23g)
Scheme 3. Reagents and conditions: (a) NH2(CH2)2OTBDMS, ethanol, 60 °C; (b) triphosgene, DIPEA, DCM; (c) TBAF, THF, rt; (d) PPh3, CBr4, DCM, 0 °C to rt; (e) 3-methylphenol
(for 23a); (f) (i) 3-methylthiophenol, K2CO3, DMF, 80 °C; (ii) KHSO5, MeOH/H2O (for 23b); (g) appropriate R1R2NH, ethanol 60 °C (for 23c, 23d, 23e and 23f); (h) (i)
propylamine, Na2CO3, DMF, 80 °C; (ii) propionyl chloride, triethylamine, DCM, rt (for 23g).

4656
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
Table 1
Inhibition of 17b-HSD3 in homogenized and intact cells by various tertiary-amine derivatives of ADT (compounds 7a–j)a
O
H
H
H
R
OH
H
#
R
Log P
Homogenate inhibition
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
Intact cells inhibition
calcb
at 0.01
l
M (%)
at 0.1
85
lM (%)
at 1
lM (%)
at 0.1
lM (%)
at 1
lM (%)
Ph
N
N
7a
6.1
42
90
86
94
Ph
7b
7c
5.8
4.7
28
0
79
10
89
60
48
nd
92
14
N
7d
5.7
13
70
85
53
90
N
7e
7f
4.8
6.7
9
19
82
69
90
nd
nd
23
52
N
Ph
40
N
7g
7h
7i
4.7
3.8
5.9
0
0
4
0
69
40
89
nd
2
20
nd
68
N
N
N
N
13
78
nd
Ph
Ph
N
N
7j
5.6
35
72
90
37
80
N
N
a
For the transformation of [14C]-4-androstene-3,17-dione (50 nM) into [14C]-testosterone at the indicated concentration of tested compound. See Section 4 for the details
of the assay. Error 10%. nd: not determined.
b
The Log P values were calculated (Log P calc) using the CS Chemdraw Ultra software version 5.0.
compound. In the series of acyclic derivatives, compound 7a was
the most active inhibitor (42% at 0.01 M and 85% at 0.1 M).
For the cyclic derivatives, compounds 7f and 7j were the best
inhibitors with 40% and 35% inhibition at 0.01 M, respectively.
interesting point is the negative effect of the pyridine ring (com-
l
l
pound 10g) on the inhibition of 17b-HSD3 pointing out to a poor
tolerance for polar substituent. This result was in accordance with
tertiary amine series where compounds with Log P values <5.0
gave poor enzyme inhibitions and was in agreement with our pre-
vious results reported for 3b-substituted ADT derivatives, which
clearly showed the importance of a non-polar group for a good
inhibition.27 Secondly, the functional group Y (CH2, CO or SO2)
slightly and differently modulates the inhibitory activity when
the rest of the molecule is identical. As examples, the sulfonamide
derivative 14b shows a better inhibition at 0.01 lM (45%) than its
corresponding amide 12c (31%) and benzylamine 10b (18%) in the
ortho-CF3 series. On the other hand, the benzyl amine derivatives
10a gave a better inhibition at 0.01 lM (51%) than their corre-
l
Clearly, compounds 7c, 7e, 7g and 7h with Log P values <5.0 gave
the lowest values of inhibition. The piperazine derivative 7j was se-
lected from Table 1 as the more interesting compound in a drug
amenable point of view, and by considering its lower hydrophobic-
ity (Log P = 5.6) than corresponding piperidine 7f (Log P = 6.7) and
its larger potential of diversification. The piperazine derivative 7j
was however more interesting considering its lower hydrophobic-
ity than corresponding piperidine 7f as well as its larger potential
for diversification. The piperazine core is also well recognized as a
common motif in several drugs.34 For all these reasons, we selected
the piperazine core to extend our SAR study.
sponding sulfonamide 14a (21%) and amide 12d (10%) in the
meta-CF3 series, but same inhibitions were observed in the para-
CF3 series (compounds 10c, 12e and 14c).
2.2.2. Piperazine derivatives
Interesting SAR observations emerged from the piperazine
derivatives reported in Table 2. First, when different substituted
aryl groups (Z) are used within a benzylamine functionality
(Y = CH2) added on the 3b-piperazine-ADT nucleus (compounds
10a–j), we observed that inhibition values remained almost un-
changed either for compounds with aromatic bearing electron
withdrawing groups (CF3 and Cl) or electron donating group
(OCH3). In fact, only compound 10a, with a meta-CF3, gave better
inhibition of 17b-HSD3 than the reference compound 7j. Another
Although the use of trans-2,5-dimethylpiperazine moiety (X
group) did not increase the inhibition of the benzylamine or amide
series when compared to piperazine moiety, the most interesting
inhibition results were obtained in the trans-2,5-dimethylpipera-
zine derivatives having a sulfonamide function, namely com-
pounds 15b and 15c (55% and 79% of inhibition at 0.01
Thus, the sulfonamide 15c clearly gave a higher percentage of inhi-
bition (79% at 0.01 M) than its corresponding amide 13d (17%)
and amine 11c (14%). Two other important observations were also
lM).
l

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4657
Table 2
Inhibition of 17b-HSD3 in homogenized and intact cells: optimization of 3b-piperazine ADT series (compounds 10a–j, 11a–j, 12a–d, 13a–d, 14 a–c and 15a–c)a
O
H
H
H
Z
Y
X
OH
H
#
Z
Y
X
Log P
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
calcb
at 0.01
l
M (%)
at 0.1
l
M (%)
at 0.1
lM (%)
7j
CH2
CH2
N
N
N
N
5.6
6.5
36
74
38
10a
51
88
52
F3C
F3C
N
N
10b
10c
10d
CH2
CH2
CH2
6.5
6.5
8.0
18
14
3
86
81
91
20
33
CF3
N
N
N
N
nd (62)c
nd (75)c
F3CS
Cl
N
N
N
N
10e
CH2
6.1
26
85
10f
CH2
CH2
5.4
4.2
21
10
83
33
47
H3CO
N
10g
nd (63)c
N
N
N
N
CF3
10h
10i
10j
CH2
CH2
CH2
7.4
7.4
7.4
18
22
25
85
75
83
12
16
25
F3C
CF3
N
N
N
N
F3C
F3C
F3C
F3C
11a
CH2
7.1
31
89
nd (78)c
N
N
11b
11c
11d
CH2
CH2
CH2
7.1
7.1
8.6
22
14
18
85
87
88
36
N
N
N
N
N
N
CF3
F3C
36
nd (58)c
F3CS
(continued on next page)

4658
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
Table 2 (continued)
#
Z
Y
X
Log P
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
at 0.1 M (%)
calcb
at 0.01
l
M (%)
at 0.1
lM (%)
l
11e
11f
11g
CH2
CH2
CH2
6.8
6.1
4.9
26
84
nd (55)c
N
N
N
N
N
N
N
N
Cl
19
17
87
47
50
H3CO
N
nd (58)c
CF3
11h
11i
11j
CH2
CH2
CH2
8.0
8.0
8.0
16
15
25
71
71
74
23
21
16
F3C
CF3
N
N
N
N
F3C
F3C
F3C
12a
12b
CO
CO
N
N
N
N
N
N
5.0
5.1
17
19
76
75
50
nd (70)c
12c
CO
5.9
31
86
62
CF3
N
N
12d
12e
13a
CO
CO
CO
5.9
5.9
5.6
10
22
24
92
84
87
91
56
78
F3C
F3C
N
N
N
N
13b
13c
CO
CO
5.7
6.6
24
28
90
89
77
70
N
N
N
N
F3C
F3C
13d
14a
CO
6.6
5.8
17
21
91
82
61
N
N
N
N
SO2
nd (71)c
F3C
F3C
N
N
N
N
14b
14c
SO2
SO2
5.8
5.8
45
21
92
78
63
34
CF3

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4659
Table 2 (continued)
#
Z
Y
X
Log P
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
calcb
at 0.01
l
M (%)
at 0.1
l
M (%)
at 0.1
lM (%)
15a
SO2
6.5
6.5
6.5
32
91
77
N
N
N
N
N
N
F3C
15b
15c
SO2
SO2
55
79
92
92
84
47
CF3
F3C
a
For the transformation of [14C]-4-androstene-3,17-dione (50 nM) into [14C]-testosterone at the indicated concentration of the tested compound. See Section 4 for the
details of the assay. Error 10%. nd: not determined. The 2,5-dimethylpiperazine derivatives are in the trans configuration.
b
The Log P values were calculated (Log P calc) using the CS Chemdraw Ultra software version 5.0.
Tested at 1 lM.
c
made relatively to the steroid scaffold (data not shown). First, we
observed a strong negative impact on inhibition when we replaced
the C19-steroid nucleus of 7j by a C21-steroid nucleus. In fact, 17b-
HSD3 shows a clear preference for androstane over pregnane scaf-
by crossing the cell membrane. We thus used the results in intact
cells (Tables 1–3) as a second screen for selecting the best
inhibitors.
In Table 1, the results obtained in intact cells confirmed the
selection of tertiary amine 7a as the best inhibitor over 7f and 7j
fold (74% of inhibition at 0.1 lM vs 12%, respectively, when
Z = C6H5; Y = CH2 and X = piperazine). Secondly, the presence of
an insaturation at positions C4–C5 of the androstane derivative
7j had no important impact on enzyme inhibition.
(94%, 52% and 80% of inhibition at 1 lM, respectively). In Table 2,
only compounds 10a, 14b, 15b and 15c inhibited over 40% of the
activity in homogenized cells (51%, 45%, 55%, 79% of inhibition at
0.01
strated that 15b is the best candidate from this series of piperazine
ADT derivatives. Producing 84% of inhibition at 0.1 M, compound
lM, respectively), but the results in intact cells clearly demon-
2.2.3. Carbamate derivatives
We previously synthesized a small library of 3b-carbamate-ADT
derivatives that showed promising inhibitory activities on 17b-
HSD3.27 Despite the good results, the compounds possess an ester
group (see 4 and 5 in Fig. 2B) that is vulnerable to in vivo hydroly-
sis. In order to provide good inhibitors with a more stable chain on
the carbamate moiety, we synthesized a new series of analogue
compounds. We were especially interested in exploring the toler-
ance of the enzyme for different kinds of substituents (Table 3).
The first general observation that can be made about carbamate
derivatives is the good tolerance of the enzyme for all hydrophobic
chains, as represented by compounds 17a, 17b, 17d, 17f, 17g, 17h,
17i, 22 and 23a, in comparison with the weak inhibition obtained
with hydrophilic chains, as represented by compounds 17e, 21,
23b, 23c, 23e, 23f and 23g. This is particularly obvious when we
compare the alcohol 21 (Log P = 3.7) with the corresponding bro-
mide 22 (Log P = 5.0), and the p-CF3-pyridine derivative 17e
(Log P = 5.9) with the p-CF3-phenyl derivative 17d (Log P = 6.9).
The sulfone derivative 23b (Log P = 5.3) was also less potent than
corresponding ether 23a (Log P = 6.3). The addition of two methyl
groups at position C16 of the compound 17b was clearly not well
tolerated by the enzyme and, consequently, compound 18 weakly
inhibited the 17b-HSD3. Overall, a slight improvement of inhibi-
tion activity was obtained with compounds 17a, 17b, 17d, 17f,
17g and 23a relatively to reference compound 4 (D-5-4). It is also
expected that these new carbamate derivatives without an ester
side-chain as element of diversity could provide a better stability
and bioavailability.
l
15b is a more potent inhibitor in intact cells than 10a, 14b and 15c
which inhibited 52%, 63% and 47%, respectively, the transformation
of
D
4-dione into T. The carbamate derivatives represented in Ta-
ble 3 appear to be less potent inhibitors than the two other series
of compounds (Tables 1 and 2). In fact, only three compounds, 17a,
17c and 17d, inhibited over 40% of enzyme inhibition in intact cells
at the reported concentration of 0.1
lM. We however selected 17a,
which inhibited 44% of 17b-HSD3 activity at 0.1
lM in intact cells,
as a representative inhibitor of this series of carbamate-ADT
derivatives.
For purposes of comparison, we next proceeded to the IC50 val-
ues determination of the piperazine derivative 15b, the first gener-
ation inhibitor D-5-2 (5) and the natural substrate
D
4-dione used
itself as inhibitor. Being less potent in both homogenized and in-
tact cell assays, the carbamate derivative 17a was not selected
for IC50 determination as well as the tertiary amine derivative 7a
that was shown to be an androgenic compound (see Section 2.4).
From the curve of inhibition obtained in intact HEK-293 cells over-
expressing 17b-HSD3 (Fig. 3), 15b was found to be an eightfold
better inhibitor than reference compound 5 (IC50 = 6 and 51 nM,
respectively). It is also 56-fold better for inhibiting the transforma-
tion of labeled
D
D
4-dione into T (IC50 = 337 nM) than the unlabeled
4-dione used itself as an inhibitor.
2.4. Proliferative (androgenic) activity on Shionogi cells
In the context of a treatment of androgen-dependent diseases,
an important criterion to provide valuable 17b-HSD3 candidates
is their non-androgenic character or their ability not to activate
the androgen receptor (AR). To address this, we evaluated the ago-
nist (proliferative) activity on androgen-sensitive (AR+) Shionogi
cells of new inhibitors 7a, 15b and 17a in comparison with the first
generation inhibitors D-5-2 (5) and CS-213 (Fig. 4). In this assay,
the basal cell proliferation (control) was set as 100%, the potent
2.3. Inhibition of 17b-HSD3 in intact cells
After having established the inhibitory activity of all com-
pounds in homogenized HEK-293 cells, we determined their capa-
bility to exert their action in intact cells overexpressing 17b-HSD3.
This is obviously a crucial step towards further development of a
potent inhibitor because a good inhibitor must enter into the cell

4660
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
Table 3
Inhibition of 17b-HSD3 in homogenized and intact cells: optimization of 3-carbamate ADT series (compounds 17a–i, 18, 21, 22 and 23a–g)a
O
R2
R2
H
H
H
R1
N
O
H
O
#
R1
R2
Log P
Homogenate inhibition
at 0.01 M (%)
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
calcb
l
at 0.1
l
M (%)
at 1
lM (%)
at 0.1
lM (%)
O
O
4 (D-5-4)27
H
5.2
2
79
94
49
N
O
17a
17b
H
H
5.9
6.4
21
17
86
81
93
93
44
38
17c
17d
17e
H
H
H
5.8
6.9
5.9
5
25
3
84
80
65
93
nd
nd
43
43
36
H3CO
F3C
F3C
N
17f
H
H
6.9
6.9
17
16
81
80
nd
nd
35
36
CF3
F3C
F3C
17g
17h
H
7.8
12
66
nd
35
CF3
17i
18
H
5.7
7.7
13
2
75
22
78
74
29
18
CH3
16c
31
HO
Br
21
22
H
H
3.7
5.0
0
9
38
93
17
69
23a
23b
H
H
6.3
5.4
13
0
85
52
95
93
40
5
O
O2
S
23c
23d
H
H
4.9
6.3
0
0
21
93
10c
40
N
14
71
N
N
23e
H
6.1
2
21
72
2

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4661
Table 3 (continued)
#
R1
R2
Log P
Homogenate inhibition
Homogenate inhibition
Homogenate inhibition
Intact cells inhibition
calcb
at 0.01
lM (%)
at 0.1
l
M (%)
at 1
lM (%)
at 0.1
lM (%)
23f
H
H
5.8
4.9
0
10
62
8c
N
O
23g
0
35
80
25
N
a
For the transformation of [14C]-4-androstene-3,17-dione (50 nM) into [14C]-testosterone at the indicated concentration of tested compound. See Section 4 for the details
of the assay. Error 10%. nd: not determined.
b
The Log P values were calculated (Log P calc) using the CS Chemdraw Ultra software version 5.0.
Tested in another experiment.
c
androgen dihydrotestosterone (0.1
l
M) stimulated the cell prolif-
to the inhibitor D-5-2, which did not significantly stimulate cell
proliferation, the inhibitor CS-213 is fully androgenic inducing
eration to 320% and the antiandrogen (AR antagonist) hydroxy-flu-
tamide35 (OH-Flu) did not stimulate the cell proliferation. Contrary
225% and 396% of basal cell proliferation at 0.1 and 1
tively. Clearly, the tertiary amine 7a showed a strong proliferative
effect (292% and 314% at 0.1 and 1 M, respectively). However, at
lM, respec-
l
these two concentrations, the carbamate derivative 17a and piper-
azine derivative 15b did not stimulate the proliferation of AR+ cells,
thus suggesting no androgenic activity.
3. Conclusion
Two different series of ADT derivatives at position 3, the tertiary
amines and the carbamates, were synthesized and tested as new
inhibitors of 17b-HSD3. In the first series, the piperazine-methyl
core at position 3b of ADT was selected as the most interesting
building block regarding the inhibitory activity and its capacity
to easily generate a large diversity of compounds. Different types
of substituted piperazine derivatives were then synthesized
including benzylamines, sulfonamides and amides. From these
compounds, the sulfonamide 15b was identified as a non-andro-
genic inhibitor with strong inhibitory activity (IC50 = 6 nM) for
the transformation of
D
4-dione into T in intact transfected HEK-
293 cells overexpressing 17b-HSD3. In the second series, we
introduced different substituents (amide, amines, sulfonamides,
sulfones and pyridine) on the carbamate functionality at position
3 of ADT. The most potent inhibitors were compounds with a
hydrophobic chain such as the benzylic derivative 17a. In intact
cells, however, the inhibitory activity produced by the carbamate
compounds was clearly lower than those found in the piperazine
Figure 3. Inhibition of the transformation of [14C]-4-androstene-3,17-dione ([14C]-
D
intact HEK-293 cells. See Section 4 for the details of the assay.
4-dione) (50 nM) into [14C]-testosterone ([14C]-T) by 17b-HSD3 overexpressed in
series (44% inhibition at 0.1
lM for benzyl carbamate derivative
17a vs 84% inhibition at 0.1
lM for sulfonamide piperazine 15b).
We thus chose compound 15b as the most interesting candidate
relatively to (1) its inhibitory activity on 17b-HSD3 in both homog-
enized and intact HEK-293 cells overexpressing the target enzyme
and (2) its non-androgenic activity as assessed by the proliferation
of androgen-sensitive Shionogi cells. Considering the potentially
better in vivo stability of 15b relatively to the first generation of
analogue inhibitors (Fig. 2), it will be used for further in vivo stud-
ies aimed to evaluate its capacity to reduce the formation of testos-
terone in rats or to reduce the growth of prostate cancer tumors.
4. Experimental
4.1. Chemical synthesis
General information: The reagents for chemical synthesis were
purchased from Sigma–Aldrich Canada Ltd (Oakville, ON, Canada).
The usual solvents were obtained from Fisher Scientific (Montreal,
QC, Canada) and were used as received. Anhydrous tetrahydrofu-
ran (THF) and anhydrous dichloromethane (DCM) were from
Figure 4. Proliferative effect (androgenic activity) on androgen-sensitive Shionogi
cells induced by androgen dihydrotestosterone (DHT), antiandrogen hydroxy-
flutamide (OH-Flu) and 17b-HSD3 inhibitors CS-213, D-5-2 (5), 7a, 15b and 17a.
Data are expressed as means SEM of one experiment in triplicate.

4662
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
Sigma–Aldrich. Thin-layer chromatography (TLC) and flash-col-
umn chromatography were performed on 0.20-mm Silica Gel 60
F254 plates and with Silicycle R10030B 230–400-mesh silica gel
(Quebec, QC, Canada). Nuclear magnetic resonance (NMR) spectra
were recorded at 400 MHz for 1H and 100.6 MHz for 13C with a
Bruker Avance 400 digital spectrometer (Billerica, MA, USA) The
chemical shifts (d) are expressed in ppm and referenced to acetone
(2.06 ppm, 1H), chloroform (7.26 ppm, 1H and 77.0 ppm, 13C) or
methanol (3.33 ppm, 1H). 1H NMR signals were reported as s (sin-
glet), d (doublet), t (triplet), q (quartet), m (multiplet) and broad
(br). We also used d or t for a signal that looks alike but is not nec-
essary a true d or t since it comes from a higher-order spectra in-
(s, 2H), 2.37 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.58 (q, J = 7.1 Hz,
4H); LRMS for C24H42NO2 [M+H]+ 376.2.
4.1.1.4. (3a,5a)-3-[(Dibutylamino)methyl]-3-hydroxyandro-
stan-17-one (7d). 16 mg, 22%; 1H NMR (acetone-d6) d 0.81 (s,
3H), 0.84 (s, 3H), 0.91 (t, J = 7.1 Hz, 6H), 0.80-2.10 (m, 30H), 2.32
(s, 2H), 2.37 (dd, J1 = 8.7 Hz, J2 = 18.1 Hz, 1H), 2.51 (t, J = 7.4 Hz,
4H); LRMS for C28H50NO2 [M+H]+ 432.2.
4.1.1.5. (3a,5a)-3-Hydroxy-3-(piperidin-1-ylmethyl)androstan-
17-one (7e). 25 mg, 39%; 1H NMR (acetone-d6) d 0.81 (s, 3H),
0.84 (s, 3H), 0.75-2.09 (m, 28H), 2.18 (s, 2H), 2.37 (dd, J1 = 8.6 Hz,
J2 = 18.2 Hz, 1H), 2.52 (br s, 4H); LRMS for C25H42NO2 [M+H]+ 388.2.
stead of
a first-order spectra. Low-resolution mass spectra
(LRMS) were recorded on a Perkin-Elmer Sciex API-150ex appara-
tus (Foster City, CA, USA) equipped with a turbo ion-spray source
and expressed in m/z. High-resolution mass spectra (HRMS) were
provided by Pierre Audet at the Laval University Chemistry Depart-
ment (Quebec, QC, Canada). High-performance liquid chromatog-
raphy (HPLC) analyses were carried out using a Waters system
(Milford, MA, USA) equipped with a UV detector (207 nm), a re-
4.1.1.6. (3a,5a)-3-[(4-Benzylpiperidin-1-yl)methyl]-3-hydroxy-
androstan-17-one (7f). 34 mg, 43%; 1H NMR (acetone-d6) d
0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 27H), 2.20 (m, 4H), 2.37
(dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.53 (d, J = 6.9 Hz, 2H), 2.88 (br
d, J = 11.4 Hz, 2H), 7.18 (d, J = 6.7 Hz, 3H), 7.27 (m, 2H); LRMS for
C
32H48NO2 [M+H]+ 478.1.
verse-phase column (Luna C18(2) 100A, 100 ꢀ 4.6 mm, 3
lm or
Luna Phenyl-Hexyl, 75 ꢀ 4.6 mm, 3 m) from Phenomenex (Tor-
l
4.1.1.7. (3a,5a
)-3-(1,40-Bipiperidin-10-ylmethyl)-3-hydroxyan-
rance, CA, USA) and using an appropriate system of solvents (meth-
anol and water). The HPLC purity was determined for key
representative compounds, but only compounds with purity over
90% were used in the biological screening assays (inhibition of
17b-HSD3). The names of new compounds were obtained using -
ACD/Labs (Chemist’s version) software (Toronto, ON, Canada).
drostan-17-one (7g). 28 mg, 36%; 1H NMR (acetone-d6) d 0.81
(s, 3H), 0.84 (s, 3H), 0.75-2.15 (m, 33H), 2.21 (s, 2H), 2.25 (m,
2H), 2.38 (dd, J1 = 8.8 Hz, J2 = 18.2 Hz, 1H), 2.47 (m, 4H), 2.93 (br
d, J = 11.5 Hz, 2H); LRMS for C30H51N2O2 [M+H]+ 471.3.
4.1.1.8. (3a,5a)-3-Hydroxy-3-[(4-methylpiperazin-1-yl)methyl]-
androstan-17-one (7h). 29 mg, 43%; 1H NMR (acetone-d6) d
0.81 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 22H), 2.16 (s, 3H), 2.23 (s,
2H), 2.34 (br m, 4H), 2.37 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.58
(br s, 4H); LRMS for C25H43N2O2 [M+H]+ 403.2.
4.1.1. General procedure for synthesis of 3b-substituted-3a-
hydroxy-androstan-17-one derivatives (7a–j)
To a solution of oxirane 631 (50 mg, 0.165 mmol) in anhydrous
ethanol (3 mL) was added the appropriate amine (0.5 mmol) and
the solution was stirred for 5 h at 60 °C. The solvent was then evap-
orated and the resulting mixture was dissolved in dichloromethane
(10 mL) and methylisocyanate resin (300 mg, 1.8 mmol/g) was
added. The suspension was stirred for 2 h and filtered to give the
corresponding crude amine, which was purified by flash chroma-
tography using EtOAc/hexanes (2:8) as eluent to give the desired
amines 7a–j. All compounds were characterized by 1H NMR and
MS analyses whereas 13C NMR, HRMS and HPLC data were added
for the key representative compound 7a.
4.1.1.9. (3a,5a)-3-Hydroxy-3-[(4-phenylpiperazin-1-yl)methyl]-
androstan-17-one (7i). 54 mg, 70%; 1H NMR (acetone-d6) d 0.83
(s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 21H), 2.32 (s, 2H), 2.37 (dd,
J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.75 (m, 4H), 3.18 (m, 4H), 6.77 (t,
J = 7.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.7 Hz, 2H); LRMS
for C30H45N2O2 [M+H]+ 465.0.
4.1.1.10. (3a,5a)-3-[(4-Benzylpiperazin-1-yl)methyl]-3-hydroxy
androstan-17-one (7j). 60 mg, 76%; 1H NMR (acetone-d6) d 0.81
(s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 22H), 2.24 (s, 2H), 2.37 (dd,
J1 = 8.8 Hz, J2 = 18.3 Hz, 1H), 2.43 (br s, 4H), 2.61 (br s, 4H), 3.47 (s,
2H), 7.23 (m, 1H), 7.32 (m, 4H); LRMS for C31H47N2O2 [M+H]+ 478.9.
4.1.1.1.
(3a,5a)-3{[Benzyl(ethyl)amino]methyl}3-hydroxyan-
drostan-17-one (7a). 15 mg, 20%; 1H NMR (acetone-d6) d 0.82
(s, 3H), 0.84 (s, 3H), 1.01 (t, J = 7.1 Hz, 3H), 0.80-2.10 (m, 22H),
2.37 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 2.45 (s, 2H), 2.52 (q,
J = 7.1 Hz, 2H), 3.74 (s, 2H), 7.23 (t, J = 7.2 Hz, 1H), 7.32 (t,
J = 7.7 Hz, 2H), 7.39 (d, J = 7.5 Hz, 2H); 13C NMR (CDCl3) d 11.3,
12.1, 13.8, 20.2, 21.8, 28.4, 30.8, 31.6, 32.9, 34.0, 35.1, 35.9 (2ꢀ),
39.8, 40.8, 47.8, 49.4, 51.4, 54.3, 60.8, 65.3, 70.0, 127.1, 128.4
(2ꢀ), 128.6 (2ꢀ), 139.5, 221.6; LRMS for C29H44NO2 [M+H]+
438.2; HRMS calcd for C29H44NO2 [M+H]+ 438.3367, found
438.3374; HPLC purity = 99.2% (RT = 7.2 min, 96:4 MeOH/H2O-iso-
cratic, Luna C18 column).
4.1.2. Synthesis of intermediates 8 and 9
To a solution of oxirane 631 (1.0 g, 3.3 mmol) in anhydrous eth-
anol (15 mL) was added piperazine (573 mg, 6.7 mmol) or trans-
2,5-dimethylpiperazine (755 mg, 6.7 mmol). The solutions were
stirred for 5 h at 60 °C. The resulting solutions were poured into
water and extracted three times with EtOAc. The organic layers
were washed with brine, dried with MgSO4, filtered and evapo-
rated under reduced pressure to give crude compounds 8 and 9
which were purified by flash chromatography using EtOAc/hex-
anes (1:1) as eluent.
4.1.1.2. (3a,5a)-3-{[Benzyl(methyl)amino]methyl}-3-hydroxy-
androstan-17-one (7b). 29 mg, 41%; 1H NMR (acetone-d6) d
0.82 (s, 3H), 0.84 (s, 3H), 0.80-2.10 (m, 22H), 2.25 (s, 3H), 2.37
(dd, J1 = 9.0 Hz, J2 = 18.4 Hz, 1H), 2.41 (s, 2H), 3.63 (s, 2H), 7.24 (t,
J = 7.2 Hz, 1H), 7.32 (t, J = 7.4 Hz, 2H), 7.37 (d, J = 7.2 Hz, 2H); LRMS
for C28H42NO2 [M+H]+ 424.2.
4.1.2.1. (3a,5a)-3-Hydroxy-3-(piperazin-1-ylmethyl)androstan-
17-one (8). 750 mg, 58%; 1H NMR (MeOH-d4) d 0.84 (s, 3H), 0.89
(s, 3H), 1.01 (t, J = 7.1 Hz, 3H), 0.80-2.15 (m, 20H), 2.25 (s, 2H), 2.45
(dd, J1 = 8.6 Hz, J2 = 19.2 Hz, 1H), 2.58 (br s, 4H), 2.84 (t, J = 4.8 Hz,
4H); LRMS for C24H41N2O2 [M+H]+ 389.6.
4.1.1.3.
(3
a
,5
a
)-3-[(Diethylamino)methyl]-3-hydroxyandro-
4.1.2.2. (3a,5a)-3-{[trans-2,5-Dimethylpiperazin-1-yl]methyl}-
stan-17-one (7c). 40 mg, 65%; 1H NMR (acetone-d6) d 0.81 (s,
3H), 0.84 (s, 3H), 0.99 (t, J = 7.1 Hz, 6H), 0.75-2.08 (m, 22H), 2.30
3-hydroxyandrostan-17-one (9). 650 mg, 47%; 1H NMR (CDCl3)
d 0.77 (s, 3H), 0.85 (s, 3H), 0.99 (d, J = 6.0 Hz, 3H), 1.00 (d,

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4663
J = 6.1 Hz, 3H), 0.75-1.85 (m, 22H), 1.94 (m, 1H), 2.04 (m, 3H), 2.25
(m, 2H), 2.43 (dd, J1 = 8.6 Hz, J2 = 18.9 Hz, 1H), 2.54 (dt, J1 = 2.8 Hz,
J2 = 9.8 Hz, 1H), 2.61 (d, J = 13.9 Hz, 1H), 2.90 (m, 3H); LRMS for
4.1.3.7. (3a,5a)-3-Hydroxy-3-{[4-(pyridin-3-ylmethyl)piperazin-
1-yl]methyl}androstan-17-one (10g). 20 mg, 65%; 1H NMR
(CDCl3) d 0.76 (s, 3H), 0.85 (s, 3H), 0.78-2.10 (m, 22H), 2.27 (s, 2H),
2.43 (br m, 5H), 2.65 (br s, 4H), 3.51 (s, 2H), 7.24 (d, J = 4.9 Hz,
1H), 7.65 (d, J = 7.8 Hz, 1H), 8.51 (dd, J1 = 1.3 Hz, J2 = 4.8 Hz, 1H),
8.54 (d, J = 1.4 Hz, 1H); LRMS for C30H46N3O2 [M+H]+ 480.2.
C
26H45N2O2 [M+H]+ 417.3.
4.1.3. General procedure for the synthesis of amines 10a–j and
11a–j
To a solution of compound 8 (25 mg, 0.064 mmol) or compound
9 (30 mg, 0.072 mmol) in anhydrous dichloromethane (5 mL) was
added triethylamine (24 mg, 0.24 mmol, 33 lL) and the appropri-
ate benzyl bromide (0.12 mmol). The mixture was stirred over-
night and the resulting solution was evaporated and purified by
flash chromatography using EtOAc/hexanes (3:7) to give the ben-
zylamines 10a–j and 11a–j. All compounds were characterized
by 1H NMR and MS analyses whereas 13C NMR, HRMS and HPLC
data were added for the key representative compounds 10a and
11a.
4.1.3.8. (3a,5a)-3-({4-[2,5-Bis(trifluoromethyl)benzyl]pipera-
zin-1-yl}methyl)-3-hydroxyandrostan-17-one (10h). 27 mg,
69%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.78-2.10 (m,
22H), 2.29 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.3 Hz, 1H), 2.51 (br
s, 4H), 2.68 (br s, 4H), 3.69 (s, 2H), 7.59 (d, J = 8.1 Hz, 1H), 7.75 (d,
J = 8.2 Hz, 1H), 8.12 (s, 1H); LRMS for C33H45F6N2O2 [M+H]+ 615.5.
4.1.3.9. (3a,5a)-3-({4-[2,4-Bis(trifluoromethyl)benzyl]pipera-
zin-1-yl}methyl)-3-hydroxyandrostan-17-one (10i). 27 mg,
69%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.83-2.10 (m,
22H), 2.28 (s, 2H), 2.43 (dd, J1 = 8.5 Hz, J2 = 19.1 Hz, 1H), 2.51 (br
s, 4H), 2.67 (br s, 4H), 3.70 (s, 2H), 7.77 (d, J = 8.3 Hz, 1H), 7.88 (s,
1H), 7.98 (d, J = 8.2 Hz, 1H); LRMS for C33H45F6N2O2 [M+H]+ 615.2.
4.1.3.1.
(3a,5a)-3-Hydroxy-3-({4-[3-(trifluoromethyl)benzyl]-
piperazin-1-yl}methyl)androstan-17-one (10a). 31 mg, 89%;
1H NMR (CDCl3) d 0.76 (s, 3H), 0.86 (s, 3H), 0.75-2.10 (m,
22H), 2.27 (s, 2H), 2.44 (br m, 5H), 2.66 (br s, 4H), 3.54 (s,
2H), 7.42 (t, J = 7.6 Hz, 1H), 7.50 (br d, J = 7.5 Hz, 2H), 7.58 (s,
1H); 13C NMR (CDCl3) d 11.2, 13.8, 20.2, 21.8, 28.3, 30.8, 31.5,
32.7, 33.8, 35.1, 35.8, 35.9, 39.6, 40.7, 47.8, 51.4, 54.2, 55.7
4.1.3.10. (3a,5a)-3-({4-[3,5-Bis(trifluoromethyl)benzyl]pipera-
zin-1-yl}methyl)-3-hydroxyandrostan-17-one (10j). 29 mg,
74%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m,
22H), 2.28 (s, 2H), 2.43 (br m, 5H), 2.67 (br s, 4H), 3.59 (s, 2H),
7.77 (s, 1H), 7.79 (s, 2H); LRMS for C33H45F6N2O2 [M+H]+ 615.5.
(4ꢀ), 62.3, 69.0, 70.1, 124.0 (q, JC–C–C–F = 3.6 Hz), 124.2 (q, JC–F
=
=
272 Hz), 125.6 (q, JC–C–C–F = 3.8 Hz), 128.7, 130.6 (q, JC–C–F
32 Hz), 132.4, 139.2, 221.5; LRMS for
C
32H46F3N2O2 [M+H]+
4.1.3.11. (3a,5a)-3-({trans-2,5-Dimethyl-4-[3-(trifluoromethyl)-
547.3; HRMS calcd for C32H46F3N2O2 [M+H]+ 547.3506, found
547.3510; HPLC purity: 97.2% (RT = 5.5 min, 96:4 MeOH/H2O-
isocratic, Luna C18 column).
benzyl]piperazin-1-yl}methyl)-3-hydroxyandrostan-17-one
(11a). 25 mg, 61%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H),
0.92 (d, J = 6.2 Hz, 3H), 1.10 (d, J = 6.0 Hz, 3H), 0.80-2.10 (m,
23H), 2.32-2.60 (m, 6H), 2.91 (d, J = 11.1 Hz, 1H), 3.11 (d,
J = 13.7 Hz, 1H), 3.22 (d, J = 16.1 Hz, 1H), 4.07 (d, J = 13.7 Hz, 1H),
7.43 (m, 1H), 7.51 (d, J = 7.8 Hz, 2H), 7.58 (s, 1H); 13C NMR (CDCl3)
d 11.3, 13.8, 17.2 (ꢁ), 18.2 (ꢁ), 20.2, 21.8, 28.3, 30.8, 31.6, 32.7, 33.9,
35.1, 35.9 (2ꢀ), 39.4, 40.8, 47.8, 51.4, 54.3, 55.8, 56.3, 57.5, 59.8 (ꢁ),
63.8 (2ꢀ), 69.4, 123.7 (q, JC–C–C–F = 3.6 Hz), 125.0 (q, JC–F = 273 Hz),
125.4 (q, JC–C–C–F = 3.6 Hz), 128.6, 130.5 (q, JC–C–F = 32 Hz), 132.1,
140.3, 221.6 [ꢁ: a very weak or no signal observed although a cor-
relation in HSQC experiment]; LRMS for C34H50F3N2O2 [M+H]+
575.2; HRMS for C34H50F3N2O2 [M+H]+ 575.3819, found
575.3826; HPLC purity: 99.0% (RT = 6.6 min, 96:4 MeOH/H2O-iso-
cratic, Luna C18 column).
4.1.3.2.
(3a,5a)-3-Hydroxy-3-({4-[2-(trifluoromethyl)benzyl]-
piperazin-1-yl}methyl)androstan-17-one (10b). 33 mg, 95%;
1H NMR (CDCl3) d 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H),
2.27 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.2 Hz, 1H), 2.50 (br s, 4H),
2.66 (br, s, 4H), 3.65 (s, 2H), 7.33 (t, J = 7.7 Hz, 1H), 7.51 (d,
J = 6.7 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.7 Hz, 1H); LRMS
for C32H46F3N2O2 [M+H]+ 547.1.
4.1.3.3.
(3a,5a)-3-Hydroxy-3-({4-[4-(trifluoromethyl)benzyl]-
piperazin-1-yl}methyl)androstan-17-one (10c). 32 mg, 92%;
1H NMR (CDCl3) d 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H),
2.27 (s, 2H), 2.45 (br m, 5H), 2.65 (br s, 4H), 3.54 (s, 2H); 7.44 (d,
J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H); LRMS for C32H46F3N2O2
[M+H]+ 547.2.
4.1.3.12. (3a,5a)-3-({trans-2,5-Dimethyl-4-[2-(trifluoromethyl)-
benzyl]piperazin-1-yl}methyl)-3-hydroxyandrostan-17-one
(11b). 17 mg, 41%; 1H NMR (CDCl3) d 0.78 (s, 3H), 0.86 (s, 3H),
0.93 (d, J = 6.2 Hz, 3H), 1.05 (d, J = 6.0 Hz, 3H), 0.75-2.10 (m,
23H), 2.33-2.60 (m, 6H), 2.93 (d, J = 11.1 Hz, 1H), 3.23 (br s, 1H),
3.31 (d, J = 15.1 Hz, 1H), 4.07 (d, J = 14.8 Hz, 1H), 7.31 (t,
J = 7.7 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.87
(d, J = 7.8 Hz, 1H); LRMS for C34H50F3N2O2 [M+H]+ 575.3.
4.1.3.4. (3a,5a)-3-Hydroxy-3-[(4-{3-[(trifluoromethyl)sulfanyl]-
benzyl}piperazin-1-yl)methyl]androstan-17-one (10d). 25 mg,
68%; 1H NMR (CDCl3) d 0.76 (s, 3H), 0.86 (s, 3H), 0.78-2.10 (m,
22H), 2.27 (s, 2H), 2.45 (br m, 5H), 2.65 (br s, 4H), 3.52 (s, 2H),
7.37 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.54 (d, J = 7.7 Hz,
1H), 7.62 (s, 1H); LRMS for C32H46F3N2O2S [M+H]+ 579.3.
4.1.3.13. (3a,5a)-3-({trans-2,5-Dimethyl-4-[4-(trifluoromethyl)-
4.1.3.5. (3
a
,5
a
)-3-{[4-(3-Chlorobenzyl)piperazin-1-yl]methyl}-
benzyl]piperazin-1-yl}methyl)-3-hydroxyandrostan-17-one
(11c). 22 mg, 53%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H),
0.92 (d, J = 6.2 Hz, 3H), 1.10 (d, J = 6.0 Hz, 3H), 0.75-2.15 (m,
24H), 2.30-2.60 (m, 6H), 2.91 (d, J = 11.5 Hz, 1H), 3.11 (d,
J = 13.6 Hz, 1H), 3.20 (br s, 1H), 4.07 (d, J = 13.9 Hz, 1H), 7.37 (d,
J = 7.7 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H); LRMS
for C34H50F3N2O2 [M+H]+ 575.2.
3-hydroxyandrostan-17-one (10e). 31 mg, 94%; 1H NMR (CDCl3)
d 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.27 (s, 2H), 2.43 (br
m, 5H), 2.65 (br s, 4H), 3.46 (s, 2H), 7.19 (m, 1H), 7.23 (m, 2H), 7.33
(s, 1H); LRMS for C31H46ClN2O2 [M+H]+ 513.3.
4.1.3.6. (3a,5a)-3-Hydroxy-3-{[4-(3-methoxybenzyl)piperazin-
1-yl]methyl}androstan-17-one (10f). 21 mg, 64%; 1H NMR
(CDCl3) d 0.76 (s, 3H), 0.85 (s, 3H), 0.78-2.10 (m, 22H), 2.26 (s,
2H), 2.43 (br m, 5H), 2.65 (br s, 4H), 3.48 (s, 2H), 3.81 (s, 3H),
6.80 (d, J = 7.5 Hz, 1H), 6.89 (m, 2H), 7.23 (t, J = 8.0 Hz, 1H); LRMS
for C32H49N2O3 [M+H]+ 509.3.
4.1.3.14. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{3-[(trifluoromethyl)-
sulfanyl]benzyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-
17-one (11d). 15 mg, 34%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s,
3H), 0.92 (d, J = 6.2 Hz, 3H), 1.10 (d, J = 6.0 Hz, 3H), 0.75-2.10 (m,

4664
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
23H), 2.30-2.60 (m, 6H), 2.91 (d, J = 10.5 Hz, 1H), 3.10 (d,
J = 13.2 Hz, 1H), 3.21 (d, J = 16.2 Hz, 1H), 4.04 (d, J = 13.1 Hz, 1H),
7.36 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.53 (d, J = 8.5 Hz,
1H), 7.62 (s, 1H); LRMS for C34H50F3N2O2S [M+H]+ 607.2.
HPLC data were added for the key representative compounds 12a,
12c and 13a.
4.1.4.1. (3a,5a)-3-Hydroxy-3-{[4-(phenylcarbonyl)piperazin-1-
yl]methyl}androstan-17-one (12a). 33 mg, 64%; 1H NMR (CDCl3)
d 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m, 22H), 2.31 (s, 2H), 2.43
(dd, J1 = 8.7 Hz, J2 = 19.2 Hz, 1H), 2.57 and 2.70 (br 2s, 4H), 3.43
(br s, 2H), 3.79 (br s, 2H), 7.40 (m, 5H); 13C NMR (CDCl3) d 11.2,
13.8, 20.2, 21.7, 28.3, 30.8, 31.5, 32.4, 33.6, 35.0, 35.8, 35.9, 39.3,
40.6, 47.8, 51.4, 54.1, 55.4 (2ꢀ), 55.7 (2ꢀ), 69.1, 70.5, 127.0 (2ꢀ),
128.5 (2ꢀ), 129.8, 135.5, 170.3, 221.5; LRMS for C31H45N2O3
[M+H]+ 493.0; HRMS calcd for C31H45N2O3 [M+H]+ 493.3425, found
493.3433; HPLC purity: 94.8% (RT = 10.2 min, 75:25 to 5:95 MeOH/
H2O-linear gradient in 20 min, Luna Phenyl-Hexyl column).
4.1.3.15.
(3a,5a)-3-{[trans-4-(3-Chlorobenzyl)-2,5-dimethylpi-
perazin-1-yl]methyl}-3-hydroxyandrostan-17-one (11e). 12 mg,
31%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.93 (d,
J = 6.2 Hz, 3H), 1.09 (d, J = 6.0 Hz, 3H), 0.75-2.10 (m, 23H), 2.30-
2.62 (m, 6H), 2.89 (d, J = 11.2 Hz, 1H), 3.03 (d, J = 13.4 Hz, 1H),
3.22 (br s, 1H), 4.00 (d, J = 13.3 Hz, 1H), 7.18 (t, J = 7.2 Hz, 1H),
7.23 (s, 2H), 7.33 (s, 1H); LRMS for C33H5035ClN2O2 [M+H]+ 541.3.
4.1.3.16. (3a,5a)-3-Hydroxy-3-{[trans-4-(3-methoxybenzyl)-2,5-
dimethylpiperazin-1-yl]methyl}androstan-17-one (11f). 14 mg,
36%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d,
J = 6.0 Hz, 3H), 1.11 (d, J = 5.3 Hz, 3H), 0.75-2.10 (m, 24H), 2.30-
2.80 (m, 6H), 2.89 (d, J = 10.2 Hz, 1H), 3.07 (d J = 13.2 Hz, 1H),
3.81 (s, 3H), 4.01 (d, J = 12.8 Hz, 1H), 6.79 (m, 1H), 6.89 (m, 2H),
7.22 (m, 1H); LRMS for C34H53N2O3 [M+H]+ 537.4.
4.1.4.2. (3a,5a)-3-{[4-(Cyclohexylcarbonyl)piperazin-1-yl]methyl}-
3-hydroxyandrostan-17-one (12b). 30 mg, 58%; 1H NMR (CDCl3)
d 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m, 21H), 2.29 (s, 2H), 2.43 (m,
2H), 2.60 (m, 4H), 2.92 (br s, 1H), 3.49 (br s, 2H), 3.61 (br s, 2H);
LRMS for C31H51N2O3 [M+H]+ 499.3.
4.1.3.17. (3a,5a)-3-{[trans-2,5-Dimethyl-4-(pyridin-3-ylmethyl)-
4.1.4.3. (3a,5a)-3-Hydroxy-3-[(4-{[2-(trifluoromethyl)phenyl]-
piperazin-1-yl]methyl}-3-hydroxyandrostan-17-one (11g). 10 mg,
27%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.92 (d, J = 6.0 Hz,
3H), 1.12 (d, J = 5.4 Hz, 3H), 0.75-2.10 (m, 22H), 2.30-2.68 (m, 7H),
2.90 (d, J = 11.0 Hz, 1H), 3.10 (m, 2H), 4.02 (d, J = 13.2 Hz, 1H), 7.25
(s, 1H), 7.64 (m, 1H), 8.52 (m, 2H); LRMS for C32H50N3O2 [M+H]+
508.3.
carbonyl}-piperazin-1-yl)methyl]androstan-17-one (12c). 30 mg,
52%; 1H NMR (CDCl3) d 0.76 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m,
22H), 2.30 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.1 Hz, 1H), 2.53 (m,
2H), 2.70 (br s, 2H), 3.18 (t, J = 5.0 Hz, 2H), 3.82 (br m, 2H), 7.32
(d, J = 7.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H),
7.71 (d, J = 7.8 Hz, 1H); 13C NMR (CDCl3) d 11.2, 13.8, 20.2, 21.7,
28.3, 30.8, 31.5, 32.3, 33.6, 35.1, 35.8, 35.9, 39.3, 40.6, 41.9, 47.3,
47.8, 51.4, 54.1, 55.1, 55.2, 69.0, 70.6, 123.6 (q, JC–F = 274 Hz),
126.6 (q, JC–C–C–F = 4.4 Hz), 126.7 (q, JC–C–F = 32 Hz), 127.2, 129.2,
132.2, 134.7, 167.3, 221.4; LRMS for C32H44F3N2O3 [M+H]+ 561.1;
HRMS calcd for C32H44F3N2O3 [M+H]+ 561.3299, found 561.3304;
HPLC purity: 96.7% (RT = 10.7 min 75:25 to 5:95 MeOH/H2O-linear
gradient in 20 min, Luna Phenyl-Hexyl column).
4.1.3.18. (3a,5a)-3-({trans-4-[2,5-Bis(trifluoromethyl)benzyl]-
2,5-dimethylpiperazin-1-yl}methyl)-3-hydroxyandrostan-17-
one (11h). 46 mg, 98%; 1H NMR (CDCl3) d 0.78 (s, 3H), 0.86 (s,
3H), 0.95 (d, J = 6.1 Hz, 3H), 1.03 (d, J = 6.0 Hz, 3H), 0.75-2.10
(m, 23H), 2.35-2.60 (m, 6H), 2.95 (d, J = 11.6 Hz, 1H), 3.18 (br s,
1H), 3.40 (d, J = 15.8 Hz, 1H), 4.06 (d, J = 15.8 Hz, 1H), 7.58 (d,
J = 8.1 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 8.22 (s, 1H); LRMS for
C
35H49F6N2O2 [M+H]+ 643.3.
4.1.4.4. (3a,5a)-3-Hydroxy-3-[(4-{[3-(trifluoromethyl)phenyl]-
carbonyl}piperazin-1-yl)methyl]androstan-17-one (12d). 42 mg,
73%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.12 (m,
22H), 2.32 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.3 Hz, 1H), 2.59 (br
s, 2H), 2.72 (br s, 2H), 3.41 (br s, 2H), 3.80 (br s, 2H), 7.55 (m,
2H), 7.67 (s, 2H); LRMS for C32H44F3N2O3 [M+H]+ 561.4.
4.1.3.19. (3 ,5 )-3-({trans-4-[2,4-Bis(trifluoromethyl)benzyl]-
a
a
2,5-dimethylpiperazin-1-yl}methyl)-3-hydroxyandrostan-17-
one (11i). 46 mg, 98%; 1H NMR (CDCl3) d 0.78 (s, 3H), 0.86 (s, 3H),
0.94 (d, J = 6.1 Hz, 3H), 1.03 (d, J = 6.1 Hz, 3H), 0.75-2.10 (m, 23H),
2.32-2.60 (m, 6H), 2.94 (d, J = 11.0 Hz, 1H), 3.13 (br s, 1H), 3.39 (d,
J = 15.8 Hz, 1H), 4.08 (d, J = 15.4 Hz, 1H), 7.76 (d, J = 8.2 Hz, 1H),
7.86 (s, 1H), 8.08 (d, J = 8.2 Hz, 1H); LRMS for C35H49F6N2O2
[M+H]+ 643.3.
4.1.4.5. (3a,5a)-3-Hydroxy-3-[(4-{[4-(trifluoromethyl)phenyl]-
carbonyl}piperazin-1-yl)methyl]androstan-17-one (12e). 44 mg,
76%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m,
22H), 2.32 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.3 Hz, 1H), 2.57 (br
s, 2H), 2.73 (br s, 2H), 3.39 (br s, 2H), 3.80 (br s, 2H), 7.51 (d,
J = 8.0 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H); LRMS for C32H44F3N2O3
[M+H]+ 561.2.
4.1.3.20. (3a,5a)-3-({trans-4-[3,5-Bis(trifluoromethyl)benzyl]-
2,5-dimethylpiperazin-1-yl}methyl)-3-hydroxyandrostan-17-
one (11j). 20 mg, 43%; 1H NMR (CDCl3) d 0.78 (s, 3H), 0.86 (s, 3H),
0.94 (d, J = 6.1 Hz, 3H), 1.09 (d, J = 6.1 Hz, 3H), 0.75-2.12 (m, 24H),
2.35-2.57 (m, 6H), 2.93 (d, J = 11.4 Hz, 1H), 3.18 (d, J = 11.4 Hz, 1H),
4.09 (d, J = 13.3 Hz, 1H), 7.76 (s, 1H), 7.80 (s, 2H); LRMS for
4.1.4.6. (3a,5a)-3-{[trans-2,5-Dimethyl-4-(phenylcarbonyl)pip-
erazin-1-yl]methyl}-3-hydroxyandrostan-17-one (13a). 25 mg,
47%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 1.27 (m, 3H),
1.41 (d, J = 6.8 Hz, 3H), 0.75-2.00 (m, 23H), 2.10 (m, 2H), 2.40 (m,
3H), 2.90 (br s, 1H), 3.06 (d, J = 8.8 Hz, 1H), 3.50 (br s, 1H), 7.36
(m, 5H); 13C NMR (CDCl3) d 8.6, 11.2, 13.8, 16.4 (ꢁ), 20.2, 21.7,
28.3, 30.8, 31.5, 32.5, 33.8, 35.0, 35.8, 35.9, 39.4, 40.7, 47.8, 51.4
(2ꢀ), 51.7 (2ꢀ) (ꢁ), 54.0, 55.1, 65.8, 70.9, 126.5, 128.5 (2ꢀ), 129.3
(2ꢀ), 136.4, 171.2, 221.5 [ꢁ: a very weak or no signal observed
C
35H49F6N2O2 [M+H]+ 643.4.
4.1.4. General procedure for synthesis of amides 12a–e and
13a–d
To a solution of compound 8 (40 mg, 0.103 mmol) or compound
9 (25 mg, 0.060 mmol) in anhydrous dichloromethane (3 mL) was
added triethylamine (4.0 equiv) and the appropriate acyl chloride
(2.0 equiv). The solution was then stirred for 3 h at room tempera-
ture. The resulting solution was evaporated and purified by flash
chromatography using EtOAc/hexanes (7:3 to 9:1) to give the cor-
responding amides 12a–e and 13a–d. All compounds were charac-
terized by 1H NMR and MS analyses whereas 13C NMR, HRMS and
although
C
a
correlation in HSQC experiment]; LRMS for
33H49N2O3 [M+H]+ 521.4; HRMS calcd for C33H49N2O3 [M+H]+
521.3738, found 521.3745; HPLC purity: 96.4% (RT = 12.1 min,
70:30 to 5:95 MeOH/H2O-linear gradient in 20 min, Luna Phenyl-
Hexyl column).

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4665
4.1.4.7. (3
a
,5a)-3-{[trans-4-(Cyclohexylcarbonyl)-2,5-dimethyl-
4.1.5.4. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{[3-(trifluoromethyl)-
piperazin-1-yl]methyl}-3-hydroxyandrostan-17-one (13b). 14 mg,
44%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s, 3H), 1.27 (m, 6H),
0.80-2.00 (m, 22H), 2.10 (m, 2H), 2.41 (m, 3H), 3.02 (br m, 3H),
3.46 and 3.60 (2d, J = 11.8 Hz, 1H), 4.02, 4.28 and 4.70 (3 m, 1H);
LRMS for C33H55N2O3 [M+H]+ 527.3.
phenyl]sulfonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-
17-one (15a). 30 mg, 67%; 1H NMR (CDCl3) d 0.75 (s, 3H), 0.85 (s,
3H), 1.00 (d, J = 6.4 Hz, 3H), 1.14 (d, J = 6.8 Hz, 3H), 0.75-2.18 (m,
22H), 2.38 (m, 3H), 2.69 (s, 1H), 2.95 (m, 1H), 3.07 (dd,
J1 = 3.5 Hz, J2 = 11.9 Hz, 1H), 3.39 (s, 2H), 4.10 (m, 1H), 7.66 (t,
J = 7.8 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H),
8.06 (s, 1H); LRMS for C33H47F3N2O4SNa [M+Na]+ 647.4.
4.1.4.8. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{[3-(trifluoromethyl)-
phenyl]carbonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-
17-one (13c). 27 mg, 77%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s,
3H), 1.26 (m, 3H), 1.43 (d, J = 6.4 Hz, 3H), 0.78-2.00 (m, 22H), 2.11
(m, 2H), 2.41 (m, 3H), 2.95 (br s, 2H), 3.07 (d, J = 10.4 Hz, 1H), 3.55
(br s, 1H), 7.55 (d, J = 4.5 Hz, 2H), 7.61 (s, 1H), 7.68 (m, 1H); LRMS
for C34H48F3N2O3 [M+H]+ 589.3.
4.1.5.5. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{[2-(trifluoromethyl)-
phenyl]sulfonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-
17-one (15b). 29 mg, 63%; 1H NMR (CDCl3) d 0.75 (s, 3H), 0.85 (s,
3H), 0.89 (m, 3H), 1.19 (m, 3H), 0.75-1.98 (m, 19H), 2.08 (m, 2H),
2.33 (t, J = 13.2 Hz, 2H), 2.43 (dd, J1 = 8.8 Hz, J2 = 19.2 Hz, 1H),
2.82 (s, 1H), 2.90 (m, 1H), 3.09 (dd, J1 = 3.3 Hz, J2 = 11.6 Hz, 1H),
3.35 (m, 2H), 3.52 (d, J = 13.0 Hz, 1H), 4.05 (m, 1H), 7.69 (m, 2H),
7.89 (m, 1H), 8.18 (m, 1H); 13C NMR (CDCl3) d 8.6, 11.2, 13.8,
15.6, 20.2, 21.7, 28.3, 30.7, 31.5, 32.4, 33.8, 35.0, 35.8, 35.9, 39.3,
40.7, 46.0, 47.8, 49.5, 51.4, 52.4, 54.2, 54.7, 65.7, 70.9, 122.6 (q,
JC–F = 274 Hz), 127.5 (q, JC–C–F = 33 Hz), 128.5 (q, JC–C–C–F = 6.4 Hz),
131.9, 132.1, 132.5, 139.3, 221.5; LRMS for C33H47F3N2O4SNa
[M+Na]+ 647.2; HRMS calcd for C33H48F3N2O4S [M+H]+ 625.3281,
found 625.3291; HPLC purity: 98.9% (RT = 16.1 min, 70:30 to
5:95 MeOH/H2O-linear gradient in 20 min, Luna Phenyl-Hexyl
column).
4.1.4.9. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{[4-(trifluoromethyl)-
phenyl]carbonyl}piperazin-1-yl]methyl}-3-hydroxyandrostan-
17-one (13d). 31 mg, 89%; 1H NMR (CDCl3) d 0.77 (s, 3H), 0.86 (s,
3H), 1.27 (m, 3H), 1.42 (m, 3H), 0.80-1.98 (m, 22H), 2.06 (m, 2H),
2.41 (m, 3H), 2.95 (br s, 2H), 3.05 (br s, 1H), 3.58 (br s, 1H), 7.46
(d, J = 7.9 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H); LRMS for C34H48F3N2O3
[M+H]+ 589.2.
4.1.5. General procedure for the synthesis of sulfonamides 14a–
c and 15a–c
To a solution of compound 8 (30 mg, 0.077 mmol) or compound
9 (30 mg, 0.072 mmol) in anhydrous dichloromethane (3 mL) was
added triethylamine (33 mL, 24 mg, 0.24 mmol) and the appropri-
ate sulfonyl chloride (0.12 mmol). The solution was then stirred for
3 h at room temperature. The resulting solution was evaporated
and purified by flash chromatography using EtOAc/hexanes (3:7
to 1:1) to give the corresponding sulfonamides 14a–c and 15a–c.
All compounds were characterized by 1H NMR and MS analyses
whereas 13C NMR, HRMS and HPLC data were added for the key
representative compounds 14a, 15b and 15c.
4.1.5.6. (3a,5a)-3-{[trans-2,5-Dimethyl-4-{[2-trifluoromethyl)-
phenyl]sulfonyl}piperazin-1-yl] methyl}-3-hydroxyandrostan-
17-one (15c). 35 mg, 78%; 1H NMR (CDCl3) d 0.75 (s, 3H), 0.85
(s, 3H), 1.01 (d, J = 5.9 Hz, 3H), 1.14 (d, J = 6.7 Hz, 3H), 0.75-1.98
(m, 20H), 2.09 (m, 2H), 2.38 (m, 3H), 2.68 (s, 1H), 2.95 (d,
J = 6.6 Hz, 1H), 3.07 (dd, J1 = 3.6 Hz, J2 = 11.8 Hz, 1H), 3.40 (s, 2H),
4.05 (br s, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.93 (d, J = 8.3 Hz, 2H); 13C
NMR (CDCl3) d 9.1, 11.2, 13.8, 14.9, 20.2, 21.7, 28.3, 30.7, 31.5,
32.4, 33.7, 35.0, 35.8, 35.9, 39.3, 40.7, 46.1, 47.8, 49.9, 51.4, 53.0,
54.2, 54.6, 65.6, 71.0, 123.2 (q, JC–F = 273 Hz), 126.1, 126.2 (q, JC–
C–C–F = 3.6 Hz), 127.4 (2ꢀ), 134.1 (q, JC–C–F = 33 Hz), 144.2, 221.5;
LRMS for
33H48F3N2O4S [M+H]+ 625.3281, found 625.3287; HPLC purity:
C
33H47F3N2O4SNa [M+Na]+ 647.4; HRMS calcd for
4.1.5.1. (3a,5a)-3-Hydroxy-3-[(4-{[3-(trifluoromethyl)phenyl]-
C
sulfonyl} piperazin-1-yl)methyl]androstan-17-one (14a). 29 mg,
63%; 1H NMR (CDCl3) d 0.74 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m,
21H), 2.28 (s, 2H), 2.43 (dd, J1 = 8.7 Hz, J2 = 19.3 Hz, 1H), 2.50 (s,
1H), 2.72 (t, J = 4.7 Hz, 4H), 3.05 (br s, 4H), 7.73 (t, J = 7.8 Hz, 1H),
7.90 (d, J = 7.8 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 8.01 (s, 1H); 13C
NMR (CDCl3) d 11.2, 13.8, 20.2, 21.7, 28.2, 30.7, 31.5, 32.2, 33.6,
35.0, 35.8, 35.9, 39.1, 40.5, 46.3 (2ꢀ), 47.8, 51.4, 54.1, 54.7 (2ꢀ),
68.8, 70.6, 123.2 (q, JC–F = 273 Hz), 124.7 (q, JC–C–C–F = 3.7 Hz),
129.6, 130.0, 130.9, 131.9 (q, JC–C–F = 33 Hz), 137.0, 221.4; LRMS
for C31H43F3N2O4SNa [M+Na]+ 619.5; HRMS calcd for C31H44F3N2
O4S [M+H]+ 597.2968, found 597.2974; HPLC purity: 98.4%
(RT = 15.4 min, 70:30 to 5:95 MeOH/H2O-linear gradient in
20 min, Luna Phenyl-Hexyl column).
98.7% (RT = 17.0 min, 70:30 to 5:95 MeOH/H2O-linear gradient in
20 min, Luna Phenyl-Hexyl column).
4.1.6. General procedure for synthesis of 3-carbamate-
androsterone derivatives 17a–i
To a solution of oxirane 631 (75 mg, 0.25 mmol) in ethanol
(5 mL) was added the appropriate primary amine (0.75 mmol)
and the solution was stirred at 70 °C overnight. The resulting solu-
tion was evaporated and purified by flash chromatography using
EtOAc/hexanes (7:3 to 9:1) to give the corresponding secondary
amines of general structure 16 in good yields (70–90%). To the sec-
ondary amine, typically 0.22 mmol, in anhydrous dichloromethane
(7 mL) at 0 °C under atmosphere of argon was added diisopropyl-
amine (0.66 mmol) and triphosgene (0.11 mmol). The solution
was stirred at room temperature overnight. The resulting solution
was poured into water, extracted with dichloromethane, filtered
on a phase separator (Biotage, Uppsala, Sweden) and then evapo-
rated. The crude compounds were then purified by flash chroma-
tography using EtOAc/hexanes (1:9 to 3:7) to give the
corresponding carbamates 17a–i. All compounds were character-
ized by 1H NMR and MS analyses whereas 13C NMR, HRMS and
HPLC data were added for the representative compound 17a.
4.1.5.2. (3a,5a)-3-Hydroxy-3-[(4-{[2-(trifluoromethyl)phenyl]-
sulfonyl}piperazin-1-yl)methyl]androstan-17-one (14b). 33 mg,
72%; 1H NMR (CDCl3) d 0.75 (s, 3H), 0.85 (s, 3H), 0.75-2.12 (m,
21H), 2.29 (s, 2H), 2.43 (dd, J1 = 8.6 Hz, J2 = 19.2 Hz, 1H), 2.69 (m,
5H), 3.26 (br s, 4H), 7.72 (m, 2H), 7.92 (d, J = 9.1 Hz, 1H), 8.10 (d,
J = 9.1 Hz, 1H); LRMS for C31H43F3N2O4SNa [M+Na]+ 619.2.
4.1.5.3. (3a,5a)-3-Hydroxy-3-[(4-{[4-(trifluoromethyl)phenyl]-
sulfonyl}-piperazin-1-yl)methyl]androstan-17-one (14c). 26 mg,
56%; 1H NMR (CDCl3) d 0.74 (s, 3H), 0.84 (s, 3H), 0.75-2.10 (m, 23H),
2.27 (s, 2H), 2.43 (dd, J1 = 8.6 Hz, J2 = 19.2 Hz, 1H), 2.50 (s, 1H), 2.72
(br t, J = 4.7 Hz, 4H), 3.06 (br s, 4H), 7.83 (d, J = 8.4 Hz, 1H), 7.89 (t,
J = 8.3 Hz, 1H); LRMS for C31H43F3N2O4SNa [M+Na]+ 619.3.
4.1.6.1. (3R,5S,8R,9S,10S,13S,14S)-30-Benzyl-10,13-dimethyltet-
radecahydro-20H spiro[cyclopenta [ ] phenanthrene-3,50-[1,3]-
a
oxazolidine]-20,17(2H)-dione (17a). 20 mg, 18%; 1H NMR (ace-
tone-d6) d 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H), 2.37 (dd,

4666
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
J1 = 8.2 Hz, J2 = 17.7 Hz, 1H), 3.16 (s, 2H), 4.39 (s, 2H), 7.31 (m, 3H),
7.38 (m, 1H); 13C NMR (CDCl3) d 11.4, 13.8, 20.2, 21.7, 27.8, 30.6,
31.5, 32.8, 33.8, 35.0, 35.4, 35.8, 39.4, 40.8, 47.7, 48.1, 51.3, 53.8,
55.6, 78.9, 127.9, 128.0, 128.8 (2ꢀ), 129.7, 135.9, 157.6, 221.3;
(dd, J1 = 8.8 Hz, J2 = 18.3 Hz, 1H), 3.23 (s, 2H), 3.53 (m, 1H); LRMS
for C27H41NO3Na [M+Na]+ 450.4.
4.1.7. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-10,13,16,16-
tetramethyl-30-(4-methylbenzyl) tetradecahydro-20H-
LRMS for
C
28H37NO3Na [M+Na]+ 458.3; HRMS calcd for
C
28H38NO3 [M+H]+ 436.2846, found 436.2854; HPLC purity: 98.7%
spiro[cyclopenta[a
]phenanthrene-3,50-[1,3]oxazolidine]-
(RT = 13.9 min, 75:25 to 5:95 MeOH/H2O-linear gradient in
20 min, Luna Phenyl-Hexyl column).
20,17(2H)-dione (18)
To a solution of compound 17b (100 mg, 0.22 mmol) in anhy-
drous THF (15 mL) under an argon atmosphere was added NaH
60% in oil (89 mg, 2.2 mmol). The solution was stirred at room tem-
4.1.6.2. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-(4-methyl-
benzyl)tetradecahydro-20H-spiro [cyclopenta[
a
]phenanthrene-
perature for 1 h. Methyl iodide (110 lL, 250 mg, 1.76 mmol) was
3,50-[1,3]oxazolidine]-20,17(2H)-dione (17b). 34 mg, 30%; 1H
NMR (acetone-d6) d 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 21H),
2.32 (s, 3H), 2.37 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 3.13 (s, 2H),
4.34 (s, 2H), 7.19 (s, 4H); LRMS for C29H39NO3Na [M+Na]+ 472.2.
then added and the solution was refluxed overnight. The resulting
solution was poured into water and extracted three times with
EtOAc, washed with brine and dried with MgSO4. The crude com-
pound was purified by flash chromatography using EtOAc/hexanes
(2:8) to give compound 18 (32 mg, 30%). 1H NMR (CDCl3) d 0.77 (s,
3H), 0.87 (s, 3H), 1.03 (s, 3H), 1.16 (s, 3H), 0.80-1.85 (m, 20H), 2.34
(s, 3H), 3.03 (s, 2H), 4.37 (s, 2H), 7.15 (s, 4H); LRMS for C31H43NO3-
Na [M+Na]+ 500.2.
4.1.6.3. (3R,5S,8R,9S,10S,13S,14S)-30-(4-Methoxybenzyl)-10,13-
dimethyltetradecahydro-20H-spiro[cyclopenta[
a]phenanthrene-
3,50-[1,3]oxazolidine]-20,17(2H)-dione (17c). 18 mg, 16%; 1H
NMR (acetone-d6) d 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.10 (m, 22H),
2.37 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 3.12 (s, 2H), 3.79 (s, 3H),
4.31 (s, 2H), 6.93 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H); LRMS
for C29H40NO4Na [M+Na]+ 488.3.
4.1.8. Synthesis of (3a,5a)-3-{[(2{[tert-butyl(dimethyl)silyl]-
oxy}ethyl)amino]methyl}-3-hydroxyandrostan-17-one (19)
To a solution of oxirane 631 (0.96g, 3.18 mmol) in ethanol
(50 ml) was added 2-{[tert-butyl(dimethyl)silyl] oxy}ethanamine
(1.75 g, 10.0 mmol) and the mixture was refluxed for 5 h. The
resulting solution was then evaporated to dryness and purified by
flash chromatography using EtOAc/hexanes (2:8) to give compound
19 (1.0 g, 66%). 1H NMR (acetone-d6) d 0.08 (s, 6H), 0.81 (s, 6H), 0.91
(s, 9H), 0.80-2.10 (m, 20H), 2.22 and 2.38 (2 m, 2H), 2.48 (s, 2H),
2.72 (t, J = 5.5 Hz, 2H), 3.25 (m, 1H), 3.72 (t, J = 5.5 Hz, 2H), 3.80 (t,
J = 6.0 Hz, 1H); LRMS for C28H52NO3Si [M+H]+ 478.2.
4.1.6.4. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-[4-(trifluo-
romethyl)benzyl]tetradecahydro-20H-spiro[cyclopenta[
a]phen-
anthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (17d). 55 mg,
43%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.87 (s, 3H), 0.80-2.10
(m, 21H), 2.38 (dd, J1 = 8.8 Hz, J2 = 18.2 Hz, 1H), 3.23 (s, 2H), 4.51
(s, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H); LRMS for
C
29H36F3NO3Na [M+Na]+ 526.1.
4.1.6.5. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-{[(6-trifluo-
romethyl)pyridine-3-yl]methyl} tetradecahydro-20H-spiro-
4.1.9. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-30(2-{[tert-butyl-
(dimethyl)silyl]oxy}ethyl)-10,13-dimethyltetradecahydro-20
[cyclopenta[
a
]phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-
H-spiro[cyclopenta[
17(2H)-dione (20)
a
]phenanthrene-3,50-[1,3]oxazolidine]-20,
dione (17e). 18 mg, 14%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.87
(s, 3H), 0.80-2.10 (m, 21H), 2.38 (dd, J1 = 8.8 Hz, J2 = 18.2 Hz, 1H),
3.30 (s, 2H), 4.58 (s, 2H), 7.87 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz,
1H), 8.73 (s, 1H); LRMS for C28H35F3N2O3Na [M+Na]+ 527.4.
To a solution of compound 19 (1.0 g, 2.21 mmol) in anhydrous
dichloromethane (100 mL) at 0 °C under an argon atmosphere
was added diisopropylamine (760 lL, 564 mg, 4.4 mmol) and tri-
phosgene (325 mg, 1.1 mmol). The solution was stirred for 8 h at
room temperature. The resulting solution was poured into water,
extracted two times with dichloromethane and dried over a phase
separator syringe (Biotage) and finally evaporated to dryness. The
crude compound was purified by flash chromatography using
EtOAc/hexanes (2:8) to give compound 20 (370 mg, 36%). 1H
NMR (acetone-d6) d 0.09 (s, 6H), 0.85 (s, 3H), 0.89 (s, 3H), 0.91 (s,
9H), 0.80-2.07 (m, 22H), 2.38 (dd, J1 = 8.6 Hz, J2 = 18.3 Hz, 1H),
3.30 (t, J = 5.3 Hz, 2H), 3.40 (s, 1H), 3.78 (t, J = 5.4 Hz, 2H); LRMS
for C29H49NO4SiNa [M+Na]+ 526.5.
4.1.6.6. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-[2-(trifluo-
romethyl)benzyl]tetradecahydro-20H-spiro[cyclopenta[
a]phen-
anthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (17f). 62 mg,
49%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.88 (s, 3H), 0.82-2.10
(m, 21H), 2.38 (dd, J1 = 8.7 Hz, J2 = 18.3 Hz, 1H), 3.23 (s, 2H), 4.61
(s, 2H), 7.57 (m, 2H), 7.74 (m, 2H); LRMS for C29H36F3NO3Na
[M+Na]+ 526.4.
4.1.6.7. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-[3-(trifluo-
romethyl)benzyl]tetradecahydro-20H-spiro[cyclopenta[
a]phen-
anthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (17g). 30 mg,
24%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.87 (s, 3H), 0.82-2.10
(m, 21H), 2.38 (dd, J1 = 8.7 Hz, J2 = 18.2 Hz, 1H), 3.23 (s, 2H), 4.52
(s, 2H), 7.65 (m, 4H); LRMS for C29H36F3NO3Na [M+Na]+ 526.0.
4.1.10. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-30-(2-
hydroxyethyl)-10,13-dimethyltetradecahydro-20H-spiro
[cyclopenta[
dione (21)
a
]phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-
To a solution of compound 20 (360 mg, 0.71 mmol) in anhy-
drous THF (70 mL) was added a solution (1.0 M) of tetrabutylam-
monium fluoride (TBAF) in THF (1.05 mL, 1.05 mmol) at room
temperature under an argon atmosphere. The solution was stirred
at room temperature for 1 h. The resulting mixture was poured
into water, extracted two times with EtOAc and dried with Na2SO4,
filtered and finally evaporated to dryness. The crude compound
was purified by flash chromatography using EtOAc to give com-
pound 21 (225 mg, 81%). 1H NMR (acetone-d6) d 0.85 (s, 3H),
0.89 (s, 3H), 0.80-2.08 (m, 23H), 2.37 (dd, J1 = 8.6 Hz, J2 = 18.3 Hz,
1H), 3.29 (t, J = 5.3 Hz, 2H), 3.40 (s, 1H), 3.67 (t, J = 5.6 Hz, 2H);
LRMS for C23H35NO4Na [M+Na]+ 412.2.
4.1.6.8. (3R,5S,8R,9S,10S,13S,14S)-30-[3,5-Bis(trifluoromethyl)-
benzyl]-10,13-dimethyltetradecahydro-20H-spiro[cyclopenta[
a]-
phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (17h). 28 mg,
20%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.87 (s, 3H), 0.81-2.10 (m,
21H), 2.38 (dd, J1 = 8.7 Hz, J2 = 17.7 Hz, 1H), 3.31 (s, 2H), 4.64 (s,
2H), 8.00 (s, 3H); LRMS for C30H35F6NO3Na [M+Na]+ 594.2.
4.1.6.9. (3R,5S,8R,9S,10S,13S,14S)-30-Cyclohexyl-10,13-dimethyl-
tetradecahydro-20H-spiro [cyclopenta[
oxazolidine]-20,17(2H)-dione (17i).
a
]phenanthrene-3,50-[1,3]
19 mg, 18%; 1H NMR
(acetone-d6) d 0.84 (s, 3H), 0.88 (s, 3H), 0.80-2.08 (m, 31H), 2.38

R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
4667
4.1.11. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-30-(2-bromoethyl)-
10,13-dimethyltetradecahydro-20H-spiro[cyclopenta[
poured into water and extracted three times with diethylether.
The combined organic layer was successively washed with brine,
dried over Na2SO4, filtered and evaporated to dryness under vac-
uum. The resulting crude compound was purified by flash chroma-
tography using either dichloromethane/methanol/triethylamine
(90:9:1) (23c, 23e and 23f) or EtOAc/hexanes 1:1 (23d) to give
the corresponding compounds 23c–23f.
a
]
phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (22)
To a solution of compound 21 (225 mg, 0.58 mmol) in anhydrous
dichloromethane (50 mL) was added triphenylphosphine (303 mg,
1.16 mmol) and carbon tetrabromide (383 mg, 1.16 mmol) at 0 °C
under an atmosphere of argon. The mixture was stirred for 1 h at
room temperature. The resulting solution was washed with water,
and the organic phase was dried using a phase separator syringe
(Biotage). The crude compound was purified by flash chromatogra-
phy using EtOAc/hexanes (4:6) to give compound 22. 235 mg, 90%;
1H NMR (acetone-d6) d 0.85 (s, 3H), 0.89 (s, 3H), 0.82-2.08 (m,
21H), 2.38 (dd, J1 = 8.6 Hz, J2 = 18.3 Hz, 1H), 3.41 (s, 2H), 3.63 (s,
4H); LRMS for C23H3479BrNO3Na [M+Na]+ 474.1.
4.1.14.1. (3R,5S,8R,9S,10S,13S,14S)-10,13-Dimethyl-30-[2-(piperi-
din-1-yl)ethyl]tetradecahydro-20H-spiro[cyclopenta[
a]phenan-
threne-3,50-[1,3]oxazolidine]-20,17(2H)-dione (23c). 7 mg, 22%;
1H NMR (acetone-d6) d 0.84 (s, 3H), 0.89 (s, 3H), 0.80-2.08 (m,
24H), 2.26 (m, 2H), 2.38 (dd, J1 = 8.8 Hz, J2 = 18.3 Hz, 1H), 3.18
(m, 4H), 3.42 (s, 2H), 3.46 (t, J = 6.5 Hz, 2H), 3.80 (t, J = 6.5 Hz,
2H); LRMS for C28H47N3O3 [M+NH4]+ 474.2.
4.1.12. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-30-
[2-(3-methylphenoxy) ethyl]tetradecahydro-20H-spiro
4.1.14.2. (3R,5S,8R,9S,10S,13S,14S)-30-{2-[Ethyl(phenyl)ami-
no]ethyl}-10,13-dimethyltetrahydrodecahydro-20H-spiro[cyclo-
[cyclopenta[
dione (23a)
a
]phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-
penta[a
]phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione
To a solution 3-methylphenol (22 lL, 22 mg, 0.200 mmol) and
(23d). 16 mg, 47%; 1H NMR (acetone-d6) d 0.85 (s, 3H), 0.88 (s,
3H), 1.03 (t, J = 7.1 Hz, 3H), 0.82-2.07 (m, 22H), 2.38 (dd,
J1 = 8.7 Hz, J2 = 18.3 Hz, 1H), 2.53 (q, J = 7.1 Hz, 1H), 2.60 (t,
J = 6.1 Hz, 2H), 3.28 (s, 2H), 3.32 (m, 2H), 3.59 (s, 2H), 7.23 (t,
J = 7.2 Hz, 1H), 7.30 (t, J = 7.3 Hz, 2H), 7.36 (t, J = 7.3 Hz, 2H); LRMS
for C32H46N2O3Na [M+Na]+ 529.4.
K2CO3 (28 mg, 0.200 mmol) in anhydrous DMF (2 mL) previously
stirred 10 min at 70 °C was added compound 22 (30 mg,
0.067 mmol). The mixture was stirred overnight at 70 °C under
an argon atmosphere. The resulting solution was poured into a
solution of NaOH 1.0 N and extracted three times with diethyl-
ether. The combined organic layer was successively washed with
brine, dried over Na2SO4, filtered, and evaporated to dryness under
vacuum. The resulting crude compound was purified by flash chro-
matography using EtOAc/hexanes (2:8) to give compound 23a.
11 mg, 34%; 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.88 (s, 3H),
0.80-2.07 (m, 21H), 2.29 (s, 3H), 2.37 (dd, J1 = 8.7 Hz, J2 = 18.8 Hz,
1H), 3.45 (s, 2H), 3.59 (m, 2H), 4.15 (t, J = 5.3 Hz, 2H), 6.77 (m,
3H), 7.16 (t, J = 7.8 Hz, 1H); LRMS for C30H41NO4Na [M+Na]+ 502.4.
4.1.14.3. (3R,5S,8R,9S,10S,13S,14S)-30-{2-[Cyclohexyl(ethyl)amino]
ethyl}-10,13-dimethyltetradecahydro-20H-spiro[cyclopenta[
a]
phenanthrene-3,50-[1,3]oxazolidine]-20,17(2H)-dione (23e). 13 mg,
39%; 1H NMR (CDCl3) d 0.81 (s, 3H), 0.86 (s, 3H), 1.35 (t,
J = 7.2 Hz, 3H), 0.78-2.10 (m, 29H), 2.23 (br dd, J1 = 2.7 Hz,
J2 = 11.8 Hz, 1H), 2.35 (br d, J = 11.9 Hz, 1H), 2.43 (dd, J1 = 8.6 Hz,
J2 = 19.3 Hz, 1H), 3.17 (br t, J = 11.5 Hz, 1H), 3.35 (m, 6H), 3.80
(m, 2H); LRMS for C31H54N3O3 [M+NH4]+ 516.4.
4.1.13. Synthesis of (3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-30-
{2-(3-methylphenyl) sulfonyl]ethyl}tetradecahydro-20H-
4.1.14.4. (3R,5S,8R,9S,10S,13S,14S)-30-[2-(Dipropylamino)ethyl]-
spiro[cyclopenta[a
]phenanthrene-3,50-[1,3]oxazolidine]-20,17
10,13-dimethyltetradecahydro-20H-spiro[cyclopenta[
a
]phenan-
threne-3,50-[1,3]oxazolidine]-20,17(2H)-dione (23f). 13 mg, 40%;
1H NMR (acetone-d6)
0.84 (s, 3H), 0.89 (s, 3H), 0.94 (t,
(2H)-dione (23b)
To a solution 3-methylbenzenthiol (41 mg, 0.33 mmol) and
K2CO3 (46 mg, 0.33 mmol) in anhydrous DMF (3 mL) first stirred
10 min at 70 °C was added compound 22 (50 mg, 0.11 mmol). The
mixture was stirred 3 h at 70 °C under an argon atmosphere. The
resulting solution was poured into water and extracted three times
with diethylether. The combined organic layer was successively
washed with brine, dried over Na2SO4, filtered, and evaporated to
dryness under vacuum to give the corresponding thioether deriva-
tives as crude compound. The crude thioether compound (45 mg)
was then diluted in methanol/water (1:1) and oxone (112 mg)
was added. The solution was stirred at room temperature over-
night. The resulting solution was poured into water, extracted three
times with EtOAc and the combined organic layer was successively
washed with brine and dried over Na2SO4, filtered, and evaporated
to dryness under vacuum. The resulting crude compound was puri-
fied by flash chromatography using EtOAc/hexanes (1:1) to give the
corresponding sulfone 23b (9 mg, 16% for two steps); 1H NMR (ace-
tone-d6) d 0.84 (s, 3H), 0.86 (s, 3H), 0.80-2.07 (m, 21H), 2.38 (dd,
J1 = 8.7 Hz, J2 = 8.3 Hz, 1H), 2.46 (s, 3H), 3.30 (s, 2H), 3.50 (t,
J = 6.6 Hz, 2H), 3.61 (m, 2H), 7.57 (m, 2H), 7.77 (m, 2H); LRMS for
d
J = 7.4 Hz, 3H), 0.78-2.10 (m, 28H), 2.38 (dd, J1 = 8.7 Hz,
J2 = 18.2 Hz, 1H), 3.23 (m, 4H), 3.42 (s, 2H), 3.48 (t, J = 6.6 Hz,
2H), 3.74 (t, J = 6.9 Hz, 2H); LRMS for C29H52N3O3 [M+NH4]+ 490.5.
4.1.15. Synthesis of N-{(3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-
20,17-dioxohexadecahydro-30H-spiro[cyclopenta[
a]phenanth-
rene-3,50-[1,3]oxazolidine-30-yl]ethyl}-N-propylpropanamide
(23g)
To a solution of compound 22 (30 mg, 0.067 mmol) in anhy-
drous DMF (3 mL) was added K2CO3 (21 mg, 0.20 mmol) and N-
propylamine (16 lL, 12 mg, 0.20 mmol). The mixture was heated
at 80 °C for 5 h and then poured into water and extracted with
diethylether. The combined organic layer was successively
washed with brine, dried over Na2SO4, filtered, and evaporated
to dryness under vacuum. The resulting secondary amine was
used directly without further purification for the next steps. To
the crude compound (30 mg) in anhydrous dichloromethane
(3 mL) was added triethylamine (27
lL, 21 mg) and propionyl
30H41NO5SNa [M+Na]+ 550.2.
chloride (13 L, 13 mg). The solution was stirred at room temper-
l
C
ature for 4 h under an atmosphere of argon. The resulting solu-
tion was diluted with dichloromethane (15 mL), washed with
water, dried using a phase separator syringe (Biotage), and evap-
orated to dryness. The resulting crude compound was purified by
flash chromatography using EtOAc/hexanes (1:1) to give the com-
pound 23g (11 mg, 34%); 1H NMR (acetone-d6) d 0.84 (s, 3H), 0.88
(s, 3H), 0.91 (t, J = 7.4 Hz, 3H), 1.04 (t, J = 7.4 Hz, 3H), 0.82-2.07
4.1.14. General procedure for the synthesis of 3-carbamate-
androsterone derivatives 23c–f
To a solution of compound 22 (30 mg, 0.067 mmol) in anhy-
drous DMF (2 mL) was added the appropriate secondary amine
(0.20 mmol) and sodium carbonate (21 mg, 0.20 mmol). The mix-
ture was stirred at 70 °C for 3 h and the resulting solution was

4668
R. Maltais et al. / Bioorg. Med. Chem. 19 (2011) 4652–4668
(m, 23H), 2.34 (m, 3H), 3.32 (m, 6H), 3.53 (m, 2H); LRMS for
29H46N2O4Na [M+Na]+ 509.1.
Supplementary data
C
Supplementary data (1H NMR spectrum of 15b and assignment
of protons, 13C NMR (APT) spectrum of 15b and assignment of car-
bons, and 2D NMR (COSY, TOCSY, NOESY, HSQC and HMBC) spectra
of 15b, the best 17b-HSD3 inhibitor resulting from our SAR study)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.06.003.
4.2. Biological assays
4.2.1. Inhibition of 17b-HSD3 in homogenized cells
The inhibitory activity of each compound was determined fol-
lowing a procedure described in our previous published articles.29
The HEK-293 cells overexpressing 17b-HSD3 were briefly homoge-
nized and used for the enzymatic assay. The transformation of 50
References and notes
nM of [4-14C]-4-androstene-3,17-dione ([14C]- 4-dione) into
D
1. http://www.cancer.org/Cancer-ProstateCancer/DetailedGuide/prostate-cancer-
key-statistics.
[
14C]-testosterone ([14C]-T) in presence of NADPH in excess
(5 mM) and ethanol only (control) or an inhibitor dissolved in eth-
anol was measured and used to determine the percentage of inhi-
bition at a given concentration.
2. Group. VACUR. Surg. Gynecol. Obset. 1967, 124, 1011.
3. Seidenfeld, J.; Samson, D. J.; Hasselblad, V.; Aronson, N.; Albertsen, P. C.;
Bennett, C. L.; Wilt, T. J. Ann. Intern. Med. 2000, 132, 566.
4. Santen, R. J. J. Clin. Endo. Metab. 1992, 75, 685.
5. Hedlund, P. O. Prostate Suppl. 2000, 10, 32.
6. Wysowski, D. K.; Freiman, J. P.; Tourtelot, J. B.; Hroton, M. L. Ann. Intern. Med.
1993, 118, 860.
7. Shahinian, V. B.; Kuo, Y. F.; Freeman, J. L.; Goodwin, J. S. N. Eng. J. Med. 2005,
352, 154.
8. Tammela, T. J. Steroid Biochem. Mol. Biol. 2004, 92, 287.
9. Laplante, Y.; Poirier, D. Steroids 2008, 73, 266.
10. Inano, H.; Tamaoki, B. I. Steroids 1986, 48, 1.
11. Luu-The, V.; Zhang, Y.; Poirier, D.; Labrie, F. J. Steroid Biochem. Mol. Biol. 1995,
55, 581.
12. Penning, T. Endocr. Relat. Cancer 1996, 3, 41.
13. Moeller, G.; Adamski, J. Mol. Cell. Endocrinol. 2009, 301, 7.
14. Mohler, M. L.; Narayanan, R.; He, Y.; Miller, D. D.; Dalton, J. T. Recent Pat. Endocr.
Metab. Immune Drug Discov. 2007, 1, 103.
4.2.2. Inhibition of 17b-HSD-3 in intact cells
HEK-293 cells were seeded at 200,000 cells/well in a 12-well
plate (BD Falcon) at 37 °C under 95% air 5% CO2 humidified
atmosphere in minimum essential medium (MEM) containing
non-essential amino acids (0.1 mM), glutamine (2 mM), sodium
pyruvate (1 mM), 10% foetal bovine serum, penicillin (100 IU/mL)
and streptomycin (100
for 17b-HSD3 was transfected using the Exgen 500 procedure
(Fermentas, Burlington, ON, Canada) with 2 g of recombinant
lg/mL). The expression vector encoding
l
plasmid per well. For the inhibitory activity assay, a final concen-
tration of 50 nM of [4-14C]-4-androstene-3,17-dione in ethanol
(53.6 mCi/mmol, Perkin Elmer Life Sciences Inc., Boston, MA,
USA) and an ethanolic solution of inhibitor (0.5% v/v) were added
to freshly cultured medium and incubated for 1 h. Each inhibitor
was assessed in triplicate at tested concentrations. After incuba-
tion, culture medium was removed and radiolabeled steroids were
extracted and quantified as described for the enzymatic assay in
homogenized cells.29 Percentage of transformation and then the
IC50 value was calculated as previously reported.29
15. Day, J. M.; Tutill, H. J.; Purohit, A.; Reed, M. J. Endocr. Relat. Cancer 2008, 15, 665.
16. Labrie, F. Mol. Cell. Endocrinol. 1991, 78, C113.
17. Poirier, D. Drug Dev. Res. 2008, 69, 304.
18. Luu-The, V.; Belanger, A.; Labrie, F. Best Pract. Res. Clin. Endocrinol. Metab. 2008,
22, 207.
19. Koh, E.; Noda, T.; Kanya, J.; Namiki, M. Prostate 2002, 53, 154.
20. Deqtyar, V. G.; Babkina, T. V.; Kazantseva, I. A.; Morozov, A. P.; Trapeznikova,
M. F.; Kushlinski, N. E. Bull. Exp. Biol. Med. 2005, 139, 715.
21. Montgomery, R. B.; Mostaghel, E. A.; Vessella, R.; Hess, D.; Kalhorn, T. F.;
Higano, C. S.; True, L. D.; Nelson, P. S. Cancer Res. 2008, 68, 4447.
22. Locke, J. A.; Guns, E.; Lubik, A. A.; Adomat, H. H.; Hendy, S. C.; Wood, C. A.;
Ettinger, S. L.; Gleave, M. E.; Nelson, C. C. Cancer Res. 2008, 68, 6407.
23. Mostaghel, E. A.; Page, S. T.; Lin, D. W.; Fazli, L.; Coleman, I. M.; True, L. D.;
Knudsen, B.; Hess, D. L.; Nelson, C. C.; Matsumoto, A. M.; Bremner, W. J.;
Gleave, M. E.; Nelson, P. Cancer Res. 2007, 67, 5033.
24. Page, S. T.; Lin, D. W.; Mostaghel, E. A.; Hess, D. L.; True, L. D.; Amory, J. K.;
Nelson, P. S.; Matsumoto, A. M.; Bremner, W. J. J. Clin. Endocrinol. Metab. 2006,
91, 3850.
25. Wang, L. G.; Mencher, S. K.; McCarron, J. P.; Ferrari, A. C. Oncol. Rep. 2004, 11,
1325.
4.2.3. Proliferative activity on androgen-sensitive (AR+)
Shionogi cells
The proliferative (androgenic) activity was determined on
androgen-sensitive Shionogi cells following a previously pub-
lished35 procedure that is well established in our laboratory. Inhib-
itors and reference compounds were tested at two concentrations
of 0.1 and 1.0 lM. The androgenicity was reported as % of cell pro-
liferation (versus the control). The basal cell proliferation (control)
was fixed at 100%.
26. Leibowitz, R. L.; Tucker, S. J. Oncologist 2001, 6, 177.
27. Maltais, R.; Luu-The, V.; Poirier, D. J. Med. Chem. 2002, 45, 640.
28. Tchedam-Ngatcha, B.; Laplante, Y.; Labrie, F.; Luu-The, V.; Poirier, D. Mol. Cell.
Endocrinol. 2006, 248, 225.
29. Tchedam-Ngatcha, B.; Luu-The, V.; Labrie, F.; Poirier, D. J. Med. Chem. 2005, 48,
5257.
30. Maltais, R.; Poirier, D. Tetrahedron Lett. 1998, 39, 4151.
31. Maltais, R.; Luu-The, V.; Poirier, D. Bioorg. Med. Chem. 2001, 9, 3101.
32. Berube, M.; Laplante, Y.; Poirier, D. Med. Chem. 2006, 2, 329.
33. Berube, M.; Poirier, D. J. Enzyme Inhib. Med. Chem. 2007, 22, 201.
34. http://en.wikipedia.org/wiki/Piperazine.
Acknowledgments
This work was supported by the Canadian Institutes of Health
Research (CIHR). Careful reading of the manuscript by Ms. Miche-
line Harvey is greatly appreciated.
35. Roy, J.; Breton, R.; Martel, C.; Labrie, F.; Poirier, D. Bioorg. Med. Chem. 2007, 15,
3003.