Synthesis, structural analysis and antiproliferative activity of some novel D-homo lactone androstane derivatives

Article abstract of DOI:10.1039/c3ra41336e

An efficient synthesis of several A,B-modified D-homo lactone androstane derivatives is reported. The synthetic scheme shows the transformation of 17-oxa-D-homoandrost-5-en-16-on-3β-yl acetate 1 into the 5α-hydroxy-17-oxa-D-homoandrostane-6,16-dion-3β-yl acetate (4). After the dehydration of 4, the newly synthesized 6-keto-androst-4-ene-3β-yl acetate derivative 5 was oximinated to give the 6-hydroximino derivative 6, which was converted to A,B-condensed isoxazole derivatives 7 and 8. Compound 4 was also converted (via 6(E)- and 6(Z)-hydroximino derivatives 9 and 10) to the B-seco-cyano derivative 11 under a Beckmann fragmentation, while compound 5 was transformed to the 4β,5β-epoxy derivative 12. Structures were confirmed by IR, ¹H NMR, ¹³C NMR, and HRMS, and for 7 and 8 by X-ray crystallography. All compounds were tested in vitro on six malignant cell lines (MCF-7, MDA-MB-231, PC-3, HeLa, HT-29, K562) and one non-tumor MRC-5 cell line. Significant antiproliferative activity was observed for specific compounds against prostate (PC-3), cervical (HeLa) and colon (HT-29) cancer cells, while no compounds showed antiproliferative activity to non-cancerous control cells (MRC-5). Interestingly, 1-8 displayed selective antiproliferative activity against estrogen-independent (ER-, MDA-MB-231) breast cancer cells over estrogen-dependent (ER+, MCF-7) breast cancer cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra41336e

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PAPER
Synthesis, structural analysis and antiproliferative
activity of some novel D-homo lactone androstane
derivatives3
Cite this: RSC Advances, 2013, 3,
10385
a
a
b
b
´
´
´
´
Marina P. Savic, Evgenija A. Djurendic,* Edward T. Petri, Andjelka Celic, Olivera
c
d
d
´
ˇ a
R. Klisuric, Marija N. Sakac, Dimitar S. Jakimov, Vesna V. Kojic and Katarina M.
´
ˇ a
Penov Gasi
An efficient synthesis of several A,B-modified D-homo lactone androstane derivatives is reported. The
synthetic scheme shows the transformation of 17-oxa-D-homoandrost-5-en-16-on-3b-yl acetate 1 into the
5a-hydroxy-17-oxa-D-homoandrostane-6,16-dion-3b-yl acetate (4). After the dehydration of 4, the newly
synthesized 6-keto-androst-4-ene-3b-yl acetate derivative 5 was oximinated to give the 6-hydroximino
derivative 6, which was converted to A,B-condensed isoxazole derivatives 7 and 8. Compound 4 was also
converted (via 6(E)- and 6(Z)-hydroximino derivatives 9 and 10) to the B-seco-cyano derivative 11 under a
Beckmann fragmentation, while compound 5 was transformed to the 4b,5b-epoxy derivative 12.
Structures were confirmed by IR, 1H NMR, 13C NMR, and HRMS, and for 7 and 8 by X-ray crystallography.
All compounds were tested in vitro on six malignant cell lines (MCF-7, MDA-MB-231, PC-3, HeLa, HT-29,
K562) and one non-tumor MRC-5 cell line. Significant antiproliferative activity was observed for specific
compounds against prostate (PC-3), cervical (HeLa) and colon (HT-29) cancer cells, while no compounds
showed antiproliferative activity to non-cancerous control cells (MRC-5). Interestingly, 1–8 displayed
selective antiproliferative activity against estrogen-independent (ER2, MDA-MB-231) breast cancer cells
over estrogen-dependent (ER+, MCF-7) breast cancer cells.
Received 19th March 2013,
Accepted 18th April 2013
DOI: 10.1039/c3ra41336e
www.rsc.org/advances
compounds by conversion of functional group(s) to a hetero-
cyclic moiety can result in dramatically altered pharmacological
1. Introduction
Hormone-responsive cancers, including endometrial, ovarian,
prostate and breast cancers, comprise over 30% of all newly
diagnosed cancers.1 In addition, steroidal hormones have been
proposed to modulate the proliferation of other cancers, such as
colorectal2 and cervical cancer.3 Synthetic and natural steroidal
estrogen, androgen, and progesterone derivatives influence the
growth and progression of hormone-sensitive cancers, and
modulation of the levels of circulating sex hormones represents
a major therapeutic strategy in cancer treatment.1 We are
interested in the synthesis and characterization of heterocyclic
steroidal compounds for potential applications in the treatment
of steroid-responsive tumors.4–7 Modification of steroidal
properties, and steroidal heterocycles have applications in the
treatment of a range of disorders, including breast and prostate
cancer.8–10 Numerous steroidal heterocycles have been synthe-
sized with antitumor activity.11,12 D-Homo lactone testosterone
derivatives, such as Testolactone, represent one of the first
steroidal breast cancer treatments in clinical use,13 while A- and
B-ring modified steroids have been reported with high anti-
proliferative activity against human tumor cells.6,7,14–17 In
particular, B-ring oxygenated steroids displayed strong anti-
proliferative activity toward several human cancer cells, includ-
ing selectivity towards leukemia cell lines.17 Steroidal
heterocycles with isoxazole modifications have also been
synthesized with antitumor properties,18 and are convenient
intermediates for the synthesis of multifunctional com-
pounds.19 Clinically, steroidal isoxazoles, such as Danazol are
important in the treatment of endometriosis,20 and inhibit
endometrial cancer proliferation in vitro.21,22 Other isoxazole
derivatives have been reported with wide-ranging pharmacolo-
gical properties, including antibacterial and antiviral activities.23
Because of their pharmacological potential, we are interested
in developing convenient synthetic routes to obtain D-homo
lactone steroidal heterocycles with A,B-ring modifications. We
aDepartment of Chemistry, Biochemistry and Environmental Protection, Faculty of
´
Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia.
E-mail: evgenija.djurendic@dh.uns.ac.rs
bDepartment of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg
´
Dositeja Obradovica 2, 21000 Novi Sad, Serbia
cDepartment of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja
´
Obradovica 4, 21000 Novi Sad, Serbia
dOncology Institute of Vojvodina, Institutski put 4, 21204 Sremska Kamenica, Serbia
CCDC 922750 and 922751. For crystallographic data in CIF or other electronic
3
format see DOI: 10.1039/c3ra41336e
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Scheme 1 Reagents and reaction conditions: (a) MCPBA, NaHCO3, CH2Cl2, 0 uC, 1 h; (b) CrO3, acetone, water, 0 uC, 30 min A rt, 40 min; (c) MCPBA, CH2Cl2, 0 uC, 90 min
A rt, 60 min, then CrO3, rt 70 min; (d) SOCl2, abs. pyridine, 0 uC, 2 h, HCl (1 : 1); (e) NH2OH?HCl, CH3COONa, C2H5OH, rt, 24 h; (f) I2, KI, NaHCO3, THF–water (4 : 1), reflux,
5.5 h, HCl (1 : 1); (g) NH2OH?HCl, CH3COONa, C2H5OH, 60 uC, 1 h; (h) Ac2O, abs. pyridine, 70 uC, 2h, then 0 uC HCl (1 : 1); (i) H2O2, NaOH, CH3OH, 0 uC, 3 h, HCl (1 : 1).
have previously reported syntheses of steroidal D-homo lactone
derivatives with marked antiproliferative activity against human
cancer cells.6,24 Here we present an efficient synthesis of several
new A,B-modified D-homo lactone androstanes, including A,B-
fused isoxazole derivatives. Synthesized products were char-
acterized by spectroscopic methods, X-ray crystallography and
computational methods, and evaluated for antiproliferative
activity against six different human cancer cell lines: PC-3 (AR2
prostate cancer), MCF-7 (ER+ breast adenocarcinoma), MDA-
MB-231 (ER2 breast adenocarcinoma), HeLa (cervical cancer),
HT-29 (colon cancer) and K562 (chronic myelogenous leuke-
mia), and non-cancerous cells (MCR-5). Results were rationa-
lized using ligand-based inverse docking.
2. Results and discussion
2.1. Synthetic studies
Scheme 1 outlines the synthetic procedures used to obtain
compounds 1–12. Compounds 1–4 were synthesized as
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described in our previous work.5,25 In this paper we report a
rapid and simple one-pot procedure26 which was modified
from our previous work25 for the synthesis of compound 4
from starting compound 1. Treatment of 4 with thionyl
chloride (absolute pyridine, 0 uC for 2 h) gave 17-oxa-D-
homoandrost-4-ene-6,16-dion-3b-yl acetate (5). Reaction com-
pletion was monitored by NMR: the doublet signal at 6.14 ppm
in the 1H-NMR spectra of compound 5 is indicative of the
presence of an H-4 proton, and a clear C-4 signal was observed
at 129.83 ppm by 13C-NMR. The hydroximino derivative 6 was
then obtained by the reaction of compound 5 with hydro-
xylamine hydrochloride in 95% ethanol, in the presence of
sodium acetate at room temperature for 24 h. The presence of
a 6(E)-hydroximino substituent on compound 6 was confirmed
by comparing spectroscopic data (of 6) with reported values for
closely related steroidal compounds.27–29
One aim of the present study was to fuse an isoxazole moiety
with the A,B-ring of a D-homo lactone steroid. 3b-Acetoxy
isoxazole derivative 7 and 3b-hydroxy isoxazole derivative 8
were prepared by the reaction of compound 6 with iodine and
potassium iodide30,31 in THF–H2O under reflux for 5.5 h.
Reaction completion was monitored by comparison of the
NMR spectra of 6 with those of reaction products 7 and 8; 1H-
NMR spectra of pure 7 and 8 did not contain signals at 10.83
ppm (assigned to 6(E)-hydroximino in 6) or 5.53 ppm (H-4 in
6), respectively. The structures of A,B-fused isoxazole D-homo
androstane derivatives 7 and 8 were then unambiguously
confirmed by single crystal X-ray diffraction (Fig. 1, Table 1).
Interestingly, analysis of the crystal lattice packing in the
crystal structure of compound 8 revealed intermolecular O–
…
…
H
O and O–H N hydrogen bonds with ordered water,
mediated by N1 of the A,B-isoxazole moiety and O1 of the C3
hydroxyl group (Fig. 2, Table 2). Consistent with this,
molecular electrostatic potential (MEP) maps calculated from
the crystal structures of 7 and 8 suggest that isoxazole addition
introduces a significant negative partial charge to the A,B-ring
(Fig. 3).
An alternative reaction protocol for compound 4 was also
explored. The reaction of 4 with hydroxylamine hydrochloride
in 95% ethanol in the presence of sodium acetate at 60 uC for 1
h afforded a mixture of 6(Z)-(9) and 6(E)-hydroximino (10)
derivatives in the ratio E : Z = 1.47 : 1, which could not be
separated. This mixture was used for the synthesis of a new
B-seco-cyano derivative 11, which was synthesized by a
Beckmann fragmentation of 9 and 10 with acetic anhydride
in absolute pyridine at 70 uC for 2 h. The structure of
compound 11 was confirmed spectroscopically by IR and
NMR: IR absorption bands at 2245 cm21 and 1732 cm21 are
consistent with the presence of cyano and keto functions,
while 13C-NMR signals at 117.26 ppm and 215.66 ppm support
the presence of a cyano and C5LO group, respectively.
Finally, the reaction of a 6-keto derivative 5 with 30%
hydrogen peroxide under alkaline conditions in methanol at 0
uC for 3 h gives the 4b,5b-epoxy-3b-hydroxy derivative 12. The
structure of compound 12 was confirmed spectroscopically by
IR and NMR. 1H NMR revealed a doublet at 3.32 ppm assigned
to the H-4 proton, which in compound 5 is shifted to 6.14
ppm. Moreover, no signal was observed at 170.63 ppm from
the 3b-acetoxy function in compound 5 by 13C NMR, while the
presence of a broad IR band at 3446 cm21 confirmed the
hydroxy group in compound 12. The b-configuration of the 4,5-
1
epoxy group in compound 12 was assigned by H-NMR NOE:
irradiation of the H-4 proton at 3.32 ppm resulted in NOE
enhancement at H-3a (4.07 ppm), also irradiation of H-1a (2.16
ppm) gave an NOE enhancement at H-4 (3.32 ppm). The same
4b,5b-configuration of the epoxide was confirmed in
4b,5b-epoxy-cholest-4-en-6-one-3b-yl acetate.32
2.2. Antiproliferative activity
Synthesized compounds were evaluated for antiproliferative
activity against PC-3 (AR2 prostate cancer), MCF-7 (ER+ breast
adenocarcinoma), MDA-MB-231 (ER2 breast adenocarci-
noma), HeLa (cervical cancer), HT-29 (colon cancer) and
K562 (chronic myelogenous leukemia), as well as control
non-cancerous MRC-5 (normal fetal lung fibroblast) cells.
Antiproliferative activity was evaluated in vitro using the SRB
assay,33 following 48 h treatment with test compounds as
previously described.25 Results were compared with the non-
selective antiproliferative drug, Doxorubicin, as well as a
clinically approved androstane derivative (Formestane) used in
the treatment of ER+ breast cancer, and are presented in
Table 3 as IC50 values (mM). All of the newly synthesized
compounds were non-toxic to normal MRC-5 cells, whereas
the Doxorubicin was highly toxic to these cells, consistent with
the severe side effects associated with Doxorubicin chemother-
apy. As can be seen, strong antiproliferative activity was
observed against prostate cancer (PC-3), cervical cancer (HeLa)
Fig. 1 X-ray crystal structures of A,B-isoxazole D-homo lactone androstane
derivatives 7 and 8. (A) ORTEP drawing of the molecular structure of compound
7 with labeled non-H atoms. Displacement ellipsoids are shown at 50%
probability, and H atoms are drawn as spheres of arbitrary radii. (B) ORTEP
drawing of the molecular structure of compound 8 with labeled non-H atoms.
Displacement ellipsoids are shown at 50% probability level and H atoms are
drawn as spheres of arbitrary radii. A hydrogen bond between 8 and an ordered
water is shown as a dashed line.
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Table 1 X-ray data and crystal structure refinementa
Compound No.
7
8
Empirical formula
Formula weight
Temperature
C21 H27 N O5
373.44
293(2) K
1.54180 Å
C19 H27 N O5
349.42
293(2) K
Wavelength
0.71073 Å
Crystal system
Space group
Unit cell dimensions
Orthorhombic
P 21 21 21
a = 7.6964(6) Å
b = 9.0385(9) Å
c = 28.1815(19) Å
1960.4(3) Å3
Orthorhombic
P 21 21 21
a = 7.6411(3) Å
b = 8.3284(4) Å
c = 27.4713(13) Å
Volume
1748.2(2) Å3
Z
4
4
Density (calculated)
Absorption coefficient
F(000)
Crystal size
h range for data collection
Index ranges
1.265 mg m23
1.328 mg m23
0.096 mm21
0.734 mm21
800
752
0.232 6 0.114 6 0.041 mm3
3.14 to 74.74u
29 ¡ h ¡ 8
0.237 6 0.162 6 0.045 mm3
2.97 to 24.99u
29 ¡ h ¡ 9
210 ¡ k ¡ 10
234 ¡ l ¡ 34
4426
26 ¡ k ¡ 9
232 ¡ l ¡ 31
5375
Reflections collected
Independent reflections
Completeness to h = 67.50u
Absorption correction
Max. and min. transmission
Refinement method
3150 [R(int) = 0.0171]
99.8%
Semi-empirical from equivalents
1.00000 and 0.96820
Full-matrix least-squares on F2
3150/0/264
2871 [R(int) = 0.0258]
99.8%
Semi-empirical from equivalents
1.00000 and 0.98354
Full-matrix least-squares on F2
2871/0/256
Data/restraints/parameters
Goodness-of-fit on F2
1.028
1.075
Final R indices [I . 2s(I)]
R indices (all data)
Extinction coefficient
R1 = 0.0448, wR2 = 0.1108
R1 = 0.0711, wR2 = 0.1413
0.0024(4)
R1 = 0.0465, wR2 = 0.0901
R1 = 0.0571, wR2 = 0.0946
Largest diff. peak and hole
0.251 and 20.256 e Å23
0.128 and 20.155 e Å23
a
Computer programs: CrysAlisPro, Oxford Diffraction Ltd., Version 1.171.34.36 (release 02-08-2010 CrysAlis171.NET) (compiled Aug 2 2010,
13:00:58), SIR92 (Altomare, 1993), SHELXL97 (Sheldrick, 1997).
and colon cancer (HT-29) cells. Consistent with these results,
steroidal compounds have been shown to modulate the
proliferation of colorectal and cervical cancer,1,3 and steroid-
based anti-androgen drugs can interfere with androgen
receptor function, leading to targeted apoptosis and inhibition
of androgen-dependent prostate cancer growth.34 Compound
2, which contains a 5a,6a-epoxy function, displayed the
strongest antiproliferative activity against prostate cancer cells
(IC50 2.64 mM), while compound 4 (obtained from mixture of 2
and 3) was only slightly toxic (IC50 66.87 mM), suggesting the
importance of the 5a,6a-epoxy group on the A,B-ring. In
support of this, other steroidal 5a,6a-epoxides have also been
reported with significant antiproliferative activity against PC-3
tumor cells.14 Furthermore, compound 12, which contains
4b,5b-epoxy and 6-keto functions, was nontoxic to PC-3 cells,
possibly suggesting a specific role for the 5a,6a-epoxy group in
PC-3 antiproliferative activity. Along the same lines, com-
Unexpectedly, compounds 1–8 displayed selective antiproli-
ferative activity against estrogen receptor negative (ER2, MDA-
MB-231) over estrogen receptor positive (ER+, MCF-7) breast
cancer cell lines (Table 3). Of these, compounds 2, 6 and 8
showed significant antiproliferative activity (IC50 12.98 mM,
11.88 mM and 11.24 mM, respectively) against MDA-MB-231
breast cancer cells. MDA-MB-231 breast adenocarcinoma cells
are triple-negative,35 they are associated with poorer prog-
nosis, and no targeted therapies have been developed to date
for the treatment of triple-negative breast cancers.36
Identification of compounds which selectively inhibit MDA-
MB-231 cell growth may therefore be important for the
development of more effective breast cancer chemotherapies.
In an attempt to provide a theoretical rationale for these
results, we conducted in silico ligand-based virtual screening
using the program idTarget.37 Ligand-based inverse screening
for protein targets (inverse-docking) has been used to suggest
possible molecular mechanisms for the function of new
compounds, and to identify possible off-target pathways for
known drugs.38–41 Compound 8 was chosen for initial screen-
ing since it showed antiproliferative activity only against MDA-
MB-231 cells, and we had determined its crystal structure.
idTarget predicts protein targets for a given small molecule
using a rapid molecular docking-based scoring function which
enables molecular docking simulations to be performed
against the entire protein databank (PDB).37 idTarget identi-
pound 6, which contains
a 6(E)-hydroximino function,
displayed the strongest antiproliferative effect against colon
cancer cells (IC50 3.97 mM), while 5 was nontoxic, suggesting
the importance of the 6(E)-hydroximino in the antiproliferative
properties of 6. Only compound 12 (IC50 4.97 mM) showed
strong antiproliferative activity against cervical cancer (HeLa)
cells. Because 12 was non-toxic to all other cells, this
antiproliferative activity may be selective for cervical cancer
cells.
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Fig. 2 PLATON drawing showing the crystal packing of compound 8 along the a axis. Intermolecular hydrogen bonds are shown as dashed lines. Some atoms in the
molecule are not shown for clarity.
fied 400 proteins with an AutoDock4 binding energy of 29.5
kcal mol21 or better and a negative Z-score. These results were
then filtered to include only proteins shown in the literature to
have an increased expression in estrogen receptor negative
breast cancers versus ER+ breast cancer cells,42–44 resulting in
23 potential protein targets (Table 4) for future experimental
investigation. Importantly, based on our experimental results,
compounds 2, 6 and 8 could represent possible starting points
for the development of triple-negative specific antiproliferative
compounds.
higher concentrations (Fig. 4 A). Compounds 5 and 6 showed
similar antiproliferative activity, which was slightly higher
compared to Formestane (Fig. 4 B). Compound 7 showed
activity similar to Formestane, while compound 8 was more
toxic at concentrations above 1 mM (Fig. 4 C). Compound 12
had the lowest activity against MDA-MB-231 cells over the
whole range of concentrations (Fig. 4 D).
Considering the results of the antiproliferative activity and
IC50 values of the investigated compounds against the MDA-
MB-231 cell line (Fig. 4, Table 3), it could be concluded that
compound 8 is the most promising derivative. With the lowest
IC50 value (11.24 mM), this compound produced most of its
antiproliferative activity effects at higher concentrations (>1
mM), while at the same time was less toxic compared to other
derivatives at lower concentrations.
Compound 4 exerted the highest antiproliferative activity at
lower concentrations, while compound 2 did not show
appreciable antiproliferative activity at these concentrations.
However, both compounds 2 and 4 had similar activity at
a
…
…
Table 2 Intermolecular O–H O and O–H N hydrogen-bond parameters (Å, u)
3. Conclusions
…
…
…
…
D–H A
D–H
A
D–H
H
A
D
A
Here we report a convenient and efficient scheme for the
synthesis of A,B-modified D-homo lactone androstane deriva-
tives, including A,B-condensed isoxazoles. Structures of reac-
Compound 8
b
…
O(1)–H(1) O(5)
0.89(3)
0.78(3)
0.87(3)
1.85(3)
2.04(3)
2.07(3)
2.725(4)
2.818(4)
2.936(4)
168(3)
173(3)
175(3)
c
…
O(5)–H(5A) O(1)
d
…
O(5)–H(5B) N(1)
1
tion products were confirmed by IR, H NMR, 13C NMR, and
a
b
Symmetry transformations used to generate equivalent atoms. 21
HRMS, and by X-ray crystallography for the A,B-condensed
isoxazoles. The introduction of an isoxazole group increases
c
d
+ x,1 + y, z. 2x,21/2 + y,1/2 2 z. 1 + x,y,z.
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homo lactone androstane derivatives could be used to develop
new, selective antiproliferative agents, particularly against
ER2 breast cancers.
4. Experimental
4.1. General
Melting points were determined using a Electrothermal 9100
apparatus and are uncorrected. Infrared spectra (wave num-
bers in cm21) were recorded in KBr pellets (for crystals) on a
NEXUS 670 SP-IR spectrometer. NMR spectra were recorded
using a Bruker AC 250E spectrometer operating at 250 MHz
(proton) and 62.5 MHz (carbon) with tetramethylsilane as the
internal standard. Chemical shifts are given in ppm (d-scale).
High resolution mass spectra (HRMS) were recorded on a 6210
Time-of-Flight LC/MS Agilent Technologies (ESI+) instrument.
Chromatographic separations were performed on silica gel
columns (Kieselgel 60, 0.063–0.20 mm, Merck). All reagents
used were of analytical grade.
4.2. 5a-Hydroxy-17-oxa-D-homoandrostane-6,16-dion-3b-yl
acetate (4)
Fig. 3 Calculated molecular electrostatic potential (MEP) map of A,B-isoxazole
D-homo lactone androstane derivatives 7 (upper) and 8 (lower). MEP partial
charges are colored according to the gradient shown to the left of each
structure. Note the significant negative partial charge gained upon addition of
the isoxazole group.
In a solution of compound 1 (0.2205 g, 0.64 mmol) in
dichloromethane (3 mL) at 0 uC, MCPBA (0.1580 g, 0.92 mmol)
was added and the mixture was stirred for 1 h 30 min at 0 uC,
and after that for 1 h at room temperature. Acetone (8 mL) was
added and the mixture was cooled to 0 uC in an ice bath before
the addition of a solution of CrO3 (0.3235 g, 3.24 mmol) in
water (1 mL). The ice bath was removed and the mixture
stirred at room temperature for 20 min, then again cooled to 0
uC in the ice bath prior to the drop wise addition of a solution
of CrO3 (0.1617, 1.62 mmol) in water (0.5 mL). The ice bath
was removed and the mixture stirred at room temperature for
50 min. The reaction mixture was poured into water (25 mL)
and extracted with dichloromethane (3 6 20 mL). The organic
layer was washed with water (1 6 20 mL), 10% NaHCO3
solution (1 6 20 mL) and saturated NaCl solution (1 6 20
mL), dried (anh. Na2SO4) and evaporated to afford the crude
product, which was purified on a silica gel column (5 g,
hexane–ethyl-acetate, 2 : 1 and 1 : 1) to give pure compound 4
(0.1323 g, 54.9%, mp >300 uC after recrystallization from
hexane–ethyl-acetate). IR (KBr) nmax cm21: 3431, 2944, 1716,
1381, 1243, 1031, 754. 1H NMR (250 MHz, CDCl3): dH = 0.82 (3
H, s, H-18), 1.01 (3 H, s, H-19), 2.03 (3 H, s, CH3 from OAc),
2.60 (1 H, d, J = 5.8 Hz, H-7), 2.69 (1 H, dd, J1 = 5.6 Hz, J2 = 18.5
Hz, H-15b), 2.80 (1 H, bs, 5a-OH), 3.92 (1 H, d, J = 10.8 Hz,
the hydrogen bonding potential of the steroidal A,B-ring.
Strong antiproliferative activity was observed for three com-
pounds against prostate (PC-3), cervical (HeLa) and colon (HT-
29) cancer cells, while no compounds showed antiproliferative
activity to K562 cells and non-cancerous control cells (MRC-5).
In addition, several compounds displayed selective antiproli-
ferative activity against ER2 vs. ER+ breast cancer cells.
Steroidal isoxazole 8 showed antiproliferative activity only to
ER2 breast cancer cells and inverse docking suggests that 8
interacts with proteins differentially expressed in ER2 vs. ER+
breast cancers. Based on the present study, A,B-modified D-
Table 3 Antiproliferative activity against a panel of human cancer cell lines
IC50 (mM)
Compounds MCF-7 MDA-MB-231 PC-3 HeLa HT-29 K562 MRC-5c
H-17a), 4.00 (1 H, d, J = 10.9 Hz, H-17b), 5.03 (1 H, m, H-3). 13
C
125
225
425
5
6
7
.100 23.54
.100 12.98
.100 15.67
.100 35.47
81.32 11.88
.100 49.76
.100 11.24
79.59 .100
78.31 .100 61.16 .100 .100
2.64 .100 .100 .100 .100
66.87 .100 .100 .100 .100
45.21 97.34 .100 .100 .100
36.65 35.97 3.97 .100 .100
25.56 .100 .100 .100 .100
.100 .100 .100 .100 .100
.100 4.97 .100 .100 .100
48.36 5.55 .100 .100 .100
95.61 1.17 0.32 0.36 0.12
NMR (62.5 MHz, CDCl3): dC = 15.04 (C-18), 19.67 (C-19), 21.33
(CH3 from OAc), 26.11, 29.25, 29.64, 31.42, 32.10, 32.71, 34.21,
37.56, 39.51, 42.08, 43.11, 44.43, 70.50 (C-3), 79.63 (C-5), 80.83
(C-17), 170.28 (C16LO), 171.17 (CLO from OAc), 211.02 (C6LO).
HRMS (TOF), m/z: for C21H30O6: [M + H]+ calcd. 379.21152,
found 379.21164.
8
12
Formestanea .100 55.5
Doxorubicinb 0.75
0.12
4.3. 17-Oxa-D-homoandrost-4-ene-6,16-dion-3b-yl acetate (5)
a
Control steroidal compound in clinical use for treatment of breast
Thionyl chloride (0.3 mL, cooled to 0 uC) was added to
5a-hydroxy-17-oxa-D-homoandrostane-6,16-dion-3b-yl acetate
b
c
cancer. Control antiproliferative compound. Normal control cells.
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stirred at room temperature for 24 h. After reaction comple-
tion, water (100 mL) was added and the reaction mixture
extracted with dichloromethane (3 6 10 mL). The combined
organic extract was dried (anh. Na2SO4) and solvent was
evaporated to yield a crude product (0.835 g), which was
purified by column chromatography (10 g silica gel, hexane–
ethyl-acetate 1 : 3), affording pure compound 6 (43.2%, 0.336
g, mp 179–181 uC) after recrystallization from ethyl-acetate–
hexane. IR (KBr) nmax cm21: 3355, 2945, 1732, 1406, 1380,
1241, 1195, 1020, 754. 1H NMR (250 MHz, CDCl3): dH = 1.02 (3
H, s, H-18), 1.04 (3 H, s, H-19), 2.07 (3 H, s, CH3 from OAc),
2.83 (1 H, dd, J1 = 5.4 Hz, J2 = 18.6 Hz, H-15), 3.44 (1 H, dd, J1 =
3.2 Hz, J2 = 14.5 Hz, H-7), 3.91 (1 H, d, J = 10.8 Hz, H-17a), 3.99
(1 H, d, J = 10.8 Hz, H-17b), 5.30 (1 H, m, H-3), 5.76 (1 H, s,
Table 4 In silico ligand-based screening for possible protein targets of
compound 8
Proteina
Binding energy
PDB
ADP-ribosylation factor 1
Cyclin homolog
59-nucleotidase
Glycogen phosphorylase
14-3-3-like protein C
Phosphoglycerate kinase 2
Adenylate kinase
Cell division protein kinase 2
6-phosphofructo-2-kinase
Tyrosine-protein kinase Lyn
Dual specificity mitogen-activated
protein kinase kinase 1
210.83
210.73
210.66
210.24
29.99
29.97
29.95
29.94
29.89
29.88
29.86
1S9D
1XO2
1HO5
1KTI
2O98
2PAA
1KHT
2IW6
2DWO
2ZV9
3EQC
1
Serine/threonine-protein kinase Chk1
Pyridoxal kinase
Eukaryotic initiation factor 4A-III
Peptidyl-prolyl cis–trans isomerase A
Death-associated protein kinase 1
Dual specificity mitogen-activated
protein kinase kinase 1
29.85
29.85
29.79
29.79
29.78
29.72
2WMU
3IBQ
3EX7
1VBS
3EH9
3EQF
H-4). H NMR (250 MHz, DMSO-d6): dH = 0.89 (3 H, s, H-18),
0.90 (3 H, s, H-19), 2.01 (3 H, s, CH3 from OAc), 5.53 (1 H, s,
H-4), 10.83 (1 H, s, =NOH). 13C NMR (62.5 MHz, CDCl3): dC
=
15.00 (C-18), 18.61 (C-19), 19.09, 21.26 (CH3 from OAc), 21.45,
25.24, 27.62, 31.67, 32.38, 34.02, 34.21, 37.73, 44.88, 51.15,
69.34, 80.75 (C-17), 123.94 (C-4), 142.66 (C-5), 156.67
(C6LNOH), 170.10 (C16LO), 170.88 (CLO from OAc). HRMS
(TOF), m/z: for C21H29NO5: [M + H]+ calcd. 376.21185, found
376.21186.
Phosphatidylinositol 3-kinase
catalytic subunit type 3
Ribosomal protein S6 kinase alpha-1
Serine/threonine-protein kinase
Aldose reductase
Cyclin-dependent protein kinase PHO85
Fatty acid-binding protein
29.7
3LS8
29.67
29.62
29.58
29.57
29.51
2Z7S
2RIO
2IKH
2PMI
3FR2
4.5. Isoxazolo[59,49,39 : 4,5,6]-17-oxa-D-homoandrost-4-en-16-
on-3b-yl acetate (7) and 3b-hydroxy-isoxazolo[59,49,39 : 4,5,6]-
17-oxa-D-homoandrost-4-en-16-one (8)
a
Proteins identified by in silico screening to bind compound 8
which are reported to have increased expression in ER-negative (MB-
MDA-231) versus ER-positive (MCF-7) breast cancer cells.
Iodine (0.165 g, 0.65 mmol), potassium iodide (0.280 g, 1.6
mmol), sodium hydrogencarbonate (0.20 g, 2.3 mmol) and
water (3 mL) were added to 6(E)-hydroximino-17-oxa-D-homo-
androst-4-en-16-on-3b-yl acetate (6, 0.238 g, 0.63 mmol)
dissolved in tetrahydrofuran (10 mL), and the reaction mixture
was stirred under reflux for 5.5 h. When the reaction was
completed, the reaction mixture was poured into water (20
mL), acidified with HCl (1 : 1), and extracted with dichlor-
omethane (4 6 10 mL). The combined organic extract was
washed with a 5% solution of sodium thiosulfate and brine,
dried and the solvent was removed to give the crude product
(0.178 g). The pure compound 7 (0.066 g, 27.9%, mp 225–227
uC) was obtained in the form of white crystals after purification
by column chromatography (5 g silica gel, hexane–ethyl-
acetate 1 : 1 and 1 : 2). Pure compound 8 was obtained in a
yield of 6.2% (0.013 g, mp 150–152 uC). Compound 7: IR (KBr)
nmax cm21: 3058, 2943, 1736, 1475, 1427, 1405, 1380, 1355,
(4, 0.173 g, 0.47 mmol) dissolved in absolute pyridine (7 mL,
cooled to 0 uC) and the reaction mixture was stirred at 0 uC for
2 h. When the reaction was complete, the mixture was poured
into water (10 mL), acidified with HCl (1 : 1) and extracted
with dichloromethane (4 6 10 mL). The combined organic
extract was dried (anh. Na2SO4) and solvent was evaporated to
yield a crude product (0.086 g). Pure compound 5 (0.062 g,
37.7%, mp 168–170 uC) was obtained in the form of white
crystals after purification by column chromatography (1 g
silica gel, hexane–ethyl-acetate 1 : 1). IR (KBr) nmax cm21: 2947,
1
1734, 1691, 1637, 1406, 1237, 1134, 1164, 1061. H NMR (250
MHz, CDCl3): dH = 1.03 (3 H, s, H-18), 1.05 (3 H, s, H-19), 2.07
(3 H, s, CH3 from OAc), 2.61 (1 H, dd, J1 = 3.9 Hz, J2 = 14.0 Hz,
H-15), 2.68 (1 H, d, J = 12.6 Hz, H-7), 3.92 (1 H, d, J = 10.8 Hz,
H-17a), 4.01 (1 H, d, J = 10.9 Hz, H-17b), 5.34 (1 H, m, H-3),
6.14 (1 H, d, J = 3.6 Hz, H-4). 13C NMR (62.5 MHz, CDCl3): dC =
14.95 (C-18), 18.96 (C-19), 19.53, 21.11 (CH3 from OAc), 24.00,
31.41, 32.29, 33.86, 34.05, 34.52, 37.90, 44.00, 44.90, 49.74,
68.97 (C-3), 80.59 (C-17), 129.83 (C-4), 146.57 (C-5), 169.74
(C16LO), 170.63 (CLO from OAc), 200.24 (C6LO). HRMS (TOF),
m/z: for C21H28O5: [M + H]+ calcd. 361.20095, found 361.20081.
1
1235, 1195, 1107, 1051, 1030, 735. H NMR (250 MHz, CDCl3):
dH = 1.08 (3 H, s, H-18), 1.22 (3 H, s, H-19), 2.14 (3 H, s, CH3
from OAc), 2.82 (1 H, dd, J1 = 5.8 Hz, J2 = 18.5 Hz, H-15), 3.03 (1
H, dd, J1 = 7.1 Hz, J2 = 17.8 Hz, H-7), 3.92 (1 H, d, J = 10.9 Hz,
H-17a), 4.01 (1 H, d, J = 10.9 Hz, H-17b), 5.86 (1 H, t, H-3, J = 8.7
Hz). 13C NMR (62.5 MHz, CDCl3): dC = 14.97 (C-18), 19.04,
19.93 (C-19), 20.96 (CH3 from OAc), 24.77, 26.52, 31.84, 32.61,
32.65, 32.93, 33.61, 36.48, 45.46, 48.29, 65.09 (C-3), 80.52 (C-
17), 125.12 (C-5), 157.87 (C-6), 161.42 (C-4), 169.83 (C16LO),
170.39 (CLO from OAc). HRMS (TOF), m/z: for C21H27NO5: [M +
H]+ calcd. 374.19620, found 374.19580. Compound 8: IR (KBr)
nmax cm21: 3409, 2939, 1727, 1405, 1381, 1339, 1233, 1195,
1051, 1028. 1H-NMR (250 MHz, Py-d5): dH = 0.89 (3 H, s, H-18),
1.05 (3 H, s, H-19), 2.89 (1 H, dd, J1 = 5.9 Hz, J2 = 18.5 Hz, H-15),
3.00 (1 H, dd, J1 = 7.2 Hz, J2 = 17.7 Hz, H-7), 3.86 (1 H, d, J = 5.5
4.4. 6(E)-Hydroximino-17-oxa-D-homoandrost-4-en-16-on-3b-yl
acetate (6)
Hydroxylamine-hydrochloride (1.10 g, 15.3 mmol) and sodium
acetate (0.464 g, 5.66 mmol) were dissolved in 95% ethanol (30
mL) and added to a solution of compound 5 (0.746 g, 2.07
mmol) in 95% ethanol (50 mL). The reaction mixture was
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Fig. 4 Antiproliferative activity of selected synthesized compounds against MDA-MB-231 cells. For- Formestane; DOX- Doxorubicin.
Hz, H-17a), 3.90 (1 H, d, J = 10.7 Hz, H-17b), 5.10 (1 H, m, H-3),
8.52 (1 H, s, OH). 13C NMR (62.5 MHz, Py-d5): 16.54 (C-18),
21.94 (C-19), 26.84, 32.63, 32.95, 34.05, 34.57, 34.75, 35.49,
38.63, 47.25, 50.02, 50.33, 65.67 (C-3), 82.37 (C-17), 131.68 (C-
5), 160.35 (C-6), 168.82 (C-4), 171.85 (C16LO). HRMS (TOF), m/z:
for C19H25NO4: [M + H]+ calcd. 332.18563, found 332.18589.
755. 1H NMR (250 MHz, Py-d5): dH = 0.81 and 0.86 (6 H, 2s, 2H-
18), 0.84 and 0.93 (6 H, 2s, 2H-19), 1.95 and 2.00 (6 H, 2s, 2CH3
from 2OAc), 3.86 (2 H, m, 2H-17), 5.62 and 5.79 (2 H, m, 2H-3),
7.77 (1 H, s, 5a-OH), 12.66 (1 H, s, =NOH). 13C NMR (62.5 MHz,
Py-d5): dH = 15.28 (2C-18), 16.41 (2C-19), 21.57, 22.74 (CH3
from OAc), 22.79 (CH3 from OAc), 24.89, 28.40, 31.36, 33.33,
34.30, 35.82, 37.10, 39.26, 41.47, 42.83, 43.90, 45.04, 72.70 and
73.25 (2C-3), 77.55 and 81.01 (2C-5), 82.26 and 82.41 (2C-17),
161.44 (C6LNOH), 171.44 and 171.87 (2CLO from 2OAc).
4.6. 6(E)-Hydroximino-5a-hydroxy-17-oxa-D-homoandrostan-
16-on-3b-yl acetate (9) and 6(Z)-hydroximino-5a-hydroxy-17-
oxa-D-homoandrostan-16-on-3b-yl acetate (10)
4.7. 6-Nitrile-17-oxa-5,6-seco-D-homoandrostane-5,16-dion-
3b-yl acetate (11)
Hydroxylamine-hydrochloride (1.75 g, 25.36 mmol) and
sodium acetate (0.74 g, 9.02 mmol) dissolved in 95% ethanol
(20 mL) were added to compound 4 (1.189 g, 3.15 mmol)
dissolved in 95% ethanol (140 mL) and the reaction mixture
was stirred at 60 uC for 1 h. After reaction completion, water
(50 mL) was added and the mixture was extracted with
dichloromethane (5 6 20 mL). The combined organic extract
was dried (over anh. Na2SO4) and evaporated to give a crude
product (1.119 g), which was purified by column chromato-
graphy (11 g silica gel, hexane–ethyl-acetate 1 : 1, 1 : 4),
affording a mixture of compounds 9 and 10 with a combined
yield of 25.2% (0.312 g, mp 284–286 uC). The mixture of
compounds 9 and 10: IR (KBr) nmax cm21: 3435, 3017, 2945,
1716, 1574, 1474, 1405, 1381, 1367, 1244, 1196, 1163, 1031,
Acetic anhydride (2 mL) was added to a mixture of 6(E)-
hydroximino-5a-hydroxy-17-oxa-D-homoandrostan-16-on-3b-yl
acetate (9) and 6(Z)-hydroximino-5a-hydroxy-17-oxa-D-homo-
androstan-16-on-3b-yl acetate (10, 0.17 g, 0.43 mmol) in
absolute pyridine (4 mL), and the reaction mixture was stirred
at 70 uC for 2 h. After reaction completion, the mixture was
poured into cold water (15 mL) and acidified with HCl (1 : 1),
then extracted with dichloromethane (5 6 5 mL). The extract
was dried (anh. Na2SO4) and evaporated to give a crude
product (0.160 g), which was further purified by column
chromatography (5 g silica gel, hexane–ethyl-acetate, 1 : 1,
1 : 3), affording compound 11 in the form of an oil (0.045 g,
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27.8%). IR (film) nmax cm21: 2947, 2245, 1732, 1638, 1474,
1441, 1406, 1367, 1243, 1197, 1031, 755. 1H NMR (250 MHz,
CDCl3): dH = 1.02 (1 H, s, H-18), 1.03 (1 H, s, H-19), 2.04 (3 H, s,
CH3 from OAc), 2.66 (3 H, m, H-4, H-7, H-15a), 3.37 (1 H, dd, J1
= 4.4 Hz, J2 = 10.5 Hz, H-15b), 3.95 (1 H, d, J = 10.9 Hz, H-17a),
4.03 (1 H, d, J = 10.9 Hz, H-17b), 5.43 (1 H, bs, H-3). 13C NMR
(62.5 MHz, CDCl3): dC = 14.84 (C-18), 16.92 (C-19), 17.41, 20.53,
20.72, 21.18, 24.98, 31.59, 32.33, 33.88, 36.67, 40.81, 41.51,
43.52, 52.10, 72.92 (C-3), 80.17 (C-17), 117.26 (C;N), 168.96
(C16LO), 170.05 (CLO from OAc), 215.66 (C5LO). HRMS (TOF),
m/z: for C21H29NO5: [M + Na]+ calcd. 398.19379, found
398.19353.
4.11. Determination of antiproliferative activity
Antiproliferative activities were measured against six different
human cancer cell lines: PC-3 (AR2 prostate cancer), MCF-7
(ER+ breast adenocarcinoma), MDA-MB-231 (ER2 breast
adenocarcinoma), HeLa (cervical cancer), HT-29 (colon cancer)
and K562 (chronic myelogenous leukemia) and one human
non-tumor cell line (normal fetal lung fibroblasts MRC-5).
Cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) with 4.5% glucose. Media were supplemented with
10% fetal calf serum (FCS, NIVNS) and antibiotics: 100 IU
mL21 penicillin and 100 lg mL21 streptomycin (ICN Galenika).
All cell lines were cultured in flasks (Costar, 25 cm2) at 37 uC in
a 100% humidity atmosphere supplemented with 5% CO2.
Only viable cells were used in the assays, and cell viability was
determined by the dye exclusion assay using trypan blue.
Antiproliferative activity was evaluated using a colorimetric
sulforhodamine B (SRB) assay.33 Briefly, single cell suspen-
sions were plated onto 96-well microtiter plates (Costar, flat
bottom): 5 6 103 cells per 180 mL of medium. Plates were pre-
incubated for 24 h at 37 uC, 5% CO2. Test substances were
added to all the wells (except controls) at concentrations
ranging from 1028 to 1024 mol L21. After the incubation
period (48 h/37 uC/5% CO2), the SRB assay was carried out as
follows: 50 mL of 80% trichloroacetic acid (TCA) was added to
all the wells; after 1 h incubation, the plates were washed with
distilled water, and 75 mL of 0.4% SRB was added to all the
wells; 30 min later the plates were washed with citric acid (1%)
and dried at room temperature. Finally, 200 mL of 10 mmol
Tris (pH = 10.5) base was added to all the wells. Absorbance (l
4.8. 4b,5b-Epoxy-3b-hydroxy-17-oxa-D-homoandrostane-6,16-
dione (12)
Sodium hydroxide (3 mL, 4 mol dm23) and 30% hydrogen
peroxide (4.5 mL) were added to a solution of 17-oxa-D-
homoandrost-4-ene-6,16-dion-3b-yl acetate (5, 0349 g, 0.96
mmol) in methanol (10 mL), and the reaction mixture was
stirred for 3 h at 0 uC. Upon reaction completion, the mixture
was acidified with HCl (1 : 1) and extracted with dichloro-
methane (4 6 10 mL). The extract was dried (anh. Na2SO4),
and evaporated to give a crude product (0.278 g), which was
further purified by column chromatography (6 g silica gel,
hexane–ethyl-acetate, 6 : 1 and 1 : 5), affording compound 12
(38.0%, 0.123 g, mp 253–255 uC). IR (KBr) nmax cm21: 3446,
2948, 1723, 1456, 1406, 1383, 1268, 1245, 1196, 1060, 1036,
926. 1H NMR (250 MHz, CDCl3): dH = 1.05 (3 H, s, H-18), 1.06 (3
H, s, H-19), 2.67 (1 H, dd, J1 = 3.4 Hz, J2 = 9.3 Hz, H-15), 2.72 (1
H, d, J = 3.2 Hz, H-7), 3.32 (1 H, d, J = 3.1 Hz, H-4a), 3.92 (1 H,
d, J = 10.9 Hz, H-17a), 4.03 (1 H, d, J = 11.0 Hz, H-17b), 4.07 (1
H, s, H-3a). 13C NMR (62.5 MHz, CDCl3): dC = 14.87 (C-18),
18.18 (C-19), 19.65, 25.53, 29.22, 31.49, 32.33, 33.94, 35.13,
37.70, 44.33, 44.79, 47.08, 62.85 (C-4), 64.63 (C-3), 69.20 (C-5),
80.51 (C-17), 169.54 (C16LO), 203.78 (C6LO). HRMS (TOF), m/z:
for C19H26O5: [M + H]+ calcd. 335.18530, found 335.18451.
540/690 nm) was measured using
a microplate reader
(Multiscan MCC340, Labsystems). Wells containing complete
media only were used as blanks.
Antiproliferative activity was calculated according to the
formula:
ꢀ
ꢁ
1{Atest
100
Acontrol
4.9. X-ray crystal structure determination
and expressed as a percent of antiproliferative activity (CI%).
Data Analysis Two independent experiments were set out in
quadruplicate for each concentration of the test compounds.
Reported IC50 values define the dose of compound that
inhibits cell growth by 50%. The IC50 of the test compounds
was determined by median effect analysis.
X-ray diffraction data for compounds 7 and 8 were collected at
room temperature on an Oxford Diffraction Gemini
S
diffractometer. Data reduction for compounds was performed
with the program package CrysAlis RED.45 Space group
determinations were based on the analysis of the Laue class
and systematically absent reflections. Structures were solved
by direct methods using SIR92.46 Structures were refined using
full-matrix least-squares. Non-hydrogen atoms were refined
anisotropically for both compounds, H atoms were treated by a
mixture of independent and constrained refinement. All
calculations were performed using SHELXL97,47 PARST48 and
PLATON,49 as implemented in the WINGX50 system of
programs. Crystal data and refinement parameters are shown
in Table 1.
4.12. Ligand-based reverse molecular docking
Ligand-based virtual screening was conducted in silico against
the entire protein data bank (PDB) using the program
idTarget.37 idTarget requires the three-dimensional structure
of an input ligand molecule and performs rapid molecular
docking against protein structures in the PDB using MEDock,
followed by rescoring using an Autodock4 scoring function.37
Proteins were considered to be possible ligand targets if the
Autodock4 binding energy was stronger than 29.5 kcal mol21
and the idTarget Z-score was negative. This preliminary list
was then further filtered by comparison with published lists of
proteins shown to have increased expression in estrogen
receptor negative breast cancers versus ER+ breast cancer
cells.42–44
4.10. Molecular electrostatic potential map calculations
Molecular electrostatic potential (MEP) maps were calculated
from the crystal structures of 7 and 8 using a GAMESS ab initio
quantum chemistry package51 (RHF/3-21G level of theory) and
were visualized using the program MOLEKEL.52
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´
´
ˇ
´
R. Klisuric, N. Andric and R. Kovacevic, Steroids, 2005,
70, 47.
Acknowledgements
We would like to thank the Ministry of Education, Science and
Technological Development of the Republic of Serbia for
financial support (Grant No. 172021).
´
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25 E. A. Djurendic, M. P. Savic, O. R. Klisuric, M. N. Sakac, G.
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M. Bogdanovic, D. S. Jakimov and K. M. Penov Gasi, Struct.
Chem., 2012, 23, 1761.
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26 A. Rosado-Abon, M. Romero-Avila and M. A. Iglesias-
Arteaga, ARKIVOC, 2010, 110.
27 J. G. Cui, L. Fan, L. L. Huang, H. L. Liu and A. M. Zhou,
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