Synthesis of 17β-N-arylcarbamoylandrost-4-en-3-one derivatives and their anti-proliferative effect on human androgen-sensitive LNCaP cell line

Article abstract of DOI:10.1016/j.ejmech.2016.05.059

In this study, we report the synthesis and anti-proliferative effect of a set of eight androst-4-ene-3-one derivatives with different arylcarbamoyl groups at C-17. The novel compounds were prepared from commercially available 3β-hydroxy-5-pregnen-20-one a

Full text of DOI:10.1016/j.ejmech.2016.05.059

Accepted Manuscript
Synthesis of 17β-N-arylcarbamoylandrost-4-en-3-one derivatives and their anti-
proliferative effect on human androgen-sensitive LNCaP cell line
Francisco Cortés-Benítez, Marisa Cabeza, María Teresa Ramírez Apan, Berenice
Alvarez-Manrique, Eugene Bratoeff
PII:
S0223-5234(16)30457-3
10.1016/j.ejmech.2016.05.059
EJMECH 8651
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2015
Revised Date: 23 May 2016
Accepted Date: 26 May 2016
Please cite this article as: F. Cortés-Benítez, M. Cabeza, M.T. Ramírez Apan, B. Alvarez-Manrique, E.
Bratoeff, Synthesis of 17β-N-arylcarbamoylandrost-4-en-3-one derivatives and their anti-proliferative
effect on human androgen-sensitive LNCaP cell line, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.05.059.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of 17
β
-N-arylcarbamoylandrost-4-en-3-one derivatives and
their anti-proliferative effect on human androgen-sensitive LNCaP cell
line
Francisco Cortés-Beníteza, Marisa Cabezab*, María Teresa Ramírez Apanc, Berenice
Alvarez-Manriquea, Eugene Bratoeffa†
aDepartamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de
México, México D. F. 04510, México.
bDepartamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad
Xochimilco, México D. F. 04960, México.
cUnidad de Pruebas Biológicas, Instituto de Química, Universidad Nacional Autónoma de
México, México D. F. 04510, México.
† Deceased
Francisco Cortés-Benítez: franciscoqfb@comunidad.unam.mx
Marisa Cabeza: marisa@correo.xoc.uam.mx
María Teresa Ramírez Apan: mtrapan@unam.mx
Berenice Álvarez-Manrique: alvarez.m.berenice@gmail.com
*Address correspondence to:
Marisa Cabeza
Departamento de Sistemas Biológicos
Universidad Autónoma Metropolitana Unidad Xochimilco
Calzada del Hueso No. 1100,
México, D.F. 04960, México.
Phone: +52(55)54837260
E-mail address: marisa@correo.xoc.uam.mx

ACCEPTED MANUSCRIPT
Abstract
In this study, we report the synthesis and anti-proliferative effect of a set of eight
androst-4-ene-3-one derivatives with different arylcarbamoyl groups at C-17. The novel
compounds were prepared from commercially available 3β-hydroxy-5-pregnen-20-one and
evaluated against the androgen-sensitive human prostate adenocarcinoma LNCaP cell line.
The cancerous cells were exposed to 50 μM of each compound and the proliferating agent
testosterone (T) or dihydrotestosterone (DHT). The most potent compounds from this assay
were further tested against the androgen-insensitive PC3 cell line. We also demonstrate the
activity of these compounds on rat peripheral blood mononuclear cells for comparison.
Both 17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-4-ene-3-one (6f) and
17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-4-ene-3-one (6g) exhibited a higher growth
inhibitory effect than commercially available drugs finasteride, flutamide and ketoconazole
on LNCaP cells in the presence and absence of androgens. In addition, 6f and 6g
demonstrated high potency on PC3 cells suggesting an androgen-independent anti-
proliferative effect. Moreover, the novel compounds showed a small effect on rat
mononuclear cells, an indication of low toxicity.

ACCEPTED MANUSCRIPT
Keywords
Androst-4-en-3-one derivatives; LNCaP cells; PC3 cells; rat peripheral blood
mononuclear cells (PBMC); prostate cancer; sulforhodamine B assay.
List of abbreviations: 5αR, 5α-reductase enzyme; AR, Androgen Receptor; BPH,
Benign Prostatic Hyperplasia; DHEA, dehydroepiandrosterone; DHT, 5α-
dihydrotestosterone; LNCaP, androgen-sensitive human prostate adenocarcinoma
cell line; PC3 androgen-insensitive human prostate adenocarcinoma cell line;
PBMC, rat peripheral blood mononuclear cells; PCa, prostate cancer; T,
testosterone.

ACCEPTED MANUSCRIPT
1. Introduction
Prostate cancer (PCa) is one of the most frequent malignant tumors in the world and
is the second leading cause of death in men [1]. The androgen receptor (AR) signaling
pathway plays a central role in PCa development and progression [2] and the initial growth
of prostate carcinomas are dependent of two principal androgens: testosterone (T) and its
metabolite, 5α-dihydrotestosterone (DHT, Figure 1) which is the most potent natural
androgen [3, 4]. In healthy and cancerous prostate cells, T is transformed to DHT by the
activity of the 5α-reductase (5αR) enzyme [5]. Once DHT is formed, it binds to the AR
promoting its activation. The active form of AR undergoes dimerization, phosphorylation,
and translocation to the nucleus, and then binds to the androgen response element which
activates the transcription of certain genes that include transcription co-regulators and the
transcriptional machinery [6].
OH
OH
H
H
H
H
H
H
O
O
H
Testosterone
5 -dihydrotestosterone (DHT)
Figure 1. The principal androgens: testosterone and 5α-dihydrotestosterone (DHT)
Since steroids have an important role as physiological regulators of cell growth and
survival, the inhibition of the androgenic pathway has been widely explored for the
treatment and prevention of PCa [7]. Currently, there are several examples of synthetic
steroidal compounds that inhibit the mechanism of action of natural hormones [8-11]. Thus,
these compounds have exhibited a remarkable anti-proliferative effect on wild-type as well

ACCEPTED MANUSCRIPT
as transfected prostate cancer LNCaP cells [12-16], which is a widely used and well
established cell line model to study PCa [17]. In previous studies, patents and patent
applications such as CN 102532236 [18] and WO2003029268 A1 [19] discloses the strong
activity of several androstene 17β-carboxamides as androgen receptor modulators and anti-
5αR compounds for treating androgen-dependent diseases. Afterwards, C.Amaral et al. [20]
synthesized androstene 17β-carboxamides with lipophilic amides at the C-17 position as
5αR inhibitors. These steroidal compounds decreased the cell viability of LNCaP cells
stimulated with T or DHT. Interestingly, their study suggests an additional mechanism
related to the cytotoxic effect aside from the anti-5αR activity. On the other hand, our
research group has demonstrated that the presence of different substituted aromatic esters
and carbamate moieties attached to the C-17 of progesterone can modulate their anti-
proliferative activity in a panel of different cancer cell lines [21].
On the basis of the facts described in these references and in order to obtain the
steroidal structure required for anti-proliferative effect on prostate cancer cells and also to
see whether this effect is exerted by an androgen-dependent or androgen-independent
effect, we describe herein the synthesis of eight new androst-4-en-3-one derivatives (6a-h)
containing different aromatic amides at C-17β (Figure 2) and their anti-proliferative effect
on LNCaP (androgen-sensitive) cells. The most potent compounds were further selected
and evaluated on PC-3 (androgen-insensitive) cells which do not express AR and their
proliferation is independent of androgen [22].
In view of the therapeutic importance of the substituent at C-17β, we proposed
steroidal compounds with various substituted 17β-N-phenylcarbamoyl groups: steroidal
derivatives 6a and 6b have electron donating groups (p-OCH3, p-CH3), 6c has an

ACCEPTED MANUSCRIPT
unsubstituted phenyl ring, 6d-f have electronegative groups (p-F, p-Cl and bis-3,5-CF3),
and derivatives 6g and 6h contain 2-aminothiazole and indoline at C-20, respectively.
H
O
H
O
N
N
6a:
6b:
6c:
X = OCH3
X = CH3
X = H
S
N
H
H
X
H
H
H
H
6d:
6e:
X = F
O
O
X = Cl
6a-e
6g
H
O
O
N
N
CF3
H
H
F3C
H
H
H
H
O
O
6f
6h
H
O
N
O
N
N O
O
H
N
F3C
H
O
O
N
N
O
H
H
H
O2N
O
N
Cl
Cl
H
Ketoconazole
Finasteride
Flutamide
Figure 2. 17β-N-arylcarbamoylandrost-4-en-3-one derivatives 6a-h, flutamide, finasteride
and ketoconazole.
The newly designed steroidal compounds (6a-h in Figure 2) were tested at a
concentration of 50 μM for their ability to inhibit the growth of LNCaP cells. In order to
observe the potency of the synthesized compounds, even in the presence of proliferating
agents, these cells were stimulated with T or DHT as well. In addition, considering that the
efficacy of a drug candidate in a short lapse of time is important, we decided to study the
growth inhibitory effect of all compounds at 48 h. Flutamide, finasteride and ketoconazole
were utilized as reference controls. Flutamide is a non-steroidal compound used as an
antiandrogen drug in the treatment of prostate cancer [23] by blocking the AR and
competing with T and DHT [24]. In previous studies, flutamide has demonstrated an anti-

ACCEPTED MANUSCRIPT
proliferative effect on different prostate cancer cell lines in vitro [25]. The second,
finasteride, is a 5αR inhibitor mainly used in BPH [26]. However, it has demonstrated
inhibitory effects on LNCaP cells through inhibition of the 5αR enzyme type 1 [27]. The
third, ketoconazole is an anti-fungal agent extensively used as a treatment in Castration-
Resistant Prostate Cancer (CRPC) patients [8]. This drug has been demonstrated to inhibit
the CYP17 enzyme which is involved in the steroid biosynthesis pathway of important
androgens such as DHEA and androstenedione [28]. Interestingly, ketoconazole has not
only shown a direct anti-proliferative activity on LNCaP cells, but also on the androgen-
insensitive PC-3 cell line [29].
Due to the toxicity of a drug candidate being equally as important as its inhibition
potential, anti-proliferative assays were also carried out in rat peripheral blood mononuclear
cells (PBMC) as a cytotoxicity model in this study [30].

ACCEPTED MANUSCRIPT
2. Results and discussion.
2.1 Chemistry
The synthetic route for steroidal derivatives 6a-h is shown in Scheme 1. Briefly, 3β-
hydroxy-5-pregnen-20-one 1 was reacted with bromine and sodium hydroxide to form the
carboxylic acid derivative 2 (yield of 90%). The acetylation of the hydroxyl group at C-3
with acetic anhydride and pyridine then afforded compound 3 (yield of 92 %). The acyl
chloride at C-17 was generated using thionyl chloride and was reacted immediately with
the respective arylamine (or indoline for compound 4h) in the presence of triethylamine in
CH2Cl2 to afford derivatives 4a-h in acceptable yields (42-68%). The hydrolysis of the
acetate group at C-3 was produced by a methanolic solution of potassium hydroxide to
obtain the steroids 5a-h in high yields (86-94%). The resulting secondary alcohol at C-3
was oxidized to the α,β-unsaturated ketone by Oppenauer oxidation using aluminum
isopropoxide and 1-methyl-4-piperidone in refluxing toluene. The desired products 6a-h
were isolated in moderate yields (45-71%).
The structure of all obtained 17β-N-arylcarbamoylandrost-4-en-3-one derivatives
1
13
(6a-h) was determined on the basis of their spectral data (IR, H-NMR and C-NMR). In
1
the H-NMR spectra, there are representative signals in the aromatic region from 6.5-8.0
ppm. The two doublets (J = 8-9 Hz) characteristic of a para substituted pattern are
presented by compounds 6a, 6b, and 6e, whereas a triplet and two doublets at 7.0 and 7.5
ppm with J = 8.8 and 4.8 Hz, respectively, for 6d were found, these signals corresponding
to JH-H and JH-F coupling constants. The mono, 1,2 di-subsituted and 1,3,4 tri-substituted
pattern was observed for 6c, 6h, and 6f, respectively. Two double signals (J = 3.5 Hz) for
the thiazole ring were also situated downfield for 6g at δ 7.0 and 7.4 ppm. On the other

ACCEPTED MANUSCRIPT
hand, the broad signal (NH) for the compounds 6a-f is present at about δ 7.1-7.6 ppm; the
same signal was shifted to δ 10.0 ppm by the thiazole ring in compound 6g.
When Oppenauer oxidation was carried out, the absorption of the 3β-OH (~3400
cm-1) group in the IR spectra of the 5a-h series was missing. The broad multiplet for H-3
1
and doublet for H-6 around δ 3.5 and 5.6 ppm, respectively in H-NMR, along with the
singlet for C-3 in 13C-NMR at about 74 ppm were all absent as well. Instead, a new
1
absorption at 1660 cm-1 in the IR spectra, a new H-4 singlet at δ 5.7 ppm in H-NMR, as
13
well as new signals for C-3, C-4 and C-5 in C-NMR at about δ 199, 127 and 171 ppm,
respectively, all appeared for compounds 6a-h indicating the formation of the α,β-
unsaturated ketone at C-3.

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: (a) NaOH, Br2, Dioxane, <10°C, 9 h; (b) Acetic
anhydride, pyridine, r.t, 8 h; (c) 1. SOCl2, CH2Cl2, r.t., 2 h; 2. Arylamine (indoline for 4h),
TEA, CH2Cl2, 1 h; (d) KOH 10% in MeOH, r.t. 50 min; (e) Al(i-OPr)3, 1-methyl-4-
piperidone, toluene, reflux 7 h.
2.2 Anti-proliferative effect on prostate cancer cells
The activity of the synthesized compounds 6a-h, flutamide, finasteride and
ketoconazole were first tested against LNCaP cells stimulated by 0.1 μM of T or DHT
(Table 1). In this assay, the basal anti-proliferative effect (androgen-free media) was set to

ACCEPTED MANUSCRIPT
0%, the androgens testosterone and DHT (at 0.1 μM) exhibiting a proliferative effect of
72.1% and 75.3%, respectively (anti-proliferative effect of -72.1% and -75.3%, respectively
using the equation described in section 3.3.1).
At a concentration of 50 µM, the antiandrogen flutamide inhibited the androgen-
induced growth by T and DHT with a percentage of 29.6% and 25.1%, respectively. The
inhibitory effect of flutamide on androgen-stimulated LNCaP cells can be explained due to
flutamide’s competitive binding to AR with T and DHT [31].
On the other hand, finasteride inhibited the cell growth of LNCaP cells that were not
androgen-stimulated (34.6%). This activity could be associated with its known alternative
effect to induce apoptosis via proteins of the Bcl-2 and caspase family [32]. Additionally,
the low anti-proliferative effect of finasteride on T-stimulated LNCaP cells as well as the
lack of effectiveness on DHT-stimulated cells, could be explained by the fact that this
steroid displays its inhibitory effect on these cells by blocking 5αR activity after 9 days of
treatment with T [31] instead of the 48 h covered by our method.
In contrast, ketoconazole shows the highest activity of the three marketed drugs,
with values of 82.7%, 76.0% and 68.2% on non-stimulated, T and DHT-stimulated LNCaP
cells, respectively; its inhibitory effect is also clearly better in the absence of androgens.
This activity could be explained due to the fact that ketoconazole is capable of decreasing
DNA synthesis and inducing cell cycle arrest in prostate cancer cells [29].
Interesting SAR observations emerged from the arylcarbamoyl compounds (6a-h).
The first general observation that can be made about these steroids is that they are able to
decrease LNCaP cell viability when these cells were androgen-stimulated or not, suggesting

ACCEPTED MANUSCRIPT
that they induce their ant-proliferative effect by a mechanism not related to the androgen-
signaling pathways. However, when 6b-d were evaluated against androgen-stimulated and
non-stimulated LNCaP cells, we observed that their anti-proliferative effect was slightly
higher on T-stimulated cells, whereas 6a and 6h acted better on non-stimulated cells (48.7
and 69.1%, respectively). Thus, it cannot be discarded that compounds 6b-d may be acting
as anti-5αR. This hypothesis is supported by studies carried out by C. Amaral, who found
that the most potent 5αR inhibitors exerted significant growth inhibitory effect on T-treated
LNCaP cells after 48 hours [20].
In addition to the T-stimulated LNCaP assay for the 6a-e derivatives, we found that
the para-substituted compounds 6a, 6b and 6d having p-OCH3, p-CH3 and p-F,
respectively, are almost equally potent (38.6, 43.3 and 44.3%, respectively), whereas the p-
Cl compound (6e) exhibited the least activity. Interestingly, compound 6c, with an
unsubstituted phenyl ring, produced the highest anti-proliferative effect of this series
(66.2%). Furthermore, 6c was as potent as its rigid indoline analog counterpart (6h) in the
same assay. Therefore, these results suggest that the presence of substituents attached to the
para-site of the phenylcabamoyl group has a negative impact on the anti-proliferative
activity on T-treated LNCaP cells.

ACCEPTED MANUSCRIPT
Table 1. Percentage growth inhibitory effect of steroidal derivatives 6a-h, as well as
flutamide, finasteride and ketoconazole at 50 µM on LNCaP and PBMC cells.
LNCaP
% Cell growth inhibition at 50
μM
Rat PBMC
% Cell
growth
Molecular Properties
Compound
R
inhibition at
50 μM
(Mean±S.D.)
(Mean±S.D.)
Androgen-
LogP TPSA nON nOHNH free media [0.1 µM]
calca calcb
T
DHT
[0.1 µM]
5.12 55.40
4
1
48.7±4.3 38.6±13.9 29.7±7.1
7.9±0.8
6a
6b
5.51 46.17
3
1
25.6±2.6
43.3±0.6
27.5±8.6
13.0±1.5
6c
5.06 46.17
5.23 46.17
3
3
1
1
40.9±2.3
29.1±0.7
66.2±6.3 59.4±17.7
8.7±2.6
4.1±2.5
6d
44.3±0.3
1.6±7.6
29.0±3.0
13.4±10.4
100±8.6
6e
6f
5.74 46.17
6.78 46.17
3
3
1
1
-6.1±0.4
NA
100.0±3.2
9.1±1.0
100±18.8
4.12 59.06
5.30 37.38
4
3
1
0
100±2.9
16.8±2.4
NA
6g
6h
100±9.9
100±4.2
69.1±4.0
49.9±1.0
52.6±0.9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-3.4±0.8
34.6±5.6
82.7±0.9
29.6±1.0 25.11±1.3
NA
3.7±1.6
ND
Flutamide
Finasteride
Ketoconazole
21.1±0.9
76.0±5.5
NA
68.2±6.1

ACCEPTED MANUSCRIPT
*NA=Null activity
**ND = Not determined
a
The molecular properties of all compounds were calculated by Molinspiration
(http://www.molinspiration.com/). miLog P, molinspiration predicted LogP; TPSA, Topological
Surface Area; nON, no. of hydrogen bond acceptors and nOHNH, no. of hydrogen bond donors.
The most surprising aspect of the data is the complete inhibition of the stimulatory
growth effect of T and DHT by derivatives 6f and 6g. These compounds also displayed the
strongest anti-proliferative effect (100%) in androgen-free media. Considering these the
best compounds that exhibited the greatest effect in the three assays, we proceeded to
determine their concentration at which the growth of 50% of LNCaP cells in absence of T
or DHT was inhibited. Moreover, we tested 6f and 6g on the androgen-insensitive PC-3 cell
line in order to detect whether the anti-proliferative activity is exerted by an androgen-
independent effect; ketoconazole was used for comparison in both cell lines (Table 2).
First, we observed that compound 6f is a better cell growth inhibitor than 6g (GI50 = 12.8 ±
0.7 µM and 16.0±1.8 µM, respectively) on the LNCaP cell line, and both compounds are
more potent than ketoconazole (GI50 = 25.0 ± 5.7 µM). Secondly, 6g exhibited the best anti-
proliferative effect on PC3 cells (GI50 = 6.6 ± 0.7 µM) as compared to 6f (GI50 = 14.2 ± 1.6
µM) and ketoconazole (GI50 = 11.7 ± 0.6 µM). These results suggest that 6f and 6g are able
to decrease cell proliferation of prostate cancer cells by an androgen independent manner.
Interestingly, this effect was greater on the PC-3 cell line which is widely used as a model
of castration resistant prostate cancer [22].

ACCEPTED MANUSCRIPT
Table 2. Concentration of compounds which inhibits growth of 50% (GI50) of
LNCaP and PC3 cells in free steroid media by steroids 6f, 6g and ketoconazole
Compound LNCaP GI50 (µM) PC3 GI50 (µM)
(Mean±S.D.)
12.8 ± 0.7
16.0 ± 1.8
25.0 ± 5.7
(Mean±S.D.)
14.2 ± 1.6
6.6 ± 0.7
6f
6g
11.7 ± 0.6
Ketoconazole
The activity displayed by these steroids may be explained by the fact that 6f, with
two trifluoromethyl groups conferring a greater lipophilic character (LogP = 6.78) to the
molecule, has a greater ability to cross the membrane of the cell. In contrast, 6g has a sp2
nitrogen on the thiazole ring which facilitates the formation of hydrogen bonds (nON = 4)
with different residues of amino acids in the target-proteins; this interaction promotes the
stability of this molecule as compared to the rest of the synthesized steroids. Moreover, this
compound has the highest topological surface area (TPSA = 59.06). Briefly, TPSA is based
on the summation of tabulated surface contributions of polar fragments in a molecule. This
parameter has been shown to correlate well with passive molecular transport through the
biological membranes [33]. Thus, we consider that the increase in the Log P as well as
TPSA values has a positive impact on the anti-proliferative effect for this series.
2.3 Cytotoxicity effect on PBMC cells.
To investigate whether the synthesized compounds may be acting as cytotoxic
agents on healthy cells, PBMC cells were treated with the same tested compounds for

ACCEPTED MANUSCRIPT
LNCaP cells (Table 1). The commercially available drugs finasteride and flutamide showed
3.69% of cell growth inhibition and no effect, respectively. For the novel synthetic steroidal
derivatives, 6g exhibited the strongest effect in this assay (16.8%). Compounds 6a-d and 6f
demonstrated low inhibitory anti-proliferative effects, whereas 6e and 6h were ineffective
against these cells. Therefore, the very slight anti-proliferative activity on rat PBMC,
suggests that these compounds show low toxicity on healthy cells.

ACCEPTED MANUSCRIPT
3. Experimental protocols
3.1. General
Reagents and solvents purchased were laboratory grade or higher. Melting points
were determined on a Fisher-Johns melting point apparatus and are uncorrected. IR spectra
were recorded on a Perkin-Elmer 200s spectrometer. Low resolution mass spectra (LRMS)
were obtained with a HP5985-B spectrometer and the high resolution mass spectra
(HRMS) were obtained using a JEOL JMS-AX505 HA mass spectrometer operated in
positive mode. 1H NMR and 13C NMR were recorded at 400 MHz for 1H and 100 MHz for
13C using Varian Gemini 200 and VRX-300 spectrometers, respectively. Chemical shifts
are given in ppm relative to that of Me4Si (δ=0) in CDCl3 (the abbreviations of signal
patterns are as follows: s, singlet; d. doublet; t, triplet; m, multiplet). The reactions were
monitored by TLC on Kieselgel-G (Merck Si 254 F) layers (0.25 mm thick); Flash
chromatography was performed using Merck silica gel 60, 40-63 µm.
3.2 Synthesis
For the synthesis of the steroidal compounds 6a-h (Scheme 1), the commercially
available 3β-hidroxy-5-pregnen-20-one 1 was used. Compounds 2 and 3 were prepared by
the procedure described in reference [34]. The synthesis of 6a-h derivatives is briefly
described below.
3.2.1. General method for the synthesis of 3β-acetoxy-17β-N-
(arylcarbamoyl)androst-5-ene derivatives (4a-h)

ACCEPTED MANUSCRIPT
0.5 g (1.5 mmol) of 3β-acetoxyandrost-5-ene-17β-carboxylic acid (3) was dissolved
in 20 mL of methylene chloride, 0.5 mL of previously distilled thionyl chloride was added
dropwise with constant stirring and the mixture was stirred at room temperature for 2 hours,
the solvent and the excess of thionyl chloride was distilled off. The acid chloride was
dissolved again with 10 mL of methylene chloride and cooled in an ice bath. 0.5 mL of
triethylmamine and 1.2 mmol of the desired arylamine were then slowly added. The
resulting yellow solution was stirred at room temperature for 1.5 h, the solvent was then
distilled and 30 mL of hot hexane was added to the oily residue. The resulting precipitate
was filtered and recrystallized from methylene chloride-methanol.
3.2.1.1. 3β-acetoxy-17β-N-(4-methoxyphenylcarbamoyl)androst-5-ene (4a)
Yield: 65%, mp 218-221°C, IR (FT-IR) cm-1: 3415, 2904, 2964, 2830, 2854, 1730,
1
1671, 1513. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 2.0 (3H, s,
COOCH3), 3.8 (3H, s, PhOCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-
6), 6.8 (2H, d, J = 8.9, H-3´ and H-5´ aromatic). 6.9 (1H, sb, PhNH), 7.4 (2H, d, J = 8.9 Hz,
H-2´ and H-6´ aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.5 (C-19), 58.0 (PhOCH3),
74.0 (C-3), 114.2 (C-3´ and C-5´, aromatic), 121.9 (C-2´and C-6´, aromatic), 122.5 (C-6),
131.4 (C-1´, aromatic), 139.8 (C-5), 156.4 (C-4´, aromatic), 170.7 (carbonyl ester), 171.1
(carbonyl amide). LRMS: 465 [M]+
3.2.1.2. 3β-acetoxy-17β-N-(4-methylphenylcarbamoyl)androst-5-ene (4b)
Yield: 68%, mp 211-215°C, IR (FT-IR) cm-1: 3417, 2967, 2942, 2908, 2894, 2870,
2856, 2828, 1729, 1681, 1512, 1248. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-
19), 2.0 (3H, s, COOCH3), 2.3 (3H, s, PhCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and

ACCEPTED MANUSCRIPT
J2= 2 Hz, H-6), 7.1 (3H, m, PhNH, H-2´ and H-6´ aromatic), 7.4 (2H, d, J = 8.0 Hz, H-3´
and H-5´ aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.4 (C-19), 21.9 (PhCH3), 74.0 (C-
3), 120.0 (C-2´ and C-6´, aromatic), 122.4 (C-6), 129.6 (C-3´and C-5´, aromatic), 135.7 (C-
1´, aromatic), 133.7 (C-4´, aromatic) 139.8 (C-5), 170.7 (carbonyl ester), 171.1 (carbonyl
amide). LRMS: 449 [M]+
3.2.1.3. 3β-acetoxy-17β-N-(phenylcarbamoyl)androst-5-ene (4c).
Yield: 67%, mp. 231-233°C. IR (FT-IR) cm-1: 3333, 2938, 2905, 2871, 2851, 1730,
1687, 1498, 1240. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 2.0 (3H, s,
COOCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.1 (1H, t, J = 7.4
Hz, H-4´, aromatic), 7.2 (1H, sb, PhNH), 7.3 (2H, t, J = 7.0 Hz, H-3´ and H-5´, aromatic),
13
7.5 (2H, d, J = 7.0 Hz, H-2´ and H-6´, aromatic). C NMR (CDCl3) δ: 13.3 (C-18), 19.4
(C-19), 74.0 (C-3), 119.9 (C-2´ and C-6´, aromatic), 122.4 (C-6), 124.1 (C-4´), 129.1 (C-
3´and C-5´, aromatic), 138.4 (C-1´, aromatic), 139.8 (C-5), 170.8 (carbonyl ester), 171.3
(carbonyl amide). LRMS: 435 [M]+
3.2.1.4. 3β-acetoxy-17β-N-(4-fluorophenylcarbamoyl)androst-5-ene (4d).
Yield: 60%, mp 230-233°C, IR (FT-IR) cm-1: 3398, 3039, 2941, 2909, 2874, 2854,
2826, 1728, 1681, 1507, 832. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 2.0
(3H, s, COOCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (2H,
ddd, JH-F = 2.9, 6.3 and 10.4 Hz, H-2´ and H-6´, aromatic), 7.1 (1H, sb, PhNH), 7.5 (2H,
dd, JH-F = 4.8 and 9.0 Hz, H-2´ and H-6´, aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.5
(C-19), 74.0 (C-3), 115.7 (d, JC-F = 22.4 Hz, C-3´ and C-5´, aromatic), 121.8 (d, JC-F = 7.8
Hz, C-2´and C-6´, aromatic), 122.4 (C-6), 134.2 (d, JC-F = 2.9 Hz, C-1´, aromatic), 139.8

ACCEPTED MANUSCRIPT
(C-5), 159.3 (d, JC-F = 243 Hz, C-4´, aromatic), 170.8 (carbonyl ester), 171.3 (carbonyl
amide). LRMS: 453 [M]+
3.2.1.5. 3β-acetoxy-17β-N-(4-chlorophenylcarbamoyl)androst-5-ene (4e).
Yield: 67%, mp 263-265°C, IR (FT-IR) cm-1: 3395, 3028, 2946, 2946, 2927, 2910,
2875, 2853, 1724, 1685, 1510, 1253. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-
19), 2.0 (3H, s, COOCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.2
(1H, sb, PhNH), 7.3 (2H, d, J = 8.8 Hz, H-2´ and H-6´, aromatic), 7.5 (2H, d, J = 8.8 Hz,
H-3´ and H-5´, aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.4 (C-19), 74.0 (C-3), 121.2
(C-2´ and C-6´, aromatic), 122.4 (C-6), 129.0 (C-3´and C-5´, aromatic), 136.8 (C-1´,
aromatic), 139.8 (C-5), 170.8 (carbonyl ester), 171.4 (carbonyl amide). MS: 470 [M]+
3.2.1.6. 3β-acetoxy-17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-5-
ene (4f).
Yield: 42%, mp 123-125 °C, IR (FT-IR) cm-1: 3347, 3112, 2943, 2905, 2851, 1732,
1710, 1380, 1276, 1129. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 2.0 (3H,
s, COOCH3), 4.7 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.6 (1H, s, H-4´,
aromatic), 8.0 (1H, sb, PhNH), 8.1 (2H, s, H-2´ and H-6´, aromatic). 13C NMR (CDCl3) δ:
13.5 (C-18), 19.4 (C-19), 74.3 (C-3), 117.4 (q, JC-F = 3.8 Hz, C-1´), 119.4 (d, JC-F = 3.2 Hz,
C-2´ and C-6´, aromatic), 122.4 (C-6), 126.0 (q, JC-F = 272 Hz, CF3), 132.3 (q, JC-F = 33.4
Hz, C-3´and C-5´, aromatic), 139.9 (C-4´), 140.0 (C-5), 171.4 (carbonyl ester), 172.1
(carbonyl amide). LRMS: 571 [M]+.
3.2.1.7. 3β-acetoxy-17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-5-ene (4g)

ACCEPTED MANUSCRIPT
Yield: 60%, mp 152-154 °C, IR (FT-IR) cm-1: 3251, 3157, 2941, 2905, 2850, 2730,
1
1731, 1682, 1557, 1247, 1029. H NMR (CDCl3) δ: 0.7 (3H, s, H-18), 1.0 (3H, s, H-19),
2.0 (3H, s, COOCH3), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H,
d, J = 3.6 Hz, H-5´, thiazole), 7.4 (1H, d, J = 3.6 Hz, H-4´, thiazole), 10.9 (1H, sb,
thiazole-NH). 13C NMR (CDCl3) δ: 13.5 (C-18), 19.4 (C-19), 74.0 (C-3), 113.6 (C-5´,
thiazole), 122.3 (C-6), 136.7 (C-4´, thiazole), 139.9 (C-5), 159.5 (C-2´, thiazole), 170.7
(carbonyl ester), 171.3 (carbonyl amide). LRMS: 442 [M]+.
3.2.1.8. 3β-acetoxy-17β-(2,3-dihydro-1H-indol-1-ylcarbonyl)androst-5-ene (4h)
Yield: 54%, mp 158-160, IR (FT-IR) cm-1: 3041, 2941, 2907, 2870, 2850, 2819,
1732, 1646, 1249, 748. 1H NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-19), 2.0 (3H, s,
COOCH3), 3.1 (2H, t, J = 8.4 Hz, CH2, indoline), 4.1 (2H, dq, J1 = 9.1 Hz, J2 = 18.6 Hz,
CH2, indoline), 4.6 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H, m, H-
7´, indoline), 7.2 (2H, m, H-5´ and H-6´, indoline), 8.3 (1H, d, J = 7.9 Hz, H-4´, indoline).
13C NMR (CDCl3) δ: 14.0 (C-18), 19.4 (C-19), 28.3 (CH2, indoline), 48.8 (CH2, indoline),
71.8 (C-3), 117.6 (C-7´, indoline), 122.5 (C-6), 123.5 (C-4´, indoline), 124.6 (C-5´,
indoline), 127.5 (C-6´, indoline), 131.5 (C-3a, indoline), 139.8 (C-5), 143.6 (C-7a,
indoline), 170.7 (carbonyl ester), 172.5 (carbonyl amide). LRMS: 461 [M]+.
3.2.2. General method for the synthesis of 3β-hydroxy-17β-N-
(arylcarbamoyl)androst-5-ene derivatives (5a-h).
0.8 mmol of the corresponding 3β-acetoxy-17β-N-phenylcarbamoylandrost-5-ene
derivative (4a-h) was suspended in 15 mL of methanol and 2 mL of a 10 % methanolic
solution of potassium hydroxide was added, this mixture was stirred at room temperature

ACCEPTED MANUSCRIPT
for 50 minutes and then poured into 50 mL of cold water. The white solid formed was
collected by vacuum filtration and recrystallized from methanol-water.
3.2.2.1. 3β-hydroxy-17β-N-(4-methoxyphenylcarbamoyl)androst-5-ene (5a)
Yield: 94%, mp 145-148°C, IR (FT-IR) cm-1: 3414, 3300, 2967, 2935, 2902, 2852
2829, 1669, 1513, 1109. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 3.5 (1H,
m, H-3), 3.8 (3H, s, PhOCH3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 6.8 (2H, d, J = 8.7
Hz, H-3´ and H-5´ aromatic), 6.9 (1H, sb, PhNH), 7.4 (2H, d, J = 8.8 Hz, H-2´ and H-6´
aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.5 (C-19), 58.1 (PhOCH3), 71.9 (C-3),
114.3 (C-3´ and C-5´, aromatic), 121.5 (C-6), 121.9 (C-2´and C-6´, aromatic), 131.3 (C-1´,
aromatic), 140.9 (C-5), 156.4 (C-4´, aromatic), 171.1 (carbonyl amide). LRMS: 423 [M]+
3.2.2.2. 3β-hydroxy-17β-N-(4-methylphenylcarbamoyl)androst-5-ene (5b)
Yield: 93%, mp 145-148°C, IR (FT-IR) cm-1: 3414, 3300, 2969, 2968, 2904, 2859,
2869, 2827, 1679, 1511. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 2.3 (3H,
s, PhCH3), 3.5 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 6.9 (1H, sb, PhNH),
7.1 (2H, d, J = 8.1 Hz, H-2´ and H-6´ aromatic), 7.4 (2H, d, J = 8.2 Hz, H-3´ and H-5´
aromatic). 13C NMR (CDCl3) δ: 13.3 (C-18), 19.4 (C-19), 21.2 (PhCH3), 71.9 (C-3), 120.1
(C-2´ and C-6´, aromatic), 121.5 (C-6), 129.6 (C-3´and C-5´, aromatic), 133.8 (C-4´,
aromatic), 135.6 (C-1´, aromatic), 140.9 (C-5), 171.1 (carbonyl amide). LRMS: 407 [M]+.
3.2.2.3. 3β-hydroxy-17β-N-(phenylcarbamoyl)androst-5-ene (5c)
Yield: 91% mp 224-226°C, IR (FT-IR) cm-1: 3413, 3308, 3052, 2968, 2938, 2904,
1
2893, 2858, 2827, 1682, 1519. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19),

ACCEPTED MANUSCRIPT
3.5 (1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H, sb, PhNH), 7.1 (1H, t,
J = 7.4 Hz, H-4´, aromatic), 7.3 (2H, t, J = 7.0 Hz, H-3´ and H-5´, aromatic), 7.5 (2H, d, J =
13
7.0 Hz, H-2´ and H-6´, aromatic). C NMR (CDCl3) δ: 13.3 (C-18), 19.5 (C-19), 71.8 (C-
3), 119.9 (C-2´ and C-6´, aromatic), 121.5 (C-6), 124.2 (C-4´), 129.1 (C-3´and C-5´,
aromatic), 138.1 (C-1´, aromatic), 140.9 (C-5), 171.3 (carbonyl amide). LRMS: 393 [M]+.
3.2.2.4. 3β-hydroxy-17β-N-(4-fluorophenylcarbamoyl)androst-5-ene (5d)
Yield: 93%, mp 236-239°C, IR (FT-IR) cm-1: 3415, 3299, 2968, 2938, 2902, 2858,
1
2827, 1678, 1510, 1098, 837. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 3.5
(1H, m, H-3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 6.9 (3H, m, PhNH, H-2´ and H-6´),
13
7.5 (2H, dd, J1 = 4.8 Hz, J2 = 9.0 Hz, H-3´ and H-5´, aromatic). C NMR (CDCl3) δ: 13.3
(C-18), 19.6 (C-19), 71.8 (C-3), 115.7 (d, JC-F = 22.4 Hz, C-3´ and C-5´, aromatic), 121.5
(C-6), 121.8 (d, JC-F = 7.8 Hz, C-2´and C-6´, aromatic), 134.1 (d, JC-F = 2.9 Hz, C-1´,
aromatic), 140.9 (C-5), 159.4 (d, JC-F = 243 Hz, C-4´, aromatic), 171.3 (carbonyl amide).
LRMS: 411 [M]+
3.2.2.5. 3β-hydroxy-17β-N-(4-chlorophenylcarbamoyl)androst-5-ene (5e)
Yield: 91%, mp 214-216°C, IR (FT-IR) cm-1: 3411, 2963, 2937, 2901, 2850, 1684,
1
1493, 1091, 830. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 3.5 (1H, m, H-
3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H, sb, PhNH), 7.3 (2H, d, J = 8.8 Hz, H-
2´ and H-6´, aromatic), 7.5 (2H, d, J = 8.8 Hz, H-3´ and H-5´, aromatic). 13C NMR (CDCl3)
δ: 13.3 (C-18), 19.6 (C-19), 71.9 (C-3), 121.2 (C-2´ and C-6´, aromatic), 121.5 (C-6), 129.1
(C-3´and C-5´, aromatic), 136.7 (C-1´, aromatic), 140.9 (C-5), 171.3 (carbonyl amide).
LRMS: 427 [M]+ 15% RA, 429 [M+2]+ 5% RA.

ACCEPTED MANUSCRIPT
3.2.2.6. 3β-hydroxy-17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-
5-ene (5f)
Yield: 89%, mp 138-141 °C, IR (FT-IR) cm-1: 3317, 3105, 2941, 2856, 1660, 1622,
1380, 1275, 1129. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.0 (3H, s, H-19), 3.5 (1H, m, H-
3), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.2 (1H, sb, PhNH), 7.6 (1H, s, H-4´,
aromatic), 8.0 (2H, s, H-2´ and H-6´, aromatic). 13C NMR (CDCl3) δ: 13.4 (C-18), 19.6 (C-
19), 71.9 (C-3), 117.4 (q, JC-F = 3.8 Hz, C-1´), 119.4 (d, JC-F = 3.2 Hz, C-2´ and C-6´,
aromatic), 122.4 (C-6), 126.0 (q, JC-F = 272 Hz, CF3), 132.3 (q, JC-F = 33.4 Hz, C-3´and C-
5´, aromatic), 139.5 (C-4´), 140.9 (C-5), 171.9 (carbonyl amide). LRMS: 529 [M]+
3.2.2.7. 3β-hydroxy-17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-5-ene (5g)
Yield: 86%, mp 203-205 °C, IR (FT-IR) cm-1: 3364, 3251, 3168, 3128, 3073, 2904.
2931, 1682, 1643, 1057, 715. 1H NMR (CDCl3) δ: 0.7 (3H, s, H-18), 1.0 (3H, s, H-19), 3.5
(1H, m, H-3), 5.3 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H, d, J = 3.6 Hz, H-5´,
thiazole), 7.4 (1H, d, J = 3.6 Hz, H-4´, thiazole), 11.2 (1H, sb, thiazole-NH). 13C NMR
(CDCl3) δ: 13.5 (C-18), 19.4 (C-19), 74.0 (C-3), 113.6 (C-5´, thiazole), 122.3 (C-6), 136.7
(C-4´, thiazole), 139.9 (C-5), 159.5 (C-2´, thiazole), 172.5 (carbonyl amide). LRMS: 400
[M]+.
3.2.2.8. 3β-hydroxy-17β-(2,3-dihydro-1H-indol-1-ylcarbonyl)androst-5-ene (5h)
Yield: 91%, mp 161-163, IR (FT-IR) cm-1: 3483, 3041, 2975, 2947, 2921, 2878,
1
2851, 2822, 1632, 1593, 1249, 751. H NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.0 (3H, s, H-
19), 3.1 (2H, t, J = 8.4 Hz, CH2, indoline), 3.5 (1H, m, H-3), 4.1 (2H, dq, J1 = 9.1 Hz, J2 =
18.6 Hz, CH2, indoline), 5.4 (1H, dd, J1= 4 Hz and J2= 2 Hz, H-6), 7.0 (1H, m, H-7´,

ACCEPTED MANUSCRIPT
indoline), 7.2 (2H, m, H-5´ and H-6´, indoline), 8.3 (1H, d, J = 7.9 Hz, H-4´, indoline). 13C
NMR (CDCl3) δ: 14.1 (C-18), 19.5 (C-19), 28.3 (CH2, indoline), 48.8 (CH2, indoline), 71.8
(C-3), 117.7 (C-7´, indoline), 121.5 (C-6), 123.6 (C-4´, indoline), 124.6 (C-5´, indoline),
127.5 (C-6´, indoline), 131.5 (C-3a, indoline), 140.1 (C-5), 143.6 (C-7a, indoline), 172.5
(carbonyl amide). LRMS: 419 [M]+.
3.2.3. General method for the synthesis of 17β-N-(arylcarbamoyl)androst-4-
ene-3-one derivatives (6a-h).
A
solution of 0.5 mmol of the corresponding 3β-hydroxy-17β-N-
(phenylcarbamoyl)androst-5-ene derivative (5a-h) in 30 mL of toluene and 2 mL of 1-
methyl-4-piperidone was refluxed under a Dean-Stark trap until approximately 5 mL of
distillate had been collected. To the solution was added 0.6 mmol of aluminum
isopropoxide and 5 mL more of distillate collected. The mixture was refluxed for 6 h,
cooled, and the excess of toluene was distilled, the gummy residue was diluted with 30 mL
of ethyl acetate, washed with four 30 mL portions of 1 M hydrochloric acid and 30 mL of
brine, and dried over anhydrous Na2SO4. The desired product was purified by silica column
chromatography eluted with hexane-ethyl acetate (7:3).
3.2.3.1. 17β-N-(4-methoxyphenylcarbamoyl)androst-4-ene-3-one (6a)
Yield: 62%, mp 112-113°C, IR (FT-IR) cm-1: 3330, 2935, 2852, 1657, 1410, 1230,
1
828. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 3.8 (3H, s, PhOCH3), 5.7
(1H, s, H-4), 6.8 (2H, d, J = 9.0 Hz, H-3´ and H-5´ aromatic), 7.1 (1H, sb, PhNH), 7.4 (2H,
d, J = 9.0 Hz, H-2´ and H-6´ aromatic). 13C NMR (CDCl3) δ: 13.5 (C-18), 17.5 (C-19), 57.8
(PhOCH3), 71.9 (C-3), 114.2 (C-3´ and C-5´, aromatic), 121.9 (C-2´and C-6´, aromatic),

ACCEPTED MANUSCRIPT
124.0 (C-4), 131.4 (C-1´, aromatic), 156.4 (C-4´, aromatic), 171.0 (C-5), 171.4 (carbonyl
amide), 199.8 (C-3). HRMS (FAB+): calc. for C27H35NO3 422.5717 [M+H]+, found
422.5721.
3.2.3.2. 17β-N-(4-methylphenylcarbamoyl)androst-4-ene-3-one (6b)
Yield: 64%, mp 208-209°C, IR (FT-IR) cm-1: 3331, 3114, 3024, 2937, 2877, 2849,
1
1660, 1515, 816. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 2.3 (3H, s,
PhCH3), 5.7 (1H, s, H-4), 7.1 (1H, sb, PhNH), 7.1 (2H, d, J = 8.2 Hz, H-2´ and H-6´
13
aromatic), 7.4 (2H, d, J = 8.3 Hz, H-3´ and H-5´ aromatic). C NMR (CDCl3) δ: 13.5 (C-
18), 17.5 (C-19), 20.3 (PhCH3), 120.0 (C-2´ and C-6´, aromatic), 124.1 (C-4), 129.6 (C-
3´and C-5´, aromatic), 133.8 (C-4´, aromatic), 135.6 (C-1´, aromatic), 171.1 (C-5), 171.2
(carbonyl amide). HRMS (FAB+): calc. for C27H35NO2 [M+H]+ 406.5723, found 406.5728.
3.2.3.3. 17β-N-(phenylcarbamoyl)androst-4-ene-3-one (6c)
Yield: 64%, mp 203-205°C, IR (FT-IR) cm-1: 3331, 3031, 2943. 2926, 2910,
2847,1715, 1678, 1517.84.1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 5.7
(1H, s, H-4), 7.1 (1H, t, J = 7.3 Hz, H-4´, aromatic), 7.2 (1H, sb, PhNH), 7.3 (2H, t, J = 7.8
13
Hz, H-3´ and H-5´, aromatic), 7.5 (2H, t, J = 7.9 Hz, H-2´ and H-6´, aromatic). C NMR
(CDCl3) δ: 13.5 (C-18), 17.5 (C-19), 119.9 (C-2´ and C-6´, aromatic), 124.1 (C-4´), 124.2
(C-4), 129.1 (C-3´and C-5´, aromatic), 138.2 (C-1´, aromatic), 171.1 (C-5), 171.2 (carbonyl
amide), 199.7 (C-3). HRMS (FAB+): calc. for C26H34NO2 [M+H]+ 392.2590, found
392.2591.
3.2.3.4. 17β-N-(4-fluorophenylcarbamoyl)androst-4-ene-3-one (6d)

ACCEPTED MANUSCRIPT
Yield: 65%, mp 218-219°C, IR (FT-IR) cm-1: 3314, 3145, 3062, 2925, 2854, 1660,
1539, 1508, 1210, 830. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 5.8 (1H, s,
H-4), 7.0 (2H, t, J = 8.6 Hz, H-3´ and H-5´, aromatic), 7.1 (1H, sb, PhNH), 7.5 (2H, dd, J1
= 4.8 Hz, J2 = 8.8 Hz, H-2´ and H-6´, aromatic). 13C NMR (CDCl3) δ: 13.5 (C-18), 17.5 (C-
19), 115.7 (d, J = 22.4 Hz, C-3´ and C-5´, aromatic), 121.8 (d, J = 7.8 Hz, C-2´and C-6´,
aromatic), 124.1 (C-4), 134.2 (C-1´, aromatic), 159.4 (d, J = 243 Hz, C-4´, aromatic), 171.0
(C-5), 171.1 (carbonyl amide), 199.7 (C-3). HRMS (FAB+): calc. for C26H33FNO2 [M+H]+
410.5361, found 410.5681.
3.2.3.5. 17β-N-(4-chlorophenylcarbamoyl)androst-4-ene-3-one (6e)
Yield: 68%, mp 234-237°C, IR (FT-IR) cm-1: 3331, 3026, 1680, 1658, 1715, 1491,
1
828, 817. H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 5.7 (1H, s, H-4), 7.3
(3H, m, PhNH, H-2´ and H-6´, aromatic), 7.5 (2H, d, J = 8.7 Hz, H-3´ and H-5´, aromatic).
13C NMR (CDCl3) δ: 13.5 (C-18), 17.5 (C-19), 121.2 (C-2´ and C-6´, aromatic), 124.2 (C-
4), 129.1 (C-3´and C-5´, aromatic), 136.8 (C-1´, aromatic), 171.2 (C-5), 171.3 (carbonyl
amide), 199.8 (C-3). HRMS (FAB+): calc. for C26H33ClNO2 [M+H]+ 426.2200, found
426.2195.
3.2.3.6. 17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-4-ene-3-one
(6f)
Yield: 65%, mp 214-216 °C, IR (FT-IR) cm-1: 3316, 3105, 2941, 2856, 1660, 1642,
1380, 1275, 1129. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 5.8 (1H, s, H-
4), 7.6 (1H, sb, PhNH), 7.6 (1H, s, H-4´, aromatic), 8.2 (2H, s, H-2´ and H-6´, aromatic).
13C NMR (CDCl3) δ: 13.6 (C-18), 17.4 (C-19), 117.4 (q, JC-F = 3.8 Hz, C-1´), 119.5 (d, JC-F

ACCEPTED MANUSCRIPT
= 3.2 Hz, C-2´ and C-6´, aromatic), 124.1 (C-4), 126.0 (q, JC-F = 272 Hz, CF3), 132.4 (q,
JC-F = 33.4 Hz, C-3´and C-5´, aromatic), 139.9 (C-1´), 171.7 (C-5), 171.8 (carbonyl amide),
200.3 (C-3). HRMS (FAB+): calc. for C28H32F6NO2 [M+H]+ 528.2337, found 528.2332.
3.2.3.7. 17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-4-ene-3-one (6g)
Yield: 71%, mp 209-211°C, IR (FT-IR) cm-1: 3229, 3132, 3061, 2980, 2964, 2933,
2923, 2884, 2869, 2854, 1681, 1666, 1584, 870. 1H NMR (CDCl3) δ: 0.8 (3H, s, H-18), 1.2
(3H, s, H-19), 5.7 (1H, s, H-4), 7.0 (1H, d, J = 3.5 Hz, H-5´, thiazole), 7.4 (1H, d, J = 3.5
Hz, H-4´, thiazole, 10.1 (1H, sb, thiazole-NH). 13C NMR (CDCl3) δ: 13.5 (C-18), 17.5 (C-
19), 113.7 (C-5´, thiazole), 124.2 (C-4), 136.9 (C-4´, thiazole), 158.9 (C-2´, thiazole), 170.8
(C-5), 170.9 (carbonyl amide), 199.6 (C-3). HRMS (FAB+): calc. for C23H30N2O2S [M+H]+
399.5615, found 399.5619.
3.2.3.8. 17β-(2,3-dihydro-1H-indol-1-ylcarbonyl)androst-4-en-3-one (6h)
Yield: 45%, mp 234-235 °C, IR (FT-IR) cm-1: 3026, 2940, 2907, 2856, 2840, 1666,
1
1645, 1595, 764. H NMR (CDCl3) δ: 0.9 (3H, s, H-18), 1.2 (3H, s, H-19), 3.1 (2H, t, J =
8.4 Hz, CH2, indoline), 4.1 (2H, dq, J1 = 9.1 Hz, J2 = 18.6 Hz, CH2, indoline) 5.7 (1H, s,
H-4), 7.0 (1H, m, H-7´, indoline), 7.2 (2H, m, H-5´ and H-6´, indoline), 8.3 (1H, d, J = 7.9
Hz, H-4´, indoline). 13C NMR (CDCl3) δ: 14.1 (C-18), 17.4 (C-19), 28.2 (CH2, indoline),
48.7 (CH2, indoline), 117.6 (C-7´, indoline), 123.6 (C-4´, indoline), 124.0 (C-4), 124.6 (C-
5´, indoline), 127.5 (C-6´, indoline), 131.5 (C-3a, indoline), 143.6 (C-7a, indoline), 171.2
(C-5), 172.2 (carbonyl amide), 199.6 (C-3). HRMS (FAB+): calc. for C28H35NO2 [M+H]+
418.2746, found 418.2745.
3.3 Anti-proliferative assay