Synthesis and in vitro antiproliferative evaluation of d-secooxime derivatives of 13β- and 13α-estrone

Article abstract of DOI:10.1016/j.steroids.2014.08.015

d-Secooximes were synthesized from the d-secoaldehydes in the 13β- and 13α-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide-alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-d-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC₅₀ values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade.

Full text of DOI:10.1016/j.steroids.2014.08.015

Steroids 89 (2014) 47–55
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and in vitro antiproliferative evaluation of D-secooxime
derivatives of 13b- and 13
a
-estrone
a
a
a
a
Erzsébet Mernyák a, , Gabriella Fiser , Johanna Szabó , Brigitta Bodnár , Gyula Schneider ,
⇑
Ida Kovács b, Imre Ocsovszki c, István Zupkó b, János Wölfling a
a Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
b Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
c Department of Biochemistry, University of Szeged, Dóm tér 9, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2014
Received in revised form 8 July 2014
Accepted 6 August 2014
Available online 20 August 2014
D
-Secooximes were synthesized from the D-secoaldehydes in the 13b- and 13a-estrone series. The oximes
were modified at three sites in the molecule: the oxime function was transformed into an oxime ether,
oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed
in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide–alkyne
cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-D-secooximes and benzyl azides. All the products were
evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent
cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC50
values, better than that of the reference agent cisplatin. The structural modifications led to significant
differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents
resulted in a cell cycle blockade.
Keywords:
Secoestrone
13a-Estrone
Oxime
Antitumor activity
Cell cycle blockade
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
have no significant uterotropic activity. These results suggest that
inversion at C-13 in the estrane skeleton could be a powerful strat-
Various types of substituted estrone analogs or homoestrones
have been synthesized as cytotoxic or cytostatic (antiproliferative)
anticancer agents [1–4]. One of the major requirements for the
pharmacological use of antitumor estrone derivatives is that they
must be devoid of estrogenic activity [5]. Natural 13b-estra-
1,3,5(10)-trienes possess a rigid steroidal framework with an aro-
matic A ring, while the B ring has a half-chair or sofa conformation,
and the C ring possesses a chair conformation. The well-defined
distances of the oxygen functionalities in estrone or estradiol are
important for their estrogen receptor-binding capabilities and their
biological activities. In contrast with the natural derivatives, the
egy in the design of non-estrogenic enzyme inhibitors. Addition-
ally, -homologization or substitution at C-2 of the estrane
skeleton usually leads to the complete loss of hormonal behaviour
[8].
2-Ethoxyestradiol is potent both as a cytotoxic agent and as a
tubulin polymerization inhibitor [9]. Its oximation at C-6 increased
its cytotoxicity from micromolar to nanomolar IC50 values, but
without an antitubulin effect. We recently synthesized 16-oxime
D
derivatives of 13b- and 13a-estrone, determined their cytotoxic
effects on human cancer cell lines and suggested their possible
mechanism of action [10]. The 16-oxime of estrone selectively
epimeric 13a-estra-1,3,5(10)-trienes have
a
relatively flexible
inhibits the proliferation of HeLa cells (IC50 = 4.41 lM), results in
molecular framework and different distances between the oxygen
functions, which influence their receptor binding affinities [6].
Poirier et al. recently reported the impact of estradiol structural
modifications (inversion of the configuration at C-13 and/or
C-17) on the in vitro and in vivo estrogenic activity [7]. It has been
found that the 13-epimers have a relative binding affinity for estro-
gen receptor alpha that is 1–2% of that of 17b-estradiol and they
cell cycle arrest in the S phase and significantly increases cas-
pase-3 activity, confirming the induction of programmed cell
death. Derivatization of the oxime function, etherification of the
3-phenolic group and inversion at C-13 led to steroids with
different antiproliferative properties. One 13
a derivative, the
16-oxime propionate in the 3-benzyl ether series, proved to be as
potent as the reference agent cisplatin, but with much better
tumor selectivity. These promising results encouraged us to con-
tinue our investigations in the field of tumor-selective estrone
oxime derivatives.
⇑
Corresponding author. Fax: +36 (62)544199.
E-mail address: bobe@chem.u-szeged.hu (E. Mernyák).
http://dx.doi.org/10.1016/j.steroids.2014.08.015
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

48
E. Mernyák et al. / Steroids 89 (2014) 47–55
Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC) is a widely
1.00 mmol). Yield: 362 mg (93%), mp 124–126 °C, Rf = 0.42 (2%
ethyl acetate/98% dichloromethane). 1H NMR (500 MHz, CDCl3): d
[ppm] = 1.12 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 5.00 (m, 2H, 16a-
H2), 5.05 (s, 2H, OCH2), 5.85 (m, 1H, 16-H), 6.74 (d, 1H, J = 2.2 Hz,
4-H), 6.81 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.21 (d, 1H,
J = 8.6 Hz, 1-H), 7.32 (s, 1H, 17-H), 7.33 (t, 1H, J = 7.2 Hz, 40-H),
7.40 (t, 2H, J = 7.2 Hz, 30-H and 50-H), 7.45 (d, 2H, J = 7.2 Hz, 20-H
and 60-H), 7.85 (s, 1H, NOH). 13C NMR d [ppm] = 15.6 (C-18),
25.8, 27.3, 30.2, 34.1, 37.7, 40.6, 41.5 (C-13), 43.2, 47.5, 69.9
(OCH2), 112.5 (C-2), 114.5 (C-4), 115.0 (C-16a), 126.4 (C-1), 127.4
(2C, C-20 and C-60), 127.8 (C-40), 128.5 (2C: C-30 and C-50), 132.4
(C-10), 137.2 (C-10), 137.9 (C-5), 139.3 (C-16), 156.8 (C-3), 160.4
(C-17). Anal. Calcd. for C26H31NO2: C, 80.17; H, 8.02. Found: C,
79.97; H, 8.16%.
applicable and facile method for the introduction of a triazole moi-
ety into the molecules [11]. Incorporation of a triazole ring into the
estrane skeleton has an additional advantage: it may enhance the
solubility and bioavailability [12]. Triazoles are attractive units
because of their stability against metabolic degradation and their
ability to form hydrogen-bonds. We have recently reported the
synthesis of various steroidal triazoles via the CuAAC reaction
[13–17].
The aim of the present study was to synthetize
D-secooximes in
the 13b- and 13 -estrone series. Structural modifications of the
a
secooximes were additionally planned in order to investigate the
structure–activity relationships: etherification or esterification of
the oxime function, removal of the 3-benzyl group and saturation
of the propenyl side-chain. A propargyl function was incorporated
into the
D
-secooxime and triazoles were formed from the secoe-
2.1.1.3.
3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)-
strone alkyne with benzyl azides via Cu(I)-catalysed azide–alkyne
cycloaddition (CuAAC). All the synthesized compounds were tested
in vitro by means of MTT assays for their antiproliferative activity
against a panel of human adherent cell lines (HeLa, MCF-7,
A2780 and A431).
trien-13b-carbaldehyde oxime (7). From compound
3
(298 mg,
1.00 mmol). Yield: 295 mg (94%); mp 89–90 °C. 7 is identical with
the compound described in the literature, mp 90–92 °C [19]. Anal.
Calcd. for C20H27NO2: C, 76.64; H, 8.68. Found: C, 76.51; H, 8.76%.
2.1.1.4. 3-Benzyloxy-14b-(prop-2-en-yl)-des-
D
-13a-estra-1,3,5(10)-
trien-13b-carbaldehyde oxime (8). From compound
4
(374 mg,
2. Experimental
1.00 mmol). Yield: 358 mg (92%), mp 105–107 °C, Rf = 0.52 (dichlo-
romethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21 (s, 3H, 18-
H3), 2.83 (m, 2H, 6-H2), 4.98–5.06 (m, 2H, 16a-H2), 5.03 (s, 2H,
OCH2), 5.84 (m, 1H, 16-H), 6.71 (d, 1H, J = 2.2 Hz, 4-H), 6.78 (dd,
1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.00 (s, 1H, 17-H), 7.19 (d, 1H,
J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40-H), 7.38 (t, 2H, J = 7.2 Hz,
30-H and 50-H), 7.42 (d, 2H, J = 7.2 Hz, 20-H and 60-H), 7.57 (s, 1H,
NOH). 13C NMR d [ppm] = 26.4 (C-18), 27.0, 27.4, 30.3, 33.3, 39.2,
40.9 (C-13), 42.3, 43.5, 50.9, 70.0 (OCH2), 112.5 (C-2), 114.5 (C-4),
114.9 (C-16a), 126.4 (C-1), 127.4 (2C, C-20 and C-60), 127.8 (C-40),
128.5 (2C: C-30 and C-50), 132.5 (C-10), 137.3 (C-10), 137.8 (C-5),
139.4 (C-16), 156.0 (C-17), 156.8 (C-3). Anal. Calcd. for
2.1. Chemistry
Melting points (mp) were determined with a Kofler hot-stage
apparatus and are uncorrected. Elemental analyses were per-
formed with a Perkin–Elmer CHN analyzer model 2400. Thin-layer
chromatography: silica gel 60 F254
; layer thickness 0.2 mm
(Merck); detection with iodine or UV (365 nm) after spraying with
5% phosphomolybdic acid in 50% aqueous phosphoric acid and
heating at 100–120 °C for 10 min. Flash chromatography: silica
gel 60, 40–63 lm (Merck). The reactions under microwave irradia-
tion were carried out with a CEM Corporation Focused Microwave
System, Model Discover SP. 1H NMR spectra were recorded in
CDCl3 solution (if not otherwise stated) with a Bruker DRX-500
instrument at 500 MHz, with Me4Si as internal standard. 13C
NMR spectra were recorded with the same instrument at
125 MHz under the same conditions.
C26H31NO2: C, 80.17; H, 8.02. Found: C, 79.98; H, 8.17%.
2.1.2. General procedure for the hydrogenolysis and the saturation of
the alkenyl side-chain
A suspension of oxime 5 or 7 (314 mg, 1.00 mmol) or 6 or 8
(390 mg, 1.00 mmol) and Pd/C (0.60 g, 10%) in ethyl acetate
(20 mL) was subjected to 30 bar of H2 pressure at room tempera-
ture for 2 h. The catalyst was then removed by filtration through
a short pad of silica gel. After evaporation of the solvent, the crude
product was subjected to flash chromatography.
2.1.1. General procedure for the synthesis of
3-methoxy- or 3-benzyloxy-14b-(prop-2-en-yl)-des-
-trien-13 -carbaldehyde (1, 2) or from 3-methoxy- or 3-benzyloxy-
D-secooximes (5–8) from
D-estra-1,3,5(10)
a
14b-(prop-2-en-yl)-des-
D
-estra-1,3,5(10)-trien-13b-carbaldehyde (3,
4)
2.1.2.1. 3-Methoxy-14b-propyl-des-D-estra-1,3,5(10)-trien-13a-carb-
Compound 1 or 3 (298 mg, 1.00 mmol) or 2 or 4 (374 mg,
1.00 mmol) was dissolved in acetonitrile (10 mL), and water
(4 mL), hydroxylamine hydrochloride (85 mg, 1.2 mmol) and
sodium acetate (125 mg, 1.5 mmol) were added. The mixture was
stirred at room temperature for 1 h, then evaporated to half vol-
ume and diluted with water (50 mL). The precipitate that formed
was filtered off, washed with water until neutral and dried in the
air. The crude product was subjected to flash chromatography over
silica gel with dichloromethane as eluent.
aldehyde oxime (9). From compound 5 (314 mg, 1.00 mmol). Col-
umn chromatography over silica gel with dichloromethane
yielded 268 mg (85%), mp 129–131 °C, Rf = 0.48 (2% ethyl acetate/
98% dichloromethane). 1H NMR (500 MHz, DMSO-d6):
d
[ppm] = 0.83 (t, 3H, J = 6.8 Hz, 16a-H3), 0.97 (s, 3H, 18-H3), 2.78
(m, 2H, 6-H2), 3.69 (s, 3H, 3-OCH3), 6.60 (d, 1H, J = 2.2 Hz, 4-H),
6.67 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.17 (s, 1H, 17-H), 7.18
(d, 1H, J = 8.6 Hz, 1-H), 10.38 (s, 1H, NOH). 13C NMR
d
[ppm] = 14.4 and 15.4 (C-16a and C-18), 23.8, 25.7, 26.8, 29.9,
31.5, 37.1, 40.5, 40.6 (C-13), 42.7, 47.0, 54.8 (3-OCH3), 111.6 (C-
2), 113.0 (C-4), 126.3 (C-1), 131.8 (C-10), 137.3 (C-5), 157.0 (C-3),
157.8 (C-17). Anal. Calcd. for C20H29NO2: C, 76.15; H, 9.27. Found:
C, 76.32; H, 9.05%.
2.1.1.1. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13a-carbaldehyde oxime (5). From compound
1
(298 mg,
1.00 mmol). Yield: 301 mg (96%); mp 130–132 °C. 5 is identical
with the compound described in the literature, mp 131–133 °C
[18]. Anal. Calcd. for C20H27NO2: C, 76.64; H, 8.68. Found: C,
76.72; H, 8.55%.
2.1.2.2. 3-Methoxy-14b-propyl-des-D-13a-estra-1,3,5(10)-trien-13b-
carbaldehyde oxime (10). From compound 7 (314 mg, 1.00 mmol).
Column chromatography over silica gel with dichloromethane
yielded 262 mg (83%), mp 127–130 °C, Rf = 0.47 (2% ethyl
acetate/98% dichloromethane). 1H NMR (500 MHz, DMSO-d6):
2.1.1.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13a-carbaldehyde oxime (6). From compound
2
(374 mg,

E. Mernyák et al. / Steroids 89 (2014) 47–55
49
d [ppm] = 0.86 (t, 3H, J = 6.8 Hz, 16a-H3), 1.18 (s, 3H, 18-H3), 2.84
(m, 2H, 6-H2), 3.76 (s, 3H, 3-OCH3), 6.63 (d, 1H, J = 2.2 Hz, 4-H),
6.72 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-
(C-17). Anal. Calcd. for C27H33NO2: C, 80.36; H, 8.24. Found: C,
80.15; H, 8.17%.
H), 7.23 (s, 1H, 17-H), 10.39 (s, 1H, NOH). 13C NMR
d
2.1.3.2.
3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)-
[ppm] = 23.6 and 25.9 (C-16a and C-18), 26.7, 27.6, 30.2, 34.3,
38.9, 40.3, 39.7 (C-13), 42.8, 42.9, 49.6, 55.3 (3-OCH3), 112.6 (C-
2), 114.6 (C-4), 126.5 (C-1), 131.8 (C-10), 137.7 (C-5), 156.9 (C-3),
157.6 (C-17). Anal. Calcd. for C20H29NO2: C, 76.15; H, 9.27. Found:
C, 76.28; H, 9.32%.
trien-13b-carbaldehyde oxime benzyl ether (14). From compound 7
(314 mg, 1.00 mmol). Yield: 331 mg (82%), oil, Rf = 0.45 (60%
dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d
[ppm] = 1.21 (s, 3H, 18-H3), 2.82 (m, 2H, 6-H2), 3.79 (s, 3H,
OCH3), 4.98 (m, 2H, 16a-H2), 5.05 (s, 2H, OCH2), 5.83 (m, 1H, 16-
H), 6.64 (d, 1H, J = 2.2 Hz, 4-H), 6.73 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz,
2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.24 (t, 1H, J = 7.2 Hz, 40-H),
7.28 (t, 2H, J = 7.2 Hz, 30-H and 50-H), 7.43 (d, 2H, J = 7.2 Hz, 20-H
and 60-H), 7.57 (s, 1H, 17-H). 13C NMR d [ppm] = 26.6 (C-18),
27.0, 27.4, 30.4, 33.3, 39.1, 41.0 (C-13), 42.3, 43.5, 50.9, 55.2
(OCH3), 75.6 (OCH2), 111.6 (C-2), 113.4 (C-4), 114.6 (C-16a),
126.4 (C-1), 127.6 and 128.2 and 128.3 (5C, C-20, C-30, C-40, C-50
and C-60), 132.4 (C-10), 137.8 (2C, C-10 and C-5), 139.7 (C-16),
155.3 (C-17), 157.5 (C-3).
2.1.2.3. 3-Hydroxy-14b-propyl-des-D-estra-1,3,5(10)-trien-13a-carb-
aldehyde oxime (11). From compound 6 (390 mg, 1.00 mmol). Col-
umn chromatography over silica gel with 30% tert-butyl methyl
ether/60% n-hexane yielded 238 mg (79%), mp 133–135 °C,
Rf = 0.48 (10% ethyl acetate/90% dichloromethane). 1H NMR
(500 MHz, DMSO-d6): d [ppm] = 0.82 (t, 3H, J = 6.8 Hz, 16a-H3),
0.97 (s, 3H, 18-H3), 2.71 (m, 2H, 6-H2), 6.42 (d, 1H, J = 2.2 Hz, 4-
H), 6.51 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.05 (d, 1H, J = 8.6 Hz,
1-H), 7.17 (s, 1H, 17-H), 9.00 (s, 1H, 3-OH), 10.38 (s, 1H, NOH).
13C NMR d [ppm] = 14.4 and 15.4 (C-16a and C-18), 23.8, 25.7,
26.9, 29.7, 31.5, 37.2, 40.6 (C-13), 40.7, 42.7, 47.0, 112.8 (C-2),
114.5 (C-4), 126.2 (C-1), 130.0 (C-10), 137.0 (C-5), 154.9 (C-3),
157.8 (C-17). Anal. Calcd. for C19H27NO2: C, 75.71; H, 9.03. Found:
C, 75.83; H, 8.98%.
Anal. Calcd. for C27H33NO2: C, 80.36; H, 8.24. Found: C, 80.25; H,
8.32%.
2.1.3.3. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13a-carbaldehyde oxime allyl ether (15). From compound
5
(314 mg, 1.00 mmol). Yield: 276 mg (78%), oil, Rf = 0.42 (60%
dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d
[ppm] = 1.06 (s, 3H, 18-H3), 2.81 (m, 2H, 6-H2), 3.75 (s, 3H,
OCH3), 4.52 (m, 2H, OCH2), 4.95 (m, 2H, 16a-H2), 5.26 (m, 2H, –
CH = CH2), 5.82 (m, 1H, 16-H), 5.96 (m, 1H, –CH@CH2), 6.60 (d,
1H, J = 2.2 Hz, 4-H), 6.70 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.17
(d, 1H, J = 8.6 Hz, 1-H). 13C NMR d [ppm] = 15.7 (C-18), 25.8, 27.4,
30.3, 34.2, 37.9, 40.9, 41.5 (C-13), 43.2, 47.4, 55.2 (OCH3), 74.4
(OCH2), 111.7 (C-2), 113.4 (C-4), 114.7 (C-16a), 117.5 (–CH@CH2),
126.4 (C-1), 132.2 (C-10), 134.3 (–CH@CH2), 137.9 (C-5), 139.7
(C-16), 157.5 (C-3), 159.3 (C-17). Anal. Calcd. for C23H31NO2: C,
78.15; H, 8.84. Found: C, 78.23; H, 8.98%.
2.1.2.4. 3-Hydroxy-14b-propyl-des-D-13a-estra-1,3,5(10)-trien-13b-
carbaldehyde oxime (12). From compound 8 (390 mg, 1.00 mmol).
Column chromatography over silica gel with 30% tert-butyl methyl
ether/60% n-hexane yielded 232 mg (77%), mp 163–167 °C,
Rf = 0.39 (2% ethyl acetate/98% dichloromethane). 1H NMR
(500 MHz, DMSO-d6): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3),
1.09 (s, 3H, 18-H3), 2.70 (m, 2H, 6-H2), 6.42 (d, 1H, J = 2.2 Hz, 4-
H), 6.51 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.03 (d, 1H, J = 8.6 Hz,
1-H), 7.38 (s, 1H, 17-H), 9.00 (s, 1H, 3-OH), 10.47 (s, 1H, NOH).
13C NMR d [ppm] = 14.3 and 26.6 (C-18 and C-16a), 24.6, 26.8,
26.9, 29.9, 31.4, 38.7, 40.2, 42.3, 42.9, 50.7, 112.8 (C-2), 114.5 (C-
4), 126.2 (C-1), 130.2 (C-10), 137.0 (C-5), 153.5 (C-17), 154.8 (C-
3). Anal. Calcd. for C19H27NO2: C, 75.71; H, 9.03. Found: C, 75.79;
H, 9.12%.
2.1.3.4.
3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)-
trien-13b-carbaldehyde oxime allyl ether (16). From compound 7
(314 mg, 1.00 mmol). Yield: 286 mg (81%), oil, Rf = 0.43 (60%
dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d
[ppm] = 1.20 (s, 3H, 18-H3), 2.83 (m, 2H, 6-H2), 3.78 (s, 3H,
OCH3), 4.50 (m, 2H, OCH2), 4.96–5.06 (overlapping multiplets,
2H, 16a-H2), 5.15–5.28 (overlapping multiplets, 2H, –CH@CH2),
5.85 (m, 1H, 16-H), 5.95 (m, 1H, –CH@CH2), 6.62 (d, 1H,
J = 2.2 Hz, 4-H), 6.71 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d,
1H, J = 8.6 Hz, 1-H), 7.56 (s, 1H, 17-H). 13C NMR d [ppm] = 26.6
(C-18), 27.1, 27.4, 30.4, 33.4, 39.2, 40.9 (C-13), 42.4, 43.6, 50.9,
55.1 (3-OCH3), 74.3 (OCH2), 111.7 (C-2), 113.4 (C-4), 114.7
(C-16a), 117.4 (–CH@CH2), 126.4 (C-1), 132.4 (C-10), 134.2
(–CH@CH2), 137.8 (C-5), 139.7 (C-16), 154.8 (C-17), 157.5 (C-3).
Anal. Calcd. for C23H31NO2: C, 78.15; H, 8.84. Found: C, 78.06; H,
8.92%.
2.1.3. General procedure for the synthesis of oxime ethers (13–16)
To a solution of oxime 5 or 7 (314 mg, 1.00 mmol) in acetoni-
trile (10 mL), water (4 mL), O-benzylhydroxylamine hydrochloride
(160 mg, 1.00 mmol) or O-allylhydroxylamine hydrochloride
(112 mg, 1.00 mmol) and sodium acetate (105 mg, 1.25 mmol)
were added. The mixture was stirred at room temperature for
6 h, then evaporated to half volume and extracted with dichloro-
methane. The organic phase was washed with water until neutral,
dried over sodium sulfate and evaporated to dryness. The residue
obtained was subjected to flash chromatography on a silica gel col-
umn with dichloromethane as eluent.
2.1.3.1. 3-Methoxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13 -carbaldehyde oxime benzyl ether (13). From compound
a
5
2.1.4. General procedure for the synthesis of oxime esters (17–20)
To a solution of oxime 5 or 7 (314 mg, 1.00 mmol) or 6 or 8
(390 mg, 1.00 mmol) in pyridine (5 mL), acetic anhydride (2 mL,
21.2 mmol) was added. The mixture was stirred at room tempera-
ture for 1 h, then poured into water (50 mL) and extracted with
dichloromethane. The organic phase was washed with water until
neutral, dried over sodium sulfate and evaporated to dryness. The
residue obtained was subjected to flash chromatography on a silica
gel column with dichloromethane as eluent.
(314 mg, 1.00 mmol). Yield: 347 mg (86%), oil, Rf = 0.44 (60%
dichloromethane/40% n-hexane). 1H NMR (500 MHz, CDCl3): d
[ppm] = 1.08 (s, 3H, 18-H3), 2.84 (m, 2H, 6-H2), 3.78 (s, 3H,
OCH3), 4.90–4.95 (m, 2H, 16a-H2), 5.08 (s, 2H, OCH2), 5.79 (m,
1H, 16-H), 6.63 (d, 1H, J = 2.2 Hz, 4-H), 6.72 (dd, 1H, J = 8.6 Hz,
J = 2.2 Hz, 2-H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.30 (t, 1H, J = 7.2 Hz,
40-H), 7.32 (s, 1H, 17-H), 7.34–7.39 (overlapping multiplets, 4H,
20-H, 30-H, 50-H, 60-H). 13C NMR d [ppm] = 15.7 (C-18), 25.8, 27.4,
30.3, 34.1, 37.9, 40.8, 42.0 (C-13), 43.2, 47.4, 55.2 (OCH3), 75.6
(OCH2), 111.7 (C-2), 113.5 (C-4), 114.7 (C-16a), 126.3 (C-1), 127.2
(2C, C-20 and C-60), 127.7 (C-40), 128.3 (2C: C-30 and C-50), 132.2
(C-10), 137.3 (C-10), 137.8 (C-5), 139.6 (C-16), 157.5 (C-3), 159.5
2.1.4.1. 3-Methoxy-14b-(prop-2-en-yl)-des-
13 -carbaldehyde oxime acetate (17). From compound 5 (314 mg,
1.00 mmol). Yield: 263 mg (74%), oil, Rf = 0.63 (2% ethyl acetate/
D-estra-1,3,5(10)-trien-
a

50
E. Mernyák et al. / Steroids 89 (2014) 47–55
98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21
(s, 3H, 18-H3), 2.16 (s, 3H, Ac-CH3), 2.85 (m, 2H, 6-H2), 3.78 (s,
3H, 3-OCH3), 4.96–5.03 (m, 2H, 16a-H2), 5.83 (m, 1H, 16-H), 6.63
(d, 1H, J = 2.2 Hz, 4-H), 6.73 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H),
7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.55 (s, 1H, 17-H). 13C NMR d
[ppm] = 15.3 and 19.7 (C-18 and Ac-CH3), 25.6, 27.3, 30.2, 34.2,
37.3, 40.5, 42.3 (C-13), 43.1, 47.4, 55.2 (3-OCH3), 111.7 (C-2),
113.5 (C-4), 115.3 (C-16a), 126.3 (C-1), 132.0 (C-10), 137.8 (C-5),
139.0 (C-16), 157.6 (C-3), 167.0 (C-17), 168.9 (Ac-CO). Anal. Calcd.
for C22H29NO3: C, 74.33; H, 8.22. Found: C, 74.42; H, 8.37%.
reactor. The mixture was heated for 6 min at 100 °C and then
transferred into water and extracted with dichloromethane. The
organic phase was washed with water until neutral, dried over
sodium sulfate and evaporated to dryness. The residue obtained
was subjected to flash chromatography on a silica gel column with
80% dichloromethane/20% n-hexane as eluent.
2.1.5.1. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13 -carbonitrile (21). From compound 6 (390 mg, 1.00 mmol).
a
Yield: 286 mg (77%), mp 78–82 °C, Rf = 0.54 (70% dichlorometh-
ane/30% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s,
3H, 18-H3), 2.84 (m, 2H, 6-H2), 5.04 (s, 2H, OCH2), 5.06–5.20 (2ꢀ
m, 2ꢀ 1H, 16a-H2), 5.95 (m, 1H, 16-H), 6.72 (d, 1H, J = 2.2 Hz,
4-H), 6.80 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.16 (d, 1H,
J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40-H), 7.39 (t, 2H, J = 7.2 Hz,
30-H and 50-H), 7.43 (d, 2H, J = 7.2 Hz, 20-H, 60-H). 13C NMR d
[ppm] = 17.3 (C-18), 25.1, 26.9, 30.0, 35.5, 37.4 (C-13), 37.7, 39.8,
42.8, 47.2, 69.9 (OCH2), 112.6 (C-2), 114.6 (C-4), 116.2 (C-16a),
126.1 (C-17), 126.3 (C-1), 127.4 (2C, C-20 and C-60), 127.9 (C-40),
128.5 (2C, C-30 and C-50), 131.4 (C-10), 137.1 (C-10), 137.6 (C-5),
137.8 (C-16), 157.0 (C-3). Anal. Calcd. for C26H29NO: C, 84.06; H,
7.87. Found: C, 83.92; H, 7.96%.
2.1.4.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-D-estra-1,3,5(10)-trien-
13 -carbaldehyde oxime acetate (18). From compound 6 (390 mg,
a
1.00 mmol). Yield: 354 mg (82%), oil, Rf = 0.67 (2% ethyl acetate/
98% dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.21
(s, 3H, 18-H3), 2.16 (s, 3H, Ac-CH3), 2.84 (m, 2H, 6-H2), 4.98 (m,
2H, 16a-H2), 5.04 (s, 2H, OCH2), 5.83 (m, 1H, 16-H), 6.72 (d, 1H,
J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d,
1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40-H), 7.38 (t, 2H,
J = 7.2 Hz, 30-H and 50-H), 7.42 (d, 2H, J = 7.2 Hz, 20-H and 60-H),
7.56 (s, 1H, 17-H). 13C NMR d [ppm] = 15.4 and 19.7 (C-18 and
Ac-CH3), 25.6, 27.3, 30.2, 34.2, 37.3, 40.5, 42.3 (C-13), 43.2, 47.4,
70.0 (OCH2), 112.5 (C-2), 114.5 (C-4), 115.3 (C-16a), 126.4 (C-1),
127.4 (2C: C-20 and C-60), 127.9 (C-40), 128.5 (2C: C-30 and C-50),
132.2 (C-10), 137.2 (C-10), 137.8 (C-5), 139.0 (C-16), 156.9 (C-3),
167.0 (Ac-CO), 167.1 (C-17). Anal. Calcd. for C28H33NO3: C, 77.93;
H, 7.71. Found: C, 78.03; H, 7.85%.
2.1.5.2. 3-Benzyloxy-14b-(prop-2-en-yl)-des-
D
-13a-estra-1,3,5(10)-
trien-13b-carbonitrile (22). From compound
8
(390 mg, 1.00
mmol). Yield: 275 mg (74%), mp 61–62 °C, Rf = 0.56 (70% dichloro-
methane/30% n-hexane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.45
(s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 5.05 (s, 2H, OCH2), 5.11 (m, 2H,
16a-H2), 5.95 (m, 1H, 16-H), 6.72 (d, 1H, J = 2.2 Hz, 4-H), 6.79 (dd,
1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.32 (t,
1H, J = 7.2 Hz, 40-H), 7.39 (t, 2H, J = 7.2 Hz, 30-H and 50-H), 7.43 (d,
2H, J = 7.2 Hz, 20-H and 60-H). 13C NMR d [ppm] = 25.9 (C-18),
26.8, 27.8, 30.1, 34.1, 38.9, 39.4 (C-13), 42.8, 42.9, 49.6, 69.9
(OCH2), 112.6 (C-2), 114.5 (C-4), 115.9 (C-16a), 123.5 (C-17),
126.4 (C-1), 127.4 (2C, C-20 and C-60), 127.8 (C-40), 128.5 (2C: C-
30 and C-50), 131.6 (C-10), 137.2 (C-10), 137.5 (C-160), 137.7 (C-5),
156.9 (C-3). Anal. Calcd. for C26H29NO: C, 84.06; H, 7.87. Found:
C, 84.22; H, 7.65%.
2.1.4.3.
3-Methoxy-14b-(prop-2-en-yl)-des-D-13a-estra-1,3,5(10)-
trien-13b-carbaldehyde oxime acetate (19). From compound
7
(314 mg, 1.00 mmol). Yield: 270 mg (76%), mp 53–55 °C, Rf = 0.37
(dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s,
3H, 18-H3), 2.11 (s, 3H, Ac-H3), 2.85 (m, 2H, 6-H2), 3.78 (s, 3H,
3-OCH3), 4.99–5.07 (m, 2H, 16a-H2), 5.81 (m, 1H, 16-H), 6.62 (d,
1H, J = 2.2 Hz, 4-H), 6.72 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18
(d, 1H, J = 8.6 Hz, 1-H), 7.87 (s, 1H, 17-H). 13C NMR
d
[ppm] = 19.6 and 26.0 (Ac-CH3 and C-18),, 27.0, 27.4, 30.2, 33.1,
39.0, 41.8 (C-13), 42.2, 43.4, 50.7, 55.2 (3-OCH3), 111.7 (C-2),
113.5 (C-4), 115.6 (C-16a), 126.4 (C-1), 132.0 (C-10), 137.7 (C-5),
138.6 (C-16), 157.6 (C-3), 163.2 (C-17), 168.9 (Ac-CO). Anal. Calcd.
for C22H29NO3: C, 74.33; H, 8.22. Found: C, 74.45; H, 8.16%.
2.1.6. Synthesis of the 3-(prop-2-inyloxy)-14b-propyl-des-
D-estra-
1,3,5(10)-trien-13 -carbaldehyde oxime (23)
a
Compound 11 (302 mg, 1.00 mmol) was dissolved in acetone
(10 mL), propargyl-bromide (0.17 mL (80 wt.% in toluene),
1.5 mmol) and potassium carbonate (968 mg, 7 mmol) were
added. The reaction mixture was stirred at 70 °C for 24 h, the sol-
vent was evaporated off, and the residue was subjected to flash
chromatography over silica gel with 2% ethyl acetate/98% dichloro-
methane as eluent. Yield: 302 mg (89%), oil, Rf = 0.46 (2% ethyl ace-
2.1.4.4. 3-Benzyloxy-14b-(prop-2-en-yl)-des-
trien-13b-carbaldehyde oxime acetate (20). From compound
D-13a-estra-1,3,5(10)-
8
(390 mg, 1.00 mmol). Yield: 367 mg (85%), mp 60–62 °C, Rf = 0.45
(dichloromethane). 1H NMR (500 MHz, CDCl3): d [ppm] = 1.32 (s,
3H, 18-H3), 2.12 (s, 3H, Ac-H3), 2.85 (m, 2H, 6-H2), 5.01 (m, 2H,
16a-H2), 5.03 (s, 2H, OCH2), 5.83 (m, 1H, 16-H), 6.71 (d, 1H,
J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.19 (d,
1H, J = 8.6 Hz, 1-H), 7.32 (t, 1H, J = 7.2 Hz, 40-H), 7.38 (t, 2H,
J = 7.2 Hz, 30- H and 50-H), 7.43 (d, 2H, J = 7.2 Hz, 20-H and 60-H),
7.87 (s, 1H, 17-H). 13C NMR d [ppm] = 19.6 and 26.0 (Ac-CH3 and
C-18), 27.0, 27.3, 30.2, 33.1, 39.0, 41.7 (C-13), 42.1, 43.4, 50.7,
70.0 (OCH2), 112.5 (C-2), 114.5 (C-4), 115.5 (C-16a), 126.4 (C-1),
127.4 (2C: C-20 and C-60), 127.8 (C-40), 128.5 (2C: C-30 and C-50),
132.3 (C-10), 137.2 (C-10), 137.7 (C-5), 138.6 (C-16), 156.9 (C-3),
163.2 (C-17), 168.8 (Ac-CO). Anal. Calcd. for C28H33NO3: C, 77.93;
H, 7.71. Found: C, 77.85; H, 7.89%.
tate/98% dichloromethane). 1H NMR (500 MHz, CDCl3):
d
[ppm] = 0.89 (t, 3H, J = 6.8 Hz, 16a-H3), 1.07 (s, 3H, 18-H3), 2.51
(s, 1H, C„CH), 2.86 (m, 2H, 6-H2), 4.66 (s, 2H, OCH2), 6.70 (d, 1H,
J = 2.2 Hz, 4-H), 6.79 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.21 (d,
1H, J = 8.6 Hz, 1-H), 7.30 (s, 1H, 17-H). 13C NMR d [ppm] = 14.5
and 15.4 (C-16a and C-18), 24.3, 25.9, 27.2, 30.5, 32.2, 37.4, 40.9,
41.3 (C-13), 43.3, 47.9, 55.7 (OCH2), 75.3 (C„CH), 79.9 (C„CH),
112.5 (C-2), 114.5 (C-4), 126.5 (C-1), 133.2 (C-10), 137.9 (C-5),
155.5 (C-3), 160.5 (C-17). Anal. Calcd. for C22H29NO2: C, 77.84; H,
8.64. Found: C, 77.65; H, 8.82%.
2.1.5. General procedure for the synthesis of seconitriles (21, 22)
Compound 6 or 8 (390 mg, 1.00 mmol) was dissolved in dichlo-
romethane (30 mL), and acetic anhydride (1 mL, 10.6 mmol), 4-
(dimethylamino)pyridine (183 mg, 1.50 mmol) and silica gel (1 g)
were added. The mixture was homogenized, the solvent was evap-
orated and the residue was placed into a pressure tube equipped
with a stirrer bar and was inserted into the cavity of the microwave
2.1.7. General procedure for the synthesis of the triazoles 25
To a stirred solution of the appropriate terminal alkyne (23,
1.0 mmol) in toluene (10 mL), benzyl azide (24, 1.0 mmol), tri-
phenylphosphine (52 mg, 0.2 mmol), copper(I) iodide (19 mg,
0.1 mmol) and N,N-diisopropylethylamine (0.52 mL, 3 mmol) were
added. The reaction mixture was refluxed for 2 h, the solvent
was evaporated off, and the residue was subjected to flash

E. Mernyák et al. / Steroids 89 (2014) 47–55
51
chromatography over silica gel with 10% ethyl acetate/90% dichlo-
romethane as eluent.
A2780 cells (isolated from ovarial cancer) were maintained in
RPMI medium supplemented with 10% FBS, 1% AAM and 1%
L-glutamine. All cell lines were purchased from the European Col-
2.1.7.1.
3-(1-Benzyl-1H-1,2,3-triazol-4-yl-methyloxy)-14b-propyl-
lection of Cell Cultures (Salisbury, U.K.). For pharmacological inves-
tigations, 10 mM stock solutions of the tested compounds were
prepared with dimethyl sulfoxide (DMSO). The highest applied
DMSO concentration of the medium (0.3%) did not have any
substantial effect on the determined cellular functions. All the
chemicals, if otherwise not specified, were purchased from
Sigma–Aldrich Ltd. (Budapest, Hungary). The antiproliferative
effects were determined in vitro on the four cell lines: HeLa,
A431, MCF-7 and A2780. The cells were grown in a humidified
atmosphere of 5% CO2 at 37 °C. Cells were seeded onto 96-well
plates at a density of 5000 cells/well and allowed to stand over-
night, after which the medium containing the tested compound
was added. After a 72-h incubation, viability was determined by
des- -estra-1,3,5(10)-trien-carbaldehyde oxime (25a). From com-
D
pound 23 (340 mg, 1.00 mmol) and benzyl azide (24a, 133 mg,
1.0 mmol). Yield: 435 mg (92%), mp 67–69 °C, Rf = 0.40 (10% ethyl
acetate/90% dichloromethane). 1H NMR (500 MHz, CDCl3):
d
[ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.06 (s, 3H, 18-H3), 2.83
(m, 2H, 6-H2), 5.16 (s, 2H, OCH2), 5.53 (s, 2H, NCH2), 6.69 (d, 1H,
J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H), 7.18 (d,
1H, J = 8.6 Hz, 1-H), 7.26–7.29 (overlapping multiplets, 3H:
30,40,50-H), 7.37 (s, 1H, 17-H), 7.38 (d, 2H, J = 7.3 Hz, 20,60-H), 7.52
(s, 1H, C@CH). 13C NMR d [ppm] = 14.5 and 15.4 (C-16a and C-
18), 24.3, 25.9, 27.2, 30.4, 32.2, 37.4, 40.9, 41.3 (C-13), 43.3, 47.9,
54.3 (NCH2), 62.2 (OCH2), 112.5 (C-2), 114.4 (C-4), 122.5 (C@CH),
126.5 (C-1), 128.1 (2C) and 129.1 (2C): C-20,30,50,60, 128.8 (C-40),
132.9 (C-10), 134.4 (C-10), 137.9 (C-5), 149.0 (C@CH), 156.2 (C-3),
160.5 (C-17). Anal. Calcd. for C29H36N4O2: C, 73.70; H, 7.68. Found:
C, 73.86; H, 7.54%.
the addition of 20 lL of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide] solution (5 mg/mL). The precipitated for-
mazan crystals were solubilized in DMSO and the absorbance was
determined at 545 nm with an ELISA reader [20]. Two independent
experiments were performed with 5 parallel wells; cisplatin, an
agent administered clinically in the treatment of certain gyneco-
logical malignancies, was used as a positive control. Sigmoidal
dose–response curves were fitted to the measured data. Calcula-
tions of IC50 values and statistical analyses (ANOVA) were per-
formed by means of GraphPad Prism 4.0 (GraphPad Software;
San Diego, CA, USA).
2.1.7.2.
14b-propyl-des-
3-(1-(4-Prop-2-ylbenzyl-1H-1,2,3-triazol-4-yl-methyloxy)-
-estra-1,3,5(10)-trien-carbaldehyde oxime (25b).
D
From compound 23 (340 mg, 1.00 mmol) and 4-prop-2-ylbenzyl
azide (24b, 176 mg, 1.0 mmol). Yield: 464 mg (90%), mp 59–
62 °C, Rf = 0.44 (10% ethyl acetate/90% dichloromethane). 1H NMR
(500 MHz, CDCl3): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.06
(s, 3H, 18-H3), 1.25 (overlapping multiplets, 6H, 2ꢀ prop-2-yl-
CH3), 2.83 (m, 2H, 6-H2), 5.17 (s, 2H, OCH2), 5.49 (s, 2H, NCH2),
6.69 (d, 1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-
H), 7.18 (d, 1H, J = 8.6 Hz, 1-H), 7.20–7.24 (overlapping multiplets,
4H, 20,30,50,60-H), 7.29 (s, 1H, (17-H), 7.53 (s, 1H, C@CH). 13C NMR d
[ppm] = 14.5 and 15.4 (C-16a and C-18), 23.9 (2C: 2ꢀ prop-2-yl-
CH3), 24.3, 25.9, 27.2, 30.4, 32.1, 33.8, 37.4, 40.9, 41.3 (C-13),
43.3, 47.9, 54.1 (NCH2), 62.1 (OCH2), 112.5 (C-2), 114.4 (C-4),
122.4 (C@CH), 126.5 (C-1), 127.2 (2C) and 128.2 (2C): C-20,30,50,60,
131.6 (C-10), 132.9 (C-10), 137.9 (C-5), 145.3 (C@CH), 149.7 (C-
40), 156.0 (C-3), 160.4 (C-17). Anal. Calcd. for C32H42N4O2: C,
74.67; H, 8.22. Found: C, 74.75; H, 8.36%.
2.3. Cell cycle analysis by flow cytometry
Cell cycle analysis by means of flow cytometry was performed
in order to characterize the cellular DNA content of treated
A2780 cells. After treatment for 24 or 48 h, cells (200,000/condi-
tion) were trypsinized (Gibco BRL, Paisley, U.K.), washed with
phosphate-buffered saline (PBS) and fixed in 1.0 mL of cold 70%
ethanol for 30 min on ice. After two washing steps in cold PBS,
DNA was stained with propidium iodide (10
lg/mL) in the pres-
ence of RNA-ase (50 g/mL). The samples were then analyzed with
l
CyFlow (Partec GmbH, Münster, Germany). In each analysis, 20,000
events were recorded, and the percentages of the cells in the differ-
ent cell-cycle phases (subG1, G1, S and G2/M) were calculated by
using ModFit LT (Verity Software House, Topsham, ME, USA) [21].
2.1.7.3. 3-(1-(4-Nitrobenzyl-1H-1,2,3-triazol-4-yl-methyloxy)-14b-
propyl-des- -estra-1,3,5(10)-trien-carbaldehyde oxime (25c). From
D
compound 23 (340 mg, 1.00 mmol) and 4-nitrobenzyl azide (24c,
178 mg, 1.0 mmol). Yield: 481 mg (93%), mp 76–79 °C, Rf = 0.33
(10% ethyl acetate/90% dichloromethane). 1H NMR (500 MHz,
CDCl3): d [ppm] = 0.88 (t, 3H, J = 6.8 Hz, 16a-H3), 1.05 (s, 3H, 18-
H3), 2.83 (m, 2H, 6-H2), 5.19 (s, 2H, OCH2), 5.65 (s, 2H, NCH2),
6.69 (d, 1H, J = 2.2 Hz, 4-H), 6.77 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-
H), 7.19 (d, 1H, J = 8.6 Hz, 1-H), 7.29 (s, 1H, (17-H), 7.40 (d, 2H,
J = 8.6 Hz, 20-H and 60-H), 7.61 (s, 1H, C@CH), 8.22 (d, 2H,
J = 8.6 Hz, 30-H and 50-H). 13C NMR d [ppm] = 14.5 and 15.4 (C-
16a and C-18), 24.3, 26.0, 27.2, 30.4, 32.1, 37.4, 40.9, 41.3 (C-13),
43.3, 47.9, 53.1 (NCH2), 62.0 (OCH2), 112.4 (C-2), 114.4 (C-4),
122.7 (C@CH), 124.3 (2C) and 128.6 (2C): C-20,30,50,60, 126.6 (C-1),
133.0 (C-10), 138.0 (C-5), 141.5 and 145.6 and 148.1 (C-10 and
C-40 and C@CH), 156.0 (C-3), 160.4 (C-17). Anal. Calcd. for
3. Results and discussion
We earlier developed an efficient route for the synthesis of a
D-secoaldehyde of estrone 3-methyl (1) or 3-benzyl ether (2)
[22,23]. This key compound, containing an aldehyde function and
a propenyl side-chain in trans stereochemistry, proved to be a
useful intermediate in the synthesis of
The similar secoaldehydes in the 13 -estrone series (3, 4) with cis
functional groups were later synthesized and used in cyclization
reactions [23,24]. The additional structural modifications of the
epimeric secoaldehydes (1–4) may lead to structurally diverse
secoestrones.
D-homoestrone derivatives.
a
C29H35N5O4: C, 67.29; H, 6.82. Found: C, 67.41; H, 6.95%.
In the present work we first set out to synthetize oximes (5–8)
from the secoaldehydes of 13b- and 13a-estrone 3-methyl- or
2.2. Cell cultures and antiproliferative assays
3-benzyl ether (1–4). The latter protecting group was used because
of the possibility of its easy removal. We earlier reported the syn-
Human cancer cell lines (HeLa, MCF-7 and A431, isolated from
cervical adenocarcinoma, breast adenocarcinoma and skin epider-
moid carcinoma, respectively) and noncancerous human foreskin
fibroblasts were maintained in minimal essential medium supple-
mented with 10% fetal bovine serum (FBS), 1% non-essential
amino-acids and an antibiotic–antimycotic mixture (AAM).
thesis and Lewis aci
D
-induced intramolecular 1,3-dipolar cycload-
-estrone 3-methyl
ditions of -secooximes of estrone and 13
D
a
ether, furnishing isoxazolidines [18,19]. Electrophile-induced nit-
rone formation and the subsequent 1,3-dipolar cycloadditions of
the cyclic nitrones with C@C or C@N dipolarophiles were addition-
ally presented [19,25,26]. Here, we synthesized the oximes (5–8)

52
E. Mernyák et al. / Steroids 89 (2014) 47–55
Table 1
Synthesis of D-secooximes (5–8) and their derivatives (9–23, 25).
Entry
Starting compound
Reagent
Time (h)
Product
Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
2
3
4
5
7
6
8
5
7
5
7
5
6
7
8
6
NH2OHꢁHCl/NaOAc
1
1
1
1
2
2
2
2
6
6
6
6
1
1
1
1
5
6
7
8
96
93
94
92
85
83
79
77
86
82
78
81
74
82
76
85
77
74
89
92
90
93
NH2OHꢁHCl/NaOAc
NH2OHꢁHCl/NaOAc
NH2OHꢁHCl/NaOAc
Pd/C, 30 bar H2
9
Pd/C, 30 bar H2
Pd/C, 30 bar H2
Pd/C, 30 bar H2
O-Benzylhydroxylamine hydrochloride/NaOAc
O-Benzylhydroxylamine hydrochloride/NaOAc
O-Allylhydroxylamine hydrochloride/NaOAc
O-Allylhydroxylamine hydrochloride/NaOAc
Ac2O/py
10
11
12
13
14
15
16
17
18
19
20
21
22
23
25a
25b
25c
Ac2O/py
Ac2O/py
Ac2O/py
Ac2O/DMAP silica gel, mw, 100 °C
Ac2O/DMAP silica gel, mw, 100 °C
Propargyl bromide/K2CO3
Benzyl azide/CuI/PPh3/DIPEA
4-(Prop-2-yl)benzyl azide/CuI/PPh3/DIPEA
4-Nitrobenzyl azide CuI/PPh3/DIPEA
6 min
6 min
2 h
2 h
2 h
2 h
8
11
23
23
23
O
17
N
OH
H
NH2OH.HCl/ NaOAc
13
H
16
16a
H
H
H
RO 3
13β-Me
13α-Me
5, 6
7, 8
13β-Me
1, 2
3, 4 13α-Me
H2, Pd/C
H2, Pd/C
R = Me
R = Bn
1, 3, 5, 7
2, 4, 6, 8
N
OH
N
OH
H
H
H
H
H
H
MeO
HO
13β-Me
10 13α-Me
13β-Me
13α-Me
11
12
9
Scheme 1. Oximation of D-secoaldehydes (1–4) and saturation of the alkenyl side-chain of the D-secooximes (5–8).
from the
D
-secoaldehydes (1–4) by using hydroxylamine hydro-
tested. The antiproliferative effects of these latter compounds (9,
10) were similar to those of their unsaturated counterparts 5 and
7, and it may therefore be stated that the saturation of the alkenyl
side-chain did not modulate the biological activity of these secoe-
strones. In the 13b-estrone series, removal of the benzyl ether pro-
tecting group resulted in compound 11, with a complete loss of the
antiproliferative properties. In contrast, the presence of the pheno-
chloride and sodium acetate (Table 1, entries 1–4, Scheme 1).
The first structural modification of the oximes (5–8) comprised
the saturation of the propenyl side-chain and the simultaneous
removal of the benzyl protecting group (Table 1, entries 5–8,
Scheme 1). In the case of the 3-methyl ethers (5, 7), only the satu-
ration of the alkenyl side-chain occurred (Table 1, entries 5 and 6).
Compounds 5–12 and the starting
D
-secoaldehydes (1–4) were
lic OH group (in compound 12) in the 13a-estrone series, improved
tested by means of MTT assays on a panel of human adherent can-
cer cell lines: HeLa, MCF-7, A2780 and A431 (Table 2). The oximes
in the 13b-estrone series were active against all these cell lines, but
the protecting group at C-3 moderately influenced the cytostatic
behavior. Secooximes 5 and 6 displayed submicromolar IC50 values
against A2780 cells. Compound 5 gave similar low IC50 values on
all these cell lines, but compound 6, bearing a benzyl ether protect-
ing group exhibited higher cancer selectivity. The epimeric deriva-
tives of the secooximes (7, 8) had no impact on the proliferation of
the tested cells. It can be concluded that the orientation of the
angular methyl group greatly affects the antiproliferative proper-
the growth-inhibitory effect, especially on HeLa cells.
Etherifications of the oximes were carried out in order to
examine whether the oxime OH function is necessary for the
cytostatic potential. Epimeric oximes 5 and 7 bearing a 3-methyl
ether group were chosen as model compounds, and were treated
with O-benzyl- or O-allylhydroxylamine hydrochloride in the pres-
ence of sodium acetate (Table 1, entries 9–12, Scheme 2). Oxime
ethers 13–16 were obtained in excellent yields and were subjected
to MTT assays. None of these derivatives inhibited the proliferation
of the tested cancer cells (Table 2). These results suggest that the
free oxime OH group is essential for the antiproliferative effect.
In view of our recently published results [10] that the oxime
ties of the D-secooximes (5–8). The secoaldehydes (1–4) appeared
to be totally inactive. In order to examine the influence of satura-
tion of the side-chain on the cytostatic behavior, 3-methyl ethers
of secooximes with a propyl side-chain (9, 10) were additionally
propionate in the 13a-estrone series results in cell cycle blockade
and induces apoptosis, we now set out to synthetize oxime esters
as well. Secooximes (5–8) were treated with acetic anhydride in

E. Mernyák et al. / Steroids 89 (2014) 47–55
53
Table 2
N
OH
N
OAc
Experimentally determined IC50 values of the synthesized D-secoestrone derivatives
1–22.
Ac2O/py
H
H
H
H
Compound
Calculated IC50 valuesa
(lM)
13β-Me
17, 18
RO
HeLa
A2780
A431
MCF-7
5, 6
7, 8
13β-Me
13α-Me
19, 20 13α-Me
1
2
3
4
5
>30
>30
>30
>30
1.2
>30
>30
>30
>30
0.9
>30
>30
>30
>30
0.8
>30
>30
>30
>30
2.6
R = Me
R = Bn
5, 7, 17, 19
6, 8, 18, 20
Scheme 3. Synthesis of
D
-secooxime acetates (17–20) from the
D-secooximes (5–8).
6
7
8
9
7.1
1.4
0.9
>30
>30
>30
2.1
>30
>30
1.7
>30
>30
0.7
>30
>30
0.9
N
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
25a
25b
25c
Cisplatin
>30
>30
12.1
>30
>30
>30
>30
0.8
13.3
>30
>30
>30
>30
1.7
>30
>30
21.1
>30
>30
>30
>30
0.4
>30
>30
28.4
>30
>30
>30
>30
0.9
>30
>30
28.0
>30
>30
>30
>30
0.6
Ac2O/DMAP
silica gel,
C
N
H
100 oC, mw
H
H
H
BnO
13β-Me
13α-Me
21
22
13β-Me
13α-Me
6
8
Scheme 4. Synthesis of D-seconitriles (21, 22) from the D-secooximes (6, 8) by
3.8
4.7
9.8
microwave (mw) irradiation.
>30
>30
>30
>30
1.9
10.1
7.7
8.8
>30
>30
>30
>30
1.5
8.1
4.4
0.9
>30
>30
>30
>30
1.0
4.5
2.1
8.0
on the biological activity. There are already literature examples
of the synthesis and biological screening of secoestrone 16-nitriles
with different functions at C-17 and protecting groups at C-3
[27,28]. It was reported that none of the seconitriles exerted estro-
genic activity, but some of them expressed moderate antiestrogen-
ic effects. This potential was regularly higher for the 3-benzyl
ethers than for the seconitriles bearing a 3-OH group. In the light
of these literature data, compounds 6 and 8 served as starting ste-
roids for the formation of 17-nitriles. Water was eliminated from
the oxime function of compounds 6 and 8 under microwave irradi-
ation on silica gel as a solid support, with acetic anhydride and
4-(dimethylamino)pyridine as reagents (Table 1, entries 17 and
18, Scheme 4). The nitriles (21, 22) were formed in excellent yields
and were subjected to MTT assays, which revealed the lack of inhi-
bition of cell growth (Table 2).
12.7
5.3
5.7
a
Mean values from two independent determinations with 5 parallel wells;
standard deviation <15%.
pyridine, leading to oxime acetates (17–20, Table 1, entries 13–16,
Scheme 3). Compounds 17 and 18 greatly inhibited the prolifera-
tion of the tested cancer cells, while their epimeric counterparts
19 and 20 surprisingly appeared to be inactive (Table 2). Sec-
ooxime acetate 3-methyl ether (17) was the most potent, with sub-
micromolar IC50 values (IC50 = 0.4–0.9
l
M), and did not exhibit
Based on our earlier promising results in the field of steroidal
triazoles [13–17], finally, we planned to introduce a triazole moi-
ety into the D-secooximes in the 13b-estrone series. Incorporation
selectivity toward the different tumor cells. Cancer selectivity of
this analog was additionally tested by determining its action on
growth of skin fibroblast. Compound 17 exerted less than 30% inhi-
of an alkyne function into the steroid was achieved by the reaction
of 11 with propargyl bromide and potassium carbonate (Table 1,
entry 19, Scheme 5). The resulting secoestrone alkyne (23) was
then reacted with benzyl azides (24a–c) under ‘click’ reaction con-
ditions (CuI as a catalyst, triphenylphosphane as a stabilizing
ligand, N,N-diisopropylethylamine as a base, toluene as a solvent,
Table 1, entries 20–22, Scheme 5). All the reactions proceeded with
full conversion of the starting compounds 23 and 24, no side prod-
ucts were formed. The triazoles (25) were subjected to MTT assays,
which revealed that all the compounds (25a–c) greatly inhibited
the cell proliferation with no selectivity toward the different tumor
bition of proliferation at 10 lM (data not presented) which indi-
cates considerable selectivity towards cancer cells. Compound 18
was slightly less potent than its 3-methoxy derivative (17), but dis-
played greater selectivity: its activity against A2780 and on A431
was one order of magnitude better than that against HeLa and
MCF-7 cells. It can be concluded that the determining structural
moiety in the case of the oxime esters is the b-orientation of the
angular methyl group.
Additionally we set out to synthesize seconitriles from the oxi-
mes and to investigate the impact of this structural modification
N
O
CH2
BnONH2.HCl,
NaOAc
H
13β-Me
13
14 13α-Me
N
OH
H
AllylONH2.HCl,
NaOAc
H
H
N
O
CH2CH CH2
MeO
13β-Me
13α-Me
5
7
H
13β-Me
13α-Me
15
16
Scheme 2. Synthesis of D-secooxime O-allyl or O-benzyl ethers (13–16) from the D-secooximes (5, 7).

54
E. Mernyák et al. / Steroids 89 (2014) 47–55
action was detected at 1 lM after 24 h of treatment, but 3 lM
N
OH
HC
K2CO3
C
CH2 Br
resulted in an increased population in the S phase at the expense
of the cells in the G2/M phase (Fig. 1). Since DNA duplication is
the most prominent feature of the S phase, it seems reasonable that
the treated A2780 cells cannot achieve the synthesis of DNA, lead-
ing to a decreased G2/M population. Potent antiproliferative com-
pounds selected from a set of estrone-16-oximes disturbed the
DNA synthesis too [10]. After the longer incubation, the accumula-
tion of hypodiploid (subG1) cells was observed, which is a generally
acknowledged morphological marker of apoptosis. Our results
therefore indicate that compound 6 induces a cell cycle disturbance
in the S phase, which may initiate the apoptotic machinery.
It can be concluded that the inversion at C-13 has a great impact
on the cytostatic properties: the 13b derivatives proved to be the
more potent in most cases. Oxime ethers or nitriles were less active
than oxime esters. The functional group at C-3 also affected the
antiproliferative behavior. 3-Methyl ethers displayed lower cancer
selectivity, but a higher growth-inhibitory effect than those of their
3-benzyl ether counterparts. Besides its potency, compound 6
exhibited considerable tumor selectivity, which could be attributed
to the cell cycle disturbance and apoptosis induction. The pre-
sented results may be regarded as evidence that these are novel
H
H
H
C
O
O
HO
HC
11
23
CH2 N3
R
24
N
N
C
N
CH
25
H2C
R
H
24, 25
1'
2'
3'
a
b
c
6'
5'
prop-2-yl
NO2
4'
R
Scheme 5. Synthesis of triazoles (25) from the 3-(prop-2-yniloxy)-
(23) by azide-alkyne click reaction.
D-secooxime
D-secoestrone-based antiproliferative compounds suitable for fur-
cells. The growth-inhibitory activity was influenced by the nature
of the p-substituent of the N-benzyl ring. The unsubstituted com-
ther development.
pound (25a) was the most potent with IC50 = 1.0–1.9 lM values.
It can be concluded that the difference in the antiproliferative
potential of compounds 25 was the most outstanding on HeLa
and A2780 cell lines.
In order to acquire experimental data concerning the possible
mechanism of the active substances, compound 6 (as one of the
most potent and most cancer-selective) was chosen for additional
cell cycle analyses. A2780 cells were exposed to 1 or 3 lM of the
test compound for 24 or 48 h and the cell cycle distribution of the
treated cells was determined by flow cytometry. No substantial
Acknowledgments
The authors thank the Hungarian Scientific Research Fund
(OTKA K101659 and K109293) and the New Hungary Development
Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0047) for financial sup-
port. This research was supported by the European Union and
the State of Hungary, co-financed by the European Social Fund in
the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 ‘National
Excellence Program’.
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