Synthesis of unprecedented steroidal spiro heterocycles as potential antiproliferative drugs

Article abstract of DOI:10.1016/j.ejmech.2017.10.063

Herein we report the straightforward preparation of novel conformationally-restricted steroids from trans-androsterone and estrone, decorated with spiranic oxazolidin-2-one or 2-aminooxazoline motifs at C-17 as potential antiproliferative agents. Such unp

Full text of DOI:10.1016/j.ejmech.2017.10.063

Accepted Manuscript
Synthesis of unprecedented steroidal spiro heterocycles as potential antiproliferative
drugs
Laura L. Romero-Hernández, Penélope Merino-Montiel, Socorro Meza-Reyes, José
Luis Vega-Baez, Óscar López, José M. Padrón, Sara Montiel-Smith
PII:
S0223-5234(17)30865-6
10.1016/j.ejmech.2017.10.063
EJMECH 9855
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2017
Revised Date: 9 October 2017
Accepted Date: 22 October 2017
Please cite this article as: L.L. Romero-Hernández, Pené. Merino-Montiel, S. Meza-Reyes, José.Luis.
Vega-Baez, Ó. López, José.M. Padrón, S. Montiel-Smith, Synthesis of unprecedented steroidal spiro
heterocycles as potential antiproliferative drugs, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.10.063.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis of unprecedented steroidal spiro heterocycles
as potential antiproliferative drugs
Laura L. Romero-Hernández,a Penélope Merino-Montiel,a* Socorro Meza-Reyes,a
José Luis Vega-Baez,a Óscar López,b José M. Padrón,c and Sara Montiel-Smitha*
aFacultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, 72570
Puebla, Pue., Mexico.
b
Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Apartado 1203, E-
41071 Seville, Spain.
cBioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Centro de
Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, c/ Astrofísico
Francisco Sánchez 2, 38206 La Laguna, Spain.
*Corresponding authors. Tel.: +52-222-2295500 ext. 7382; fax: +52-222-2443106
E-mail addresses: penelope.merino@correo.buap.mx (P. Merino-Montiel) and maria.montiel@correo.buap.mx (S.
Montiel-Smith)

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Abstract
Herein we report the straightforward preparation of novel conformationally-restricted
steroids from trans-androsterone and estrone, decorated with spiranic oxazolidin-2-one or
2-aminooxazoline motifs at C-17 as potential antiproliferative agents. Such unprecedented
pharmacophores were accessed using an aminomethylalcohol derivative at C-17 as the key
intermediate; reaction of such functionality with triphosgene, or conversion into N-
substituted thioureas, followed by an intramolecular cyclodesulfurization reaction promoted
by yellow HgO, furnished such spirocycles in excellent yields.
Title compounds were tested in vitro against a panel of six human tumor cell lines, named
A549 (non-small cell lung), HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell
lung), T-47D (breast) and WiDr (colon), and the results were compared with steroidal
chemotherapeutic agents (abiraterone and galeterone); the A-ring of the steroidal backbone,
the nature of the heterocycle and the N-substituents proved to be essential motifs for
establishing structure-activity relationships concerning not only the potency but also the
selectivity against tumor cell lines. Estrone derivatives, particularly those bearing a spiranic
2-aminooxazoline scaffold were found to be the most active compounds, with GI50 values
ranging from the low micromolar to the submicromolar level (0.34-1.5 µM). Noteworthy,
the lead compounds showed a remarkable increase in activity against the resistant cancer
cell lines (T-47D and WiDr) compared to the anticancer reference drugs (up to 120-fold).

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Keywords: steroids; oxazoline; oxazolidine; heterocycles; antiproliferative activity.
Abbreviations: COSY (Correlation Spectroscopy), DEPT (Distortionless Enhancement by
Polarization Transfer), EI (Electronic Impact), ESI (ElectroSpray Ionization), FAB (Fast
Atom Bombardment), FDA (Food and Drug Administration), GI50 (Growth Inhibition of
50%), HMBC (Heteronuclear Multiple Bond Correlation), HSQC (Heteronuclear Single
Quantum Correlation), NOESY (Nuclear Overhauser Effect Spectroscopy)

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1.
Introduction
Heterocyclic moieties are considered as privileged structures [1], endowed with a pivotal
importance from a biological and pharmaceutical point of view [2-6]. They are promising
targets in the drug discovery process [7-12], and are subsequently ubiquitous motifs in a
plethora of commercially-available drugs for the treatment of numerous diseases; in fact, it
is estimated that roughly 59% of small drugs approved by the US FDA contain a nitrogen-
based heterocycle [13].
Heterocycles exhibit a great variety of pharmaceutical properties, including antidepressant
[14], antimicrobial [15, 16], antibacterial [17], and anticancer [18, 19], among others [20].
The broad spectrum of biological activities of such molecules has encouraged the synthesis
of new compounds with improved properties; heterocycles are widely-used for the isosteric
replacement of a series of functional groups, as H-acceptors and H-donors, as metal
chelators, or for modulating the polarity of the drug, thus enhancing its bioavailability [21].
In this context, the conjugation of a heterocyclic motif with a steroidal framework has
allowed a synergic effect of both biologically-active pharmacophores, leading to unique
properties, such as anti-inflammatory [22], antibacterial [23, 24], antiproliferative [25-28],
aromatase [29] and 5α-reductase [30] inhibitors (these enzymes being involved in breast
and prostate cancer processes, respectively). Moreover, the highly lipophilic nature of the
steroidal skeleton might allow the penetration of the drug into the cytosol through the
lipidic membrane, thus improving its bioavailability.
Until now, interesting steroidal heterocycles have been synthesized and are being used in
clinical trials against different types of cancer. Abiraterone [31, 32] (17-(3-pyridyl) ring)
and galeterone [33, 34] (17-(1-benzimidazole) substituent) are clear examples of this kind
of drugs used in the treatment of prostate cancer, acting as potent and selective 17α-
hydroxylase/17,20 lyase inhibitors to block androgen synthesis [35]. Shi and co-workers
developed a new family of spirooxiindoles having the same steroidal nucleus as abiraterone
and galeterone; such compounds exhibited a good antiproliverative profile, where multiple
ROS processes seem to be involved in the tumor cells death [36]. Although a vast number
of compounds and/or therapies have been developed against cancer in the last few years, a
search of new compounds is still a key target in Medicinal Chemistry.

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Therefore, stimulated by the activities shown by heterocyclic-containing compounds,
herein we have focused on the design and synthesis of novel spiranic steroidal heterocycles
derived from trans-androsterone and estrone as potential antiproliferative agents; the
steroidal backbone, the nature of the heterocyclic motif, and substituents were modified in
order to provide the structural diversity required for establishing structure-activity
relationships.
2.
Results and discussion
2.1.
Chemistry
Incorporation of heterocycles into steroidal frameworks has usually been reported in the
pendant side-chain [37], or by fusing or joining the heterocyclic moiety mainly to the A
[38,39], B [40] or D [41-43] rings. Conversely, although the restriction of conformational
flexibility has been demonstrated to enhance some biological properties, like the receptor
selectivity and the binding affinity [44,45], the number of synthetic spiranic steroids is
relatively scarce [46-49]. Taking this into consideration, we envisioned the possibility of
preparing spiranic steroids bearing a oxazolidin-2-one or a 2-aminooxazoline scaffold
starting from two structurally-related steroids (trans-androsterone and estrone). The
synthesis of oxazolines, oxazolidines and related structures has been widely described in
the literature [50], including steroidal derivatives [51,52].
The key step to synthesize these heterocycles on the steroidal backbone was the access to
an aminomethyl alcohol on C-17; we designed the conversion of this functionality into the
general structures depicted in Figure 1, upon its transformation into transient isocyanates
and thioureas.

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Figure 1. Synthesis of oxazolidin-2-one (i) and 2-aminooxazolines (ii-iv) from aminoalcohol A.
The synthesis of the aminoalcohol was accomplished in 3 steps from trans-androsterone
and 4 steps from estrone. Trans-androsterone (1) was treated with trimethylsulfonium
iodide under basic conditions to obtain epoxide 2 [53] in a total stereoselective fashion via
a Corey-Chaykovsky reaction. Nucleophilic opening of epoxide 2 was carried out using
sodium azide in the presence of boric acid to get azide 3 in a quantitative yield (Scheme 1);
the absolute configuration of the new chiral carbon was assigned for compound 3 as the
(S)-diastereomer using a 1D-NOESY experiment. The spatial correlation in NOESY was
clearly observed between the protons CH2N3 and the protons H-12ax and H-16α, and the
evidence in the configuration was given by the correlation with H-14 (Figure 2).
16a
12b
14
3.0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
{1.97,3.23}
{1.11,3.23}
CH2N3
{1.15,3.52}
CH2N3
2.2
2.1
2.0
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
f2 (ppm)
Figure 2. NOESY correlations between CH2N3 and H-12ax, H-14 and H-16α in 3. Methyl group at
C-13 has been omitted for clarity.
Azide 3 was reduced by catalytic hydrogenation at 1 atm and 3 h to get the
aminomethylalcohol 4, which in turn was treated with triphosgene, as a secure alternative
to hazardous phosgene, in a MeOH-CH2Cl2 mixture to obtain transient isocyanate 5; this
compound underwent a spontaneous cyclization involving the nucleophilic attack of the
free OH to the heterocumulene, affording the spirooxazolidin-2-one (spirocarbamate) 6

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(52%); the use of MeOH as solvent led also to carbamate 7 as a by-product (25%). Hence
both products were tested to observe the differences in biological activity (Scheme 1).
When the reaction was made using DMF as solvent, it did not proceed, even when the
temperature and the base were changed.
Scheme 1. Synthesis of oxazolidin-2-ones (6,16) from trans-androsterone (1) and estrone (10).
The same reaction sequence was applied to get the estrone-derived spirocarbamate 16;
changing the nature of the A-ring (aromatic for estrone) might modulate the biological
properties and thus, afford valuable structure-activity relationships.
For this purpose, the free OH on C-3 was first protected as its benzyl ether, and then, the
functionalization of the C-17 was accomplished. Epoxide 12 [54] was obtained in a
stereoselective fashion, using KOtBu to form the sulfur ylide. Crystallization of 12 in a
hexane-EtOAc mixture allowed the determination of the absolute configuration (S) of the
new asymmetric carbon by single crystal X-ray diffraction (Cambridge Crystallographic
Data Centre 1561885) (Figure 3).

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Figure 3. X-ray crystal structure of 12.
The nucleophilic opening of 12 was carried out using sodium azide in DMF as indicated for
the trans-androsterone derivative, but with a longer reaction time. The structure of azide 13
obtained by single crystal X-ray diffraction (hexane-EtOAc mixture), showed the
configuration of C-17 as S (Figure 4) (Cambridge Crystallographic Data Centre 1561886).
Figure 4. X-ray crystal structure of 13.
Reduction of azide 13 under catalytic hydrogenation conditions also eliminated the benzyl
group at C-3, giving amine 14 [54] in a 77% yield after 4 steps. Reaction between 14 and
triphosgene gave isocyanate 15, which was not isolated and spontaneously evolved to the
final compound 16 and the by-product 17 in a 3:1 ratio (Scheme 1).
Aminoalcohols from trans-androsterone and estrone were also used to obtain 2-
aminooxazolines. Derivatives 4 and 14 were treated separately with different
isothiocyanates (butyl, cyclohexyl and phenyl) under basic conditions, giving thioureas 8
and 18 (Scheme 2). The cyclodesulfurization reaction of thioureas promoted by yellow

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HgO afforded 2-aminooxazolines 9 and 19 with good to excellent yields (Scheme 2); a
transient carbodiimide was reported [55] to be detected by TLC in the conversion of
glycopyranosyl thioureas into the corresponding ureas, where a desulfurization reaction
with HgO of the former was the key step. The same procedure was latter used for preparing
spiranic isoureas and guanidines on a carbohydrate template [56]. Accordingly, it can be
assumed that herein, HgO promotes the formation of a steroidal carbodiimide, which
undergoes a nucleophilic attack of the free OH on C-17, thus leading to the spirocyclic
moiety. To the best of our knowledge this is the first example of a spirocyclization reaction
in steroids starting from thioureas.
Changes in the 13C-NMR resonances of C-2’ (roughly 180 ppm for 8, 18 to roughly 160
ppm for 9, 19) supports such transformation).
Scheme 2. Synthesis of 2-aminooxazolines (9,19) from trans-androsterone (1) and estrone (10).
2.2. Antiproliferative activity
As a model to study the antiproliferative activity of compounds 3,4, 6-9, 13, 14, 16-19, we
selected the screening protocol of the National Cancer Institute [57]. The steroidal
derivatives were tested against a panel of six human solid tumor cell lines A549 (lung),
HBL-100 (breast), HeLa (cervix), SW1573 (lung), T-47D (breast) and WiDr (colon). The
results, expressed as 50% growth inhibition (GI50), are given in Table 1. The anticancer
drugs abiraterone and galeterone were also tested for comparison purposes.
According to the obtained GI50 values we can infer some structure activity relationships.
Taking the data as a whole (Figure 5), we can observe that compounds from the estrone

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series (13-19) were more potent than those from the androsterone set (3-9). In contrast, the
GI50 ranges are larger in the estrone derivatives (denoting differences in selectivity against
the cell lines) than in the androsterone compounds (indicating similar sensitivity of the cells
to the compounds). The most potent compounds in both series were the 2-aminooxazoline
derivatives 9 and 19 (low micromolar to submicromolar range), whilst compound 3 resulted
inactive against all cell lines. When considering the side chain of the 2-aminooxazoline
derivatives, the aliphatic substituents butyl (9a, 19a) and cyclohexyl (9b, 19b) are preferred
over the aromatic phenyl group (19c). A similar activity pattern based on the side chain was
observed for thioureas 8 and 18. In both series, the order of activity according to the
substituent at C-17 was established as 2-aminooxazoline (9, 19) > spirocarbamate (6, 16) >
thiourea (8, 18).
Noteworthy, the lead compounds 19a-b showed more potent activity against the resistant
cancer cell lines T-47D and WiDr, with GI50 values in the submicromolar range (0.34-0.71
µM). The relevance of this result is best understood if the GI50 values of the reference
anticancer drugs abiraterone and galeterone against the same cell lines are compared.
In addition to tumor cell lines, we tested the most potent compounds of the series (6, 9a-b,
19a-c) against the (non-tumor) human fibroblasts cell line BJ-hTert in order to look for
selectivity. Unfortunately, the normal cell line was sensitive to the compounds. This effect
was more pronounced for the reference drugs, especially for abiraterone.
Table 1. Antiproliferative values of steroidal derivatives against human cancer cell lines.
GI50 (µM)
Compound
A549
>100
HBL-100
>100
HeLa
SW1573
T-47D
WiDr
BJ-hTert
>100
>100
>100
>100
25 ± 3.9
3.8 ± 1.9
39 ± 13
3
4
17 ± 2.3
20 ± 1.9
22 ± 4.0 19 ± 4.7
21 ± 0.5
4.5 ± 1.6
41 ± 3.8
5.8 ± 1.0
18 ± 0.03
34 ± 5.1
1.2 ± 0.3
1.2 ± 0.2
6
3.7 ± 0.8 10 ± 4.5 4.0 ± 0.6 6.0 ± 3.2
<1
38 ± 8.2
12 ± 1.5
16 ± 4.7
29 ± 3.7
99 ± 1.3
13 ± 2.5
18 ± 3.4
27 ± 5.9
70 ± 27 92 ± 9.5
14 ± 2.1 9.4 ± 2.4
21 ± 0.3 15 ± 0.3
27 ± 7.6 27 ± 6.7
7
8a
8b
8c
9a
9b
4.5 ± 0.8
18 ± 1.8
17 ± 6.5
0.87 ± 0.21
1.4 ± 0.4 2.1 ± 0.3 1.6 ± 0.1 1.2 ± 0.1
1.5 ± 0.2 2.0 ± 0.1 1.6 ± 0.1 1.5 ± 0.3
<1
0.96 ± 0.21 1.2 ± 0.3

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[a]
9c
13 ± 4.0
22 ± 2.8
29 ± 5.3
27 ± 1.5 43 ± 10
18 ± 4.8
6.8 ± 0.6 4.0 ± 0.9 3.5 ± 0.2 5.9 ± 0.4
13
2.9 ± 0.4
4.1 ± 0.6
30 ± 0.5
4.4 ± 0.7
4.3 ± 1.1
3.9 ± 0.5
3.9 ± 0.9
5.7 ± 2.2
2.4 ± 0.2
22 ± 2.2
9.2 ± 1.7
4.9 ± 1.0
4.4 ± 0.8
2.8 ± 0.2
4.5 ± 0.2
39 ± 2.6
34 ± 3.7
14
16
5.4 ± 0.6 6.3 ± 0.5 1.3 ± 0.1
17
15 ± 0.1
14 ± 5.7
21 ± 4.8
18a
4.8 ± 1.8 7.2 ±2.0 4.2 ± 0.5
4.5 ± 1.7 5.1 ± 1.4 3.3 ± 0.6
18b
15 ± 2.6
18c
9.8 ± 3.0
6.3 ± 1.8
19a
1.4 ± 0.1 2.9 ± 0.4 2.1 ± 0.9 2.2 ± 0.8 0.45 ± 0.09 0.34 ± 0.13
1.1 ± 0.1 1.5 ± 0.2 1.3 ± 0.3 1.5 ± 0.2 0.71 ± 0.43 0.34 ± 0.21
<1
19b
<1
19c
2.6 ± 0.2 4.4 ± 0.6 3.3 ± 1.6 3.4 ± 0.7
2.6 ± 0.7
24 ± 4.5
2.1 ± 0.1
2.6 ± 0.8
42 ± 7.7
2.7 ± 0.2
1.4 ± 0.6
4.5 ± 1.0
5.0 ± 1.3
95 ± 8.0
>100
85 ± 8.9
Abiraterone
7.9 ± 0.5
10 ± 0.9
Galeterone 3.9 ± 1.3
5.3 ± 0.4 3.9 ± 0.5
[a] Not soluble under the assay conditions
Figure 5. GI50 range plot of tested compounds.
3.
Conclusions

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A straightforward methodology has been developed to access the hitherto unknown spiranic
steroids bearing oxazolidin-2-one and 2-aminooxazoline motifs at C-17, starting from
trans-androsterone and estrone. The key intermediate was an aminomethyl alcohol
functionality on C-17, which upon reaction with triphosgene, or with an alkyl/aryl
isothiocyanate, followed by a cyclodesulfurization reaction, furnished title heterocyclic
scaffolds.
Title compounds were tested as potential antiproliferative agents, and the order of activity
was found to be aminooxazoline > spirocarbamate > thiourea. The lead compounds,
bearing a spiranic aminooxazoline motif on estrone backbone, exhibited GI50 values in the
low micromolar to submicromolar range (0.34-1.5 µM), with particular increase in activity
against drug-resistant cell lines compared to steroidal chemotherapeutic agents (abiraterone
and galeterone).
4.
Experimental section
4.1. Materials and methods
4.1.1. General methods
Melting points were measured by the open capillary tube method on a Melt-temp apparatus
and were not corrected. Optical rotations were measured on a Jasco P-2000 polarimeter. IR
spectra were acquired on a Nicolet FT-IR 380 spectrophotometer (ꢀ̅
max, cm-1). NMR spectra
were recorded on a Bruker Ascend 500 MHz instrument. Chemical shifts are reported in
ppm (δ) and spectra were referenced to the residual protonated solvents: CDCl3 (7.26 and
77.16 ppm), CD3OD (3.31 and 49.0 ppm) and acetone-d6 (2.05 and 29.84 ppm) for 1H and
1
13C NMR respectively. Coupling constants (J) are expressed in Hertz (Hz). H and 13C
signals were assigned using 1D and 2D NMR experiments (DEPT, COSY, HSQC, HMBC
and NOESY). High resolution mass spectra were obtained by EI, ESI and FAB using a
Hewlett Packard 5989 A spectrometer coupled to a Hewlett Packard 5990 II gas
chromatographer, a Micromass AutoSpec-Q and a JEOL JMS-700 MStation mass

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spectrometers. The X-ray measurements were performed on a Xcalibur, Atlas, Gemini
diffractometer. Column chromatography was performed using Merck silica gel 60 (230-400
mesh). TLC was performed using aluminum pre-coated silica gel plates 60 F254; spots were
visualized by UV light and charring with 5% H2SO4 (aq), 10% vanillin in EtOH containing
1% of H2SO4 or with 0.1% ninhydrin in EtOH.
4.1.2. Antiproliferative activity
The in vitro antiproliferative activity was evaluated using the sulforhodamine B (SRB)
assay with slight modifications [58]. Briefly, pure compounds were initially dissolved in
DMSO at 400 times the desired final maximum test concentration. Cells were inoculated
onto 96-well plates in a volume of 100 µL per well at densities of 2 500 (A549, HBL-100,
HeLa and SW1573), 5 000 (T-47D and WiDr) or 10 000 (BJ-hTert) cells per well, based on
their doubling times. Control cells were exposed to an equivalent concentration of DMSO
(0.25% v/v, negative control). Each agent was tested in triplicate at different dilutions in the
range of 1–100 µM. The drug treatment was started on day 1 after plating. Drug incubation
times comprised 48 h, after which time cells were precipitated with 25 µL ice-cold 50%
(w/v) trichloroacetic acid and fixed for 60 min at 4 ºC. Then the SRB assay was performed.
The optical density (OD) of each well was measured at 530 nm, using BioTek’s
PowerWave XS microplate reader. Values were corrected for background OD from wells
containing only medium. The percentage growth (PG) was calculated in relation to
untreated control cells (C) at each of the drug concentration levels based on the difference
in OD at the start (T0) and end of drug exposure (T), according to NCI formulas Therefore,
if T is greater than or equal to T0, the calculation is 100 x [(T–T0)/(C–T0)]. If T is less than
T0, denoting cell killing, the calculation is 100 x [(T–T0)/(T0)]. The effect is defined as
percentage of growth, where 50% growth inhibition (GI50) represents the concentration at
which PG is +50. With these calculations, a PG value of 0 represents no difference from the
start of drug exposure, while negative PG values denote net cell death.
4.2 Chemistry

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4.2.1 (17S)-Spiro[5α-androstan-17,2'-oxiran]-3β-ol (2) [53]. To a solution of SMe3I (2.3 g,
11.27 mmol) in dry DMF (10 mL) was added NaH (1.0 g, 25.0 mmol, 60% in mineral oil) under Ar.
The mixture was stirred for 20 min at room temperature and cooled to 0 °C. After that, a solution of
trans-androsterone (1) (1.0 g, 3.44 mmol) in dry DMF (10 mL) was added. The mixture was stirred
at 0 °C for 15 min and then at room temperature for 6 h. After that, it was cooled to 0°C and MeOH
and H2O were carefully added, the corresponding mixture was diluted with EtOAc and the organic
layer was separated. Then it was washed with brine (4×10 mL), dried over anhydrous MgSO4,
filtered and concentrated to dryness. Column chromatography (9:1 hexane-EtOAc) gave 2 as a
white solid (0.74 g, 71%); mp 165-167 °C.
ꢂ
+4 (c 1.0, CHCl3). IR (cm-1): 3452 (OH), 2917-
ꢁ ꢃꢅꢆ
ꢄ
1
2850 (C-H aliphatic), 1033 (C-O). H NMR (500 MHz, CDCl3) δ 3.54 (m, 1H, H-3), 2.86 (d, 1H,
Jgem = 5.1 Hz, CH2a oxirane), 2.56 (d, 1H, Jgem = 5.1 Hz, CH2b oxirane), 1.94 (ddd, 1H, JH,H = 14.2
Hz, JH,H = 11.7 Hz, JH,H = 2.0 Hz, H-16a), 1.76 (m, 1H, H-2a), 1.75 (m, 1H, H-16b), 1.69 (m, 1H,
H-7a), 1.67 (m, 1H, H-15a), 1.66 (m, 1H, H-1a), 1.53 (m, 1H, H-4a), 1.52 (m, 1H, H-11a), 1.42 (m,
1H, H-8), 1.36 (m, 1H, H-2b), 1.35 (m, 1H, H-15b), 1.33 (m, 1H, H-12a), 1.25 (m, 3H, H-4b, H-6a,
H-6b), 1.23 (m, 1H, H-11b), 1.20 (m, 1H, H-14), 1.07 (m, 1H, H-5), 0.98 (m, 1H, H-12b), 0.92 (m,
1H, H-1b), 0.87 (m, 1H, H-7b), 0.83 (s, 3H, H-18), 0.78 (s, 3H, H-19), 0.62 (ddd, 1H, JH,H = 12.3
13
Hz, JH,H = 10.7 Hz, JH,H = 4.1 Hz, H-9). C NMR (125.7 MHz, CDCl3) δ 71.4 (C-3), 70.7 (C-17),
54.5 (C-9), 53.8 (CH2 oxirane), 53.0 (C-14), 45.0 (C-5), 40.3 (C-13), 38.3 (C-4), 37.1 (C-1), 35.8
(C-8), 35.7 (C-10), 34.1 (C-12), 31.6 (x2) (C-2, C-7), 29.2 (C-16), 28.7 (C-6), 23.7 (C-15), 20.7 (C-
11), 14.5 (C-18), 12.4 (C-19). EI-MS m/z 304 ([M]+, 14%); HREI-MS calcd for C20H32O2 ([M]+):
304.2397, found: 304.2406.
4.2.2 17α-Azidomethyl-5α-androstane-3β,17β-diol (3). To a solution of 2 (0.94 g, 3.09 mmol) in
dry DMF (28 mL) were added NaN3 (1.99 g, 30.61 mmol) and H3BO3 (0.8 g, 12.94 mmol) under
Ar. The mixture was refluxed for 2 h, poured into water and extracted with EtOAc (3×20 mL). The
organic layer was washed with brine (4×15 mL), dried over anhydrous MgSO4, filtered and
concentrated to dryness. Column chromatography (4:1 hexane-EtOAc) gave compound 3 as a white
solid (1.07 g, quant.); mp 175-177 °C.
ꢂ
-13 (c 0.2, CHCl3). IR (cm-1): 3411, 3327 (OH), 2940-
ꢁ ꢃꢅꢇ
ꢄ
2847 (C-H aliphatic), 2107 (N=N=N), 1318, 1036 (C-O, C-N). 1H NMR (500 MHz, CDCl3) δ 3.57
(m, 1H, H-3), 3.52 (dd, 1H, Jgem = 12.1 Hz, JH,H = 1.3 Hz, CH2aN3), 3.23 (d, 1H, Jgem = 12.1 Hz,
CH2bN3), 1.97 (ddd, 1H, JH,H = 14.7 Hz, JH,H = 9.7 Hz, JH,H = 7.0 Hz, H-16a), 1.79 (m, 1H, H-2a),
1.70 (m, 1H, H-1a), 1.66 (m, 1H, H-7a), 1.65 (m, 1H, H-16b), 1.59 (m, 1H, H-12a), 1.58 (m, 1H, H-
11a), 1.56 (m, 2H, H-15a, H-4a), 1.45 (m, 1H, H-8), 1.39 (m, 1H, H-2b), 1.30 (m, 1H, H-11b), 1.29
(m, 1H, H-15b), 1.28 (m, 1H, H-4b), 1.27 (m, 2H, H-6), 1.15 (m, 1H, H-12b), 1.10 (m, 1H, H-14),

ACCEPTED MANUSCRIPT
1.08 (m, 1H, H-5), 0.95 (td, 1H, JH,H = 13.5 Hz, JH,H = 3.8 Hz, H-1b), 0.87 (s, 3H, H-18), 0.83 (m,
1H, H-7b), 0.81 (s, 3H, H-19), 0.59 (ddd, 1H, JH,H = 12.3 Hz, JH,H = 10.8 Hz, JH,H = 4.0 Hz, H-9).
13C NMR (125.7 MHz, CDCl3) δ 83.6 (C-17), 71.3 (C-3), 58.5 (CH2N3), 54.3 (C-9), 51.4 (C-14),
46.1 (C-13), 45.0 (C-5), 38.2 (C-4), 37.1 (C-1), 36.3 (C-8), 35.7 (C-10), 34.8 (C-16), 32.1 (C-12),
31.9 (C-7), 31.6 (C-2), 28.6 (C-6), 23.7 (C-15), 20.9 (C-11), 14.3 (C-18), 12.4 (C-19). HRFAB-MS
calcd for C20H32N3O2 ([M-H]+): 346.2489, found: 346.2491.
4.2.3 17α-Aminomethyl-5α-androstane-3β,17β-diol (4). A solution of 3 (1.07 g, 3.08 mmol) in a
7:3 MeOH-CH2Cl2 mixture (60 mL) was hydrogenated over 20% Pd(OH)2/C (0.64 g) at 1 atm and
room temperature for 3 h. The catalyst was removed by filtration through a pad of Celite® and the
filtrate was concentrated to dryness. The residue was washed with cooled EtOAc to give 4 as a
+11 (c 0.2, MeOH). IR (cm-1): 3288 (OH),
ꢁ ꢃꢅꢇ
white solid (0.97 g, 98%); mp 260 °C (dec.).
ꢂ
ꢄ
1
2921-2862 (C-H aliphatic), 1612 (NH), 1037 (C-O, C-N). H NMR (500 MHz, MeOD-CDCl3) δ
3.51 (m, 1H, H-3), 2.97 (dd, 1H, Jgem = 12.6 Hz, JH,H = 1.2 Hz, CH2aNH2), 2.92 (d, 1H, Jgem = 12.6
Hz, CH2bNH2), 1.90 (ddd, 1H, JH,H = 16.1 Hz, JH,H = 9.5 Hz, JH,H = 6.6 Hz, H-16a), 1.76 (m, 1H, H-
2a), 1.71 (m, 1H, H-1a), 1.70 (m, 1H, H-7a), 1.69 (m, 1H, H-16b), 1.66 (m, 1H, H-15a), 1.62 (m,
1H, H-11a), 1.61 (m, 1H, H-12a), 1.52 (m, 1H, H-4a), 1.49 (m, 1H, H-8), 1.39 (m, 1H, H-2b), 1.37
(m, 1H, H-15b), 1.35 (m, 1H, H-11b), 1.28 (m, 2H, H-6), 1.27 (m, 1H, H-4b), 1.14 (m, 1H, H-12b),
1.10 (m, 1H, H-5), 0.97 (td, 1H, JH,H = 14.0 Hz, JH,H = 4.1 Hz, H-1b), 0.89 (s, 3H, H-18), 0.84 (s,
3H, H-19), 0.65 (td, 1H, JH,H = 11.7 Hz, JH,H = 3.9 Hz, H-9). 13C NMR (125.7 MHz, MeOD-CDCl3)
δ 81.6 (C-17), 71.5 (C-3), 55.2 (C-9), 52.0 (C-14), 47.0 (C-13), 46.4 (CH2NH2), 45.9 (C-5), 38.5
(C-4), 37.9 (C-1), 37.3 (C-8), 36.4 (C-10), 33.3 (C-16), 32.6 (C-7), 32.4 (C-12), 31.7 (C-2), 29.5 (C-
6), 24.3 (C-15), 21.7 (C-11), 14.5 (C-18), 12.7 (C-19). FAB-MS m/z 322 ([M+H]+, 32%); HRFAB-
MS calcd for C20H36NO2 ([M+H]+): 322.2741, found: 322.2749.
4.2.4 (17S)-2’-Oxo-spiro[3
β
-hydroxy-5
α-androstan-17,5'-oxazolidine] (6) and methyl N-
[(3 ,17 -dihydroxy-5 -androstan-17α
β
β
α
-yl)methyl]carbamate (7). To a solution of 4 (0.1 g, 0.31
mmol) in a MeOH-CH2Cl2 mixture (2:1, 15 mL) were added saturated aqueous NaHCO3 (5 mL)
and triphosgene (37 mg, 0.12 mmol) at 0 °C. The suspension was vigorously stirred for 25 min at 0
°C, filtered and the solvent was evaporated. Column chromatography (1:1 hexane-EtOAc) gave 6
ꢁ ꢃꢅꢅ
(56 mg, 52%) and 7 (29 mg, 25%) as white solids. Data for compound 6: mp 220 °C (dec.).
ꢂ
-
ꢄ
28 (c 0.4, MeOH). IR (cm-1): 3427 (NH), 3200 (OH), 2925-2854 (C-H aliphatic), 1729 (C=O),
1284-1045 (C-O, C-N). 1H NMR (500 MHz, MeOD-CDCl3) δ 3.57 (d, 1H, J4’a,4’b = 9.1 Hz, H-4’a),

ACCEPTED MANUSCRIPT
3.42 (m, 1H, H-3), 3.11 (d, 1H, J4’b,4’a = 9.1 Hz, H-4’b), 2.14 (ddd, 1H, J16a,16b = 14.9 Hz, J16a,H
=
12.8 Hz, J16a,H = 3.7 Hz, H-16a), 1.73 (ddd, 1H, J16b,16a = 14.9 Hz, J16b,H = 9.3 Hz, J16b,H = 5.8 Hz, H-
16b), 1.65 (m, 1H, H-2a), 1.59 (m, 1H, H-1a), 1.57 (m, 1H, H-7a), 1.52 (m, 1H, H-15a), 1.49 (m,
1H, H-11a), 1.47 (m, 1H, H-12a), 1.42 (m, 1H, H-4a), 1.32 (m, 1H, H-8), 1.27 (m, 1H, H-2b), 1.23
(m, 1H, H-15b), 1.18 (m, 1H, H-11b), 1.18 (m, 1H, H-12b), 1.16 (m, 2H, H-6a, H-6b), 1.15 (m, 1H,
H-4b), 0.97 (m, 1H, H-5), 0.84 (m, 1H, H-14), 0.83 (m, 1H, H-1b), 0.80 (s, 3H, H-18), 0.74 (m, 1H,
13
H-7b), 0.69 (s, 3H, H-19), 0.49 (m, 1H, H-9). C NMR (125.7 MHz, MeOD-CDCl3) δ 160.4 (C-
2’), 92.8 (C-17), 70.6 (C-3), 53.9 (C-9), 49.4 (C-4’), 49.3 (C-14), 45.3 (C-13), 44.7 (C-5), 37.5 (C-
4), 36.9 (C-1), 35.7 (C-8), 35.4 (C-10), 35.3 (C-16), 31.4 (C-7), 31.0 (C-12), 30.8 (C-2), 28.3 (C-6),
22.4 (C-15), 20.4 (C-11), 14.2 (C-18), 12.1 (C-19). FAB-MS m/z 348 ([M+H]+, 17%); HRFAB-MS
calcd for C21H34NO3 ([M+H]+): 348.2533, found: 348.2541. Data for compound 7: mp 75-77 °C.
-1
ꢁ ꢃꢅꢆ
ꢂ
-3 (c 0.1, CHCl3). IR (cm ): 3404 (OH), 2920-2851 (C-H aliphatic), 1702 (C=O), 1038 (C-O,
ꢄ
1
C-N). H NMR (500 MHz, CDCl3) δ 5.19 (m, 1H, NH), 3.66 (s, 3H, OCH3), 3.58 (m, 1H, H-3),
3.31 (dd, 1H, Jgem = 13.4 Hz, J = 8.1 Hz, CH2aNH), 3.18 (d, 1H, Jgem = 13.4 Hz, CH2bNH2), 1.90
(ddd, 1H, JH,H = 14.1 Hz, JH,H = 9.9 Hz, JH,H = 6.8 Hz, H-16a), 1.79 (m, 1H, H-2a), 1.68 (td, 1H, JH,H
= 13.2 Hz, JH,H = 3.4 Hz, H-1a), 1.64 (m, 1H, 7a), 1.56 (m, 1H, H-15a), 1.56 (m, 1H, H-11a), 1.54
(m, 1H, H-4a), 1.52 (m, 1H, H-12a), 1.48 (m, 1H, H-16b), 1.42 (m, 1H, H-8), 1.38 (m, 1H, H-2b),
1.28 (m, 1H, H-11b), 1.27 (m, 1H, H-4b), 1.25 (m, 2H, H-6a, H-6b), 1.23 (m, 1H, H-15b), 1.22 (m,
1H, H-12b), 1.21 (m, 1H, H-14), 1.08 (m, 1H, H-5), 0.95 (td, 1H, JH,H = 13.2 Hz, JH,H = 3.6 Hz, H-
1b), 0.84 (m, 1H, H-7b), 0.84 (s, 3H, H-18), 0.80 (s, 3H, H-19), 0.61 (td, 1H, JH,H = 11.2 Hz, JH,H
=
13
4.1 Hz, H-9). C NMR (125.7 MHz, CDCl3) δ 158.1 (C=O), 83.6 (C-17), 71.3 (C-3), 54.2 (C-9),
52.4 (OCH3), 50.8 (C-14), 47.1 (CH2NH), 45.8 (C-13), 44.9 (C-5), 38.2 (C-4), 37.1 (C-1), 36.4 (C-
8), 35.6 (C-10), 34.2 (C-16), 31.8 (C-7), 31.7 (C-12), 31.5 (C-2), 28.7 (C-6), 23.6 (C-15), 20.9 (C-
11), 14.2 (C-18), 12.4 (C-19). FAB-MS m/z 380 ([M+H]+, 13%); HRFAB-MS calcd for C22H38NO4
([M+H]+): 380.2796, found: 380.2795.
4.2.5 General procedure for the synthesis of steroidal thioureas 8
To a solution of 4 (0.2 g, 0.62 mmol) in MeOH (10 mL) were added the corresponding
isothiocyanate (0.75 mmol) and Et3N (50 µL, 0.36 mmol). The corresponding mixture was stirred
during the time and at the temperature indicated in each case and the solvent was evaporated to
dryness. The residue was purified by flash column chromatography to give compounds 8a-c as
white solids.

ACCEPTED MANUSCRIPT
4.2.5.1 17α-[(3’-Butylthioureido)methyl]-5α-androstane-3β,17β-diol (8a). Butyl isothiocyanate
was used (90 µL) and the reaction proceeded for 3 days at 45 °C. Column chromatography (3:2
-1
ꢁ ꢃꢅꢆ
hexane-EtOAc) afforded 8a (0.20 g, 74%); mp 134-136 °C.
ꢂ
-7 (c 1.0, MeOH). IR (cm ):
ꢄ
3279 (OH), 2929-2857 (C-H aliphatic), 1551 (C-NH), 1522, 1348, 1037 (C-O, C-N). 1H NMR (500
MHz, MeOD) δ 3.96 (brs, 1H, CH2aNH), 3.52 (m, 1H, H-3), 3.48 (m, 2H, NH-CH2-CH2), 3.34 (brs,
1H, CH2bNH), 1.89 (m, 1H, H-16a), 1.76 (m, 1H, H-2a), 1.74 (m, 1H, H-1a), 1.71 (m, 1H, H-7a),
1.61 (m, 1H, H-11a), 1.60 (m, 1H, H-15a), 1.59 (m, 1H, H-12a), 1.56 (m, 3H, NH-CH2-CH2, H-
16b), 1.53 (m, 1H, H-4a), 1.49 (m, 1H, H-8), 1.40 (m, 1H, H-2b), 1.38 (m, 3H, CH2-CH3, H-11b),
1.32 (m, 1H, H-14), 1.30 (m, 3H, H-6, H-12b), 1.29 (m, 2H, H-4b, H-15b), 1.14 (m, 1H, H-5), 1.02
(m, 1H, H-1b), 0.95 (t, 3H, JH,H = 7.4 Hz, CH2-CH3), 0.94 (m, 1H, H-7b), 0.87 (s, 3H, H-18), 0.86
13
(s, 3H, H-19), 0.70 (td, 1H, JH,H = 11.7 Hz, JH,H = 3.5 Hz, H-9). C NMR (125.7 MHz, MeOD) δ
184.3 (C=S), 84.4 (C-17), 71.8 (C-3), 55.7 (C-9), 52.2 (C-14), 51.6 (CH2NH), 47.2 (C-13), 46.2 (C-
5), 45.1 (NH-CH2-CH2), 38.9 (C-4), 38.3 (C-1), 37.7 (C-8), 36.7 (C-10), 33.9 (C-16), 33.0 (C-7),
32.7 (C-12), 32.4 (NH-CH2-CH2), 32.1 (C-2), 29.9 (C-6), 24.6 (C-15), 22.0 (C-11), 21.1 (CH2-
CH3), 14.9 (C-18), 14.2 (CH2-CH3), 12.8 (C-19). FAB-MS m/z 437 ([M+H]+, 75%); HRFAB-MS
calcd for C25H45N2O2S ([M+H]+): 437.3196, found: 437.3210.
4.2.5.2 17
α
-[(3’-Cyclohexylthioureido)methyl]-5
α
-androstane-3
β
,17β
-diol
(8b).
Cyclohexyl
isothiocyanate was used (0.11 mL) and the reaction proceeded for 56 h at 45 °C. Column
ꢁ ꢃꢅꢆ
-7 (c 0.9,
ꢄ
chromatography (3:2 hexane-EtOAc) afforded 8b (0.2 g, 70%); mp 165-167 ºC.
ꢂ
MeOH). IR (cm-1): 3253 (OH), 2923-2854 (C-H aliphatic), 1554 (C-NH), 1523, 1348, 1024 (C-O,
1
C-N). H NMR (500 MHz, MeOD) δ 4.06 (brs, 1H, H-1’’), 3.98 (brs, 1H, CH2aNH), 3.52 (m, 1H,
H-3), 3.33 (m, 1H, CH2bNH), 1.97 (m, 2H, H-2’’a, H-6’’a), 1.89 (m, 1H, H-16a), 1.76 (m, 1H, H-
2a), 1.73 (m, 2H, H-3’’a, H-5’’a), 1.72 (m, 1H, H-1a), 1.70 (m, 1H, H-7a), 1.62 (m, 1H, H-4’’a),
1.60 (m, 1H, H-11a), 1.59 (m, 1H, H-15a), 1.57 (m, 1H, H-12a), 1.53 (m, 1H, H-16b), 1.52 (m, 1H,
H-4a), 1.47 (m, 1H, H-8), 1.39 (m, 1H, H-2b), 1.37 (m, 2H, H-3’’b, H-5’’b), 1.33 (m, 1H, H-11b),
1.30 (m, 1H, H-14), 1.29 (m, 1H, H-12b), 1.28 (m, 3H, H-6, H-15b), 1.27 (m, 1H, H-4b), 1.20 (m,
3H, H-2’’b, H-6’’b, H-4’’b), 1.12 (m, 1H, H-5), 0.99 (m, 1H, H-1b), 0.92 (m, 1H, H-7b), 0.87 (s,
13
3H, H-18), 0.85 (s, 3H, H-19), 0.69 (td, 1H, JH,H = 11.6 Hz, JH,H = 3.5 Hz, H-9). C NMR (125.7
MHz, MeOD) δ 183.1 (C=S), 84.3 (C-17), 71.6 (C-3), 55.4 (C-9), 53.7 (C-1’’), 51.9 (C-14), 51.4
(CH2NH), 47.0 (C-13), 46.0 (C-5), 38.7 (C-4), 38.1 (C-1), 37.5 (C-8), 36.5 (C-10), 33.7 (x2) (C-2’’,
C-6’’), 33.7 (C-16), 32.8 (C-7), 32.5 (C-12), 31.9 (C-2), 29.7 (C-6), 26.5 (C-4’’), 25.9 (x2) (C-3’’,
C-5’’), 24.5 (C-15), 21.8 (C-11), 14.7 (C-18), 12.7 (C-19). HRFAB-MS calcd for C27H47N2O2S
([M+H]+): 463.3353, found: 463.3386.

ACCEPTED MANUSCRIPT
4.2.5.3 17α-[(3’-Phenylthioureido)methyl]-5α-androstane-3β,17β-diol (8c). Phenyl isothiocyanate
was used (90 µL) and the reaction proceeded for 30 min at room temperature. Column
ꢁ ꢃꢅꢆ
-11 (c 0.5,
ꢄ
chromatography (7:3 hexane-EtOAc) afforded 8c (0.23 g, 81%); mp 280 °C (dec.).
ꢂ
MeOH). IR (cm-1): 3204 (OH), 2920-2840 (C-H aliphatic), 1598 (C=C), 1528 (C-NH), 1523, 1026
1
(C-O, C-N). H NMR (500 MHz, MeOD) δ 7.36 (m, 4H, Ar-Ho, Ar-Hm), 7.22 (m, 1H, Ar-Hp),
4.04 (d, 1H, Jgem = 13.2 Hz, CH2aNH), 3.52 (m, 1H, H-3), 3.40 (d, 1H, Jgem = 13.2 Hz, CH2bNH),
1.90 (m, 1H, H-16a), 1.77 (m, 1H, H-2a), 1.73 (m, 1H, H-1a), 1.72 (m, 1H, H-7a), 1.63 (m, 1H, H-
15a), 1.62 (m, 1H, H-11a), 1.58 (m, 1H, H-16b), 1.56 (m, 1H, H-12a), 1.53 (m, 1H, H-4a), 1.50 (m,
1H, H-8), 1.40 (m, 1H, H-2b), 1.35 (m, 1H, H-11b), 1.33 (m, 1H, H-14), 1.30 (m, 4H, H-6a, H-6b,
H-15b, H-12b), 1.28 (m, 1H, H-4b), 1.15 (m, 1H, H-5), 1.01 (m, 1H, H-1b), 0.95 (m, 1H, H-7b),
0.85 (s, 6H, H-18, H-19), 0.70 (td, 1H, JH,H = 11.3 Hz, JH,H = 3.2 Hz, H-9). 13C NMR (125.7 MHz,
MeOD) δ 182.2 (C=S), 139.3 (Ar-Cipso), 130.5 (x2) (Ar-C), 127.0 (Ar-Cp), 125.5 (x2) (Ar-C),
84.3 (C-17), 71.8 (C-3), 55.7 (C-9), 52.2 (CH2NH), 52.2 (C-14), 47.1 (C-13), 46.2 (C-5), 38.9 (C-
4), 38.2 (C-1), 37.7 (C-8), 36.7 (C-10), 34.1 (C-16), 33.0 (C-7), 32.9 (C-12), 32.1 (C-2), 29.9 (C-6),
24.6 (C-15), 22.0 (C-11), 14.8 (C-18), 12.7 (C-19). HRFAB-MS calcd for C27H41N2O2S ([M+H]+):
457.2883, found: 457.2892.
4.2.6 General procedure for the synthesis of steroidal spiranic isoureas 9
To a solution of the corresponding thiourea 8 (0.25 mmol) in dry THF (10 mL) was added yellow
HgO (4.50 mmol for compounds 8a,b and 2.29 mmol for compound 8c) under Ar. The mixture was
stirred at room temperature in the dark during the time indicated in each case, filtered over a pad of
Celite® and evaporated to dryness. The residue was washed with hexane or purified by flash
column chromatography, as indicated in each case, to give compounds 9a-c as white foams.
4.2.6.1 (17S)-2’-n-Butylamino-spiro[3β-hydroxy-5α-androstan-17,5'-oxazoline] (9a). Thiourea 8a
was used (0.11 g) and the reaction proceeded for 6 days to give 9a (0.10 g, 99%). IR (cm-1): 3226
1
(OH), 2928-2856 (C-H aliphatic), 1650 (C=N), 1046 (C-O, C-N). H NMR (500 MHz, CDCl3) δ
4.05 (brs, 1H, NH), 3.81 (d, 1H, J4’a,4’b = 12.2 Hz, H-4’a), 3.51 (m, 1H, H-3), 3.28 (d, 1H, J4’b,4’a
=
12.2 Hz, H-4’b), 3.10 (m, 2H, H-1’’), 2.14 (ddd, 1H, JH,H = 14.4 Hz, JH,H = 12.5 Hz, JH,H = 3.6 Hz,
H-16a), 1.74 (m, 2H, H-16b, H-2a), 1.65 (m, 1H, H-1a), 1.63 (m, 1H, H-7a), 1.55 (m, 1H, H-15a),
1.53 (m, 1H, H-11a), 1.50 (m, 1H, H-4a), 1.45 (m, 2H, H-2’’), 1.39 (m, 1H, H-12a), 1.34 (m, 1H,
H-2b), 1.33 (m, 1H, H-8), 1.29 (m, 2H, H-3’’), 1.28 (m, 1H, H-12b), 1.23 (m, 2H, H-15b, H-4b),
1.22 (m, 3H, H-11b, H-6), 1.04 (m, 1H, H-5), 0.91 (m, 1H, H-1b), 0.90 (m, 1H, H-14), 0.86 (t, 3H,

ACCEPTED MANUSCRIPT
J4’’,3’’ = 7.4 Hz, H-4’’), 0.82 (s, 3H, H-18), 0.81 (m, 1H, H-7b), 0.76 (s, 3H, H-19), 0.56 (td, 1H, JH,H
= 11.9 Hz, JH,H = 4.0 Hz, H-9). 13C NMR (125.7 MHz, CDCl3) δ 160.8 (C-2’), 96.0 (C-17), 70.8 (C-
3), 60.5 (C-4’), 54.2 (C-9), 49.6 (C-14), 45.0 (C-13), 45.0 (C-5), 42.4 (C-1’’), 38.1 (C-4), 37.2 (C-
1), 35.9 (C-8), 35.6 (C-10), 35.2 (C-16), 32.0 (C-2’’), 31.6 (C-7), 31.5 (C-2), 31.2 (C-12), 28.5 (C-
6), 22.6 (C-15), 20.6 (C-11), 20.0 (C-3’’), 14.6 (C-18), 13.8 (C-4’’), 12.4 (C-19). HRFAB-MS calcd
for C25H43N2O2 ([M+H]+): 403.3319, found: 403.3313.
4.2.6.2. (17S)-2’-Cyclohexylamino-spiro[3
β
-hydroxy-5
α
-androstan-17,5'-oxazoline] (9b). Thiourea
ꢁ ꢃꢅꢆ
-21 (c 1.0,
ꢄ
8b was used (0.12 g) and the reaction proceeded for 3 days to give 9b (0.1 g, 93%).
ꢂ
CHCl3). IR (cm-1): 3286 (OH), 2924-2853 (C-H aliphatic), 1654 (C=N), 1044 (C-O, C-N). 1H NMR
(500 MHz, CDCl3) δ 3.84 (d, 1H, J4’a,4’b = 12.4 Hz, H-4’a), 3.56 (m, 1H, H-3), 3.35 (m, 1H, H-1’’),
3.32 (d, 1H, J4’b,4’a = 12.4 Hz, H-4’b), 2.17 (ddd, 1H, JH,H = 14.0 Hz, JH,H = 12.4 Hz, JH,H = 3.8 Hz,
H-16a), 1.96 (m, 2H, H-2’’a, H-6’’a), 1.78 (m, 2H, H-2a, H-16b), 1.69 (m, 1H, H-1a), 1.68 (m, 2H,
H-3’’a, H-5’’a), 1.67 (m, 1H, H-7a), 1.58 (m, 1H, H-15a), 1.57 (m, 1H, H-4’’a), 1.56 (m, 1H, H-
11a), 1.55 (m, 1H, H-4a), 1.43 (m, 1H, H-12a), 1.38 (m, 1H, H-2b), 1.36 (m, 1H, H-8), 1.34 (m, 1H,
H-12b), 1.31 (m, 2H, H-3’’b, H-5’’b), 1.27 (m, 2H, H-15b, H-4b), 1.25 (m, 3H, H-6a, H-6b, H-
11b), 1.13 (m, 1H, H-4’’b), 1.10 (m, 2H, H-2’’b, H-6’’b), 1.08 (m, 1H, H-5), 0.95 (m, 1H, H-1b),
0.94 (m, 1H, H-14), 0.85 (s, 3H, H-18), 0.84 (m, 1H, H-7b), 0.79 (s, 3H, H-19), 0.60 (td, 1H, JH,H
=
13
11.8 Hz, JH,H = 4.1 Hz, H-9). C NMR (125.7 MHz, CDCl3) δ 159.8 (C-2’), 95.8 (C-17), 71.2 (C-
3), 60.9 (C-4’), 54.2 (C-9), 51.5 (C-1’’), 49.6 (C-14), 45.1 (C-13), 45.0 (C-5), 38.2 (C-4), 37.2 (C-
1), 36.0 (C-8), 35.6 (C-10), 35.3 (C-16), 33.8 (C-2’’ or C-6’’), 33.7 (C-2’’ or C-6’’), 31.6 (C-7),
31.6 (C-2), 31.2 (C-12), 28.6 (C-6), 25.7 (C-4’’), 25.0 (x2) (C-3’’, C-5’’), 22.7 (C-15), 20.7 (C-11),
14.7 (C-18), 12.5 (C-19). FAB-MS m/z 429 ([M+H]+, 100%); HRFAB-MS calcd for C27H45N2O2
([M+H]+): 429.3476, found: 429.3508.
4.2.6.3. (17S)-2’-Phenylamino-spiro[3β-hydroxy-5α-androstan-17,5'-oxazoline] (9c). Thiourea 8c
was used (0.12 g) and the reaction proceeded for 19 h. Column chromatography (CH2Cl2
→ 20:1
-1
ꢁ ꢃꢅꢆ
CH2Cl2-Isopropanol) afforded 9c (0.10 g, 97%).
ꢂ
+2 (c 1.0, CHCl3). IR (cm ): 3282 (OH),
ꢄ
2920-2853 (C-H aliphatic), 1658 (C=N), 1594-1447 (C=C), 1040 (C-O, C-N), 748-691 (=C-H). 1H
NMR (500 MHz, CDCl3) δ 7.27 (m, 4H, Ar-Ho, Ar-Hm), 7.0 (m, 1H, Ar-Hp), 4.85 (brs, 1H, NH),
3.91 (d, 1H, J4’a,4’b = 11.1 Hz, H-4’a), 3.59 (m, 1H, H-3), 3.40 (d, 1H, J4’b,4’a = 11.1 Hz, H-4’b), 2.29
(ddd, 1H, J16a,16b = 14.6 Hz, J16a,H = 12.4 Hz, J16a,H = 3.7 Hz, H-16a), 1.88 (ddd, 1H, J16b,16a = 14.6
Hz, J16b,H = 9.5 Hz, J16b,H = 5.8 Hz, H-16b), 1.80 (m, 1H, H-2a), 1.72 (m, 1H, H-1a), 1.71 (m, 1H,
H-7a), 1.65 (m, 1H, H-15a), 1.60 (m, 1H, H-11a), 1.57 (m, 2H, H-12a, H-4a), 1.43 (m, 1H, H-8),

ACCEPTED MANUSCRIPT
1.41 (m, 1H, H-2b), 1.36 (m, 1H, H-12b), 1.35 (m, 1H, H-15b), 1.31 (m, 1H, H-11b), 1.30 (m, 1H,
H-4b), 1.28 (m, 2H, H-6), 1.10 (m, 1H, H-5), 0.99 (m, 1H, H-14), 0.97 (m, 1H, H-1b), 0.96 (s, 3H,
H-18), 0.87 (qd, 1H, JH,H = 12.1 Hz, JH,H = 5.3 Hz, H-7b), 0.82 (s, 3H, H-19), 0.63 (td, 1H, JH,H
=
11.6 Hz, JH,H = 3.9 Hz, H-9). 13C NMR (125.7 MHz, CDCl3) δ 157.1 (C-2’), 129.1 (x3) (Ar-C),
122.7 (x2) (Ar-C), 120.0 (Ar-C), 96.5 (C-17), 71.2 (C-3), 56.4 (C-4’), 54.1 (C-9), 49.9 (C-14), 45.4
(C-13), 44.9 (C-5), 38.2 (C-4), 37.1 (C-1), 36.0 (C-8), 35.6 (C-10), 35.4 (C-16), 31.6 (C-7), 31.5 (C-
2), 31.4 (C-12), 28.5 (C-6), 22.8 (C-15), 20.6 (C-11), 14.8 (C-18), 12.5 (C-19). HRFAB-MS calcd
for C27H39N2O2 ([M+H]+): 423.3006, found: 423.3004.
4.2.7. (17S)-Spiro[3-benzyloxyestra-1,3,5(10)-trien-17,2'-oxirane] (12). SMe3I (0.63 g, 3.09 mmol)
and KOtBu (0.47 g, 4.24 mmol) were added to a solution of 11 (0.69 g, 1.91 mmol) in dry DMF (16
mL) under Ar. The reaction was stirred at 0 °C for 10 min and then at room temperature for 45 min.
After this time, cold water was added and the product was extracted with diethyl ether. The organic
phase was washed with brine (3×20 mL), dried over anhydrous MgSO4, filtered and concentrated to
dryness. Column chromatography (19:1 hexane-EtOAc) gave compound 12 as white crystals (0.68
-1
ꢁ ꢃꢅꢇ
g, 95%); mp 154-156 °C (lit. 155-157 °C) [54].
ꢂ
+45 (c 0.4, CHCl3). IR (cm ): 3027 (C-H
ꢄ
1
aromatic), 2919-2862 (C-H aliphatic), 1602-1495 (C=C), 1233-1016 (C-O). H NMR (500 MHz,
CDCl3) δ 7.45 (m, 2H, Ar-Ho), 7.40 (m , 2H, Ar-Hm), 7.34 (m, 1H, Ar-Hp), 7.21 (d, 1H, J1,2 = 8.5
Hz, H-1), 6.80 (dd, 1H, J2,1 = 8.5 Hz, J2,4 = 2.7 Hz, H-2), 6.75 (d, 1H, J4,2 = 2.7 Hz, H-4), 5.05 (s,
2H, CH2-Ph), 2.98 (d, 1H, Jgem = 5.1 Hz, CH2a oxirane), 2.90 (m, 2H, H-6), 2.66 (d, 1H, Jgem = 5.1
Hz, CH2b oxirane), 2.32 (m, 1H, H-11a), 2.24 (m, 1H, H-9), 2.06 (m, 1H, H-16a), 1.96 (m, 1H, H-
7a), 1.87 (m, 1H, H-16b), 1.86 (m, 1H, H-15a), 1.55 (m, 1H, H-12a), 1.53 (m, 1H, H-8), 1.53 (m,
1H, H-15b), 1.52 (m, 1H, H-14), 1.50 (m, 1H, H-11b), 1.40 (m, 1H, H-7b), 1.28 (m, 1H, H-12b),
13
0.94 (s, 3H, H-18). C NMR (125.7 MHz, CDCl3) δ 156.9 (C-3), 138.1 (C-5), 137.4 (Ar-Cipso),
132.8 (C-10), 128.7 (x2) (Ar-Cm), 128.0 (Ar-Cp), 127.6 (x2) (Ar-Co), 126.4 (C-1), 114.9 (C-4),
112.4 (C-2), 70.7 (C-17), 70.0 (CH2-Ph), 53.8 (CH2 oxirane), 51.9 (C-14), 44.0 (C-9), 40.5 (C-13),
39.0 (C-8), 34.0 (C-12), 29.9 (C-6), 29.2 (C-16), 27.3 (C-7), 26.1 (C-11), 23.4 (C-15), 14.4 (C-18).
HRESI-MS calcd for C26H31O2 ([M+H]+): 375.2319, found: 375.2311.
4.2.8. 17α-Azidomethyl-3-benzyloxyestra-1,3,5(10)-trien-17β-ol (13). To a solution of 12 (1.84 g,
4.91 mmol) in dry DMF (50 mL) were added NaN3 (3.19 g, 49.07 mmol) and H3BO3 (1.28 g, 20.70
mmol) under Ar. The mixture was refluxed for 4 h, poured into water and extracted with diethyl
ether (4×25 mL). The organic phase was washed with brine (3×15 mL), dried over anhydrous
MgSO4, filtered and concentrated to dryness. Column chromatography (19:1 hexane-EtOAc) gave

ACCEPTED MANUSCRIPT
ꢁ ꢃꢅꢇ
+26 (c 1.0,
ꢄ
compound 13 as white crystals (1.95 g, 95%); mp 92-93 °C (lit 96-98 °C) [54].
ꢂ
CHCl3). IR (cm-1): 3434 (OH), 3020 (C-H aromatic), 2928-2865 (C-H aliphatic), 2093 (N=N=N),
1
1609-1450 (C=C), 1237-1018 (C-O, C-N). H NMR (500 MHz, CDCl3) δ 7.46 (m, 2H, Ar-Ho),
7.41 (m, 2H, Ar-Hm), 7.35 (m, 1H, Ar-Hp), 7.22 (d, 1H, J1,2 = 8.6 Hz, H-1), 6.82 (dd, 1H, J2,1 = 8.6
Hz, J2,4 = 2.6 Hz, H-2), 6.75 (d, 1H, J4,2 = 2.6 Hz, H-4), 5.06 (s, 2H, CH2-Ph), 3.62 (d, 1H, Jgem
=
12.1 Hz, CH2aN3), 3.32 (d, 1H, Jgem = 12.1 Hz, CH2bN3), 2.89 (m, 2H, H-6a, H-6b), 2.35 (m, 1H, H-
11a), 2.19 (m, 1H, H-9), 2.08 (ddd, 1H, JH,H = 15.8 Hz, JH,H = 9.2 Hz, JH,H = 5.8 Hz, H-16a), 1.91
(m, 1H, H-7a), 1.78 (dt, 1H, JH,H = 12.5 Hz, JH,H = 3.1 Hz, H-12a), 1.74 (m, 1H, H-16b), 1.72 (m,
1H, H-15a), 1.55 (m, 1H, H-11b), 1.53 (m, 1H, H-8), 1.45 (m, 1H, H-15b), 1.42 (m, 1H, H-12b),
1.38 (m, 1H, H-14), 1.35 (m, 1H, H-7b), 0.95 (s, 3H, H-18). 13C NMR (125.7 MHz, CDCl3) δ 156.8
(C-3), 138.0 (C-5), 137.3 (Ar-Cipso), 132.6 (C-10), 128.6 (x2) (Ar-Cm), 128.0 (Ar-Cp), 127.5 (x2)
(Ar-Co), 126.4 (C-1), 114.8 (C-4), 112.3 (C-2), 83.6 (C-17), 70.0 (CH2Ph), 58.4 (CH2N3), 50.3 (C-
14), 46.2 (C-13), 43.8 (C-9), 39.4 (C-8), 34.8 (C-16), 32.0 (C-12), 29.8 (C-6), 27.5 (C-7), 26.2 (C-
11), 23.3 (C-15), 14.2 (C-18). HRESI-MS calcd for C26H31N3NaO2 ([M+Na]+): 440.2308, found:
440.2298.
4.2.9. 17α-Aminomethylestra-1,3,5(10)-triene-3,17β-diol (14). A solution of 13 (0.5 g, 1.20 mmol)
in a 7:3 MeOH-CH2Cl2 mixture (30 mL) was hydrogenated over 10% Pd/C (0.25 g) at 1 atm and
room temperature for 25 h. The catalyst was removed by filtration through a pad of Celite® and the
filtrate was concentrated to dryness, dissolved with MeOH and precipitated with CH2Cl2. The
compound was filtered and washed with cooled diethyl ether to give 14 as a white solid (0.31 g,
-1
ꢁ ꢃꢅꢇ
86%); mp 254 °C (dec.) (lit. 242-243 °C) [54].
ꢂ
+48 (c 0.3, MeOH). IR (cm ): 3471 (NH),
ꢄ
3200 (OH), 3060 (C-H aromatic), 2920-2860 (C-H aliphatic), 1614 (NH), 1600-1494 (C=C), 1241-
1018 (C-O, C-N). 1H NMR (500 MHz, MeOD) δ 7.07 (d, 1H, J1,2 = 8.4 Hz, H-1), 6.54 (dd, 1H, J2,1
= 8.4 Hz, J2,4 = 2.6 Hz, H-2), 6.48 (d, 1H, J4,2 = 2.6 Hz, H-4), 3.04 (s, 2H, CH2NH2), 2.78 (m, 2H,
H-6a, H-6b), 2.33 (m, 1H, H-11a), 2.12 (m, 1H, H-9), 1.98 (ddd, 1H, JH,H = 14.3 Hz, JH,H = 9.2 Hz,
JH,H = 5.3 Hz, H-16a), 1.88 (m, 1H, H-7a), 1.78 (m, 1H, H-15a), 1.76 (m, 1H, H-16b), 1.74 (m, 1H,
H-12a), 1.49 (m, 1H, H-11b), 1.46 (m, 1H, H-15b), 1.45 (m, 1H, H-8), 1.44 (m, 1H, H-14), 1.40 (m,
1H, H-12b), 1.30 (m, 1H, H-7b), 0.95 (s, 3H, H-18). 13C NMR (125.7 MHz, MeOD) δ 156.0 (C-3),
138.7 (C-5), 132.2 (C-10), 127.2 (C-1), 116.1 (C-4), 113.8 (C-2), 81.9 (C-17), 51.1 (C-14), 47.5 (C-
13), 46.7 (CH2NH2), 44.9 (C-9), 41.1 (C-8), 33.6 (C-16), 32.6 (C-12), 30.6 (C-6), 28.6 (C-7), 27.4
(C-11), 24.2 (C-15), 14.6 (C-18). HRESI-MS calcd for C19H28NO2 ([M+H]+): 302.2115, found:
302.2110.

ACCEPTED MANUSCRIPT
4.2.10. (17S)-2’-Oxo-spiro[3-hydroxyestra-1,3,5(10)-trien-17,5'-oxazolidine] (16) and methyl N-
[(3,17β-dihydroxyestra-1,3,5(10)-trien-17α-yl)methyl]carbamate (17). To a solution of 14 (0.15 g,
0.51 mmol) in MeOH (20 mL) were added a saturated aqueous solution of NaHCO3 (5 mL) and
triphosgene (53 mg, 0.18 mmol) at 0 °C. The resulting suspension was vigorously stirred for 15 min
at 0 °C and then filtered. The filtrate was evaporated and the residue was purified by flash column
chromatography (7:3 hexane-EtOAc) to give 16 (96 mg, 57%) and 17 (35 mg, 19%) as white solids.
-1
ꢁ ꢃꢅꢅ
Data for compound 16: mp 270 °C (dec.).
ꢂ
-2 (c 0.6, MeOH). IR (cm ): 3371 (NH), 3259
ꢄ
(OH), 2922-2862 (C-H aliphatic), 1730 (C=O), 1618-1492 (C=C), 1285-1063 (C-O, C-N). 1H NMR
(500 MHz, MeOD-CDCl3) δ 7.07 (d, 1H, J1,2 = 8.4 Hz, H-1), 6.58 (dd, 1H, J2,1 = 8.4 Hz, J2,4 = 2.1
Hz, H-2), 6.52 (d, 1H, J4,2 = 2.1 Hz, H-4), 3.74 (d, 1H, J4’a,4’b= 9.3 Hz, H-4’a), 3.27 (d, 1H, J4’b,4’a
=
9.3 Hz, H-4’b), 2.78 (m, 2H, H-6a, H-6b), 2.31 (m, 1H, H-11a), 2.27 (m, 1H, H-16a), 2.11 (td, 1H,
JH,H = 11.2 Hz, JH,H = 4.0 Hz, H-9), 1.90 (ddd, 1H, JH,H = 14.8 Hz, JH,H = 9.4 Hz, JH,H = 5.6 Hz, H-
16b), 1.85 (m, 1H, H-7a), 1.73 (m, 1H, H-15a), 1.69 (m, 1H, H-12a), 1.51 (m, 1H, H-12b), 1.47 (m,
1H, H-11b), 1.43 (m, 1H, H-15b), 1.42 (m, 1H, H-8), 1.28 (m, 1H, H-7b), 1.18 (m, 1H, H-14), 0.94
(s, 3H, H-18). 13C NMR (125.7 MHz, MeOD-CDCl3) δ 161.2 (C-2’), 155.0 (C-3), 138.3 (C-5),
131.4 (C-10), 126.8 (C-1), 115.7 (C-4), 113.3 (C-2), 93.4 (C-17), 50.1 (C-4’), 48.8 (C-14), 46.2 (C-
13), 44.0 (C-9), 39.7 (C-8), 35.8 (C-16), 31.6 (C-12), 30.0 (C-6), 27.7 (C-7), 26.5 (C-11), 22.7 (C-
15), 14.6 (C-18). HRESI-MS calcd for C20H25NNaO3 ([M+Na]+): 350.1726, found: 350.1724. Data
-1
ꢁ ꢃꢅꢈ
for compound 17: mp 164-166 °C.
ꢂ
+49 (c 0.5, MeOH). IR (cm ): 3292 (OH), 2920-2870 (C-
ꢄ
1
H aliphatic), 1700 (C=O), 1602-1523 (C=C), 1212-1058 (C-O, C-N). H NMR (500 MHz, MeOD)
δ 7.07 (d, 1H, J1,2 = 8.4 Hz, H-1), 6.54 (dd, 1H, J2,1 = 8.4 Hz, J2,4 = 2.6 Hz, H-2), 6.48 (d, 1H, J4,2
=
2.6 Hz, H-4), 3.66 (s, 3H, OCH3), 3.36 (d, 1H, Jgem = 13.6 Hz, CH2aNH), 3.18 (dd, 1H, Jgem = 13.6
Hz, JH,H = 1.1 Hz, CH2bNH), 2.77 (m, 2H, H-6a, H-6b), 2.29 (m, 1H, H-11a), 2.12 (m, 1H, H-9),
1.91 (ddd, 1H, JH,H = 16.1 Hz, JH,H = 9.6 Hz, JH,H = 6.6 Hz, H-16a), 1.87 (m, 1H, H-7a), 1.70 (m,
1H, H-12a), 1.69 (m, 1H, H-15a), 1.59 (m, 1H, H-16b), 1.49 (m, 1H, H-14), 1.48 (m, 1H, H-12b),
1.46 (m, 1H, H-11b), 1.45 (m, 1H, H-8), 1.39 (m, 1H, H-15b), 1.30 (m, 1H, H-7b), 0.91 (s, 3H, H-
13
18). C NMR (125.7 MHz, MeOD) δ 160.3 (C=O), 155.9 (C-3), 138.8 (C-5), 132.5 (C-10), 127.2
(C-1), 116.1 (C-4), 113.7 (C-2), 84.2 (C-17), 52.7 (OCH3), 51.2 (C-14), 48.2 (CH2NH), 47.3 (C-13),
45.1 (C-9), 41.2 (C-8), 33.5 (C-16), 32.6 (C-12), 30.7 (C-6), 28.7 (C-7), 27.5 (C-11), 24.2 (C-15),
14.8 (C-18). HRESI-MS calcd for C21H29NNaO4 ([M+Na]+): 382.1989, found: 382.1987.
4.2.11. General procedure for the synthesis of steroidal thioureas 18

ACCEPTED MANUSCRIPT
To a solution of 14 (0.25 g, 0.83 mmol) in MeOH (15 mL) were added the corresponding
isothiocyanate (1.0 mmol) and Et3N (50 µL, 0.36 mmol). The corresponding mixture was stirred
during the time and at the temperature indicated in each case and, after that, it was evaporated to
dryness. The residue was purified by flash column chromatography to give compounds 18a-c as
colorless oils.
4.2.11.1
17α-[(3’-Butylthioureido)methyl]estra-1,3,5(10)-triene-3,17β-diol (18a). Butyl
isothiocyanate was used (0.12 mL) and the reaction proceeded for 4 h at 45 °C. Column
ꢁ ꢃꢅꢆ
+34 (c 0.4,
ꢄ
chromatography (hexane
→
7:3 hexane-EtOAc) afforded 18a (0.23 mg, 66%).
ꢂ
acetone). IR (cm-1): 3286 (OH), 2928-2872 (C-H aliphatic), 1551-1498 (C-NH), 1250 (C=S), 1215-
1024 (C-O, C-N). 1H NMR (500 MHz, acetone-d6) δ 7.20 (brs, 1H, NH), 7.04 (d, 1H, J1,2 = 8.4 Hz,
H-1), 6.77 (brs, 1H, NH), 6.56 (dd, 1H, J2,1 = 8.4 Hz, J2,4 = 2.6 Hz, H-2), 6.49 (d, 1H, J4,2 = 2.6 Hz,
H-4), 3.88 (brs, 1H, CH2aNH), 3.49 (brs, 1H, CH2bNH), 3.48 (m, 2H, NH-CH2-CH2), 2.72 (m, 2H,
H-6a, H-6b), 2.24 (m, 1H, H-11a), 2.05 (m, 1H, H-9), 1.95 (m, 1H, H-16a), 1.82 (m, 1H, H-7a),
1.65 (m, 1H, H-12a), 1.62 (m, 1H, H-15a), 1.59 (m, 1H, H-16b), 1.51 (m, 2H, NH-CH2-CH2), 1.47
(m, 1H, H-14), 1.46 (m, 1H, H-12b), 1.39 (m, 1H, H-8), 1.37 (m, 1H, H-11b), 1.32 (m, 2H, CH2-
CH3), 1.31 (m, 1H, H-15b), 1.24 (m, 1H, H-7b), 0.88 (s, 3H, H-18), 0.88 (t, 3H, JH,H = 7.4 Hz, CH2-
13
CH3). C NMR (125.7 MHz, acetone-d6) δ 184.5 (C=S), 155.9 (C-3), 138.3 (C-5), 131.9 (C-10),
127.0 (C-1), 115.9 (C-4), 113.5 (C-2), 83.9 (C-17), 51.4 (CH2NH), 50.5 (C-14), 46.9 (C-13), 44.8
(NH-CH2-CH2), 44.5 (C-9), 40.7 (C-8), 34.3 (C-16), 32.3 (C-12), 32.1 (NH-CH2-CH2), 30.3 (C-6),
28.3 (C-7), 27.1 (C-11), 24.0 (C-15), 20.7 (CH2-CH3), 14.7 (C-18), 14.1 CH2-CH3). FAB-MS m/z
417 ([M+H]+, 36%); HRFAB-MS calcd for C24H37N2O2S ([M+H]+): 417.2570, found: 417.2574.
4.2.11.2
Cyclohexyl isothiocyanate was used (0.14 mL) and the reaction proceeded for 22 h at room
temperature. Column chromatography (alumina, hexane 4:1 hexane-EtOAc) afforded 18b (0.27
ꢁ ꢃꢅꢆ
17
α
-[(3’-Cyclohexylthioureido)methyl]estra-1,3,5(10)-triene-3,17
β
-diol
(18b).
→
g, 73%).
ꢂ
+28 (c 0.1, acetone). IR (cm-1): 3293 (OH), 2930-2855 (C-H aliphatic), 1609-1449
ꢄ
(C=C), 1546-1498 (C-NH), 1252 (C=S), 1225-1020 (C-O, C-N). 1H NMR (500 MHz, acetone-d6) δ
7.96 (brs, 1H, NH), 7.05 (d, 1H, J1,2 = 8.5 Hz, H-1), 6.72 (brs, 1H, NH), 6.55 (dd, 1H, J2,1 = 8.5 Hz,
J2,4 = 2.6 Hz, H-2), 6.49 (d, 1H, J4,2 = 2.6 Hz, H-4), 4.10 (brs, 1H, H-1’’), 3.92 (brs, 1H, CH2aNH),
3.47 (d, 1H, Jgem = 11.4 Hz, CH2bNH), 2.73 (m, 2H, H-6a, H-6b), 2.25 (m, 1H, H-11a), 2.08 (m, 1H,
H-9), 1.96 (m, 2H, H-2’’a, H-6’’a), 1.95 (m, 1H, H-16a), 1.82 (m, 1H, H-7a), 1.67 (m, 2H, H-3’’a,
H-5’’a), 1.65 (m, 1H, H-12a), 1.63 (m, 1H, H-15a), 1.58 (m, 1H, H-16b), 1.55 (m, 1H, H-4’'a),
1.49 (m, 1H, H-12b), 1.49 (m, 1H, H-14), 1.40 (m, 1H, H-8), 1.39 (m, 1H, H-11b), 1.32 (m, 1H, H-

ACCEPTED MANUSCRIPT
15b), 1.31 (m, 2H, H-3’’b, H-5’’b), 1.26 (m, 1H, H-7b), 1.19 (m, 2H, H-2’’b, H-6’’b), 1.15 (m, 1H,
13
H-4’’b), 0.88 (s, 3H, H-18). C NMR (125.7 MHz, acetone-d6) δ 183.6 (C=S), 155.9 (C-3), 138.4
(C-5), 132.0 (C-10), 127.0 (C-1), 115.9 (C-4), 113.6 (C-2), 84.0 (C-17), 53.3 (C-1’’), 51.3
(CH2NH), 50.6 (C-14), 46.9 (C-13), 44.5 (C-9), 40.7 (C-8), 34.3 (C-16), 33.5 (C-2’’ or C-6’’), 33.5
(C-2’’ or C-6’’), 32.3 (C-12), 30.3 (C-6), 28.3 (C-7), 27.1 (C-11), 26.3 (C-4’’), 25.6 (x2) (C-3’’, C-
5’’), 24.0 (C-15), 14.7 (C-18). FAB-MS m/z 443 ([M+H]+, 15%); HRFAB-MS calcd for
C26H39N2O2S ([M+H]+): 443.2727, found: 443.2709.
4.2.11.3
17α-[(3’-Phenylthioureido)methyl]estra-1,3,5(10)-triene-3,17β-diol (18c). Phenyl
isothiocyanate was used (0.12 mL) and the reaction proceeded for 3 h at room temperature. Column
ꢁ ꢃꢅꢆ
+32 (c 0.9,
ꢄ
chromatography (hexane
→
7:3 hexane-EtOAc) afforded 18c (0.31 g, 86%).
ꢂ
acetone). IR (cm-1): 3304 (OH), 2921-2873 (C-H aliphatic), 1596-1449 (C=C), 1541-1496 (C-NH),
1246 (C=S), 1227-1022 (C-O, C-N). 1H NMR (500 MHz, acetone-d6) δ 9.11 (brs, 1H, NH), 7.43 (d,
2H, Jo-,m- = 7.4 Hz, Ar-Ho), 7.34 (t, 2H, JH,H = 7.4 Hz, Ar-Hm), 7.14 (m, 1H, Ar-Hp), 7.11 (brs, 1H,
NH), 7.05 (d, 1H, J1,2 = 8.4 Hz, H-1), 6.57 (dd, 1H, J2,1 = 8.4 Hz, J2,4 = 2.6 Hz, H-2), 6.50 (d, 1H,
J4,2 = 2.6 Hz, H-4), 4.01 (brs, 1H, CH2aNH), 3.58 (dd, 1H, Jgem = 13.3 Hz, JH,H = 2.5 Hz, CH2bNH),
2.72 (m, 2H, H-6a, H-6b), 2.25 (m, 1H, H-11a), 2.07 (td, 1H, JH,H = 11.4 Hz, JH,H = 4.4 Hz, H-9),
1.96 (m, 1H, H-16a), 1.82 (m, 1H, H-7a), 1.66 (m, 1H, H-15a), 1.65 (m, 1H, H-12a), 1.64 (m, 1H,
H-16b), 1.50 (m, 1H, H-14), 1.49 (m, 1H, H-12b), 1.39 (m, 1H, H-8), 1.38 (m, 1H, H-11b), 1.34 (m,
1H, H-15b), 1.26 (m, 1H, H-7b), 0.88 (s, 3H, H-18). 13C NMR (125.7 MHz, acetone-d6) δ 181.9
(C=S), 155.8 (C-3), 139.3 (Ar-Cipso), 138.3 (C-5), 131.9 (C-10), 130.0 (x2) (Ar-Cm), 127.0 (C-1),
126.0 (Ar-Cp), 124.5 (x2) (Ar-Co), 115.9 (C-4), 113.5 (C-2), 83.7 (C-17), 51.9 (CH2NH), 50.5 (C-
14), 46.9 (C-13), 44.4 (C-9), 40.6 (C-8), 34.5 (C-16), 32.4 (C-12), 30.3 (C-6), 28.2 (C-7), 27.0 (C-
11), 23.9 (C-15), 14.6 (C-18). FAB-MS m/z 437 ([M+H]+, 23%); HRFAB-MS calcd for
C26H33N2O2S ([M+H]+): 437.2257, found: 437.2241.
4.2.12 General procedure for the synthesis of steroidal spiranic isoureas 19
To a solution of the corresponding thiourea 18 (0.25 mmol) in dry MeOH or THF (10 mL) was
added yellow HgO (4.5 mmol for compounds 18a,b and 2.25 mmol for compound 18c) under Ar.
The mixture was stirred at room temperature in the dark during the time indicated in each case,
filtered over a pad of Celite® and evaporated to dryness. The residue was purified by flash column
chromatography and washed with cooled diethyl ether to give compounds 19a-c as colorless oils.

ACCEPTED MANUSCRIPT
4.2.12.1
(17S)-2’-Butylamino-spiro[3-hydroxyestra-1,3,5(10)-trien-17,5'-oxazoline] (19a).
Thiourea 18a was used (0.10 g) and the reaction proceeded for 4 days. Column chromatography
-1
ꢁ ꢃꢅꢆ
(CH2Cl2
→
5:1 CH2Cl2-MeOH) afforded 19a (64 mg, 68%).
ꢂ
-10 (c 0.4, CHCl3). IR (cm ):
ꢄ
3231 (OH), 2928-2867 (C-H aliphatic), 1654 (C=N), 1600-1449 (C=C), 1242-1060 (C-O, C-N). 1H
NMR (500 MHz, CDCl3) δ 7.08 (d, 1H, J1,2 = 8.4 Hz, H-1), 6.79 (brs, 1H, NH), 6.70 (dd, 1H, J2,1
=
8.4 Hz, J2,4 = 2.2 Hz, H-2), 6.62 (d, 1H, J4,2 = 2.2 Hz, H-4), 3.91 (d, 1H, J4’a,4’b = 11.7 Hz, H-4’a),
3.42 (d, 1H, J4’b,4’a = 11.7 Hz, H-4’b), 3.21 (m, 2H, H-1’’), 2.79 (m, 2H, H-6), 2.30 (m, 1H, H-16a),
2.27 (m, 1H, H-11a), 2.04 (m, 1H, H-9), 1.89 (ddd, 1H, JH,H = 14.7 Hz, JH,H = 9.4 Hz, JH,H = 5.6 Hz,
H-16b), 1.84 (m, 1H, H-7a), 1.71 (m, 1H, H-15a), 1.58 (m, 1H, H-12a), 1.53 (m, 2H, H-2’’), 1.47
(m, 1H, H-12b), 1.46 (m, 1H, H-11b), 1.42 (m, 1H, H-15b), 1.41 (m, 1H, H-8), 1.34 (m, 2H, H-3’’),
1.27 (m, 1H, H-7b), 1.09 (m, 1H, H-14), 0.91 (s, 3H, H-18), 0.89 (t, 3H, J4’’,3’’= 7.4 Hz, H-4’’). 13C
NMR (125.7 MHz, CDCl3) δ 161.1 (C-2’), 155.4 (C-3), 137.8 (C-5), 130.5 (C-10), 126.3 (C-1),
115.8 (C-4), 113.3 (C-2), 97.6 (C-17), 57.5 (C-4’), 48.4 (C-14), 45.6 (C-13), 43.5 (C-9), 42.5 (C-
1’’), 39.2 (C-8), 35.1 (C-16), 31.9 (C-2’’), 31.1 (C-12), 29.7 (C-6), 27.3 (C-7), 26.0 (C-11), 22.3 (C-
15), 19.9 (C-3’’), 14.5 (C-18), 13.8 (C-4’’). FAB-MS m/z 383 ([M+H]+, 80%); HRFAB-MS calcd
for C24H35N2O2 ([M+1]+): 383.2693, found: 383.2700.
4.2.12.2
(17S)-2’-Cyclohexylamino-spiro[3-hydroxyestra-1,3,5(10)-trien-17,5'-oxazoline]
(19b). Thiourea 18b was used (0.11 g) and the reaction proceeded for 7 days. Column
ꢁ ꢃꢅꢆ
-19 (c
ꢄ
chromatography (CH2Cl2
→
20:1 CH2Cl2-Isopropanol) afforded 19b (95 mg, 92%).
ꢂ
0.7, CHCl3). IR (cm-1): 3284 (OH), 2924-2854 (C-H aliphatic), 1653 (C=N), 1600-1448 (C=C),
1
1240-1016 (C-O, C-N). H NMR (500 MHz, CDCl3) δ 7.10 (d, 1H, J1,2 = 8.5 Hz, H-1), 6.68 (dd,
1H, J2,1 = 8.5 Hz, J2,4 = 2.2 Hz, H-2), 6.61 (d, 1H, J4,2 = 2.2 Hz, H-4), 3.93 (d, 1H, J4’a,4’b = 12.2 Hz,
H-4’a), 3.41 (d, 1H, J4’b,4’a = 12.2 Hz, H-4’b), 3.41 (m, 1H, H-1’’), 2.80 (m, 2H, H-6), 2.29 (m, 1H,
H-11a), 2.28 (m, 1H, H-16a), 2.10 (td, 1H, JH,H = 10.7 Hz, JH,H = 3.0 Hz, H-9), 1.97 (m, 2H, H-2’’a,
H-6’’a), 1.86 (m, 1H, H-16b), 1.85 (m, 1H, H-7a), 1.70 (m, 1H, H-15a), 1.67 (m, 2H, H-3’’a, H-
5’’a), 1.59 (m, 1H, H-12a), 1.57 (m, 1H, H-4’’a), 1.54 (m, 1H, H-12b), 1.46 (m, 1H, H-11b), 1.42
(m, 1H, H-8), 1.40 (m, 1H, H-15b), 1.29 (m, 3H, H-7b, H-3’’b, H-5’’b), 1.15 (m, 1H, H-14), 1.13
(m, 3H, H-4’’b, H-2’’b, H-6’’b), 0.92 (s, 3H, H-18). 13C NMR (125.7 MHz, CDCl3) δ 160.2 (C-2’),
155.6 (C-3), 137.8 (C-5), 130.6 (C-10), 126.4 (C-1), 115.9 (C-4), 113.4 (C-2), 96.2 (C-17), 59.7 (C-
4’), 51.5 (C-1’’), 48.4 (C-14), 45.5 (C-13), 43.6 (C-9), 39.3 (C-8), 35.2 (C-16), 33.7 (C-2’’ or C-
6’’), 33.6 (C-2’’ or C-6’’), 31.2 (C-12), 29.8 (C-6), 27.3 (C-7), 26.2 (C-11), 25.6 (C-4’’), 24.8 (x2)
(C-3’’, C-5’’), 22.3 (C-15), 14.6 (C-18). FAB-MS m/z 409 ([M+H]+, 100%); HRFAB-MS calcd for
C26H37N2O2 ([M+H]+): 409.2850, found: 409.2813.

ACCEPTED MANUSCRIPT
4.2.12.3
(17S)-2’-Phenylamino-spiro[3-hydroxyestra-1,3,5(10)-trien-17,5'-oxazoline] (19c).
Thiourea 18c was used (0.11 g) and the reaction proceeded for 21 h. Column chromatography
ꢁ ꢃꢅꢆ
+22 (c 0.2, CHCl3). IR
ꢄ
(CH2Cl2
→
20:1 CH2Cl2-Isopropanol) afforded 19c (96 mg, 96%).
ꢂ
(cm-1): 3280 (OH), 2923-2871 (C-H aliphatic), 1656 (C=N), 1592-1447 (C=C), 1224-1062 (C-O, C-
1
N). H NMR (500 MHz, CDCl3) δ 7.20 (m, 4H, Ar-Ho, Ar-Hm), 7.02 (d, 1H, J1,2 = 8.5 Hz, H-1),
6.93 (m, 1H, Ar-Hp), 6.59 (dd, 1H, J2,1 = 8.5 Hz, J2,4 = 1.9 Hz, H-2), 6.51 (d, 1H, J4,2 = 1.9 Hz, H-
4), 5.54 (brs, 1H, NH), 3.83 (d, 1H, J4’a,4’b = 11.0 Hz, H-4’a), 3.34 (dd, 1H, J4’b,4’a = 11.0 Hz, H-4’b),
2.72 (m, 2H, H-6a, H-6b), 2.28 (ddd, 1H, J16a,16b = 14.8 Hz, J16a,H = 12.6 Hz, J16a,H = 3.5, H-16a),
2.21 (m, 1H, H-11a), 2.04 (m, 1H, H-9), 1.84 (ddd, 1H, J16b,16a = 14.8 Hz, J16b,H = 9.4 Hz, J16b,H = 5.8
Hz, H-16b), 1.79 (m, 1H, H-7a), 1.66 (m, 1H, H-15a), 1.62 (m, 1H, H-12a), 1.45 (m, 1H, H-12b),
1.41 (m, 1H, H-11b), 1.37 (m, 2H, H-15b, H-8), 1.23 (m, 2H, H-7b, OH), 1.11 (m, 1H, H-14), 0.90
13
(s, 3H, H-18). C NMR (125.7 MHz, CDCl3) δ 157.3 (C-2’), 154.5 (C-3), 138.0 (C-5), 131.3 (C-
10), 129.1 (x3) (Ar-C), 126.5 (C-1), 122.8 (Ar-Cp), 120.5 (x2) (Ar-C), 115.7 (C-4), 113.2 (C-2),
96.4 (C-17), 56.3 (C-4’), 48.7 (C-14), 45.7 (C-13), 43.6 (C-9), 39.2 (C-8), 35.2 (C-16), 31.4 (C-12),
29.7 (C-6), 27.3 (C-7), 26.1 (C-11), 22.4 (C-15), 14.7 (C-18). FAB-MS m/z 403 ([M+H]+, 28%);
HRFAB-MS calcd for C26H31N2O2 ([M+H]+): 403.2380, found: 403.2376.
Acknowledgements
The authors thank CONACYT-México for scholarship to LLRH, and financial support (grant
240329 to SMS). We also thank Dr. Angel Mendoza for X-ray data collection.
References
[1] C.D. Duarte, E.J. Barreiro, C.A. Fraga, Privileged structures: a useful concept for the rational design of
new lead drug candidates, Mini Rev Med Chem. 7 (2007) 1108–1119.
[2] J. Akhtar, A.A. Khan, Z. Ali, R. Haider, M. S. Yar, Structure-activity relationship (SAR) study and design
strategies of nitrogen-containing heterocyclic moieties for their anticancer activities, Eur. J. Med. Chem.
125 (2017) 143–189.
[3] T. Shiro, T. Fukaya, M. Tobe, The chemistry and biological activity of heterocycle-fused quinolinone
derivatives: A review, Eur. J. Med. Chem. 97 (2015), 397–408.
[4] M.L. Fascio, M.I. Errea, N.B. D'Accorso, Imidazothiazole and related heterocyclic systems. Synthesis,
chemical and biological properties, Eur. J. Med. Chem. 90 (2015) 666–683.

ACCEPTED MANUSCRIPT
[5] O.O. Ajani, Present status of quinoxaline motifs: Excellent pathfinders in therapeutic medicine, Eur. J.
Med. Chem. 85 (2014) 688–715.
[6] Y. Li, J. Geng, Y. Liu, S. Yu, G. Zhao, Thiadiazole-a promising structure in Medicinal Chemistry,
ChemMedChem 8 (2013) 27–41.
[7] Y. Liu, H. Hu, J. Zhou, W. Wang, Y. He, C. Wang, Application of primary halogenated hydrocarbons for
the synthesis of 3-aryl and 3-alkyl indolizines, Org. Biomol. Chem. 15 (2017) 5016–5024.
[8] L.M.T. Frija, A.J.L. Pombeiro, M.N. Kopylovich, Building 1,2,4-thiadiazole: ten years of progress, Eur. J.
Org. Chem. 2017 (2017) 2670–2682.
[9] K.M. Elattar, B.D. Mert, Recent developments in the chemistry of bicyclic 6-6 systems: chemistry of
pyrido[4,3-d]pyrimidines, RSC Adv. 6 (2016) 71827–71851.
[10] C.W. Muir, A.R. Kennedy, J.M. Redmond, A.J.B. Watson, Synthesis of functionalised 4H-quinolizin-4-
ones via tandem Horner-Wadsworth-Emmons olefination/cyclisation, Org. Biomol. Chem. 11 (2013)
3337–3340.
[11] Y. Fujiwara, J.A. Dixon, F. O'Hara, E.D. Funder, D.D. Dixon, R.A. Rodríguez, R.D. Baxter, B. Herlé, N.
Sach, M.R. Collins, Y. Ishihara, P.S. Baran, Practical and innate carbon-hydrogen functionalization of
heterocycles, Nature 492 (2012) 95–99.
[12] C.M. Marson, New and unusual scaffolds in medicinal chemistry, Chem. Soc. Rev. 40 (2011) 5514–
5533.
[13] E. Vitaku, D.T. Smith, J.T. Njardarson, Analysis of the structural diversity, substitution patterns, and
frequency of nitrogen heterocycles among U.S. FDA approved pharmaceuticals, J. Med. Chem. 57
(2014) 10257–10274.
[14] A. Zagórska, M. Kolaczkowski, A. Bucki, A. Siwek, G. Kazek, G. Satala, A.J. Bojarski, A. Partyka, A.
Wesolowska, M. Pawlowski, Structure-activity relationships and molecular studies of novel
arylpiperazinylalkyl purine-2,4-diones and purine-2,4,8-triones with antidepressant and anxiolytic-like
activity, Eur. J. Med. Chem. 97 (2015) 142–154.
[15] S.P. Wagh, Synthesis of 3,4-diylidine and N-substituted pyrrolidines and its anti-microbial activity, Am.
J. PharmTech Res. 5 (2015) 153–159.
[16] Y. Kouadri, M.R. Ouahrani, B.E. Missaoui, F. Chebrouk, N. Gherraf, Reactivity of β-cetoesters
compounds, synthesis of nitrogenated heterocycles (derivatives of tetrahydroacridin-9-ones and
pyrimidinone) and biological properties of pyrimidinone derivatives, Asian J. Chem. 27 (2015) 3675–
3680.
[17] S. Kethireddy, L. Eppakayala, T.C. Maringanti, Synthesis and antibacterial activity of novel 5,6,7,8
tetrahydroimidazo[1,2-a]pyrimidine-2-carbohydrazide derivatives, Chem. Cent. J. 9 (2015) 51–56.
[18] M. Xie, R.G. Lapidus, M. Sadowska, M.J. Edelman, R.S. Hosmane, Synthesis, anticancer activity, and
SAR analyses of compounds containing the 5:7-fused 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring
system, Bioorg. Med. Chem. 24 (2016) 2595–2602.

ACCEPTED MANUSCRIPT
[19] P. Martins, J. Jesus, S. Santos, L.R. Raposo, C. Roma-Rodrigues, P.V. Baptista, A.R. Fernandes,
Heterocyclic anticancer compounds: recent advances and the paradigm shift towards the use of
nanomedicine’s tool box, Molecules 20 (2015) 16852–16891.
[20] J.J. Chen, Q. Liu, C. Yuan, V. Gore, P. López, V. Ma, A. Amegadzie, W. Qian, T.C. Judd, A.E. Minatti,
J. Brown, Y. Cheng, M. Xue, W. Zhong, T.A. Dineen, O. Epstein, J. Human, C. Kreiman, I. Marx, M.M.
Weiss, S.A. Hitchcock, T.S. Powers, K. Chen, P.H. Wen, D.A. Whittington, A.C. Cheng, M.D.
Bartberger, D. Hickman, J.A. Werner, H.M. Vargas, N.E. Everds, S.L. Vonderfecht, R.T. Dunn 2nd, S.
Wood, R.T. Fremeau Jr., R.D. White, V.F. Patel, Development of 2-aminooxazoline 3-azaxanthenes as
orally efficacious β-secretase inhibitors for the potential treatment of Alzheimer’s disease, Bioorg. Med.
Chem. Lett. 25 (2015) 767–774.
[21] A. Gomtsyan, Heterocycles in drugs and drug discovery, Chem. Heterocycl. Comp. 48 (2012) 7–10.
[22] A.-G. E. Amr, M.M. Abdulla, Anti-inflammatory profile of some synthesized heterocyclic pyridone and
pyridine derivatives fused with steroidal structure, Bioorg. Med. Chem. 14 (2006) 4341–4352.
[23] A.V. Karnik, N.J. Malviya, A.M. Kulkarni, B.L. Jadhav, Synthesis and in vitro antibacterial activity of
novel heterocyclic derivatives of 18-nor-equilenin, Eur. J. Med. Chem. 41 (2006) 891–895.
[24] C. Lange, N. Holzhey, B. Schönecker, R. Beckert, U. Möllmann, H.-M. Dahse, ω-Pyridiniumalkylethers
of steroidal phenols: new compounds with potent antibacterial and antiproliferative activities, Bioorg.
Med. Chem. 12 (2004) 3357–3362.
[25] A. Fuentes-Aguilar, L.L. Romero-Hernández, A. Arenas-González, P. Merino-Montiel, S. Montiel-
Smith, S. Meza-Reyes, J.L. Vega-Báez, G.B. Plata, J.M. Padrón, Ó. López, J.G. Fernández-Bolaños,
New selenosteroids as antiproliferative agents, Org. Biomol. Chem. 15 (2017) 5041–5054.
[26] A. Ali, M. Asif, H. Khanam, A. Mashrai, M.A. Sherwani, M. Owais, Shamsuzzaman, Synthesis and
characterization of steroidal heterocyclic compounds, DNA condensation and molecular docking studies
and their in vitro anticancer and acetylcholinesterase inhibition activities, RSC Adv. 5 (2015) 75964–
75984.
[27] É. Frank, Z. Mucsi, I. Zupkó, B. Réthy, G. Falkay, G. Schneider, J. Wölfling, Efficient approach to
androstene-fused arylpyrazolines as potent antiproliferative agents. Experimental and theoretical studies
of substituent effects on BF3-catalyzed intramolecular [3+2] cycloadditions of olefinic
phenylhydrazones, J. Am. Chem. Soc. 131 (2009) 3894–3904.
[28] M. El-Far, G.A. Elmegeed, E.F. Eskander, H.M. Rady, M.A. Tantawy, Novel modified steroid
derivatives of androstanolone as chemotherapeutic anti-cancer agents, Eur. J. Med. Chem. 44 (2009)
3936–3946.
[29] M.M. Abdalla, M.A. Al-Omar, M.A. Bhat, A.-G.E. Amr, A.M. Al-Mohizea, Steroidal pyrazolines
evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer, Int. J. Biol.
Macromol. 50 (2012) 1127–1132.