Chemical synthesis of 2β-amino-5α-androstane-3α,17β-diol N-derivatives and their antiproliferative effect on HL-60 human leukemia cells

Article abstract of DOI:10.1016/j.bmc.2008.03.031

Even though few steroids are used for the treatment of leukemia, 2β-(4-methylpiperazinyl)-5α-androstane-3α,17β-diol (1) was recently reported for its ability to inhibit the proliferation of human leukemia HL-60 cells. With an efficient procedure that we had developed for the aminolysis of hindered steroidal epoxides, we synthesized a series of 2β-amino-5α-androstane-3α,17β-diol N-derivatives structurally similar to 1. Hence, the opening of 2,3α-epoxy-5α-androstan-17β-diol with primary and secondary amines allowed the synthesis of aminosteroids with diverse length, ramification, and functionalization of the 2β-side chain. Sixty-four steroid derivatives were tested for their capacity to inhibit the proliferation of HL-60 cells; thus obtaining first structure-activity relationship results. Ten aminosteroids with long alkyl chains (7-16 carbons) or bulky groups (diphenyl or adamantyl) have shown antiproliferative activity over 78% at 10 μM and superior to that of the lead compound. The 3,3-diphenylpropylamino, 4-nonylpiperazinyl and octylamino derivatives of 5α-androstane-3α,17β-diol inhibited the HL-60 cell growth with IC₅₀ of 3.1, 4.2 and 6.4 μM, respectively. They were also found to induce the HL-60 cell differentiation.

Full text of DOI:10.1016/j.bmc.2008.03.031

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5062–5077
Chemical synthesis of 2b-amino-5a-androstane-3a,17b-diol
N-derivatives and their antiproliferative effect on HL-60
human leukemia cells
Dominic Thibeault, Jenny Roy, Patrick DeRoy and Donald Poirier*
Medicinal Chemistry Division, Oncology and Molecular Endocrinology Laboratory,
CHUL Research Center (CHUQ—Pavillon CHUL) and Universite´ Laval, 2705 Laurier Boulevard, Que., Canada G1V 4G2
Received 16 November 2007; revised 8 March 2008; accepted 11 March 2008
Available online 14 March 2008
Abstract—Even though few steroids are used for the treatment of leukemia, 2b-(4-methylpiperazinyl)-5a-androstane-3a,17b-diol (1)
was recently reported for its ability to inhibit the proliferation of human leukemia HL-60 cells. With an efficient procedure that we
had developed for the aminolysis of hindered steroidal epoxides, we synthesized a series of 2b-amino-5a-androstane-3a,17b-diol
N-derivatives structurally similar to 1. Hence, the opening of 2,3a-epoxy-5a-androstan-17b-diol with primary and secondary amines
allowed the synthesis of aminosteroids with diverse length, ramification, and functionalization of the 2b-side chain. Sixty-four
steroid derivatives were tested for their capacity to inhibit the proliferation of HL-60 cells; thus obtaining first structure–activity
relationship results. Ten aminosteroids with long alkyl chains (7–16 carbons) or bulky groups (diphenyl or adamantyl) have shown
antiproliferative activity over 78% at 10 lM and superior to that of the lead compound. The 3,3-diphenylpropylamino, 4-nonylpip-
erazinyl and octylamino derivatives of 5a-androstane-3a,17b-diol inhibited the HL-60 cell growth with IC50 of 3.1, 4.2 and 6.4 lM,
respectively. They were also found to induce the HL-60 cell differentiation.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
acute myeloid leukemia has also increased over time
from 38% to 65%.2 But even if the success of clinical tri-
als in identifying new agents and treatment modalities
has been significant, current treatments have many lim-
itations related to their side effects and the development
of acquired drug resistance.5 The new therapeutic agents
thus needed should be more active and produce less side
effects and they also should act through a mechanism
different from that of cytotoxic agents already used.
Leukemia is a major type of cancer affecting a significant
segment of the population, and especially children. In
fact, leukemia is the most frequent childhood cancer,
with 26% of all cases, and 30% mortality.1 The Ameri-
can Cancer Society (ACS) estimated that 35,070 new
cases of leukemia would be diagnosed in the United
States in 2006, whereas about 22,280 adults and children
would die of leukemia during 2006.2 Although the inci-
dence rate for this disease remains relatively unchanged,
some success has fortunately been attained in its treat-
ment. Since the early 1950s, mortality rates for child-
hood cancer have declined by more than 50%, with
most of the improvement occurring after 1970.3 Forty
years ago,4 nearly no one could survive acute lymphoid
leukemia or ‘childhood leukemia’, but today, 86% of the
children and teens affected are still alive 5 years after the
diagnosis.2 The five-year survival rate of all people with
Up to now, few steroidal compounds have been investi-
gated for potential antileukemic activity. Notably,
corticosteroids like prednisone or prednisolone are well
documented for the treatment of lymphoid leukemia.6–10
Other steroid derivatives have been investigated for
potential inhibitory effect on leukemic cells, such as 7-
keto hybrid steroidal esters of nitrogen mustard.11
Estrogen derivatives,12 and 1,25-dihydroxyvitamin
D3 and its derivatives,14,15 have also been studied
13
and good results obtained but they do not always suffice
for the purpose of cancer therapy. Recently, He and
Jiang16 working with human myeloid leukemia HL-60
cells reported the antiproliferative properties of
2b-(4-methylpiperazinyl)-5a-androstane-3a,17b-diol, the
Keywords: Steroid; Androstane; Amine; Epoxide; Leukemia; HL-60
cells.
*
Corresponding author. Tel.: +1 418 654 2296; fax: +1 418 654
2761; e-mail: donald.poirier@crchul.ulaval.ca
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.031

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5063
aminosteroid 1 illustrated in Figure 1. This compound
and its 17-keto analogue 2 were also reported to induce
differentiation of HL-60 cells.17,18 No structure–activity
relationship (SAR) study was however conducted in or-
der to optimize the antileukemic potential of this newly
reported lead compound.
In search of a novel family of anticancer agents with
fewer side effects and/or a different mechanism of action,
we first verified the antileukemic activity of compound 1
and, second, synthesized analogous products with the
aim of improving potency following two strategies. We
first generated libraries of aminosteroids 3 using parallel
solid-phase organic synthesis,19 while a solution-phase
approach was used for the synthesis of compounds 4.
Since the synthesis of aminosteroids such as 4 presents
certain limitations, we had previously developed a new
method for the efficient aminolysis of 2,3a-steroidal
epoxides,20 which allowed us to obtain a larger series
of 2b-aminosteroids and then extend our SAR study.
In this paper, we report the chemical synthesis and char-
acterization of more than 64 compounds of general
structure 4 (Fig. 1) and their level of antiproliferative
activity in HL-60 cells.
Scheme 1. Synthesis of epoxide 8. Reagents and conditions: (a) Ac2O,
pyridine, DMAP cat., rt; (b) Br2, AcOH, rt; (c) K-selectride, ꢀ78 ꢁC,
THF; (d) Zn dust, AcOH, reflux; (e) K2CO3/H2O, MeOH, reflux; (f) m-
CPBA, CH2Cl2, 0 ꢁC.
Although the way reported above afforded epoxide 8 in
acceptable yield and purity when carefully carried out,
we also explored various other possibilities for the syn-
thesis of 8 with fewer synthetic steps and potentially bet-
ter purity.21 Shapiro’s reaction22 was first tried out as a
straightforward approach for obtaining 7 from the cor-
responding tosylhydrazone derivative of DHT (5). Var-
ious bases and conditions were then used in order to
improve the regioselectivity of the elimination step, but
a mixture of two alkenes (C-2,3 and C-3,4) was always
observed and our attempts to separate the two isomers
were unsuccessful. These elimination reactions never
afforded less than 6% of C-3,4 alkene, which was found
unsatisfactory for our purposes. A second strategy we
put to test was the elimination of a good C-3 leaving
group such as a tosylate or a bromide, easily obtained
from epiandrosterone, but at least 10% of C-3,4 isomer
was obtained in both cases. Since we were able to pro-
2. Results and discussion
2.1. Chemical synthesis
2.1.1. Synthesis of epoxide 8. The first step for generating
all aminosteroids presented here was the synthesis of
epoxide 8. It was obtained from commercially available
dihydrotestosterone (DHT; 5) by a known sequence of
reactions (Scheme 1).19 Briefly, DHT was acetylated
and brominated at C2, the ketone was reduced into a
3a-OH and bromoalcohol 6 was transformed into al-
kene 7, which after hydrolysis of the acetate and double
bond epoxidation afforded stereoselectively the 2,3a-
epoxide 8.
Figure 1. Lead compound 1, its analogue 2, and general structures of related aminosteroids 3 and 4. Illustrated only for steroids 1 and 2, the
stereogenic centers are the same for all other aminosteroids reported in this paper. The compounds synthesized in our study and represented by the
general structure 4 are given in Tables 1–4 and in Supplementary data.

5064
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
duce efficiently and rapidly a mixture of alkenes, we next
considered the possibility of isomerizing the C-3,4 al-
kene into a C-2,3 alkene. Various catalysts known to
facilitate a double bond isomerization23,24 were thus
tested with the substrate in refluxing toluene or DMF.
Although an enrichment in C-2,3 isomer (compound
7) was observed, much degradation occurred. The low
rate of isomerization and the loss of products convinced
us to generate alkene 7 with the method described above
and using the C2-bromination pathway.
of amines; we thus obtained targeted aminosteroids
21–52 in medium to high yields (35–99%) with easier
work-up procedure and purification than with Method
A. Only 3 equiv of amine with 20% molar of Gd(OTf)3
as catalyst was needed to provide the desired product by
heating (150–190 ꢁC) in a Schlenk tube the epoxide 8 for
2 h (primary amines) or overnight (secondary and cyclic
amines). In addition to cyclic amines, various aliphatic
amines were also used to generate aminosteroids and
to extend our SAR study, which would not have been
possible with the classical aminolysis procedure applied
in Method A. The regio and stereoselectivity outcome of
the aminolysis of epoxide 8 deserves some comments. In
fact, in agreement with Barton’s generalization,28,29 ring
opening of a steroidal epoxide gives trans-diaxially dis-
persed substituents. Thus, the aminolysis of 2,3a-epox-
ide 8 will generate a diaxial amino (2b-N) alcohol (3a-
OH) derivative as represented by compounds 9–53. This
regio and stereochemical aminolysis of such steroidal
epoxide was documented in the literature25–27 and con-
firmed by the X-ray analysis of compound 53.19
2.1.2. Synthesis of aminosteroids 9–53 by opening of
epoxide 8. To start our SAR study, we first added a
diversity of cyclic amines at the C2-carbon of 8 using
Method A, an aminolysis procedure known in the liter-
ature.25–27 An excess of amine (30 equiv) used as solvent
and water (18 equiv) were refluxed with epoxide 8 to af-
ford the desired products 9–20 with low to medium
yields (10–88%) after tedious purification (Scheme 2).
However, a number of amines did not react, thus limit-
ing the possibility of diversification. We then applied a
new procedure,20 Method B, which we had previously
developed for the aminolysis of a hindered steroidal
epoxide and that allows introducing a larger diversity
2.1.3. Synthesis of aminosteroids 54–71 by amidation and
reduction. The N-alkylpiperazine derivatives 54–69 were
obtained from 53 (Scheme 3). The latter was first synthe-
sized from epoxide 8 in 70% yield by the Method A of
aminolysis given above, except that we utilized a
Schlenk tube. The amidation of 53 with a carboxylic
acid, HBTU as coupling agent, and DIPEA as base gave
good yields (74–90%) of amides 54–61. The carbonyl
reduction with BH3-THF afforded the alkylamine che-
lated with boron.30 This complex was then broken in
refluxing methanol overnight to give the desired prod-
ucts 62–69 in good yields after purification. In the for-
mation of the last two compounds, 70 and 71 (Scheme
4), from the secondary amines 33 and 35, no complexa-
tion occurred during the reduction step, probably due to
high steric hindrance. A simple work-up followed by
flash chromatography gave the tertiary amines 70 and
71.
Scheme 2. Synthesis of aminosteroids 9–52 with methods A and B.
Reagents and conditions: (a) Method A: amine (30 equiv), H2O (18
equiv), reflux, 24 h; (b) Method B: amine (3 equiv), Gd(OTf)3 (0.2
equiv), toluene, Schlenk tube, 150–190 ꢁC, 2 h or overnight. The nature
of R groups is given in Tables 1–4 and their structure in Supplemen-
tary data.
Scheme 3. Synthesis of aminosteroids 53–69. Reagents and conditions: (a) Piperazine, H2O, Schlenk tube (150–190 ꢁC); (b) HBTU, carboxylic acid,
DIPEA, DMF, 0 ꢁC to rt; (c) BH3-THF, THF, 0 ꢁC to rt then reflux in MeOH overnight. The nature of the R groups is given in Tables 1, 2 and 4 and
their structure in Supplementary data.

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5065
Scheme 4. Synthesis of N,N-dialkyl aminosteroids 70 and 71. Reagents and conditions: (a) HBTU, carboxylic acid, DIPEA, DMF, 0 ꢁC to rt; (b)
BH3-THF, THF, 0 ꢁC to rt.
2.2. Biological results
Table 2. Percentage of inhibition of HL-60 cell growth by tertiary
amines (N-alkylamido or N-alkylamino derivatives of piperazine)a
HL-60 cells were incubated for 3 days at 37 ꢁC with syn-
#
R
Inhibition Inhibition
(%) at
thesized compounds in a humidified atmosphere of 95%
air/5% CO2. For screening purposes, the cell prolifera-
tion assay was performed at concentrations of 1 and
10 lM for all compounds and the results were expressed
as the percentage of cell growth inhibition (Tables 1–4).
Doxorubicin, a well-known cytotoxic agent used for the
treatment of leukemia,31 and lead compound 1 were
used as references. The antiproliferative activity of 1
that we measured (16% at 10 lM) was however lower
than expected based on published data.16 The fact that
our experimental conditions were not exactly the same
probably explains this discrepancy. Regardless of this,
we decided to begin a SAR study in order to generate
2b-aminosteroids with potentially better antiprolifera-
tive properties. Since we suspected that the methyl–pip-
erazinyl moiety was not the optimal building block for
cell growth inhibition, our strategy was to diversify the
amino group at carbon 2. We first tested the effect of
several cyclic amines and of a series piperazine deriva-
tives on cell proliferation (Table 1). At a concentration
of 1 lM, no significant antiproliferative activity was ob-
served for any compound tested. Although most of the
(%) at
10 lM
1 lM
1
48
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
Doxob
4-Methylpiperazinyl
4-Acetylpiperazinyl
0.0 4.7 16.2 6.0
0.0 6.0
0.0 1.4
0.0 2.9
0.0 7.6
0.0 3.8
0.0 6.2
0.0 5.8
0.0 7.1
4.6 2.7
1.1 2.7
7.4 1.5
5.5 2.4
4-Propanoylpiperazinyl
4-Butanoylpiperazinyl
4-iso-Butanoylpiperazinyl
4-Pentanoylpiperazinyl
4-iso-Pentanoylpiperazinyl
4-Nonanoylpiperazinyl
8.1 3.7 99.0 0.7
3.9 3.7
4-Cyclohexylcarbonylpiperazinyl 0.0 4.0
4-Benzoylpiperazinyl
4-Propylpiperazinyl
4-Butylpiperazinyl
4-iso-Butylpiperazinyl
4-Pentylpiperazinyl
4-iso-Pentylpiperazinyl
4-Nonylpiperazinyl
4-Cyclohexylmethylpiperazinyl
4-Benzylpiperazinyl
—
0.0 5.5 11.4 0.5
0.0 11.7 0.0 11.3
0.0 4.2
0.0 5.2
7.6 4.8
5.0 3.5
0.0 10.8 7.7 4.2
0.0 8.5 9.2 2.2
9.1 5.0 99.8 0.2
0.0 5.3 4.0 0.6
0.0 2.8 25.7 2.8
91.8–96.2 94.6–95.3
a The chemical structure of R group is provided in Supplementary
data. Results are the means ( SD) of triplicates.
b Doxorubicin.
Table 1. Percentage of inhibition of HL-60 cell growth by tertiary
amines (cyclic amines and N-piperazine derivatives)a
cyclic amines represented in Table 1 did not give
good results, three compounds—2b-(4-benzylpiperidi-
nyl)-5a-androstane-3a,17b-diol (15), 2b-{4-(4-nitro-
#
R
Inhibition Inhibition
(%) at
(%) at
10 lM
1 lM
phenyl)-piperazinyl}-5a-androstane-3a,17b-diol
(18),
1
4-Methylpiperazinyl
Pyrrolidinyl
0.0 4.7 16.2 6.0
and 2b-(4-piperidinylpiperidinyl)-5a-androstane-3a,17b-
diol (52)—inhibit significantly (over 50%) the cell growth
at 10 lM.
9
10
11
12
13
14
15
16
17
18
19
20
49
50
51
52
53
Doxob
0.0 4.6
0.0 0.5
0.0 7.7
9.9 4.8
3.4 9.8
4.8 6.3
Homopiperidinyl
4-Ethylpiperazinyl
4-Hydroxyethylpiperazinyl
Morpholinyl
0.8 5.5 17.7 3.4
14.9 1.7 8.7 5.7
The best results being obtained with the piperazine
derivative 18, we then extended our study by testing a
series of compounds with various alkyl chains on the
piperazine moiety. We first added different carboxylic
acids on the piperazine moiety to generate amide deriv-
atives 54–61. The corresponding amine derivatives 62–
69, without a carbonyl group, were next synthesized
and tested on HL-60 cells in the same conditions. Re-
sults reported in Table 2 are similar to those of Table
1, except for amide 59 and amine 67. Indeed, at 10 lM
the presence of a long alkyl side chain fully reduced
(>99%) the cell growth. No antiproliferative effect was
however obtained with shorter side chains.
Homopiperazinyl
4-Benzylpiperidinyl
1,2,3,4-Tetrahydroisoquinolinyl 3.4 6.2 29.6 4.8
1.0 10.5 21.4 4.3
9.9 8.7 57.9 1.7
1-Pyridin-2-yl-piperazinyl
1-(4-Nitrophenyl)-piperazinyl
4-Piperonylpiperazinyl
Pyrazolyl
0.0 2.9 26.1 4.6
12.5 1.6 92.2 1.6
0.0 2.2 29.1 2.1
8.4 9.1 12.8 4.9
7.0 2.2 36.6 5.4
0.0 5.8 10.1 2.9
4-Phenylpiperazinyl
4-Fluorophenylpiperazinyl
Piperidinyl
0.0 9.9 1.5 7.9
0.0 1.3 77.8 0.6
4-(4-Piperidinyl)piperidinyl
Piperazinyl
—
6.9 7.9
91.8–96.2
5.9 1.7
94.6–95.3
a The chemical structure of R group is provided in Supplementary
data. Results are the means ( SD) of triplicates.
b Doxorubicin.
We next elaborated and tested a third series of
compounds, which were synthesized by the opening of

5066
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
Table 3. Percentage of inhibition of HL-60 cell growth by secondary
amines (normal and branched alkyls)a
alkyl amines with an oxygen atom (ether function), and
amines containing an aromatic ring at the end of the chain
or a bulky group like adamantylmethyl. The only com-
pounds that gave a good inhibition (86% and 95%) in this
fourth screening were compounds 45 and 46, with hin-
dered and bulky building blocks 3,3-diphenylpropyl-
amine and adamantylmethylamine, respectively. Dialkyl
compounds 70 and 71 were synthesized because their cor-
responding monoalkyl chains (compounds 33 and 35)
afforded good results at 10 lM. But dialkyl compounds
70 and 71 did not afford any good inhibition. With their
two long n-alkyl chains, it is possible that these tertiary
aminosteroids remain in the phospholipid bilayer of the
membrane cells.32 On the other hand, compounds 45
and 46 are probably not retained in the phospholipid bi-
layer since their bulky groups are not structurally similar
to the long alkyl chains of compounds 70 and 71.
Although less impressive than 45 and 46, compounds
42–44 also produced some cell growth inhibition at
10 lM (27–43%). These compounds are however less po-
tent than three aminosteroids (see 3 in Fig. 1) synthesized
using solid-phase chemistry (IC50 = 0.58–2.87 lM)19 and
much less potent than doxorubicin (IC50 = 0.10 lM) used
as a reference compound.
#
R
Inhibition
(%) at
Inhibition
(%) at
1 lM
10 lM
1
4-Methylpiperazinyl
n-Propylamino
i-Propylamino
n-Butylamino
i-Butylamino
0.0 4.7
0.0 3.9
0.0 7.4
0.0 3.4
0.0 2.9
0.0 3.6
0.0 0.8
0.0 5.3
0.0 7.0
0.0 2.9
0.0 5.4
10.1 3.8
0.0 0.8
3.9 4.5
0.0 4.9
0.0 0.6
0.0 4.9
0.0 5.2
91.8–96.2
16.2 6.0
0.4 5.6
0.0 1.9
3.1 1.2
0.0 0.8
0.5 2.2
0.0 0.7
3.0 5.6
3.3 7.9
1.8 4.1
6.9 1.2
82.3 3.3
99.2 2.9
89.9 0.2
96.9 0.7
96.3 0.3
92.0 0.3
94.8 4.5
94.6–95.3
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Doxob
tert-Butylamino
n-Pentylamino
i-Pentylamino
neo-Pentylamino
tert-Pentylamino
n-Hexylamino
n-Heptylamino
n-Octylamino
n-Nonylamino
n-Decylamino
n-Undecylamino
n-Dodecylamino
n-Hexadecylamino
—
a The chemical structure of R group is provided in Supplementary
data. Results are the means ( SD) of triplicates.
b Doxorubicin.
Figure 2 summarizes the biological screening data ob-
tained with the more active aminosteroids synthesized in
our study (Tables 1–4). They can be divided in two
groups, the tertiary (15, 18, 52, 59, and 67) and secondary
(32, 33, 36, 45, and 46) amines, but they roughly induce the
same level of growth inhibition on HL-60 cells. Three rep-
resentative candidates of each group were selected for
IC50 determination, the n-octyl secondary amine 32, the
diphenyl–propyl secondary amine 45, and the n-octyl–
piperazinyl tertiary amine 67. As reported in Figure 3,
the IC50 values are similar for 45 and 67 (3.1 and
4.2 lM, respectively), but higher for 32 (6.4 lM).
Table 4. Percentage of inhibition of HL-60 cell growth by secondary
amines (cyclic and alkyl derivatives)a
#
R
Inhibition
(%) at
Inhibition
(%) at
1 lM
10 lM
1
38
4-Methylpiperazinyl
Cyclopentylamino
Cyclohexylamino
Cycloheptylamino
3-Ethoxypropylamino
3-Butoxypropylamino
2-Phenylethylamino
3-Phenylpropylamino
3,3-Diphenylpropylamino
Adamantylmethylamino
Diethylamino
0.0 4.7
0.0 5.7
0.0 3,5
0.0 8.8
0.0 2.9
4.8 4.3
9.5 1.7
0.9 3.8
12.9 2.5
6.4 3.7
0.0 3.7
9.8 2.0
11.7 5.9
91.8–96.2
16.2 6.0
0.0 7.1
0.0 2.0
0.0 4.1
10.8 1.2
26.5 1.9
42.8 4.2
37.6 2.7
95.2 0.5
85.8 0.8
6.7 3.0
41.5 1.5
7.0 1.9
94.6–95.3
39
40
41
42
43
44
After we identified the representative aminosteroids 32,
45, and 67 from the antiproliferative screening assay,
we tested their ability to induce the differentiation of
HL-60 cells. In fact, an attractive alternative to the anti-
proliferative approach is to induce cell differentiation in
order to convert malignant cells into mature resting
cells.33 Furthermore, the lead compound 1 was previ-
ously reported to induce the differentiation of HL-60
cells,16 and the stimulation of cell differentiation by a
therapeutic agent is particularly promising for the treat-
ment of human leukemias. To do so, we evaluated expres-
sion of surface antigens CD11b (both granulocyte- and
monocyte-like cell marker) and CD14 (monocyte-like cell
marker) because in leukemia, the cell differentiation be-
gins with an amplification of the expression of these
two antigens. The markers were analyzed using flow
cytometry after incubation for 72 h with medium supple-
mented with a 10 lM concentration of aminosteroids
(Fig. 4). Vitamin D3 (VD3), a chemical agent well known
to induce differentiation,13–15 served as positive control.
Treatment of HL-60 with VD3 increased CD11b and
double-marked CD11b/CD14 expressing cells to 65%
and 32%, respectively. A 3-day exposure to compound
1 induced a weak increase in the expression of CD11b
but it did not modify the levels of expression of CD14
45
46
47
70
Dinonylamino
Diundecylamino
—
71
Doxob
a The chemical structure of R group is provided in Supplementary
data. Results are the means ( SD) of triplicates.
b Doxorubicin.
epoxide 8 with normal or branched alkyl primary
amines (Table 3). Although this series of secondary ami-
nosteroids were not active at 1 lM, better results were
attained with long n-alkylamines at 10 lM (compounds
31–37). In fact, the levels of inhibition obtained with
side chains longer (7 to 16 carbons) than hexyl (6 car-
bons) were all above 82%. Similarly as observed for
the tertiary aminosteroids reported in Table 2, no inter-
esting antiproliferative effect was obtained with the sec-
ondary aminosteroids having a short alkyl group
(compounds 21–31).
In the last series of aminosteroids (Table 4), we tested
additional secondary amines such as cycloalkylamines,

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5067
Figure 2. Percentage (%) of HL-60 cell growth inhibition produced by aminosteroids selected from our screening study and that produce over 50% of
inhibition. Results are the means of triplicates ( SD). Only their 2b-groups are represented in the bottom of the histogram.
or CD11b/CD14 (double-marked) cell surface markers as
indicated by flow cytometry analysis. On the other hand,
we observed a stronger differentiation of HL-60 cells fol-
lowing treatments with 32, 45, and 67, as CD11b and
double-marked CD11b/CD14 were significantly up-regu-
lated during the treatment. The percentage of CD14 po-
sitive cells was however not affected. Compound 32
induced the expression of CD11b in 29% of cells and that
of CD11b/CD14 in 10% of cells. It was however clearly
less efficient than 45 and 67 in the induction of differenti-
ation. In fact, after incubation with 45, we measured 58%
and 39% of cells expressing CD11b and double-marked
CD11b/CD14, respectively, and these results were similar
to those obtained after treatment with 67 (55% and 32%,
respectively). Interestingly, these levels of differentiation
are close to the effects obtained with VD3.
Figure 3. Effect of increasing concentrations of aminosteroids 32, 45,
and 67 on HL-60 cell growth represented by the absorbance at 490 nm
(see Section 4). Cells (1 · 104) were incubated with various concentra-
tions of each compound for 3 days. IC50 represents the concentration
3. Conclusion
The chemical synthesis of a series of 2b-amino-5a-
androstane-3a,17b-diol N-derivatives 9–71 was per-
that inhibits 50% of the cell growth. Results are the means ( SD) of
triplicates.
Figure 4. Expression (%) of CD11b, CD14, and CD11b/CD14 cell surface markers in HL-60 cells after a 72 h-treatment with vitamin D3 (VD3) or
aminosteroid 1, 32, 45, or 67. Results are the means ( SEM) of triplicates. *,**Indicate a result significantly different from the control (CTL)
(*P < 0.05; **P < 0.01).

5068
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
formed in order to improve the antiproliferative proper-
ties on HL-60 leukemia cells of the lead compound 1,
the first representative of this family of aminosteroids.
A large series of 2b-tertiary and 2b-secondary aminos-
teroids was thus obtained from the aminolysis of epox-
ide 8 using a classical or a new methodology. It
appears that the 4-methylpiperazinyl group can be mod-
ified resulting in a better antiproliferative activity. The
tertiary aminosteroid 67, with an IC50 value of 4.2 lM,
illustrates the inhibitory effect of a long nonyl side chain
replacing the N-methyl group of 1. This finding is in
agreement with the results obtained with a series of pip-
erazinyl derivatives generated by solid-phase organic
synthesis, which clearly demonstrated that substituting
the N-methyl piperazine ring with a more complex ami-
noacid building block modulates the antiproliferative
activity.19 The 4-methylpiperazinyl moiety can also be
replaced by an appropriate secondary amine as exempli-
fied by aminosteroids 32 and 45. The diphenylpropyl
amino moiety of 45 showed better antiproliferative po-
tential than the octyl amino group of 32 (IC50 = 3.1
and 6.4 lM, respectively). In addition to their antiprolif-
erative activity, the three aminosteroids tested induce
the differentiation of HL-60 cells. Compounds 45 and
67 thus induce the same level of expression of cell mark-
ers as vitamin D3, a known differentiating agent.
nium heptamolybdate tetrahydrate. Flash column chro-
matography was performed on Silicycle R10030B 230-
´
400 mesh silica gel (Quebec, QC, Canada). Infrared
spectra (IR) were obtained from a thin film obtained
with compound usually solubilized in CDCl3 and lay-
ered over a NaCl pellet. They were recorded with a Per-
kin-Elmer series 1600 FT-IR spectrometer (Norwalk,
CT, USA) and only significant bands were reported in
cmꢀ1. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AC/F300 spectrometer (Billerica,
MA, USA), a Bruker Avance 400 digital spectrometer
or an Inova Varian AS 400 (Palo Alto, CA, USA).
The chemical shifts (d) were expressed in ppm and refer-
1
enced to CHCl3 (7.26 and 77.0 ppm) for H and 13C,
respectively. Only characteristic signals were reported.
Low-resolution mass spectra (LRMS) were recorded
on a PE Sciex API-150ex apparatus (Foster City, CA,
USA) equipped with a turbo ionspray source. High-res-
olution mass spectra (HRMS) were provided by Pierre
´
Audet at the Departement de chimie de l’Universite La-
val (Quebec, QC, Canada). The HPLC purity was deter-
´
´
mined with a Waters’ apparatus (Waters Associates
Milford, MA, USA) using a Nova Pak C18 reversed-
˚
phase column (150 mm · 3.9 mm id, 4 lm, 60 A) and
methanol and/or water containing 10 mM of ammo-
nium acetate as eluent.
We succeeded in optimizing the antiproliferative activity
of lead compound 1 by a modification of the amino
group at position 2b. This first SAR study identified
some chemical groups more suitable for a better biolog-
ical activity, but could be extended by modifying the ste-
reochemistry at C2, the positioning of the amino moiety
on the steroid nucleus, and the nature and stereochemis-
try of the substituent at C3. The steroid nucleus appears
however to be a good scaffold because the biological
activity was lost when the four-ring steroid nucleus
was replaced by a single cyclohexyl nucleus (data not re-
ported). It is however not clear yet if the tertiary and sec-
ondary aminosteroids identified in our study possess the
same mechanism of action, and additional studies will
be necessary to elucidate their mechanism of action.
Identification of this mechanism could help us to ratio-
nalize our results and design other active compounds
more efficiently.
4.1.1. Classical method for aminolysis of epoxide 8
(synthesis of 1 and 9–20). Epoxide 819 (1 mmol), the de-
sired amine (30 mmol), and H2O (18 equiv) were re-
fluxed 24 h. The reaction mixture was then cooled,
poured in water, and the precipitate was filtered. The so-
lid was dissolved in CH2Cl2 and the solution dried over
MgSO4, filtered, and evaporated to dryness. The crude
product was then purified by flash chromatography
using an appropriate mixture of MeOH:CH2Cl2.
4.1.1.1.
2b-(4-Methylpiperazinyl)-5a-androstane-
3a,17b-diol (1).16 Yield: 88%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.08; IR (film): 3369 (OH); 1H NMR
(400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.85 (s, 19-
CH3), 0.70–2.10 (residual H), 2.32 (s, 40-CH3), 2.53
(m, 2· CH2N and 2a-CH), 2.71 (m, 2· CH2N), 3.62
(t, J = 8.5 Hz, 17a-CH), 3.84 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 11.18, 17.15, 20.78, 23.31, 28.16,
30.56, 31.09, 32.70, 34.64, 35.41, 35.72, 36.88, 38.43,
42.94, 45.58, 50.84, 55.22 (4·), 56.10, 63.60 (C3),
64.37 (C2), 81.26; LRMS for C24H43N2O2 [M+H]+:
391.3 m/z; HRMS: calcd for C24H43N2O2 [M+H]+
391.33191, found 391.33211.
4. Experimental
4.1. Chemistry
Dihydrotestosterone (DHT) was purchased from Stera-
loids (Wilton, NH, USA) or Sigma–Aldrich Canada
Ltd. (Oakville, ON, Canada), while chemical reagents
were from Sigma–Aldrich Canada Ltd. The usual sol-
4.1.1.2. 2b-(Pyrrolidinyl)-5a-androstane-3a,17b-diol
(9). Yield: 41%; TLC (hexanes/acetone, 6:4): Rf = 0.2;
IR (film): 3325 (OH); 1H NMR (400 MHz, CDCl3):
0.73 (s, 18-CH3), 1.04 (s, 19-CH3), 0.70–2.10 (residual
H), 2.38 (m, 2a-CH), 2.53 (m, 2· CH2N), 3.62 (m,
17a-CH), 4.04 (m, 3b-CH); 13C NMR (75 MHz,
CDCl3): 11.15, 13.23, 20.60, 23.43 (3·), 27.86, 30.53,
31.53, 32.57, 34.89, 35.07, 36.41, 36.78, 39.35, 43.01,
51.07, 51.47 (2x), 55.40, 65.98 (C3), 67.91 (C2), 81.98;
LRMS for C23H40NO2 [M+H]+: 362.2 m/z; HRMS:
calcd for C23H40NO2 [M+H]+ 362.30536, found
362.30593.
´
vents were obtained from Fisher Scientific (Montreal,
QC, Canada) and were used as received. Tetrahydrofu-
ran (THF) was distilled from sodium/benzophenone ke-
tyl under argon. All anhydrous reactions were
performed in oven-dried glassware under positive argon
pressure. Thin-layer chromatography was performed on
´
0.20 mm silica gel 60 F254 plates (Fisher, Montreal,
Canada) and compounds were visualized using ammo-

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5069
4.1.1.3. 2b-(Homopiperidinyl)-5a-androstane-3a,17b-
diol (10). Yield: 18%; TLC (CH2Cl2/MeOH, 9:1): Rf =
0.26; IR (film): 3366 (OH); 1H NMR (400 MHz,
CDCl3): 0.74 (s, 18-CH3), 0.85 (s, 19-CH3), 0.70–2.20
(residual H), 2.51 (m, CH2N), 2.75 (m, CH2N and 2a-
CH), 3.62 (t, J = 8.5 Hz, 17a-CH), 3.74 (m, 3b-CH);
13C NMR (100.6 MHz, acetone-d6): 11.81, 17.03,
21.74, 24.14, 27.63 (2·), 29.31, 30.43 (2·), 31.07, 32.26,
34.91, 35.85, 36.51, 36.58, 37.99, 39.57, 44.00, 52.03,
52.08 (2·), 57.29, 65.41 (C3), 66.80 (C2), 81.87; LRMS
for C25H44NO2 [M+H]+: 390.0 m/z; HRMS: calcd for
C25H44NO2 [M+H]+ 390.33666, found 390.33656.
51.61, 52.23, 57.46, 66.97 (C2), 67.07 (C3), 82.47; LRMS
for C24H43N2O2 [M+H]+: 391.3 m/z; HRMS: calcd for
C24H43N2O2 [M+H]+ 391.33191, found 391.33195.
4.1.1.8. 2b-(4-Benzylpiperidinyl)-5a-androstane-3a,17b-
diol (15). Yield: 18%; TLC (hexanes/acetone, 6:4):
Rf = 0.45; IR (film): 3350 (OH); H NMR (400 MHz,
1
CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-CH3), 0.70–2.20
(residual H), 2.54 (d, J = 4.8 Hz, CH2Ph), 2.32, 2.60
and 2.90 (3m, 2a-CH and 2· CH2N), 3.62 (t,
J = 8.5 Hz, 17a-CH), 3.88 (m, 3b-CH), 7.12 (d,
J = 7.1 Hz, 2H of Ar), 7.20 (t, J = 7.3 Hz, 1H of Ar),
7.29 (d, J = 7.1 Hz, 2H of Ar); 13C NMR (100.6 MHz,
CDCl3): 11.15, 17.68, 20.97, 23.30, 28.17, 30.51, 31.00,
33.45 (2·), 35.40, 35.53, 35.88, 36.76, 37.72, 38.47,
42.73, 43.10, 44.94, 50.81, 52.32 (2·), 56.27, 63.73
(C3), 65.11 (C2), 81.78, 125.97, 128.23 (2·), 129.00
(2·), 140.06; LRMS for C31H48NO2 [M+H]+: 466.5 m/
z; HRMS: calcd for C31H48NO2 [M+H]+ 466.36796,
found 466.36801.
4.1.1.4. 2b-(4-Ethylpiperazinyl)-5a-androstane-3a,17b-
diol (11). Yield: 58%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.12; IR (film): 3396 (OH); H NMR (400 MHz,
1
CDCl3): 0.73 (s, 18-CH3), 0.84 (s, 19-CH3), 1.10 (t,
J = 7.1 Hz, 200-CH3), 0.60–2.20 (residual H), 2.43 (m,
CH2), 2.53 (m, 2 · CH2N), 2.68 (3m, 2· CH2N and
2a-CH), 3.61 (m, 17a-CH), 3.83 (m, 3b-CH); 13C
NMR (100.6 MHz, CDCl3): 11.19, 11.87, 17.28, 20.79,
23.37, 28.26, 30.65, 31.18, 32.55, 34.66, 35.47, 35.72,
36.90, 38.43, 43.00, 50.87, 52.29, 53.32 (4·), 56.15,
63.53 (C3), 64.34 (C2), 81.41; LRMS for C25H45N2O2
[M+H]+: 405.6 m/z; HRMS: calcd for C25H45N2O2
[M+H]+ 405.34756, found 405.34813.
4.1.1.9. 2b-(1,2,3,4-Tetrahydroisoquinolinyl)-5a-andro-
stane-3a,17b-diol (16). Yield: 10%; TLC (hexanes/ace-
tone, 6:4): Rf = 0.69; IR (film): 3390 (OH); H NMR
1
(400 MHz, CDCl3): 0.74 (s, 18-CH3), 0.90 (s, 19-CH3),
0.70–2.15 (residual H), 2.64 (m, 2a-CH), 2.93 (m,
NCH2–CH2–Ar), 3.63 (t, J = 8.5 Hz, 17a-CH), 3.74
and 3.90 (2d, J = 14.7 Hz, NCH2–Ar), 3.96 (m, 3b-
CH), 7.02 (m, 1H of Ar), 7.13 (m, 3H of Ar); 13C
NMR (75 MHz, CDCl3): 11.18, 17.41, 20.93, 23.31,
28.22, 29.64, 30.50, 31.11, 32.74, 34.88, 35.54, 35.84,
36.78, 38.55, 43.07, 45.62, 50.84, 51.19, 56.23, 63.97
(C3), 64.35 (C2), 81.85, 125.69, 126.23, 126.52, 128.73,
134.30 (2·); LRMS for C28H42NO2 [M+H]+: 424.0
m/z; HRMS: calcd for C28H42NO2 [M+H]+ 424.32101,
found 424.32141.
4.1.1.5. 2b-{4-(2-Hydroxyethyl)piperazinyl}-5a-andro-
stane-3a,17b-diol (12). Yield: 67%; TLC (CH2Cl2/
MeOH, 9:1): Rf = 0.07; IR (film): 3328 (OH); 1H
NMR (400 MHz, CDCl3): 0.74 (s, 18-CH3), 0.85 (s,
19-CH3), 0.70–2.20 (residual H), 2.45–2.75 (broad,
5 · CH2N and 2a-CH), 3.62 (m, 17a-CH and CH2OH),
3.84 (m, 3b-CH); 13C NMR (75 MHz, CDCl3): 11.19,
17.26, 20.90, 23.31, 28.21, 30.48, 31.13, 32.75, 34.65,
35.51, 35.77, 36.79, 38.44, 43.08, 45.69, 50.86, 53.35
(2·), 56.15, 57.60 (2·), 59.21, 63.66 (C3), 64.45 (C2),
81.83; LRMS for C25H45N2O3 [M+H]+: 421.6 m/z;
HRMS: calcd for C25H45N2O3 [M+H]+ 421.34247,
found 421.34279.
4.1.1.10. 2b-{4-(2-Pyridyl)piperazinyl}-5a-androstane-
3a,17b-diol (17). TLC (hexanes/acetone, 6:4): Rf = 0.59;
IR (film): 3401 (OH), 1596 (C@C); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.86 (s, 19-CH3),
0.65–2.15 (residual H), 2.56 (m, CH2N), 2.73 (m, 3H,
CH2N and 2a-CH), 3.53 (m, 2· CH2N), 3.62 (t,
J = 8.6 Hz, 17a-CH), 3.88 (m, 3b-CH), 6.62 (m, 2H of
Ar), 7.47 (m, 1H of Ar), 8.20 (m, 1H of Ar); 13C
NMR (75 MHz, CDCl3): 11.20, 17.26, 20.88, 23.32,
28.20, 30.48, 31.14, 32.66, 34.62, 35.49, 36.00, 36.76,
38.44, 43.10, 45.78 (2·), 48.05 (2·), 50.85, 56.11, 63.73
(C3), 64.72 (C2), 81.87, 107.09, 113.34, 137.46, 147.93,
ꢁ160.00; LRMS for C28H44N3O2 [M+H]+: 454.1 m/z;
HRMS: calcd for C28H44N3O2 [M+H]+ 454.34280,
found 454.34367.
4.1.1.6. 2b-(Morpholinyl)-5a-androstane-3a,17b-diol
(13). Yield: 42%; TLC (hexanes/acetone, 6:4):
Rf = 0.57; IR (film): 3442 (OH); H NMR (400 MHz,
1
CDCl3): 0.74 (s, 18-CH3), 0.87 (s, 19-CH3), 0.65–2.15
(residual H), 2.45 (m, CH2N), 2.63 (m, CH2N and 2a-
CH), 3.73 (m, 17a-CH, 2· CH2O), 3.86 (m, 3b-CH);
13C NMR (75 MHz, CDCl3): 11.18, 16.94, 20.89,
23.33, 28.16, 30.53, 31.18, 32.58, 34.42, 35.50, 35.84,
36.80, 38.53, 43.10, 48.88 (2·), 50.90, 56.07, 63.65
(C3), 65.02 (C2), 67.48 (2·), 81.91; LRMS for
C23H40NO3 [M+H]+: 378.3 m/z; HRMS: calcd for
C23H40NO3 [M+H]+ 378.30027, found 378.30053.
4.1.1.11. 2b-{4-(4-Nitrophenyl)-piperazinyl}-5a-andro-
stane-3a,17b-diol (18). Yield: 12%; TLC (hexanes/ace-
tone, 6:4): Rf = 0.61; IR (film): 3406 (OH), 1597, 1327
4.1.1.7. 2b-(Homopiperazinyl)-5a-androstane-3a,17b-
diol (14). Yield: 62%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.02; IR (film): 3372 (OH and NH); 1H NMR
(300 MHz, CD3OD): 0.72 (s, 18-CH3), 0.90 (s, 19-
CH3), 0.70–2.10 (residual H), 2.65–3.35 (m, 4· CH2N
and 2a-CH), 3.55 (t, J = 8.5 Hz, 17a-CH), 3.90 (m, 3b-
CH); 13C NMR (75 MHz, CD3OD): 11.76, 17.15,
22.03, 24.28, 27.82, 29.40, 30.65, 32.41, 36.68, 36.87,
37.01, 38.12, 40.13, 43.53, 44.21, 46.05, 47.92, 48.88,
1
(NO2); H NMR (400 MHz, CDCl3): 0.72 (s, 18-CH3),
0.87 (s, 19-CH3), 0.70–2.10 (residual H), 2.61 (m,
CH2N), 2.70 (m, 2a-CH), 2.79 (m, CH2N), 3.42 (m, 2·
CH2N), 3.61 (t, J = 8.5 Hz, 17a-CH), 3.91 (m, 3b-CH),
6.81 (d, J = 9.4 Hz, 2H of Ar), 8.13 (d, J = 9.3 Hz, 2H
of Ar); 13C NMR (75 MHz, CD3OD): 11.74, 14.28,
21.76, 24.30, 29.00, 30.62, 32.79, 33.90, 34.36, 36.53,

5070
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
37.36, 38.11, 40.66, 44.15, 47.10, 48.21, 51.52 (2·), 52.42,
56.84, 66.57 (C2), 66.76 (C3), 82.50, 113.62 (2·), 126.73
(2·), 139.26, 156.59; LRMS for C29H44N3O4 [M+H]+:
498.5 m/z; HRMS: calcd for C29H44N3O4 [M+H]+
498.33263, found 498.33251.
HRMS: calcd for C22H40NO2 [M+H]+ 350.30536, found
350.30647.
4.1.2.3. 2b-(iso-Propylamino)-5a-androstane-3a,17b-
diol (22). Yield: 46%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.02; IR (film): 3441 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.92 (s, 19-CH3),
0.70-2.20 (residual H), 1.34 and 1.45 (2d, J = 6.4 Hz,
2 · 20-CH3), 3.31 (m, 10-CH), 3.41 (m, 2a-CH), 3.63 (t,
J = 8.5 Hz, 17a-CH), 4.10 (m, 3b-CH); 13C NMR
(100.6 MHz, CDCl3): 11.14, 17.10, 17.77, 20.24, 21.06,
23.24, 27.87, 30.44, 30.63, 35.39, 35.91 (2·), 36.57,
38.89, 38.93, 43.04, 47.93, 50.61, 56.20, 56.76 (C2),
66.10 (C3), 81.76; LRMS for C22H40NO2 [M+H]+:
350.4 m/z; HRMS: calcd for C22H40NO2 [M+H]+
350.30536, found 350.30588.
4.1.1.12. 2b-(4-Piperonylpiperazinyl)-5a-androstane-
3a,17b-diol (19). Yield: 22%; TLC (hexanes/acetone,
6:4): Rf = 0.39; IR (film): 3378 (OH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.84 (s, 19-CH3),
0.70–2.15 (m, residual H), 2.46 (m, 2a-CH and 2·
CH2N), 2.66 (m, 2 · CH2N), 3.40 (s broad, NCH2Ar),
3.60 (t, J = 8.5 Hz, 17a-CH), 3.81 (m, 3b-CH), 5.93 (s,
OCH2O), 6.73 (m, 2H of Ar), 6.84 (s, 1H of Ar); 13C
NMR (75 MHz, CDCl3): 11.17, 17.23, 20.89, 23.33,
28.25, 30.54, 31.18, 32.86, 34.71, 35.55, 35.78, 36.85,
38.50, 43.11, 50.93, 53.44 (4·), 56.22, 62.70, 63.62
(C3), 64.46 (C2), 81.81, 100.84, 107.82, 109.49, 122.21,
131.94, 146.59, 147.61; LRMS for C31H47N2O4
[M+H]+: 511.3 m/z; HRMS: calcd for C31H47N2O4
[M+H]+ 511.35303, found 511.35339.
4.1.2.4. 2b-(n-Butylamino)-5a-androstane-3a,17b-diol
(23). Yield: 55%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.18;
IR (film): 3358 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.72 (s, 18-CH3), 0.91 (t, J = 7.3 Hz, 40-CH3),
0.95 (s, 19-CH3), 0.70–2.20 (residual H), 2.56 and 2.68
(2m, 10-CH2), 2.74 (m, 2a-CH), 3.62 (t, J = 8.5 Hz,
17a-CH), 3.81 (m, 3b-CH); 13C NMR (100.6 MHz,
CDCl3): 11.17, 13.99, 15.20, 20.44, 20.60, 23.31, 28.02,
30.47, 31.35, 32.19, 32.97, 35.14, 36.07, 36.75, 39.26,
39.49, 43.02, 47.90, 50.92, 55.56, 59.78 (C2), 69.13
(C3), 81.88; LRMS for C23H42NO2 [M + H]+: 364.4
m/z; HRMS: calcd for C23H42NO2 [M+H]+ 364.32101,
found 364.32207.
4.1.1.13. 2b-(Pyrazolyl)-5a-androstane-3a,17b-diol (20).
Yield: 76%; TLC (hexanes/acetone, 6:4): Rf = 0.57; IR
(film): 3358 (OH); H NMR (400 MHz, CDCl3): 0.72
1
(s, 18-CH3), 0.76 (s, 19-CH3), 0.70–2.20 (residual H),
3.63 (t, J = 8.5 Hz, 17a-CH), 4.53 (m, 2a-CH and 3b-
CH); 6.36 (t, J = 2.2 Hz, 1H of Ar), 7.54 (d,
J = 2.3 Hz, 1H of Ar), 7.63 (d, J = 1.8 Hz, 1H of Ar);
13C NMR (75 MHz, CDCl3): 11.15, 14.82, 20.79,
23.28, 27.89, 30.38, 31.15, 34.17, 35.22, 35.76, 36.69,
39.27, 41.57, 43.01, 50.84, 55.41, 62.22 (C3), 67.38
(C2), 81.76, 105.23, 128.76, 138.83; LRMS for
C22H35N2O2 [M+H]+: 359.2 m/z; HRMS: calcd for
C22H35N2O2 [M+H]+ 359.26930, found 359.26927.
4.1.2.5. 2b-(iso-Butylamino)-5a-androstane-3a,17b-diol
(24). Yield: 47%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.28;
IR (film): 3384 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.73 (s, 18-CH3), 0.96 (m, 19-CH3 and 2 · 30-
CH3), 0.70–2.20 (residual H), 2.48 and 2.63 (2m, 10-
CH2), 2.89 (m, 2a-CH), 3.62 (t, J = 8.5 Hz, 17a-CH),
3.92 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.17, 15.81, 20.35, 20.52, 20.74, 23.29, 27.64, 27.99,
30.45, 31.16, 33.81, 35.22, 36.09, 36.71, 39.10, 39.19,
43.03, 50.84, 55.02, 55.72, 59.99 (C2), 67.95 (C3),
81.86; LRMS for C23H42NO2 [M+H]+: 364.3 m/z;
HRMS: calcd for C23H42NO2 [M+H]+ 364.32101, found
364.32141.
4.1.2. New method for aminolysis of epoxide 8 (synthesis
of 1 and 21–52). In a Schlenk tube purged with argon
were added epoxide 8 (1 mmol) dissolved in toluene
(30 mL), the desired amine (3 mmol), and Gd(OTf)3
(0.2 mmol). The reaction mixture was heated at 150–
190 ꢁC for 2 h (primary amines) or overnight (second-
ary and cyclic amines). The mixture was then cooled,
poured on a silica gel column, and the chromatography
performed with an appropriate mixture of
MeOH:CH2Cl2.
4.1.2.6. 2b-(tert-Butylamino)-5a-androstane-3a,17b-
diol (25). Yield: 89%; TLC (CH2Cl2/MeOH, (9:1):
Rf = 0.20; IR (film): 3427 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.75 (s, 18-CH3), 0.95 (s, 19-CH3),
1.47 (s, 3 · 20-CH3), 0.60–2.20 (residual H), 3.26 (m,
2a-CH), 3.64 (t, J = 8.5 Hz, 17a-CH), 4.12 (m, 3b-
CH); 13C NMR (100.6 MHz, CDCl3): 11.15, 16.86,
20.85, 23.28, 26.86 (3·), 27.99, 30.39, 30.82, 35.40,
35.49, 36.43, 36.64, 38.96, 41.59, 43.07, 50.70, 56.25,
56.82 (C2), 59.18, 65.22 (C3), 81.81; LRMS for
C23H42NO2 [M+H]+: 364.5 m/z; HRMS: calcd for
C23H42NO2 [M+H]+ 364.32101, found 364.32143.
4.1.2.1. 2b-(4-Methylpiperazinyl)-5a-androstane-3a,
17b-diol (1).16 This compound was obtained in 88%
yield. The data were identical as reported in the previous
section.
4.1.2.2. 2b-(n-Propylamino)-5a-androstane-3a,17b-diol
(21). Yield: 84%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.12;
IR (film): 3428 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.72 (s, 18-CH3), 0.92 (t, J = 7.4 Hz, 30-CH3),
0.94 (s, 19-CH3), 0.70–2.40 (residual H), 2.58 and 2.68
(2m, 10-CH2), 2.77 (m, 2a-CH), 3.62 (t, J = 8.5 Hz,
17a-CH), 3.81 (m, 3b-CH); 13C NMR (100.6 MHz,
CDCl3): 11.17, 11.70, 15.37, 20.63, 22.92, 23.31, 28.01,
30.46, 31.40, 33.19, 35.16, 36.07, 36.74, 39.22, 39.47,
43.02, 49.78, 50.90, 55.62, 59.63 (C2), 68.95 (C3),
81.87; LRMS for C22H40NO2 [M+H]+: 350.2 m/z;
4.1.2.7. 2b-(n-Pentylamino)-5a-androstane-3a,17b-diol
(26). Yield: 48%; TLC (CH2Cl2/MeOH, (9:1): Rf = 0.24;
IR (film): 3337 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.73 (s, 18-CH3), 0.90 (t, J = 6.9 Hz, 50-CH3),
0.95 (s, 19-CH3), 0.70–2.20 (residual H), 2.60 and 2.74

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5071
(2m, 10-CH2), 2.83 (m, 2a-CH), 3.62 (t, J = 8.5 Hz, 17a-
CH), 3.87 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.17, 14.01, 15.53, 20.69, 22.49, 23.32, 28.01, 29.08,
29.34, 30.49, 31.26, 33.43, 35.20, 36.07, 36.74, 39.23,
39.28, 43.04, 48.78, 50.89, 55.66, 59.80 (C2), 68.60
(C3), 81.89; LRMS for C24H44NO2 [M+H]+: 378.4
m/z; HRMS: calcd for C24H44NO2 [M+H]+ 378.33666,
found 378.33998.
[M+H]+: 392.4 m/z; HRMS: calcd for C25H46NO2
[M+H]+ 392.35231, found 392.35533.
4.1.2.12. 2b-(n-Heptylamino)-5a-androstane-3a,17b-
diol (31). Yield: 77%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.31; IR (film): 3376 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.88 (t,
J = 6.8 Hz, 70-CH3), 0.94 (s, 19-CH3), 0.70–2.20 (resid-
ual H), 2.68 and 2.81 (2m, 10-CH2), 2.94 (m, 2a-CH),
3.63 (t, J = 8.5 Hz, 17a-CH), 3.95 (m, 3b-CH); 13C
NMR (100.6 MHz, CDCl3): 11.16, 14.06, 16.03, 20.79,
22.66, 23.29, 26.92, 27.96, 28.28, 28.96, 30.46, 31.08,
31.65, 34.17, 35.25, 36.02, 36.69, 38.96, 39.13, 43.04,
47.10, 50.81, 55.77, 59.75 (C2), 67.73 (C3), 81.84; LRMS
for C26H48NO2 [M+H]+: 406.4 m/z; HRMS: calcd for
C26H48NO2 [M+H]+ 406.36796, found 406.36919.
4.1.2.8. 2b-(iso-Pentylamino)-5a-androstane-3a,17b-
diol (27). Yield: 99%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.27; IR (film): 3416 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.74 (s, 18-CH3), 0.94 (m, 19-
CH3and 2 · 40-CH3), 0.70–2.20 (residual H), 2.88
and 3.08 (2m, 10-CH2), 3.25 (m, 2a-CH), 3.63 (t,
J = 8.5 Hz, 17a-CH), 4.18 (m, 3b-CH); 13C NMR
(100.6 MHz, CDCl3): 11.16, 17.06, 21.04, 21.99,
22.37, 23.27, 25.95, 27.93, 30.46, 30.75, 34.48,
35.41, 53.63, 35.97, 36.61, 38.19, 39.00, 43.07,
44.24, 50.67, 56.00, 59.84 (C2), 65.89 (C3), 81.78;
LRMS for C24H44NO2 [M+H]+: 378.4 m/z; HRMS:
calcd for C24H44NO2 [M+H]+ 378.33666, found
378.33690.
4.1.2.13. 2b-(n-Octylamino)-5a-androstane-3a,17b-diol
(32). Yield: 93%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.38;
IR (film): 3430 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.73 (s, 18-CH3), 0.88 (t, J = 6.8 Hz, 80-CH3),
0.91 (s, 19-CH3), 0.60–2.10 (residual H), 2.86 and 3.02
(2m, 10-CH2), 3.25 (m, 2a-CH), 3.62 (t, J = 8.1 Hz,
17a-CH), 4.08 (m, 3b-CH); 13C NMR (100.6 MHz,
CDCl3): 11.16, 14.05, 16.89, 21.13, 22.58, 23.39, 26.38,
26.49, 28.07, 28.94, 28.98, 30.44, 30.74, 31.66, 35.38,
35.53, 35.95, 36.61, 38.42, 38.95, 43.05, 45.90, 50.68,
56.00, 59.69 (C2), 66.29 (C3), 81.77; LRMS for
C27H50NO2 [M+H]+: 420.4 m/z; HRMS: calcd for
C27H50NO2 [M+H]+ 420.38361, found 420.38386.
4.1.2.9. 2b-(neo-Pentylamino)-5a-androstane-3a,17b-
diol (28). Yield: 85%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.50; IR (film): 3363 (OH and NH); 1H NMR
(400 MHz, CDCl3 + CD3OD): 0.69 (s, 18-CH3), 0.87
(s, 3 · CH3), 0.93 (s, 19-CH3), 0.65–2.10 (residual H),
2.17 and 2.48 (2d, J = 11.4 Hz, 10-CH2), 2.64 (m, 2a-
CH), 3.57 (t, J = 8.5 Hz, 17a-CH), 3.75 (m, 3b-CH);
13C NMR (100.6 MHz, CDCl3 + CD3OD): 11.02,
15.07, 20.49, 23.16, 27.52 (3·), 27.95, 29.92, 31.23,
31.28, 32.65, 35.03, 35.91, 36.64, 39.14, 39.63, 42.87,
50.82, 55.47, 59.82 (C2), 60.25, 68.60 (C3), 81.48; LRMS
for C24H44NO2 [M+H]+: 378.3 m/z; HRMS: calcd for
C24H44NO2 [M+H]+ 378.33666, found 378.33729.
4.1.2.14.
2b-(n-Nonylamino)-5a-androstane-3a,17b-
diol (33). Yield: 85%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.38; IR (film): 3383 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.87 (t, J = 6.4 Hz,
90-CH3), 0.93 (s, 19-CH3), 0.60–2.10 (residual H), 2.66
and 2.79 (2m, 10-CH2), 2.93 (m, 2a-CH), 3.61 (t,
J = 8.4 Hz, 17a-CH), 3.91 (m, 3b-CH); 13C NMR
(100.6 MHz, CDCl3): 11.18, 14.10, 15.86, 20.76, 22.64,
23.30, 27.00, 27.98, 28.56, 29.22, 29.33, 29.43, 30.44,
31.12, 31.84, 33.98, 35.24, 36.03, 36.73, 39.04, 39.14,
43.04, 47.32, 50.84, 55.74, 59.72 (C2), 68.00 (C3), 81.83;
LRMS for C28H52NO2 [M+H]+: 434.4 m/z; HRMS: calcd
for C28H52NO2 [M+H]+ 434.39926, found 434.40196.
4.1.2.10. 2b-(tert-Pentylamino)-5a-androstane-3a,17b-
diol (29). Yield: 35%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.30; IR (film): 3426 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.74 (s, 18-CH3), 0.94 (s, 19-CH3),
1.01 (t, J = 7.5 Hz, 40-CH3), 1.39 (s, 20-CH3), 1.40 (s,
20-CH3), 0.70–2.20 (residual H), 3.26 (m, 2a-CH), 3.62
(t, J = 8.5 Hz, 17a-CH), 4.17 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 7.92, 11.15, 16.91, 20.86, 23.25,
23.73, 24.06, 28.00, 30.31, 30.78, 31.84, 35.35, 35.45,
36.46, 36.60, 38.92, 41.72, 43.03, 50.63, 56.30, 56.30
(C2), 62.17, 65.02 (C3), 81.76; LRMS for C24H44NO2
[M+H]+: 378.2 m/z; HRMS: calcd for C24H44NO2
[M+H]+ 378.33666, found 378.33727.
4.1.2.15. 2b-(n-Decylamino)-5a-androstane-3a,17b-diol
(34). Yield: 88%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.42;
IR (film): 3423 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.73 (s, 18-CH3), 0.88 (t, J = 6.8 Hz, 100-CH3),
0.92 (s, 19-CH3), 0.60–2.10 (residual H), 2.81 and 2.95
(2m, 10-CH2), 3.17 (m, 2a-CH), 3.62 (t, J = 8.4 Hz, 17a-
CH), 4.04 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.16, 14.09, 16.70, 20.95, 22.65, 23.27, 26.63, 26.70,
27.93, 29.11, 29.27, 29.36, 29.48, 30.43, 30.82, 31.85,
35.22, 35.35, 35.97, 36.63, 38.49, 38.99, 43.05, 46.15,
50.71, 55.94, 59.67 (C2), 66.58 (C3), 81.77; C29H54NO2
[M+H]+: 448.5 m/z; HRMS: calcd for C29H54NO2
[M+H]+ 448.41491, found 448.41755.
4.1.2.11. 2b-(n-Hexylamino)-5a-androstane-3a,17b-diol
(30). Yield: 74%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.32;
IR (film): 3382 (OH and NH); 1H NMR (400 MHz,
CDCl3): 0.73 (s, 18-CH3), 0.88 (t, J = 6.8 Hz, 60-CH3),
0.93 (s, 19-CH3), 0.70–2.20 (residual H), 2.72 and 2.85
(2m, 10-CH2), 3.00 (m, 2a-CH) 3.62 (t, J = 8.5 Hz,
17a-CH), 3.96 (m, 3b-CH); 13C NMR (100.6 MHz,
CDCl3): 11.17, 13.98, 16.21, 20.84, 22.46, 23.29, 26.54,
27.81, 27.96, 30.45, 31.01, 31.40, 34.44, 35.29, 36.01,
36.68, 38.81, 39.09, 43.05, 46.84, 50.78, 55.82, 59.71
(C2), 67.41 (C3), 81.82; LRMS for C25H46NO2
4.1.2.16. 2b-(n-Undecylamino)-5a-androstane-3a,17b-
diol (35). Yield: 83%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.42; IR (film): 3420 (OH and NH); 1H NMR
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.87 (t,

5072
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
J = 6.4 Hz, 110-CH3), 0.92 (s, 19-CH3), 0.70–2.20 (resid-
ual H), 2.77 and 2.88 (2m, 10-CH2), 3.08 (m, 2a-CH),
3.62 (t, J = 8.3 Hz, 17a-CH), 4.00 (m, 3b-CH); 13C
NMR (75 MHz, CDCl3): 11.15, 14.11, 16.43, 20.86,
22.66, 23.25, 26.73, 27.25, 27.92, 29.19, 29.31, 29.40,
29.55 (2·), 30.38, 30.88, 31.87, 34.82, 35.29, 35.94,
36.62, 38.60, 38.99, 43.03, 46.46, 50.70, 55.84, 59.62
(C2), 66.96 (C3), 81.76; LRMS for C30H56NO2
[M+H]+: 462.4 m/z; HRMS: calcd for C30H56NO2
[M+H]+ 462.43056, found 462.43256.
(2·), 34.58, 35.31, 36.06, 36.66, 39.06, 39.64, 43.05,
50.75, 54.78, 55.91, 56.16 (C2), 67.51 (C3), 81.83; LRMS
for C25H44NO2 [M+H]+: 390.3 m/z; HRMS: calcd for
C25H44NO2 [M+H]+ 390.33666, found 390.33716.
4.1.2.21. 2b-(Cycloheptylamino)-5a-androstane-3a,17b-
diol (40). Yield: 56%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.49; IR (film): 3396 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.70 (s, 18-CH3), 0.89 (s, 19-CH3),
0.70–2.20 (residual H) 2.96 (m, 2a-CH and 10-CH),
3.58 (t, J = 8.5 Hz, 17a-CH), 3.91 (m, 3b-CH); 13C
NMR (100.6 MHz, CDCl3 + CD3OD): 11.08, 16.20,
20.77, 23.19, 23.71, 24.03, 27.60, 27.64, 27.94, 30.12,
30.95, 31.71, 33.65, 34.23, 35.22, 35.94, 36.60, 38.84,
38.93, 42.95, 50.70, 55.78, 56.40 (C2), 56.97, 66.97
(C3), 81.55; LRMS for C26H46NO2 [M+H]+: 404.4
m/z; HRMS: calcd for C26H46NO2 [M+H]+ 404.35231,
found 404.35264.
4.1.2.17. 2b-(n-Dodecylamino)-5a-androstane-3a,17b-
diol (36). Yield: 78%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.40; IR (film): 3421 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.87 (t,
J = 6.6 Hz, 120-CH3), 0.92 (s, 19-CH3), 0.60–2.10 (resid-
ual H), 2.71 and 2.84 (2m, 10-CH2), 3.00 (m, 2a-CH),
3.61 (t, J = 8.1 Hz, 17a-CH), 3.94 (m, 3b-CH); 13C
NMR (100.6 MHz, CDCl3): 11.18, 14.11, 16.13, 20.82,
22.67, 23.29, 26.88, 27.94, 27.97, 29.27, 29.34, 29.45,
29.56, 29.63, 29.64, 30.41, 31.02, 31.90, 34.39, 35.28,
36.00, 36.70, 38.82, 39.07, 43.04, 46.92, 50.79, 55.80,
59.67 (C2), 67.50 (C3), 81.79; LRMS for C31H58NO2
[M+H]+: 476.4 m/z; HRMS: calcd for C31H58NO2
[M+H]+ 476.44621, found 476.44798.
4.1.2.22.
2b-(3-Ethoxypropylamino)-5a-androstane-
3a,17b-diol (41). Yield: 39%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.38; IR (film): 3426 (OH and NH); 1H
NMR (400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.93 (s,
19-CH3), 1.21 (t, J = 8.0 Hz, 60-CH3), 0.60–2.10 (resid-
ual H), 3.06 (m, 10-CH2), 3.21 (m, 2a-CH), 3.35 (m,
10-CH2), 3.54 (m, CH2O), 3.63 (m, 17a-CH and
CH2O), 4.10 (m, 3b-CH); 13C NMR (100.6 MHz,
CDCl3): 11.21, 15.00, 16.46, 20.88, 23.28, 25.43, 27.93,
30.41, 30.89, 34.63, 35.36, 35.82, 36.64, 37.72, 38.87,
43.08, 46.46, 50.75, 55.84, 59.79 (C2), 65.37 (C3),
67.33, 69.89, 81.78; LRMS for C24H44NO3 [M+H]+:
394.4 m/z; HRMS: calcd for C24H44NO3 [M+H]+
394.33157, found 394.33273.
4.1.2.18. 2b-(n-Hexadecylamino)-5a-androstane-3a,17b-
diol (37). Yield: 82%; TLC (CH2Cl2/MeOH, 9:1): Rf =
0.44; IR (film): 3426 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.87 (t broad,
160-CH3), 0.92 (s, 19-CH3), 0.60–2.10 (residual H),
2.76 and 2.90 (2m, 10-CH2), 3.08 (m, 2a-CH), 3.62 (t,
J = 8.3 Hz, 17a-CH), 3.99 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 11.18, 14.17, 16.41, 20.88, 22.68,
23.28, 26.76, 27.37, 27.95, 29.21, 29.35, 29.42, 29.57,
29.65, 29.68, 29.70 (4·), 30.44, 30.94, 31.96, 34.79,
35.35, 36.01, 36.67, 38.69, 39.04, 43.04, 46.58, 50.78,
55.89, 59.69 (C2), 67.13 (C3), 81.82; LRMS for
C35H66NO2 [M+H]+: 532.5 m/z; HRMS: calcd for
C35H66NO2 [M+H]+ 532.50881, found 532.50954.
4.1.2.23.
2b-(3-Butoxypropylamino)-5a-androstane-
3a,17b-diol (42). Yield: 82%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.43; IR (film): 3420 (OH and NH); 1H
NMR (400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.90 (s
and t, 19-CH3 and 80-CH3), 0.60–2.10 (residual H),
3.08 (m, 10-CH2), 3.21 (m, 2a-CH), 3.33 (m, 10-CH2),
3.45 (m, 2H, CH2O), 3.62 (m, 17a-CH and CH2O),
4.08 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.22, 13.87, 16.38, 19.22, 20.84, 23.31, 25.49, 27.86,
30.38, 30.89, 31.40, 34.51, 35.30, 35.77, 36.65, 37.77,
38.79, 43.07, 46.40, 50.73, 55.75, 59.79 (C2), 65.37
(C3), 69.93, 71.79, 81.74; LRMS for C26H48NO3
[M+H]+: 422.3 m/z; HRMS: calcd for C26H48NO3
[M+H]+ 422.36287, found 422.36324.
4.1.2.19. 2b-(Cyclopentylamino)-5a-androstane-3a,17b-
diol (38). Yield: 98%; TLC (CH2Cl2/MeOH, 9:1): Rf =
0.36; IR (film): 3383 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.93 (s, 19-
CH3), 0.70–2.20 (residual H), 2.98 (m, 2a-CH), 3.36
(m, 10-CH), 3.62 (t, J = 8.5 Hz, 17a-CH), 3.94 (m,
3b-CH); 13C NMR (75 MHz, CDCl3): 11.18, 16.19,
20.81, 23.31, 23.79 (2·), 28.01, 30.50, 31.12, 31.78,
32.68, 34.00, 35.28, 36.10, 36.72, 39.11, 39.98, 43.07,
50.84, 55.85, 57.37, 58.10 (C2), 68.47 (C3), 81.88;
LRMS for C24H42NO2 [M+H]+: 376.4 m/z; HRMS:
calcd for C24H42NO2 [M+H]+ 376.32101, found
376.32138.
4.1.2.24. 2b-(2-Phenylethylamino)-5a-androstane-
3a,17b-diol (43). Yield: 81%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.60; IR (film): 3370 (OH and NH); 1H
NMR (400 MHz, CDCl3): 0.68 (s, 18-CH3), 0.85 (s,
19-CH3), 0.60–2.10 (residual H), 2.83 (m, 10-CH2 and
20-CH2), 2.98 (m, 2a-CH), 3.58 (t, J = 8.7 Hz, 17a-
CH), 3.81 (m, 3b-CH), 7.12-7.38 (m, 5H of Ar); 13C
NMR (100.6 MHz, CDCl3): 11.17, 15.45, 20.67, 23.33,
27.99, 30.55, 31.21, 33.42, 35.18, 35.55, 36.03, 36.61,
39.13, 39.19, 43.04, 48.97, 50.92, 55.65, 59.72 (C2),
68.56 (C3), 81.88, 126.51, 128.61 (2·), 128.74 (2·),
138.55; LRMS for C27H42NO2 [M+H]+: 412.3 m/z;
HRMS: calcd for C27H42NO2 [M+H]+ 412.32101, found
412.32178.
4.1.2.20. 2b-(Cyclohexylamino)-5a-androstane-3a,17b-
diol (39). Yield: 91%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.43; IR (film): 3386 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.93 (s, 19-CH3),
0.70–2.20 (residual H), 2.81 (m, 10-CH), 3.11 (m, 2a-
CH), 3.62 (t, J = 8.5 Hz, 17a-CH), 3.96 (m, 3b-CH);
13C NMR (100.6 MHz, CDCl3): 11.17, 16.49, 20.88,
23.28, 24.63, 24.85, 25.33, 27.99, 30.46, 30.97, 31.99

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5073
4.1.2.25.
2b-(3-Phenylpropylamino)-5a-androstane-
NMR (300 MHz, CDCl3): 0.75 (s, 18-CH3), 0.87 (s,
19-CH3), 2.10 (s, 200-CH3), 0.65–2.15 (residual H), 2.50
(m, CH2N), 2.70 (m, 2a-CH and CH2N), 3.56 (m, 17a-
CH and 2 · CH2NCO), 3.87 (m, 3b-CH); 13C NMR
(100.6 MHz, CDCl3): 11.18, 17.33, 20.94, 21.27, 23.29,
28.07, 30.47, 31.02, 33.28, 34.94, 35.48, 35.87, 36.71,
38.51, 41.31, 43.07, 46.18, 47.90, 48.52, 50.80, 56.09,
63.88 (C3), 64.99 (C2), 81.80, 168.86; LRMS for
C25H43N2O3 [M+H]+: 419.3 m/z; HRMS: calcd for
C25H43N2O3 [M+H]+ 419.32682, found 419.32700.
3a,17b-diol (44). Yield: 83%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.51; IR (film): 3392 (OH and NH); 1H
NMR (400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.89 (s,
19-CH3), 0.60–2.10 (residual H), 2.68 (m, 1H of 10-
CH2 and 30-CH2), 2.85 (m, 1H of 10-CH2), 2.93 (m,
2a-CH), 3.62 (t, J = 8.5 Hz, 17a-CH), 3.90 (m, 3b-
CH), 7.15–7.23 (m, 3H of Ar), 7.25–7.33 (m, 2H of
Ar); 13C NMR (100.6 MHz, CDCl3): 11.19, 15.93,
20.77, 23.30, 27.93, 29.70, 30.46, 31.10, 33.04, 34.12,
35.25, 36.00, 36.70, 39.01, 39.12, 43.05, 46.50, 50.86,
55.76, 59.74 (C2), 67.75 (C3), 81.85, 126.14, 128.44
(2·), 128.52 (2·), 140.94. LRMS for C28H44NO2
[M+H]+: 426.3 m/z; HRMS: calcd for C28H44NO2
[M+H]+ 426.33666, found 426.33731.
4.1.2.30. 2b-(4-Phenylpiperazino)-5a-androstane-3a,17b-
diol (49). Yield: 80%; TLC (hexanes/acetone, 6:4): Rf =
0.67; IR (film): 3364 (OH), 1600 (CH aromatic); 1H
NMR (400 MHz, CDCl3): 0.74 (s, 19-CH3), 0.89 (s,
19-CH3), 0.70–2.20 (residual H), 2.75 (m, CH2N), 2.93
(m, 2a-CH and CH2N), 3.28 (m, 2 · CH2NCO), 3.63
(t, J = 8.5 Hz, 17a-CH), 3.94 (m, 3b-CH), 6.91 (m, 3H
of Ar), 7.28 (m, 2H of Ar); 13C NMR (100.6 MHz,
CDCl3): 11.18, 17.43, 20.95, 23.29, 28.09, 30.48, 31.03,
33.32, 35.12, 35.48, 35.91, 36.72, 38.55, 43.07, 48.04
(2·), 49.23 (2·), 50.78, 56.16, 63.97 (C3), 64.68 (C2),
81.82, 116.44 (2·), 120.29, 129.16 (2·), 151.17; LRMS
for C29H45N2O2 [M+H]+: 453.3 m/z; HRMS: calcd for
C29H45N2O2 [M+H]+ 453.34756, found 453.34788.
4.1.2.26. 2b-(3,3-Diphenylpropylamino)-5a-androstane-
3a,17b-diol (45). Yield: 82%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.66; IR (film): 3360 (OH and NH); 1H NMR
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.89 (s, 19-CH3),
0.60–2.10 (residual H), 2.24 (m, 20-CH2), 2.52 (m, 1H
of 10-CH2), 2.67 (m, 2a-CH and 1H of 10-CH2), 3.62
(t, J = 8.5 Hz, 17a-CH), 3.71 (m, 3b-CH), 4.04 (t,
J = 7.8 Hz, 30-CH), 7.15–7.30 (m, 10H of Ar); 13C
NMR (75 MHz, CDCl3): 11.18, 15.19, 20.54, 23.31,
27.98, 30.46, 31.31, 32.89, 35.10, 35.64, 36.00, 36.72,
39.20, 39.59, 43.00, 46.46, 48.94, 50.89, 55.47, 59.69
(C2), 68.76 (C3), 81.86, 126.24 (2·), 127.76 (4·),
128.48 (4·), 144.53 (2·); LRMS for C34H48NO2
[M+H]+: 502.3 m/z; HRMS: calcd for C34H48NO2
[M+H]+ 502.36796, found 502.36818.
4.1.2.31. 2b-{4-(4-Fluorophenyl)-piperazino}-5a-andro-
stane-3a,17b-diol (50). Yield: 97%; TLC (hexanes/ace-
tone, 6:4): Rf = 0.67; IR (film): 3368 (OH); H NMR
1
(400 MHz, CDCl3): 0.74 (s, 18-CH3), 0.88 (s, 19-CH3),
0.70–2.20 (residual H), 2.66 (m, CH2N), 2.84 (m, 2a-
CH and CH2N), 3.13 (m, 2 · CH2NCO), 3.62 (m, 17a-
CH), 3.91 (m, 3b-CH), 6.87 (m, 2H of Ar), 6.96 (m,
2H of Ar); 13C NMR (100.6 MHz, CDCl3): 11.21,
17.26, 20.91, 23.31, 28.16, 30.50, 31.12, 32.92, 34.74,
35.50, 35.85, 36.77, 38.52, 43.08, 48.18 (2·), 50.62 (2·),
50.84, 56.13, 63.85 (C3), 64.60 (C2), 81.86, 115.42,
115.64, 118.00, 118.07, 147.80, 156.08 and 158.46 (C–F
coupling); LRMS for C29H44FN2O2 [M+H]+: 471.3 m/
z; HRMS: calcd for C29H44FN2O2 [M+H]+ 471.33813,
found 471.33799.
4.1.2.27. 2b-(Adamantylmethylamino)-5a-androstane-
3a,17b-diol (46). Yield: 72%; TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.67; IR (film): 3356 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.96 (s, 19-CH3),
0.60–2.00 (residual H), 2.09 (d, J = 11.6 Hz, 1H of 10-
CH2), 2.39 (d, J = 11.2 Hz, 1H of 10-CH2), 2.63 (m,
2a-CH), 3.62 (t, J = 8.7 Hz, 17a-CH), 3.74 (m, 3b-
CH); 13C NMR (100.6 MHz, CDCl3): 11.18, 15.23,
20.62, 23.34, 28.07, 28.44 (3·), 30.51, 31.42, 32.82,
33.36, 35.17, 36.18, 36.80, 37.24 (3·), 39.37, 39.95,
40.84 (3·), 43.05, 51.02, 55.59, 60.31, 61.09 (C2), 69.27
(C3), 81.96; LRMS for C30H50NO2 [M+H]+: 456.4 m/
z; HRMS: calcd for C30H50NO2 [M+H]+ 456.38361,
found 456.38410.
4.1.2.32. 2b-(Piperidinyl)-5a-androstane-3a,17b-diol
(51). Yield: 86%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.19;
IR (film): 3424 (OH); 1H NMR (300 MHz, CDCl3):
0.73 (s, 18-CH3), 0.83 (s, 19-CH3), 0.70–2.20 (residual
H), 2.34 (m, CH2N), 2.60 (m, CH2N and 2a-CH), 3.62
(t, J = 7.8 Hz, 17a-CH), 3.80 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 11.21, 17.98, 21.05, 22.32, 23.22,
23.56 (2·), 27.86, 30.36, 30.59, 34.94, 35.41, 36.16,
36.26, 36.53, 38.59, 43.03, 50.53, 51.80 (2x), 56.13,
63.78 (C3), 66.12 (C2), 81.63; LRMS for C24H42NO2
[M+H]+: 376.4 m/z; HRMS: calcd for C24H42NO2
[M+H]+ 376.32101, found 376.32177.
4.1.2.28. 2b-(Diethylamino)-5a-androstane-3a,17b-diol
(47). Yield: 67%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.38;
IR (film): 3444 (OH); 1H NMR (400 MHz, CDCl3): 0.74
(s, 18-CH3), 0.93 (s, 19-CH3), 1.47 (t, J = 7.1 Hz, 2 · 20-
CH3), 0.70–2.20 (residual H), 2.83 (m, 2a-CH), 3.05 and
3.39 (2m, 2 · 10-CH2), 3.64 (t, J = 8.5 Hz, 17a-CH), 4.10
(m, 3b-CH); 13C NMR (100.6 MHz, CDCl3): 10.23,
11.18, 11.36, 17.90, 21.08, 23.24, 27.89, 30.43, 30.57,
34.92, 35.44, 36.17, 36.31, 36.51, 38.77, 43.03, 44.27,
48.07, 50.51, 56.28, 63.70 and 63.75 (C2 and C3), 81.69;
LRMS for C23H42NO2 [M+H]+: 364.3 m/z; HRMS: calcd
for C23H42NO2 [M+H]+ 364.32101, found 364.32285.
4.1.2.33. 2b-(4-Piperidinylpiperidinyl)-5a-androstane-
3a,17b-diol (52). Yield: 73%; TLC (CH2Cl2/MeOH,
9:1): Rf = 0.08; IR (film): 3423 (OH); 1H NMR
(400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.83 (s, 19-CH3),
0.70–2.20 (residual H), 2.59 (m, CHN), 2.73 (m, 2a-
CH), 2.90 (m, 4 · CH2N), 3.62 (t, J = 8.4 Hz, 17a-
CH), 3.81 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.15, 17.33, 20.91, 23.12, 23.29, 24.17 (2·), 27.03, 27.44,
4.1.2.29.
2b-(4-Acetylpiperazino)-5a-androstane-
3a,17b-diol (48). Yield: 83%; TLC (hexanes/acetone,
6:4): Rf = 0.06; IR (film): 3384 (OH), 1630 (C@O); H
1

5074
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
28.18, 30.48, 31.08, 33.05, 34.86, 35.53, 35.76, 36.77,
38.44, 43.09, 50.20 (4·), 50.87, 56.19, 63.90, 63.90
(C3), 64.55 (C2), 81.80; LRMS for C29H51N2O2
[M+H]+: 459.4 m/z; HRMS: calcd for C29H51N2O2
[M+H]+ 459.39451, found 459.39454.
19-CH3), 0.96 (t, J = 7.4 Hz, 400-CH3), 0.60–2.20 (resid-
ual H), 2.29 (t, J = 7.5 Hz, 200-CH2), 2.55 (m broad,
CH2N), 2.75 (m broad, 2a-CH and CH2N), 3.65 (m
broad, 17a-CH and 2 · CH2NCO), 3.90 (m, 3b-CH);
13C NMR (75 MHz, CDCl3): 11.18, 13.98, 17.41,
18.66, 20.95, 23.28, 28.04, 30.46, 30.96, 33.43, 35.08
(2·), 35.46, 35.91, 36.68, 38.50, 41.03, 43.06, 45.16,
47.93, 48.58, 50.75, 56.06, 63.88 (C3), 65.13 (C2),
81.79, 171.43; LRMS for C27H47N2O3 [M+H]+: 447.3
m/z; HRMS: calcd for C27H47N2O3 [M+H]+
447.35812, found 447.35804.
4.1.3. 2b-Piperazinyl-5a-androstane-3a,17b-diol (53).19
A
solution of epoxide 8 (6.46 g, 19.4 mmol) in piperazine
(33.3 g, 388 mmol) and H2O (5 mL) was heated in a
Schlenk tube at 150–190 ꢁC overnight, then poured in
water (500 mL) and the precipitate was filtered. The so-
lid was dissolved in CH2Cl2 and the solution dried over
MgSO4, filtered and evaporated to dryness. Purification
of the crude product by flash chromatography (MeO-
H:Et3N:CH2Cl2, 14:1:85) yielded 5.76 g (70%) of com-
pound 53 as a white solid. TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.02; IR (film): 3370 (OH and NH); 1H NMR
(400 MHz, CDCl3): 0.71 (s, 18-CH3), 0.84 (s, 19-CH3),
0.65–2.15 (residual H), 2.42, 2.58 and 2.90 (3m, 2a-CH
and 4 · CH2N), 3.62 (t, J = 8.5 Hz, 17a-CH), 3.84 (m,
3b-CH); 13C NMR, 75 MHz (CDCl3): 11.18, 17.31,
20.86, 23.31, 28.23, 30.52, 31.15, 32.58, 34.67, 35.50,
35.72, 36.87, 38.40, 43.04, 46.67 (2·), 49.38 (2·), 50.87,
56.17, 63.35 (C3), 65.03 (C2), 81.60; LRMS for
C23H41N2O2 [M+H]+: 377.3 m/z; HRMS: calcd for
C23H41N2O2 [M+H]+ 377.31625, found 377.31624.
4.1.4.3. 2b-(4-iso-Butanoylpiperazinyl)-5a-androstane-
3a,17b-diol (56). Yield: 75%; TLC (hexanes/acetone,
1
6:4): Rf = 0.30; IR (film): 3402 (OH), 1624 (C@O); H
NMR (300 MHz, CDCl3): 0.73 (s, 18-CH3), 0.85 (s,
19-CH3), 1.11 (d, J = 6.6 Hz, 2 · 300-CH3), 0.60–2.20
(residual H), 2.50 (m, CH2N), 2.75 (m, 2a-CH, 200-CH
and CH2N), 3.60 (m broad, 17a-CH and 2 · CH2NCO),
3.88 (m, 3b-CH); 13C NMR (75 MHz, CDCl3): 11.18,
17.18, 19.38 (2·), 20.89, 23.28, 28.11, 29.94, 30.45,
31.07, 32.87, 34.62, 35.46, 35.79, 36.72, 38.43, 41.90,
43.06, 45.73, 48.15, 48.81, 50.81, 56.04, 63.75 (C3),
64.81 (C2), 81.79, 175.30; LRMS for C27H47N2O3
[M+H]+: 447.3 m/z; HRMS: calcd for C27H47N2O3
[M+H]+ 447.35812, found 447.35798.
4.1.4. Synthesis of N-amide derivatives of 53 (general
procedure for the synthesis of 54–61). HBTU (O-Ben-
4.1.4.4.
2b-(4-Pentanoylpiperazinyl)-5a-androstane-
3a,17b-diol (57). Yield: 80%; TLC (hexanes/acetone,
6:4): Rf = 0.31; IR (film): 3398 (OH), 1624 (C@O); H
1
zotriazol-1-yl-N,N,N0,N0-tetramethyluronium
hexa-
fluorophosphate) (1 equiv) and carboxylic acid (1.1
equiv) were dissolved in DMF at 0 ꢁC. Then, diisopro-
pylethylamine (DIPEA) was added to the mixture and
allowed to react for 5 min. Thereafter, compound 53
(1 equiv) dissolved in DMF was added to the solution
and the temperature was raised to room temperature
for 2–3 h. The resulting mixture was diluted in ethyl ace-
tate and washed with water (4·). The organic layer was
dried over MgSO4, filtered, and evaporated under re-
duced pressure to dryness. Purification of the crude
product by flash chromatography (1–4% MeOH in
CH2Cl2) yielded the desired amide as a white solid.
NMR (300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.86 (s, 19-
CH3), 0.92 (t, J = 7.3 Hz, 500-CH3), 0.60–2.20 (residual
H), 2.31 (t, J = 7.7 Hz, 200-CH2), 2.60 (m broad,
CH2N), 2.80 (m broad, 2a-CH and CH2N), 3.60 (m
broad, 17a-CH and 2 · CH2NCO), 3.91 (m, 3b-CH);
13C NMR (75 MHz, CDCl3): 11.18, 13.86, 17.40, 20.94,
22.53, 23.28, 27.33, 28.04, 30.44, 30.96, 32.90, 33.46,
35.11, 35.45, 35.91, 36.68, 38.49, 40.99, 43.06, 45.14,
47.97, 48.57, 50.75, 56.04, 63.87 (C3), 65.14 (C2), 81.76,
171.62, LRMS for C28H49N2O3 [M+H]+: 461.3 m/z;
HRMS: calcd for C28H49N2O3 [M+H]+ 461.37377, found
461.37345.
4.1.4.1. 2b-(4-Propanoylpiperazinyl)-5a-androstane-
3a,17b-diol (54). Yield: 85%; TLC (hexanes/acetone,
6:4): Rf = 0.19; IR (film): 3419 (OH), 1629 (C@O); H
4.1.4.5. 2b-(4-iso-Pentanoylpiperazinyl)-5a-androstane-
3a,17b-diol (58). Yield: 79%; TLC (hexanes/acetone,
6:4): Rf = 0.31; IR (film): 3402 (OH), 1626 (C@O); H
1
1
NMR (300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.84 (s,
19-CH3), 1.13 (t, J = 7.4 Hz, 300-CH3), 0.60–2.20 (resid-
ual H), 2.32 (q, J = 7.5 Hz, 200-CH2), 2.48 (m, CH2N),
2.69 (m, 2a-CH and CH2N), 3.49 (m, CH2NCO), 3.63
(m, 17a-CH and CH2NCO), 3.88 (m, 3b-CH); 13C
NMR (75 MHz, CDCl3): 9.45, 11.17, 17.22, 20.89,
23.28, 26.39, 28.10, 30.44, 31.05, 32.97, 34.72, 35.45,
35.80, 36.72, 38.44, 41.70, 43.06, 45.58, 48.04, 48.57,
50.81, 56.04, 63.75 (C3), 64.90 (C2), 81.79, 172.19;
LRMS for C26H45N2O3 [M+H]+: 433.3 m/z; HRMS:
calcd for C26H45N2O3 [M+H]+ 433.34247, found
433.34278.
NMR (400 MHz, CDCl3): 0.72 (s, 18-CH3), 0.84 (s,
19-CH3), 0.96 (d, J = 6.6 Hz, 2· 300-CH3), 0.60–2.10
(residual H), 2.19 (d, J = 8.2 Hz, 20-CH2), 2.43 (m,
CH2N), 2.63 (m, CH2N and 2a-CH), 3.46 (m,
CH2NCO), 3.61 (m, CH2NCO and 17a-CH), 3.86
(m, 3b-CH); 13C NMR (75 MHz, CDCl3): 11.20,
17.21, 20.91, 22.77 (2·), 23.32, 25.80, 28.17, 30.52,
31.12, 32.80, 34.59, 35.51, 35.81, 36.77, 38.46, 41.95,
42.03, 43.09, 46.31, 48.20, 48.73, 50.87, 56.10, 63.75
(C3), 64.81 (C2), 81.88, 170.97; LRMS for
C28H49N2O3 [M+H]+: 461.3 m/z; HRMS: calcd for
C28H49N2O3 [M+H]+ 461.37377, found 461.37384.
4.1.4.2. 2b-(4-Butanoylpiperazinyl)-5a-androstane-
3a,17b-diol (55). Yield: 82%; TLC (hexanes/acetone,
6:4): Rf = 0.24; IR (film): 3390 (OH), 1634 (C@O); H
NMR (300 MHz, CDCl3): 0.73 (s, 18-CH3), 0.86 (s,
4.1.4.6.
2b-(4-Nonanoylpiperazinyl)-5a-androstane-
3a,17b-diol (59). Yield: 70%; TLC (hexanes/acetone,
6:4): Rf = 0.43; IR (film): 3394 (OH), 1628 (C@O); H
NMR (400 MHz, CDCl3): 0.73 (s, 18-CH3), 0.85 (s,
1
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D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
5075
19-CH3), 0.87 (t, J = 7.0 Hz, 900-CH3), 0.65–2.15 (resid-
ual H), 2.30 (t, J = 7.7 Hz, 200-CH2), 2.41 (m, CH2N),
2.64 (m, 2a-CH and CH2N), 3.46 (m, CH2NCO), 3.62
(m, 17a-CH and CH2NCO), 3.88 (m, 3b-CH); 13C
NMR (75 MHz, CDCl3): 11.17, 14.08, 17.18, 20.88,
22.63, 23.28, 25.34, 28.13, 29.15, 29.35, 29.47, 30.46,
31.09, 31.79, 32.80, 33.30, 34.57, 35.47, 35.76, 36.73,
38.43, 41.91, 43.06, 46.07, 48.09, 48.63, 50.83, 56.07,
63.73 (C3), 64.76 (C2), 81.82, 171.68; LRMS for
C32H57N2O3 [M+H]+: 517.5 m/z; HRMS: calcd for
C32H57N2O3 [M+H]+ 517.43637, found 517.43622.
3.58 (m, 17a-CH), 3.82 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 11.21, 11.90, 17.25, 19.71, 20.80,
23.36, 28.24, 30.63, 31.15, 32.64, 34.67, 35.47, 35.73,
36.90, 38.43, 43.00, 50.86, 53.60 (4·), 56.13, 60.57,
63.56 (C3), 64.34 (C2), 81.36; LRMS for C26H47N2O2
[M+H]+: 419.3 m/z; HRMS: calcd for C26H47N2O2
[M+H]+ 419.36321, found 419.36326.
4.1.5.2. 2b-(4-Butylpiperazinyl)-5a-androstane-3a,17b-
diol (63). Yield: 47%; TLC (hexanes/acetone, 6:4):
Rf = 0.12; IR (film): 3380 (OH); H NMR (300 MHz,
1
CDCl3): 0.72 (s, 18-CH3), 0.84 (s, 19-CH3), 0.93 (t,
J = 7.3 Hz, 400-CH3), 0.60–2.20 (residual H), 2.55, 2.70
and 2.85 (3m broad, 2a-CH, 100-CH2 and 4 · CH2N),
3.61 (m, 17a-CH), 3.85 (m, 3b-CH); 13C NMR
(75 MHz, CDCl3): 11.21, 13.98, 17.25, 20.70, 20.79,
23.36, 28.23, 28.60, 30.62, 31.15, 32.65, 34.67, 35.47,
35.74, 36.89, 38.43, 43.00, 50.85, 53.60 (4·), 56.13,
58.38, 63.57 (C3), 64.34 (C2), 81.37; LRMS for
C27H49N2O2 [M+H]+: 433.4 m/z; HRMS: calcd for
C27H49N2O2 [M+H]+ 433.37886, found 433.37909.
4.1.4.7. 2b-(4-Cyclohexylcarbonylpiperazinyl)-5a-andro-
stane-3a,17b-diol (60). Yield: 84%; TLC (hexanes/ace-
tone, 6:4): Rf = 0.37; IR (film): 3382 (OH), 1628
(C@O); 1H NMR (300 MHz, CDCl3): 0.73 (s, 18-
CH3), 0.86 (s, 19-CH3), 0.60–2.10 (residual H), 2.43
(m, 200-CH), 2.40–3.20 (m broad, 2a-CH and 2 ·
CH2N), 3.65 (m broad, 17a-CH and 2 · CH2NCO),
3.93 (m, 3b-CH); 13C NMR (75 MHz, CDCl3): 11.18,
17.56, 18.59, 21.01, 23.26, 25.71 (2·), 27.98, 29.33 (2·),
30.45, 30.85, 33.92, 34.69, 35.45, 36.03, 36.63, 38.52,
40.23 (2·), 42.64, 43.06, 44.59, 48.85 (2·), 50.68, 54.51,
56.02, 63.96 (C3), 65.45 (C2), 81.75, 174.52; LRMS for
C30H51N2O3 [M+H]+: 487.5 m/z; HRMS: calcd for
C30H51N2O3 [M+H]+ 487.38942, found 487.38903.
4.1.5.3.
2b-(4-iso-Butylpiperazinyl)-5a-androstane-
3a,17b-diol (64). Yield: 35%; TLC (hexanes/acetone,
6:4): Rf = 0.43; IR (film): 3370 (OH); 1H NMR
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.84 (s, 19-CH3),
0.90 (d, J = 6.5 Hz, 2 · 300-CH3), 0.60–2.15 (residual
H), 2.20, 2.60 and 2.80 (3m broad, 2a-CH, 100-CH2
and 4 · CH2N), 3.62 (m, 17a-CH), 3.84 (m, 3b-CH);
13C NMR (75 MHz, CDCl3): 11.19, 17.41, 20.92 (3·),
23.28, 25.20, 28.13, 30.46, 31.05, 33.20, 35.03, 35.48,
35.82, 36.74, 38.49, 43.07, 50.80, 53.33 (4·), 56.18,
63.81 (C3), 64.41 (C2), 66.44, 81.79; LRMS for
C27H49N2O2 [M+H]+: 433.4 m/z; HRMS: calcd for
C27H49N2O2 [M+H]+ 433.37886, found 433.37931.
4.1.4.8.
2b-(4-Benzoylpiperazinyl)-5a-androstane-
3a,17b-diol (61). Yield: 90%; TLC (hexanes/acetone,
1
6:4): Rf = 0.29; IR (film): 3392 (OH), 1622 (C@O); H
NMR (300 MHz, CDCl3): 0.73 (s, 18-CH3), 0.85 (s,
19-CH3), 0.60–2.20 (residual H), 2.40–2.90 (m broad,
2a-CH and 2 · CH2N), 3.55 (m broad, 17a-CH and
2 · CH2NCO), 3.87 (m, 3b-CH), 7.40 (s, 5H of Ar);
13C NMR (75 MHz, CDCl3): 11.18, 17.28, 20.92,
23.28, 28.08, 30.45, 31.02, 33.15, 34.81, 35.47, 35.84,
36.72, 38.45, 42.18 (2·), 43.06, 48.07, 48.66, 50.79,
56.06, 63.78 (C3), 65.01 (C2), 81.79, 127.08 (2·),
128.51 (2·), 129.85, 135.37, 170.29; LRMS for
C30H45N2O2 [M+H]+: 481.5 m/z; HRMS: calcd for
C30H45N2O3 [M+H]+ 481.34247, found 481.34230.
4.1.5.4.
2b-(4-Pentylpiperazinyl)-5a-androstane-
3a,17b-diol (65). Yield: 62%; TLC (hexanes/acetone,
6:4): Rf = 0.38; IR (film): 3385 (OH); 1H NMR
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-
CH3), 0.88 (t, J = 6.9 Hz, 500-CH3), 0.60–2.15 (residual
H), 2.40, 2.60 and 2.75 (3m broad, 2a-CH, 100-CH2
and 4 · CH2N), 3.60, (t, J = 8.5 Hz, 17a-CH), 3.84
(m, 3b-CH); 13C NMR (75 MHz, CDCl3): 11.19,
13.98, 17.21, 20.83, 22.47, 23.32, 25.84, 28.19, 29.56,
30.53, 31.12, 32.84, 34.70, 35.47, 35.77, 36.81, 38.46,
43.03, 50.84, 53.34 (4·), 56.10, 58.46, 63.71 (C3),
64.42 (C2), 81.59; LRMS for C28H51N2O2 [M+H]+:
447.4 m/z; HRMS: calcd for C28H51N2O2 [M+H]+
447.39451, found 447.39473.
4.1.5. Synthesis of N-alkylpiperazino derivatives 62–69
(general procedure). To an amidopiperazino derivative
selected from 54 to 61 (1 equiv) dissolved in dry
THF was added at 0 ꢁC a 1.0 M solution of BH3-
THF (10 equiv) and the mixture was stirred at room
temperature for 90 min. Water was then added and
the mixture was extracted with CH2Cl2, the organic
layer dried over MgSO4, filtered and evaporated to
dryness. The crude residue was dissolved in MeOH
and heated at reflux overnight to break the boron–
nitrogen complex. The solvent was evaporated and
the amine was purified by flash chromatography (2–
7% MeOH in CH2Cl2) to yield the desired free amine
as a white solid.
4.1.5.5. 2b-(4-iso-Pentylpiperazinyl)-5a-androstane-
3a,17b-diol (66). Yield: 70%; TLC (hexanes/acetone,
6:4): Rf = 0.18; IR (film): 3382 (OH); 1H NMR
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-CH3),
0.88 (d, J = 6.4 Hz, 2 · 400-CH3), 0.65–2.15 (residual
H), 2.40, 2.55 and 2.70 (3m broad, 2a-CH, 100-CH2
and 4 · CH2N), 3.58 (t, J = 8,5 Hz, 17a-CH), 3.83 (m,
3b-CH); 13C NMR (75 MHz, CDCl3): 11.21, 17.22,
20.82, 22.63 (2·), 23.35, 26.64, 28.22, 30.61, 31.14,
32.74, 34.69, 35.15, 35.47, 35.76, 36.87, 38.46, 43.00,
50.85, 53.51 (4·), 56.12, 56.84, 63.64 (C3), 64.37 (C2),
81.42. LRMS for C28H51N2O2 [M+H]+: 447.5 m/z;
4.1.5.1. 2b-(4-Propylpiperazinyl)-5a-androstane-3a,17b-
diol (62). Yield: 66%; TLC (hexanes/acetone, 6:4):
Rf = 0.08; IR (film): 3380 (OH); H NMR (300 MHz,
1
CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-CH3), 0.88 (t,
J = 7.2 Hz, 300-CH3), 0.60–2.10 (residual H), 2.30, 2.50
and 2.70 (3m broad, 2a-CH, 100-CH2 and 4 · CH2N),

5076
D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
HRMS: calcd for C28H51N2O2 [M+H]+ 447.39451,
found 447.39431.
to room temperature for 90 min. Water was then added
and mixture was extracted with CH2Cl2, the organic
layer dried over MgSO4, filtered and evaporated to dry-
ness. Purification by flash chromatography (8% acetone
in hexanes) gave the desired amine as slight yellow oil.
4.1.5.6. 2b-(4-Nonylpiperazinyl)-5a-androstane-3a,17b-
diol (67). Yield: 44%; TLC (hexanes/acetone, 6:4):
Rf = 0.31; IR (film): 3386 (OH); H NMR (400 MHz,
1
CDCl3): 0.73 (s, 18-CH3), 0.84 (s, 19-CH3), 0.88 (t,
J = 6.9 Hz, 900-CH3), 0.65–2.10 (residual H), 2.33, 2.48
and 2.68 (3m broad, 2a-CH, 100-CH2 and 4 · CH2N),
3.61 (t, J = 8.5 Hz, 17a-CH), 3.83 (m, 3b-CH); 13C
NMR (75 MHz, CDCl3): 11.20, 14.13, 17.31, 20.84,
22.68, 23.35, 26.81, 27.59, 28.27, 29.28, 29.55 (2·),
30.56, 31.18, 31.87, 32.65, 34.69, 35.51, 35.76, 36.84,
38.46, 43.07, 50.87, 53.79 (4·), 56.16, 58.81, 63.59
(C3), 64.39 (C2), 81.75; LRMS for C32H59N2O2
[M+H]+: 503.5 m/z; HRMS: calcd for C32H59N2O2
[M+H]+ 503.45765, found 503.45720.
4.1.6.1. 2b-(Dinonylamino)-5a-androstane-3a,17b-diol
(70). Yield: 47%; TLC (CH2Cl2/MeOH, 9:1): Rf = 0.71;
IR: 3397 (OH); H NMR (400 MHz, CDCl3): 0.73 (s,
1
18-CH3), 0.84 (s, 19-CH3), 0.88 (t, J = 6.4 Hz, 2 · 90
CH3), 0.70–2.10 (residual H), 2.29 and 2.42 (2m,
2 · 10-CH2), 2.75 (m, 2a-CH), 3.62 (t, J = 8.5 Hz, 17a-
CH), 3.73 (m, 3b-CH); 13C NMR (100.6 MHz, CDCl3):
11.16, 14.08 (2·), 17.60, 20.93, 22.65 (2·), 23.35, 27.43
(2·), 28.47, 29.20 (2·), 29.36 (2·), 29.60 (4·), 30.59,
31.17, 31.88 (2·), 33.31, 34.92, 35.64, 35.72, 36.84,
38.49, 43.12, 50.22 (2·), 50.91, 56.43, 60.89 (C2), 63.96
(C3), 81.93; LRMS for C37H70NO2 [M+H]+: 560.5 m/
z; HRMS: calcd for C37H70NO2 [M+H]+ 560.54011,
found 560.54026.
4.1.5.7. 2b-(4-Cyclohexylmethylpiperazinyl)-5a-andro-
stane-3a,17b-diol (68). Yield: 78%; TLC (hexanes/ace-
tone, 6:4): Rf = 0.47; IR (film): 3380 (OH); H NMR
1
(300 MHz, CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-CH3),
0.60–2.10 (residual H), 2.16 (d, J = 6.6 Hz, 100-CH2),
2.50 and 2.75 (2m broad, 2a-CH and 4 · CH2N), 3.61
(m, 17a-CH), 3.82 (m, 3b-CH); 13C NMR (75 MHz,
CDCl3): 11.19, 17.36, 20.90, 23.30, 26.04 (2·), 26.64,
28.18, 30.48, 31.10, 31.87 (2·), 32.99, 34.81, 35.49 (2·),
35.78, 36.76, 38.45, 43.08, 50.82, 53.77 (4·), 56.17,
63.70 (C3), 64.38 (C2), 65.38, 81.80; LRMS for
C30H53N2O2 [M+H]+: 473.5 m/z; HRMS: calcd for
C30H53N2O2 [M+H]+ 473.41016, found 473.40999.
4.1.6.2. 2b-(Diundecylamino)-5a-androstane-3a,17b-
diol (71). Yield: 59%. TLC (CH2Cl2/MeOH, 9:1):
Rf = 0.73; IR (film): 3407 (OH); H NMR (400 MHz,
1
CDCl3): 0.73 (s, 18-CH3), 0.83 (s, 19-CH3), 0.87 (t,
J = 6.8 Hz, 2 · 110-CH3), 0.70-2.10 (residual H), 2.29
and 2.42 (2m, 2 · 10-CH2), 2.78 (m, 2a-CH), 3.62 (t,
J = 8.5 Hz, 17a-CH), 3.73 (m, 3b-CH); 13C NMR
(100.6 MHz, CDCl3): 11.17, 14.09 (2·), 17.61, 20.94,
22.67 (2·), 23.34, 27.42 (2·), 28.46, 29.27 (2·), 29.33
(2·), 29.43 (2·), 29.59 (2·), 29.64 (4·), 30.57, 31.16,
31.91 (2·), 33.33, 34.95, 35.64, 35.72, 36.87, 38.49,
43.11, 50.21 (2·), 50.90, 56.43, 60.88 (C2), 63.96 (C3),
81.91; LRMS for C41H78NO2 [M+H]+: 616.6 m/z;
HRMS: calcd for C41H78NO2 [M+H]+ 616.60271, found
616.60234.
4.1.5.8. 2b-(4-Benzylpiperazinyl)-5a-androstane-3a,17b-
diol (69). Yield: 46%; TLC (hexanes/acetone, 6:4):
Rf = 0.37; IR (film): 3372 (OH); H NMR (300 MHz,
1
CDCl3): 0.72 (s, 18-CH3), 0.83 (s, 19-CH3), 0.65–2.20
(residual H), 2.55 and 2.75 (2m broad, 2a-CH and
4 · CH2N), 3.53 (s, PhCH2N), 3.61 (t, J = 8.5 Hz, 17a-
CH), 3.82 (m, 3b-CH), 7.30 (s, 5H of Ar); 13C NMR
(75 MHz, CDCl3): 11.18, 17.40, 20.91, 23.29, 28.14,
30.47, 31.06, 33.12, 34.97, 35.48, 35.81, 36.75, 38.46,
43.06, 50.80, 52.98 (4·), 56.16, 62.78, 63.75 (C3), 64.41
(C2), 81.76, 127.29, 128.29 (2·), 129.29 (2·), 137.34;
LRMS for C30H47N2O2 [M+H]+: 467.5 m/z; HRMS:
calcd for C30H47N2O2 [M+H]+ 467.36321, found
467.36408.
4.2. Biological evaluation of synthesized compounds
4.2.1. Cell culture. Human promyelocytic leukemia cells
HL-60 (ATCC, Rockville, MD, USA) were routinely
grown in suspension in 90% RPMI-1640 (Sigma, Saint
Louis, MO, USA) containing L-glutamine (2 nM) and
antibiotics (100 IU penicillin/mL, 100 lg streptomycin/
mL), supplemented with 10% (v/v) foetal bovine serum
(FBS), in a 5% CO2 humidified atmosphere at 37 ꢁC.
Cells were currently maintained in continuous exponen-
tial growth with twice a week dilution of the cells in cul-
ture medium.
4.1.6. Synthesis of N,N-dialkylamino derivatives 70 and
71. HBTU (O-benzotriazol-1-yl-N,N,N0,N0-tetramethyl-
uronium hexafluorophosphate) (1 equiv) and carboxylic
acid (1.1 equiv) were dissolved in DMF at 0 ꢁC. Then,
diisopropylethylamine (DIPEA) was added in the mix-
ture and allowed to react for 5 min. Thereafter, com-
pound 33 or 35 (1 equiv) dissolved in DMF was added
to the solution and the temperature was raised to room
temperature for 2–3 h. The resulting mixture was diluted
in ethyl acetate and washed with water (4·). The organic
layer was dried over MgSO4, filtered, and evaporated
under reduced pressure to dryness. Purification of the
crude product by flash chromatography (1–2% MeOH
in CH2Cl2) yielded the amide as a pale yellow oil. This,
compound was dissolved in THF at 0 ꢁC, BH3-THF (10
equiv) was added to the solution and temperature raised
4.2.2. Cell proliferation assay. The cell proliferation
assay was performed using 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-
tetrazolium (MTS) (Cell Titer 96 Aqueous, Promega,
Madison, WI, USA), which allowed us to measure the
number of viable cells. In brief, triplicate cultures of
1 · 104 cells in a total of 100 lL medium in 96-well
microtiter plates (Becton–Dickinson Company, Lincoln
Park, NJ, USA) were incubated at 37 ꢁC, 5% CO2. Com-
pounds were dissolved in ethanol to prepare the stock
solution of 1 · 10ꢀ2 M. These compounds and doxoru-
bicin (Novapharm, Toronto, Canada) were diluted at
multiple concentrations with culture media, added to

D. Thibeault et al. / Bioorg. Med. Chem. 16 (2008) 5062–5077
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Acknowledgments
We thank The Cancer Research Society for their finan-
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and Tania Fielding for their contribution to the chemi-
cal synthesis of some compounds. Careful reading of
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appreciated.
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Supplementary data
The HPLC purity and chemical structures of aminoster-
oids tested in our study. The 13C NMR spectra of non
UV-detectable aminosteroids. Supplementary data asso-
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References and notes
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