Cycloaddition of steroidal cyclic nitrones to C=N dipolarophiles: Stereoselective synthesis and antiproliferative effects of oxadiazolidinones in the estrone series

Article abstract of DOI:10.1016/j.steroids.2013.06.009

Cyclic nitrones of estrone 3-methyl or 3-benzyl ether were reacted with phenyl isocyanate or nonsubstituted phenyl isocyanates as reactive C=N dipolarophiles, yielding condensed homosteroidal oxadiazolidinones. These dipolar cycloadditions were carried out under conventional heating or microwave irradiation. The chemo- and stereoselectivities of the reactions and the effects of the aromatic substituents on the reaction rates and yields were investigated and compared. The structures of the new products were determined by NMR (one- and two-dimensional) and MALDI-MS techniques, with C₇₀ fullerenes as matrix in the latter case. The antiproliferative properties of the synthetized compounds were determined on a panel of human adherent cell lines (HeLa, MCF7, A2780 and A431) by means of MTT assays. Some of them exhibited activities comparable to that of the reference agent cisplatin. Flow cytometry indicated that one of the most potent agents (11a) resulted in a cell cycle blockade.

Full text of DOI:10.1016/j.steroids.2013.06.009

Steroids 78 (2013) 1021–1028
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Steroids
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Cycloaddition of steroidal cyclic nitrones to C@N dipolarophiles:
Stereoselective synthesis and antiproliferative effects of
oxadiazolidinones in the estrone series
Erzsébet Mernyák a, , Judit Huber a,e, Johanna Szabó a, Gyula Schneider a, Anasztázia Hetényi b,
⇑
László Márk c,d, Gábor Maász c,d, Ágnes Berényi e, Ida Kovács e, Renáta Minorics e, István Zupkó e,
,
⇑
János Wölfling a
a Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
b Department of Medical Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
c Department of Biochemistry and Medical Chemistry, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary
d János Szentágothai Research Centre, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary
e Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6., 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:
Cyclic nitrones of estrone 3-methyl or 3-benzyl ether were reacted with phenyl isocyanate or nonsubsti-
tuted phenyl isocyanates as reactive C@N dipolarophiles, yielding condensed homosteroidal oxadiazolid-
inones. These dipolar cycloadditions were carried out under conventional heating or microwave
irradiation. The chemo- and stereoselectivities of the reactions and the effects of the aromatic substitu-
ents on the reaction rates and yields were investigated and compared. The structures of the new products
were determined by NMR (one- and two-dimensional) and MALDI-MS techniques, with C70 fullerenes as
matrix in the latter case. The antiproliferative properties of the synthetized compounds were determined
on a panel of human adherent cell lines (HeLa, MCF7, A2780 and A431) by means of MTT assays. Some of
them exhibited activities comparable to that of the reference agent cisplatin. Flow cytometry indicated
that one of the most potent agents (11a) resulted in a cell cycle blockade.
Received 19 April 2013
Received in revised form 10 June 2013
Accepted 25 June 2013
Available online 4 July 2013
Keywords:
Estrone
Oxadiazolidinone
Dipolar cycloaddition
Microwave irradiation
Steroid MALDI-TOF
Antiproliferative effect
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
metrical steroid dimer. The oxime O-benzyl ether of the estrone
3-methyl ether behaved similarly to the corresponding oxime,
The formation of nitrone dipoles is usually induced by Lewis-
acid catalysts, but literature also provides examples of the electro-
phile-induced synthesis of nitrones [1–5]. We recently described
the halogen and phenylselenyl bromide-induced formation of cyc-
leading to the steroid dimer. In the 13a-estrone series, no intermo-
lecular cycloaddition was observed; only the reduced counterparts
of the corresponding cyclic oxyiminium salts were obtained.
Moreover, steroidal nitrone 1,3-dipoles reacted stereoselectively
with N-phenylmaleimide as a C@C dipolarophile, stereoselectively
yielding condensed cycloadducts containing six-membered piperi-
dino D rings and isoxazolidine E rings [6].
We now describe the 1,3-dipolar cycloadditions of cyclic nitro-
nes of estrone 3-methyl or 3-benzyl ether with phenyl isocyanate
or its substituted derivatives as C@N dipolarophiles. Novel steroi-
dal oxadiazolidinones were primarily prepared in order to investi-
gate the chemoselectivities of the reactions and to observe the
effects of the aromatic substituents on the dipolar cycloadditions.
We were additionally interested in the investigation of the stere-
oselectivities of the reactions in comparison with our previous
findings [6,7]. The cycloadditions were carried out under conven-
tional heating or were promoted by microwave irradiation. Micro-
wave heating is a very effective and nonpolluting method of
lic nitrones derived from d-alkenyl
D-seco-oximes of estrone and
13 -estrone 3-methyl ether [6,7]. The 13b-oxime behaved as an
a
ambident nucleophile. The trapping of the intermediate halonium
or seleniranium ion proceeded via the O atom in the case of the
Z-oxime, and via the N atom in the case of the E-oxime. The oxaze-
pine derivative (as a steroidal C@N dipolarophile) and the cyclic
nitrone (a 1,3-dipole) reacted with each other in an intermolecular
1,3-dipolar cycloaddition, stereoselectively furnishing a nonsym-
⇑
Corresponding authors. Tel.: +36 62544277 (E. Mernyak); fax: +36 62 545567.
(I. Zupkó)
E-mail addresses: bobe@chem.u-szeged.hu (E. Mernyák), zupko@pharm.
u-szeged.hu (I. Zupkó).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.06.009

1022
E. Mernyák et al. / Steroids 78 (2013) 1021–1028
Table 1
activation. The key features of microwave-assisted reactions are
enhanced selectivity, improved reaction rates, milder reactions,
and the formation of cleaner products in higher yields and with
less waste relative to conventional heating.
Synthesis of steroidal oxadiazolidinones 8–11.
Entry Starting
oxime
Electrophilic
reagent
4-
Time
(h)
Product Yield (%)a
Substituent
of phenyl
isocyanate
Synthetic steroidal compounds are crucial lead molecules for
anticancer drug discovery and development. Analogs of naturally
occurring estrogens are particularly important anticancer drug
candidates. 2-Methoxyestradiol, a currently investigated metabo-
lite produced by catechol-O-methyltransferase (EC number:
2.1.1.6.), inhibits the proliferation of a broad range of cancer cell
lines [8]. A new set of estrone 16-oxime ethers with considerable
antiproliferative activities were synthetized recently. In vitro
experiments on the most promising estrone analogs with the aim
of elucidation of the mechanism of action revealed apoptosis
induction and blockade of the G1–S phase transition in the cell cy-
cle [9]. Several lines of evidence support the crucial role of ring D as
a molecular moiety determining the cell growth-inhibitory capac-
ities of estrone-based molecules [10,11]. These previous data
prompted us to investigate the possible antiproliferative properties
of these novel steroids.
1
2
3
4
5
6
7
8
1
1
1
1
1
1
2
2
2
2
2
2
NBS
NIS
NBS
NIS
NBS
NIS
NBS
NIS
NBS
NIS
NBS
NIS
H
H
OMe
OMe
Cl
Cl
H
H
3
3
0.5
0.5
2
2
3
3
8a
89 (93)
84 (90)
92 (93)
95 (96)
96 (96)
90 (93)
89 (93)
85 (89)
95 (96)
97 (98)
90 (92)
91 (93)
10a
8b
10b
8c
10c
9a
11a
9b
9
OMe
OMe
Cl
0.5
0.5
2
10
11
12
11b
9c
Cl
2
11c
a
The yields in brackets are those on the use of microwave irradiation at 100 °C
for 1 min.
B: Microwave irradiation. The reaction mixture was placed into a
pressure tube equipped with a stirrer bar and was inserted into the
cavity of the microwave apparatus. The mixture was heated at
100 °C for 1 min and then poured onto water and extracted with
diethyl ether. The organic phase was dried over anhydrous sodium
sulfate, filtered off, and evaporated. The crude product was sub-
jected to flash chromatography with dichloromethane as eluent.
2. Experimental
Melting points (mps) 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); solvent: 2% ethyl acetate/dichloromethane; detection
with iodine or UV (365 nm) after spraying with 5% phosphomolyb-
dic acid in 50% aqueous phosphoric acid and heating at
100–120 °C for 10 min. Flash chromatography: silica gel 60,
2.1.1. Reaction of 16-bromomethyl nitrone 3 or 4 with phenyl
isocyanate 7a
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) was reacted with NBS (89 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and phenyl isocyanate (0.06 ml, 0.50 mmol) were added.
8a was obtained as a white solid (method A: 227 mg, 89%,
method B: 238 mg, 93%). Mp 124–127 °C; Rf = 0.55. Anal. Calcd.
for C27H31BrN2O3: C, 63.41; H, 6.11. Found: C, 63.58; H, 6.27%. 1H
NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 3.45 (m,
1H, 16-H), 3.66–3.70 (overlapping multiplets, 2H, 16a-H2), 3.76
(s, 3H, 3-OCH3), 5.17 (s, 1H, 17a-H), 6.61 (d, 1H, J = 2.3 Hz, 4-H),
6.65 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz, 2-H), 7.03 (d, 1H, J = 8.6 Hz, 1-
H), 7.29 (d, 2H, J = 7.3 Hz, 20-H and 60-H), 7.42 (t, 1H, J = 7.3 Hz,
40-H), 7.47 (t, 2H, J = 7.3 Hz, 30-H and 50-H). 13C NMR (d, ppm):
18.6 (C-18), 25.0, 26.5, 28.3, 29.8, 35.5, 35.6, 38.8, 38.9 (C-13),
39.7, 42.7, 55.2 (3-OCH3), 63.2 (C-16), 86.0 (C-17a), 111.7 (C-2),
113.5 (C-4), 126.0 (C-1), 128.0 (2C, C-20 and C-60), 128.8 (C-40),
129.9 (2C: C-30and C-50), 131.5 (C-10), 137.5 and 138.9 (C-5 and
C-10), 157.7 (C-3), 160.6 (NCO). MS positive mode: 467 (12%,
[MꢀCO2]+), 392 (63%, [MꢀCO2ꢀC6H5]+).
40–63 lm (Merck). The reactions under microwave irradiation
were carried out with a CEM Corporation Focused Microwave Sys-
tem, Model Discover SP. 1H NMR spectra were recorded in CDCl3
solution (if not otherwise stated) with a Bruker DRX-500 instru-
ment at 500 MHz, with Me4Si as internal standard. 13C NMR spec-
tra were recorded with the same instrument at 125 MHz under
the same conditions. The mass spectrometer used was an Autoflex
II TOF/TOF (Bruker Daltonics, Bremen, Germany) operated in
reflector mode. The ions were accelerated under delayed extrac-
tion conditions (80 ns) in positive ion modes, with an acceleration
voltage of 20.00 kV. The instrument uses a 337 nm pulsed (50 Hz)
nitrogen laser. 1 ll aliquots of the standard solutions were loaded
onto the target plate (MTP 384 target plate ground steel TF, Bruker
Daltonics, Bremen, Germany) by mixing with the same volume of
a saturated matrix solution prepared by dissolving C70 fullerenes
in toluene.
2.1. General procedure for the synthesis of cyclic nitrones (3, 4, 5 and
6) and their subsequent cycloaddition with phenyl isocyanates (7)
9a was obtained as a white solid (method A: 262 mg, 89%,
method B: 274 mg, 93%). Mp 165–169 °C; Rf = 0.60. Anal. Calcd.
for C33H35BrN2O3: C, 67.46; H, 6.00. Found: C, 67.58; H, 5.92%. 1H
NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.45 (m,
1H, 16-H), 3.65–3.72 (overlapping multiplets, 2H, 16a-H2), 5.02
(s, 2H, 3-OCH2), 5.17 (s, 1H, 17a-H), 6.71 (s, 1H, 4H), 6.73 (d, 1H,
J = 8.5 Hz, 2-H), 7.03 (d, 1H, J = 8.5 Hz, 1-H), 7.30–7.33 (overlapping
multiplets, 3H, 20-H, 60-H and 400-H), 7.35–7.43 (overlapping multi-
plets, 5H, 200-H, 300-H, 500-H, 600-H, 40-H), 7.47 (m, 2H, 30-H and 50-H).
13C NMR (d, ppm): 18.6 (C-18), 24.6, 26.1, 27.9, 29.4, 35.1, 35.2,
38.4, 38.5 (C-13), 39.3, 42.3, 62.8 (C-16), 69.5 (OCH2), 85.5 (C-
17a), 112.1 (C-2), 114.2 (C-4), 126.0 (C-1), 127.4 (2C: C-200 and C-
600), 127.9 (C-400), 128.5 (2C: C-300 and C-500), 128.8 (C-40), 129.9
(2C: C-30 and C-50), 131.8 (C-10), 137.2 (C-100), 137.5 (C-5), 138.8
(C-10), 156.9 (C-3), 160.6 (NCO). MS positive mode: 543 (20%,
[MꢀCO2]+), 429 (80%, [MꢀBrꢀC6H5]+).
Oxime 1 (157 mg, 0.50 mmol) or oxime 2 (195 mg, 0.50 mmol)
was dissolved in dry acetonitrile (5 ml), the solution was cooled in
an ice-water bath (0–5 °C) and NBS (0.50 mmol) or NIS
(0.50 mmol) was added under a nitrogen atmosphere. The mixture
was stirred for 0.5 h. The solvent was evaporated off, and toluene
(5 ml) and phenyl isocyanate (0.50 mmol) or a substituted phenyl
isocyanate (0.50 mmol) were added.
A: Conventional heating. The reaction mixture was refluxed for
the time indicated in Table 1 and then poured onto water and ex-
tracted with diethyl ether. The organic phase was dried over anhy-
drous sodium sulfate, filtered, and evaporated. The crude product
was subjected to flash chromatography with dichloromethane as
eluent.

E. Mernyák et al. / Steroids 78 (2013) 1021–1028
1023
2.1.2. Reaction of 16-bromomethyl nitrone 3 or 4 with 4-
methoxyphenyl isocyanate 7b
(d, 1H, J = 8.4 Hz, 2-H), 7.06 (d, 1H, J = 9.0 Hz, 1-H), 7.25 (d, 2H,
J = 7.8 Hz, 30-H and 50-H), 7.29–7.33 (overlapping multiplets, 3H,
400-H, 20-H and 60-H), 7.37 (m, 2H, 300-H and 500-H), 7.43 (m, 2H,
200-H and 600-H).
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) was reacted with NBS (89 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and 4-methoxyphenyl isocyanate (0.07 ml, 0.50 mmol) were
added.
13C NMR (d, ppm): 18.6 (C-18), 24.9, 26.5, 28.3, 29.8, 35.4, 35.9,
38.8, 39.0 (C-13), 39.9, 42.8, 63.2 (C-16), 69.9 (OCH2), 85.9 (C-17a),
112.5 (C-2), 114.6 (C-4), 126.1 (C-1), 127.4 (2C: C-200 and C-600),
127.9 (C-400), 128.5 (2C: C-300 and C-500), 130.2 (2C: C-30 and C-50),
131.6 (C-10), 134.7 (C-40), 137.1 (C-100), 137.5 (2C: C-5 and C-10),
157.0 (C-3), 160.4 (NCO).
8b was obtained as a white solid (method A: 250 mg, 92%,
method B: 253 mg, 93%). Mp 130–133 °C; Rf = 0.55. Anal. Calcd.
for C28H33BrN2O4: C, 62.11; H, 6.14. Found: C, 62.34; H, 6.25%. 1H
NMR (d, ppm): 1.15 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 3.42 (m,
1H, 16-H), 3.65–3.72 (overlapping multiplets, 2H, 16a-H2), 3.76
(s, 3H, 3-OCH3), 3.84 (s, 3H, 40-OCH3), 5.07 (s, 1H, 17a-H), 6.61
(d, 1H, J = 2.6 Hz, 4-H), 6.66 (dd, 1H, J = 8.6 Hz, J = 2.6 Hz, 2-H),
6.96 (d, 2H, J = 8.9 Hz, 30-H and 50-H), 7.05 (d, 1H, J = 8.6 Hz, 1-H),
7.20 (d, 2H, J = 8.9 Hz, 20-H and 60-H). 13C NMR (d, ppm): 18.7 (C-
18), 25.0, 26.5, 28.4, 29.8, 35.2, 35.6, 38.8, 38.9 (C-13), 39.8, 42.8,
55.2 (3-OCH3), 55.5 (40-OCH3), 63.1 (C-16), 85.9 (C-17a), 111.7
(C-2), 113.6 (C-4), 115.2 (2C: C-30and C-50), 126.0 (C-1), 131.3
and 131.6 (C-10 and C-10), 137.5 (C-5), 157.7 (C-3), 159.7 (C-40),
160.8 (NCO). MS positive mode: 497 (7%, [MꢀCO2]+), 417 (17%,
[MꢀCO2ꢀBr]+), 392 (56%, [MꢀCO2ꢀC7H7]+), 296 (100%,
[MꢀCO2ꢀCH2BrꢀC7H7]+).
MS positive mode: 621 (100%, M+), 493 (55%, [MꢀClꢀCH2Br]+).
2.1.4. Reaction of 16-iodomethyl nitrone 5 or 6 with phenyl isocyanate
7a
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) (was reacted with NIS (113 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and phenyl isocyanate (0.06 ml, 0.50 mmol) were added.
10a was obtained as a white solid (method A: 235 mg, 84%,
method B: 252 mg, 90%). Mp 186–188 °C; Rf = 0.73. Anal. Calcd.
for C27H31IN2O3: C, 58.07; H, 5.60. Found: C, 57.95; H, 5.78%. 1H
NMR (d, ppm): 1.17 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.05 (m,
1H, 16-H), 3.51 (m, 2H, 16a-H2), 3.76 (s, 3H, 3-OCH3), 5.15 (s, 1H,
17a-H), 6.61 (d, 1H, J = 2.4 Hz, 4-H), 6.65 (dd, 1H, J = 8.6 Hz,
J = 2.4 Hz, 2-H), 7.02 (d, 1H, J = 8.6 Hz, 1-H), 7.29 (d, 2H,
J = 7.2 Hz, 20-H and 60-H), 7.41 (t, 1H, J = 7.2 Hz, 40-H), 7.47 (t, 2H,
J = 7.2 Hz, 30-H and 50-H). 13C NMR (d, ppm): 11.1 (C-16a), 18.8
(C-18), 25.0, 26.5, 29.8, 30.3, 35.5, 38.8, 39.0 (C-13), 39.8, 42.7,
55.2 (3-OCH3), 62.5 (C-16), 86.0 (C-17a), 111.7 (C-2), 113.5 (C-4),
126.0 and 128.8 (C-1 and C-40), 129.9 (2C: C-30and C-50), 131.5
(C-10), 137.5 and 138.9 (C-5 and C-10), 157.7 (C-3), 160.6 (NCO).
MS positive mode: 515 (5%, [MꢀCO2]+), 440 (31%, [M-CO2-
C6H5]+), 387 (100%, [MꢀCO2ꢀI]+).
9b was obtained as a white solid (method A: 293 mg, 95%,
method B: 296 mg, 96%). Mp 165–167 °C; Rf = 0.50. Anal. Calcd.
for C34H37BrN2O4: C, 66.12; H, 6.04. Found: C, 66.31; H, 6.15%. 1H
NMR (d, ppm): 1.15 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.43 (m,
1H, 16-H), 3.64–3.71 (overlapping multiplets, 2H, 16a-H2), 3.84
(s, 3H, 40-OCH3). 5.02 (s, 2H, 3-OCH2), 5.07 (s, 1H, 17a-H), 6.71 (s,
1H, 4-H), 6.74 (d, 1H, J = 8.4 Hz, 2-H), 6.97 (d, 2H, J = 8.6 Hz, 30-H
and 50-H), 7.05 (d, 1H, J = 8.5 Hz, 1-H), 7.20 (m, 2H, 20-H and 60-
H), 7.32 (m, 1H, 400-H), 7.38 (m, 2H, 300-H and 500-H), 7.41 (m, 2H,
200-H and 600-H. 13C NMR (d, ppm): 18.7 (C-18), 24.6, 26.1, 28.0,
29.4, 34.8, 35.2, 38.4 (2C), 39.3, 42.4, 55.1 (40-OCH3), 62.7 (C-16),
69.5 (OCH2), 85.5 (C-17a), 112.5 (C-2), 114.6 (C-4), 115.2 (2C: C-
30 and C-50), 126.0 (C-1), 127.4 (2C: C-200 and C-600), 127.9 (C-400),
128.5 (2C: C-300 and C-500), 131.2 (C-10), 131.8 (C-10), 137.2 (C-100),
137.6 (C-5), 156.9 (C-3), 159.6 (C-40), 160.8 (NCO). MS positive
mode: 573 (10%, [MꢀCO2]+), 429 (100%, [MꢀBrꢀC7H7O]+).
11a was obtained as a white solid (method A: 270 mg, 85%,
method B: 283 mg, 89%). Mp 102–107 °C; Rf = 0.60. Anal. Calcd.
for C33H35IN2O3: C, 62.46; H, 5.56. Found: C, 62.63; H, 5.75%. 1H
NMR (d, ppm): 1.17 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.05 (m,
1H, 16-H), 3.52(m, 2H, 16a-H2), 5.01 (s, 2H, 3-OCH2), 5.15 (s, 1H,
17a-H), 6.70 (s, 1H, 4H), 6.73 (d, 1H, J = 8.5 Hz, 2-H), 7.03 (d, 1H,
J = 8.5 Hz, 1-H), 7.29–7.33 (overlapping multiplets, 3H, 20-H, 60-H,
400-H), 7.36–7.43 (overlapping multiplets, 5H, 30-H, 40-H, 50-H, 300-
H and 500-H), 7.48 (m, 2H, 200-H and 600-H). 13C NMR (d, ppm):
11.1 (C-16a), 18.8 (C-18), 25.0, 26.5, 29.8, 30.3, 35.5, 38.8, 39.0
(C-13), 39.8, 42.7, 62.5 (C-16), 69.9 (OCH2), 85.9 (C-17a), 112.5
(C-2), 114.5 (C-4), 126.0 (C-1), 127.4 (2C: C-200 and C-600), 127.9
(C-400), 128.5 (2C: C-300 and C-500), 128.8 (C-40), 129.9 (2C: C-30
and C-50), 131.8 (C-10), 137.2 (C-100), 137.6 (C-5), 138.9 (C-10),
156.9 (C-3), 160.6 (NCO). MS positive mode: 591 (15%, [MꢀCO2]+),
509 (100%, [MꢀI]+).
2.1.3. Reaction of 16-bromomethyl nitrone 3 or 4 with 4-chlorophenyl
isocyanate 7c
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) was reacted with NBS (89 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and 4-chlorophenyl isocyanate (77 mg, 0.50 mmol) were added.
8c was obtained as a white solid (method A: 262 mg, 96%,
method B: 262 mg, 96%). Mp 127–130 °C; Rf = 0.77. Anal. Calcd.
for C27H30BrClN2O3: C, 62.11; H, 6.14. Found: C, 62.38; H, 6.05%.
1H NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 3.41
(m, 1H, 16-H), 3.63–3.70 (overlapping multiplets, 2H, 16a-H2),
3.76 (s, 3H, 3-OCH3), 5.14 (s, 1H, 17a-H), 6.62 (d, 1H, J = 2.2 Hz,
4-H), 6.67 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz, 2-H), 7.06 (d, 1H,
J = 8.4 Hz, 1-H), 7.24 (d, 2H, J = 8.4 Hz, 30-H and 50-H), 7.45 (d, 2H,
J = 8.4 Hz, 20-H and 60-H). 13C NMR (d, ppm): 18.6 (C-18), 24.9.
26.5. 28.3. 29.8. 35.4. 35.9. 38.8. 39.0 (C-13), 39.8. 42.8. 55.2 (3-
OCH3), 63.2 (C-16), 85.9 (C-17a), 111.7 (C-2), 113.5 (C-4), 126.0
(C-1), 130.2 (2C, C-30and C-50), 131.3 (C-10), 134.7 (C-40), 137.4
and 137.5 (C-10 and C-5), 157.7 (C-3), 160.4 (NCO). MS positive
mode: 421 (35%, [M-CO2-Br]+), 392 (100%, [M-CO2-C6H4Cl]+).
9c was obtained as a white solid (method A: 280 mg, 90%,
method B: 286 mg, 92%). Mp 170–173 °C; Rf = 0.70. Anal. Calcd.
for C33H34BrClN2O3: C, 63.72; H, 5.51. Found: C, 63.58; H, 5.36%.
1H NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.84 (m, 2H, 6-H2), 3.41
(m, 1H, 16-H), 3.63–3.71 (overlapping multiplets, 2H, 16a-H2),
5.02 (s, 2H, 3-OCH2), 5.14 (s, 1H, 17a-H), 6.71 (s, 1H, 4-H), 6.74
2.1.5. Reaction of 16-iodomethyl nitrone 5 or 6 with 4-methoxyphenyl
isocyanate 7b
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) was reacted with NIS (113 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and 4-methoxyphenyl isocyanate (0.07 ml, 0.50 mmol) were
added.
10b was obtained as a white solid (method A: 280 mg, 95%,
method B: 283 mg, 96%). Mp 139–142 °C; Rf = 0.55. Anal. Calcd.
for C28H33IN2O4: C, 57.15; H, 5.65. Found: C, 57.02; H, 5.77%. 1H
NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 3.04 (m,
1H, 16-H), 3.50 (m, 2H, 16a-H2), 3.76 (s, 3H, 3-OCH3), 3.84 (s, 3H,
40-OCH3), 5.05 (s, 1H, 17a-H), 6.62 (s, 1H, 4-H), 6.66 (d, 1H,
J = 8.6 Hz, 2-H), 6.96 (d, 2H, J = 8.4 Hz, 30-H and 50-H), 7.05 (d, 1H,
J = 8.6 Hz, 1-H), 7.19 (d, 2H, J = 8.4 Hz, 20-H and 60-H). 13C NMR d
ppm: 11.0 (C-16a), 18.8 (C-13), 25.0, 26.5, 29.8, 30.4, 35.2, 38.8,

1024
E. Mernyák et al. / Steroids 78 (2013) 1021–1028
38.9 (C-13), 39.8, 42.8; 55.2 (3-OCH3), 55.5 (40-OCH3), 62.4 (C-16),
85.9 (C-17a), 111.7 (C-2), 113.5 (C-4), 115.2 (2C: C-30and C-50),
126.0 (C-1), 131.3 and 131.5 (C-10 and C-10), 137.5 (C-5), 157.7
(C-3), 159.6 (C-40), 160.8 (NCO). MS positive mode: 440 (18%,
[MꢀCO2ꢀC7H7]+), 417 (100%, [MꢀCO2ꢀI]+).
no-acids and an antibiotic–antimycotic mixture (AAM). A2780
cells (isolated from ovarial cancer) were maintained in RPMI med-
ium supplemented with 10% FBS, 1% AAM and 1% L-glutamine. All
cell lines were purchased from the European Collection of Cell Cul-
tures (Salisbury, UK). For pharmacological investigations, 10 mM
stock solutions of the tested compounds were prepared with di-
methyl sulfoxide (DMSO). The highest applied dimethyl sulfoxide
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 overnight, after which the medium con-
taining the tested compound was added. After a 72-h incubation,
11b was obtained as a white solid (method A: 322 mg, 97%,
method B: 325 mg, 98%). Mp 119–122 °C; Rf = 0.50. Anal. Calcd.
for C34H37IN2O4: C, 61.45; H, 5.61. Found: C, 61.73; H, 5.43%. 1H
NMR (d, ppm): 1.16 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.04 (m,
1H, 16-H), 3.51(m, 2H, 16a-H2), 3.84 (s, 3H, 40-OCH3), 5.02 (s, 2H,
3-OCH2), 5.06 (s, 1H, 17a-H), 6.70 (s, 1H, 4-H), 6.73 (d, 1H,
J = 8.5 Hz, 2-H), 6.98 (m, 2H, 30-H and 50-H), 7.05 (d, 1H,
J = 8.5 Hz, 1-H), 7.19 (m, 2H, 20-H and 60-H), 7.31 (m, 1H, 400-H),
7.37 (t, 2H, J = 7.3 Hz, 300-H and 500-H), 7.41 (d, 2H, J = 7.3 Hz, 200-H
and 600-H). 13C NMR (d, ppm): 11.1 (C-16a), 18.8 (C-18), 24.9,
26.5, 29.8, 30.4, 35.2, 38.8, 38.9 (C-13), 39.8, 42.8, 55.5 (40-OCH3),
62.4 (C-16), 69.9 (OCH2), 85.9 (C-17a), 112.5 (C-2), 114.6 (C-4),
115.1 (2C: C-30 and C-50), 126.0 (C-1), 127.4 (2C: C-200 and C-600),
127.9 (C-400), 128.5 (2C: C-300 and C-500), 131.3 (C-10), 131.8 (C-10),
137.2 (C-100), 137.6 (C-5), 156.9 (C-3), 159.6 (C-40), 160.6 (NCO).
MS positive mode: 515 (42%, [MꢀCO2ꢀC7H7O]+), 497 (100%).
viability was determined by the addition of 20
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
l
l of MTT ([3-
bromide])
solution (5 mg/ml). The precipitated formazan crystals were solu-
bilized in dimethyl sulfoxide and the absorbance was determined
at 545 nm with an ELISA reader [12]. Two independent experi-
ments were performed with 5 parallel wells; cisplatin, an agent
administered clinically in the treatment of certain gynecological
malignancies, was used as a positive control. Sigmoidal dose–re-
sponse curves were fitted to the measured data. Calculations of
IC50 values and statistical analyses (t-test and ANOVA) were per-
formed by means of GraphPad Prism 4.0 (GraphPad Software;
San Diego, CA, USA).
2.1.6. Reaction of 16-iodomethyl nitrone 5 or 6 with 4-chlorophenyl
isocyanate 7c
As described in Section 2.1, oxime 1 (157 mg, 0.50 mmol) or
oxime 2 (195 mg, 0.50 mmol) was reacted with NIS (113 mg,
0.50 mmol). The solvent was evaporated off, and toluene (5 ml)
and 4-chlorophenyl isocyanate (77 mg, 0.50 mmol) were added.
10c was obtained as a white solid (method A: 267 mg, 90%,
method B: 276 mg, 93%). Mp 110–112 °C; Rf = 0.77. Anal. Calcd.
for C27H30ClIN2O3: C, 54.70; H, 5.10. Found: C, 54.61; H, 4.98%.
1H NMR (d, ppm): 1.17 (s, 3H, 18-H3), 2.86 (m, 2H, 6-H2), 3.02
(m, 1H, 16-H), 3.50 (d, 2H, J = 4.2 Hz, 16a-H2), 3.76 (s, 3H, 3-
OCH3), 5.12 (s, 1H, 17a-H), 6.62 (d, 1H, J = 2.3 Hz, 4-H), 6.67 (dd,
1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.06 (d, 1H, J = 8.5 Hz, 1-H), 7.23
(d, 2H, J = 7.7 Hz, 30-H and 50-H), 7.44 (d, 2H, J = 7.7 Hz, 20-H and
60-H). 13C NMR (d, ppm): 10.8 (C-16a), 18.8 (C-18), 24.9, 26.5,
29.8, 30.3, 35.8, 38.8, 39.1 (C-13), 39.9, 42.8, 55.2 (3-OCH3), 62.5
(C-16), 85.9 (C-17a), 111.7 (C-2), 113.5 (C-4), 126.0 (C-1), 130.2
(2C: C-30and C-50), 131.3 (C-10), 134.7 (C-40), 137.4 and 137.6 (C-
10 and C-5), 157.7 (C-3), 160.5 (NCO). MS positive mode: 440
(100%, [MꢀCO2ꢀC6H5Cl]+), 421 (37%, [MꢀCO2ꢀI]+).
2.3. Cell cycle analysis by flow cytometry
Flow cytometric analysis was performed in order to character-
ize the cellular DNA content of treated A2780 cells. After treatment
for 24 or 48 h, cells (200,000/condition) were trypsinized
(Gibco BRL, Paisley, UK), washed with phosphate-buffered sal-
ine (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
PI (10 lg/ml) in the presence of RNA-ase (50 lg/ml). The samples
were then analyzed with CyFlow (Partec GmbH, Münster, Ger-
many). In each analysis, 20,000 events were recorded, and the per-
centages of the cells in the different cell-cycle phases (subG1, G1, S
and G2/M) were calculated by using ModFit LT (Verity Software
House, Topsham, ME, USA) [13].
11c was obtained as a white solid (method A: 305 mg, 91%,
method B: 312 mg, 93%). Mp 151–155 °C; Rf = 0.70. Anal. Calcd.
for C33H34ClIN2O3: C, 59.25; H, 5.12. Found: C, 59.47; H, 5.23%.
1H NMR (d, ppm): 1.17 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.02
(m, 1H, 16-H), 3.50 (m, 2H, 16a-H2), 5.02 (s, 2H, 3-OCH2), 5.12 (s,
1H, 17a-H), 6.70 (s, 1H, 4-H), 6.74 (d, 1H, J = 8.5 Hz, 2-H), 7.06 (d,
1H, J = 8.5 Hz, 1-H), 7.23 (d, 2H, J = 7.1 Hz, 30-H and 50-H), 7.31 (t,
1H, J = 6.9 Hz, 40-H), 7.37 (m, 2H, 20-H and 60-H), 7.41 (m, 2H, 300-
H and 500-H), 7.45 (m, 2H, 200-H and 600-H). 13C NMR (d, ppm,
55 °C): 10.8 (C-16a), 18.8 (C-18), 24.9, 26.5, 29.8 30.3, 35.8, 38.7,
39.1 (C-13), 39.9, 42.8, 62.5 (C-16), 69.9 (OCH2), 85.9 (C-17a),
112.5 (C-2), 114.6 (C-4), 126.1 (C-1), 127.4 (2C: C-200 and C-600),
127.9 (C-400), 128.5 (2C: C-300 and C-500), 129.1 (2C: C-20 and C-60),
130.2 (2C: C-30 and C-50), 131.6 (C-10), 134.7 (C-40), 137.1 (C-100),
137.5 (2C: C-5 and C-10), 156.9 (C-3), 160.5 (NCO). MS positive
mode: 625 (11%, [MꢀCO2]+), 497 (100%, [MꢀCO2ꢀI]+).
3. Results and discussion
3.1. Synthesis and structure determination
Coskun and Parlar recently described the cycloaddition of acy-
clic nitrones with phenyl isocyanate [14]. The cycloadditon was
carried out in refluxing acetonitrile with a 3-fold excess of phenyl
isocyanate in reaction times of 1.5–24 h, with yields of 52–96%,
depending on the structure of the starting nitrone. It was demon-
strated that the structure of the oxime influenced the reaction rate
and the yield. When the C-phenyl oxime was substituted with
electron-withdrawing substituents on the phenyl ring, the yields
were moderate and the reaction proceeded only after prolonged
heating. When electron-donating substituents were present, the
reaction time was shorter and the yield was higher. There have
been other reports on the cycloaddition of acyclic [2,3] or cyclic
nitrones [1,2,15–17] with phenyl isocyanate, but there is no evi-
dence of such reactions on the steroid core.
2.2. Cell cultures and antiproliferative assays
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 ami-
In the present work we describe 1,3-dipolar cycloadditions of
steroidal cyclic nitrone dipoles (3–6) with substituted or nonsub-
stituted phenyl isocyanates (7). Seco-oximes of estrone 3-methyl

E. Mernyák et al. / Steroids 78 (2013) 1021–1028
1025
or 3-benzyl ether (1, 2) used for the electrophile-induced cycliza-
tion were obtained from the -seco-aldehydes using hydroxyl-
amine hydrochloride and sodium acetate [18]. -seco-aldehydes
was needed. Those promising results led us to carry out MALDI
TOF measurements of the oxadiazolidinones (8–11) with positive
mode detection. Molecular or quasimolecular ions were not
detected, because of the cleavage of carbon dioxide from the mol-
ecules. The MS spectra revealed fragment ions formed by the cleav-
age of carbon dioxide and/or the phenyl group (or substituted
phenyl group) deriving from the isocyanate and/or the appropriate
halogen atom.
D
D
are available in several steps from estrone 3-methyl or 3-benzyl
ether by employing a Grob fragmentation as the key step [19,20].
The cyclization was first carried out with two different electrophile
triggers: NBS or NIS in acetonitrile, as reported earlier [6,7]. When
TLC monitoring revealed that the starting oxime (1, 2) had been
consumed the solvent was evaporated off, toluene and 1 equiv.
of phenyl isocyanate (7) were added, and the solution was refluxed
for the time indicated in Table 1. The desired condensed homoster-
oidal oxadiazolidinone derivatives (8–11) were formed in higher
yields and shorter reaction times as compared with the literature
results [14]. An electron-donating methoxy group on the phenyl
ring (7b) promoted the reaction (Entries 3, 4, 9 and 10) and, sur-
prisingly, the dual nature of the chloro substituent (7c) also accel-
erated the reaction (Entries 5, 6, 11 and 12). The lowest extent of
reaction was observed with the unsubstituted reagent (7a, Entries
1, 2, 7 and 8). The reactions were totally chemoselective: no side-
products were formed under the reaction conditions applied. The
newly formed stereogenic centers displayed the same configura-
tions as earlier: a 16b-substituent and a 17ab-hydrogen at the
anellation of the piperidine and oxadiazolidinone rings [7].
A further aim of the present work was to compare the reaction
rates and the chemo- and stereoselectivities of the cycloadditions
carried out under reflux or with microwave irradiation. The micro-
wave-induced reactions were performed in a similar manner
(100 °C and a 1-min reaction time) independently of the nature
of the substituent in the phenyl isocyanates, yielding the oxadiazo-
lidinones (8–11) chemo- and stereoselectively. This method
greatly shortened the reaction time and improved yields were
achieved.
In the NMR spectra of the products 8–11, the proton and carbon
chemical shifts were assigned through COSY, HSQC and HMBC
experiments. The relative configurations of the products were de-
duced from the NOESY spectra, in which the cross-peaks for the
protons of 18-Me, 17a-H and 16-CH2X proved their cis arrange-
ment. The overlapping signals of C-20 and C-60 did not appear in
the 13C NMR spectrum of the compounds, recorded with the J-
MOD pulse sequence. A feasible explanation for this phenomenon
is the dynamic effect, which broadened the C-20 and C-60 signals,
which were merged therefore into the noise. To confirm this the-
ory, the spectrum of 11c was recorded at 55 °C in CDCl3 and the
signal of C-20 and C-60 appeared at 129.1 ppm, indicating that the
temperature stepped the coalescence temperature obviously over.
As a further evidence for this dynamic effect is a cross-peak be-
tween the overlapping multiplets of 20-H and 60-H and the signals
of C-20 and C-60 in the HSQC spectrum of 8a, recorded at room tem-
perature. This cross-peak indicated the chemical shift of C-20 and
C-60 at 128.0 ppm, the latter signal could not be seen on the 13C-
axis. The most stable structures the products were confirmed by
molecular modeling with all possible chiral arrangements of chiral
C atoms 17a and 16. The conformational protocol comprised a sto-
chastic search via the Merck Molecular Force Field (MMFF94), and
a subsequent minimization of the resulting low-energy conforma-
tions at the ab initio level, using the HF/6–311G⁄⁄ basis set. The
resulting structures proved to be rigid for 8–11; no minor confor-
mation was found (Scheme. 1).
3.2. Determination of the antiproliferative properties of the newly
synthetized compounds 8–11
Various types of A- and D-ring-substituted estrone analogs have
been synthetized and tested for their inhibitory effects on cell pro-
liferation [28–32]. One of the major requirements for the pharma-
cological use of antiproliferative estrone derivatives is the lack of
estrogenic activity. The introduction of a sulfamate function at po-
sition 3 or 17 or D-ring expansion drastically reduces the estroge-
neity [30]. There are only a few literature descriptions of the
antitumor behavior of
cently reported the cytostatic effect of
cells (IC50 = 5.5 M) [34]. Hillisch et al. patented the finding that
some -homoestra-1,3,5(10)-trien-3-yl 2-substituted sulfamates
D-homoestrone derivatives [33,34]. We re-
D
-homoestrone on HeLa
l
D
are potential pharmaceuticals for the treatment of tumorous dis-
eases [33]. To the best of our knowledge, there are no examples
in the literature of the antiproliferative action of condensed
D-
homoestrone derivatives containing heterocyclic D and E rings.
The introduction of N atoms into the rings of the steroidal skeleton
may influence the ability of the molecules to bind proteins since
the donor N atoms can serve as hydrogen-bond acceptor [35].
There are some literature data relating to the antimitotic prop-
erties of oxadiazoles and their partially or fully saturated
derivatives.
Tahir et al. described a novel oxadiazoline derivative (A-
204197) with antiproliferative properties. This new tubulin-bind-
ing agent is active against tumor cell lines which are resistant to
known microtubule inhibitors [36]. The oxadiazoline analog of
combrestatin A-4 containing a naphthalene ring was found to be
the promising tubulin inhibitor, with potent antiproliferative activ-
ities against three cancer cells. A molecular docking study demon-
strated the importance of the oxadiazoline moiety due to the
hydrogen-bond acceptor ability [35]. Gopalsamy et al. Reported
on a novel series of PAI-1 inhibitors (the level of PAI-1 is acutely
elevated in cancer) containing an oxadiazolidinedione moiety
[37]. An additional oxadiazolidinone derivative as a heterocyclic
acid surrogate exhibited improved pharmacokinetic properties as
a VLA-4 antagonist [38]. Ouyang et al. found that an oxadiazole
derivative caused the mitotic arrest of A431 human epidermoid
cells and cells from multidrug-resistant tumors (EC50 = 7.8 nM)
[39].
The aims of our present study included the characterization of
the antiproliferative properties of the newly synthetized com-
pounds 8–11 on a panel of human adherent cancer cell lines. These
steroidal oxadiazolidinones bearing different functional groups at
positions C-3, C-16a or C-40, displayed different growth-inhibitory
effects (Table 2). They were active exclusively against gynecologi-
cal cancer cell lines: none of them inhibited the proliferation of
A431 human epidermoid cells. It can be concluded that the substi-
tuent on C-40 has an impact on the anticancer properties, since
compounds bearing 40-methoxy groups (8–11b) appeared to be to-
Neutral steroids are difficult to analyze by desorption/ioniza-
tion methods coupled with mass spectrometry, and there have
been only a few literature reports on the analysis of derivatized
steroids through MALDI TOF mass spectrometry [21–24]. We re-
cently described the synthesis and stereochemical investigation
of N-containing heterocyclic steroids which were efficiently mea-
sured by this technique, using C70 fullerenes as matrix [25–27].
Since the N atoms are capable of protonation, no derivatization
tally inactive (IC50 > 30 lM). Modification of the phenolic ether
function led to minor changes in the antiproliferative results: 3-
benzyloxy derivatives were slightly more efficient than their 3-
methoxy counterparts. The most potent compound was the 16-
iodo-N-phenyl-3-benzyloxy derivative 11a, with IC50 = 2.19 lM

1026
E. Mernyák et al. / Steroids 78 (2013) 1021–1028
OH
N
H
H
H
H
R1O
1, 2
R1
1, 3, 5, 8, 10
2, 4, 6, 9, 11
Me
Bn
i
ii
R2
R2
O
O
N
N
+
+
CH2Br
CH2I
H
H
5, 6
3, 4
NCO
NCO
7
7
iii
iii
R2
R2
3'
4'
2'
1'
5'
O
O
6'
N
N
H
H
O
O
N
N
H
H
16a
CH2Br
CH2I
H
H
H
H
R1O
R1O
8, 9
10, 11
R2
7-11
a
b
c
H
OMe
Cl
Scheme 1. Reagents and conditions: (i) 1 equiv. of NBS; acetonitrile; N2 atmosphere; ice-water bath; 0.5 h; (ii) 1 equiv. of NIS; acetonitrile; N2 atmosphere; 0–5 °C; 0.5 h; (iii)
1 equiv. of phenyl isocyanate or substituted phenyl isocyanate; toluene; reflux; 0.5–3 h or 1 equiv. of phenyl isocyanate or substituted phenyl isocyanate; toluene; microwave
irradiation; 100 °C; 1 min.
Table 2
for A2780 ovarian carcinoma cells, a concentration at least 5 times
Experimentally determined IC50 values of the synthetized oxadiazolidinone deriva-
lower than its 50% inhibition of cell growth for the other cell lines.
Cancer selectivity is a crucial point in the design and develop-
ment of an innovative anticancer agent. As a first step to describe
this property of 11a, the viability assay was repeated on noncan-
tives 8–11.
IC50 values (
l
M)a
A431
HeLa
A2780
MCF-7
8a
10a
8b
10b
8c
10c
9a
11a
9b
11b
9c
11c
Cisplatin
11.27
6.74
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
8.81
13.78
6.28
>30
9.57
10.5
>30
cerous human skin fibroblast cells at 1 and 10
platin, 11a did not substantially disturb the proliferation of
fibroblast cells at 1 M, but its growth-inhibitory effect was signif-
lM. Similarly to cis-
l
>30
>30
>30
icantly lower at higher concentration (Table 3).
In order to shed light on the mechanism of the antiproliferative
action of 11a, cell cycle analyses were performed after 24 and 48 h
9.22
4.91
5.46
13.43
>30
13.93
4.99
3.24
2.19
>30
7.64
7.20
5.68
12.46
>30
of exposure (Fig. 1). Treatment with 3
statistically significant decrease in the population of cells in the
synthetic phase, and this became more marked at 10 M. This
lM 11a for 24 h resulted in a
>30
>30
>30
l
12.53
10.96
5.66
5.46
4.63
0.86
8.38
9.16
7.99
higher concentration led to an increase in the ratio of cells in the
G1 phase, indicating a blockade in the G1–S transition during the
cell cycle. A more marked and concentration-dependent distur-
bance in the cell distribution was evident after 48 h of exposure.
a
Mean values from 2 independent determinations with 5 parallel wells; standard
deviation <15%.

E. Mernyák et al. / Steroids 78 (2013) 1021–1028
1027
Table 3
References
Antiproliferative effects of 11a and cisplatin on noncancerous human foreskin
fibroblast cells.
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Growth inhibition (%) SEM
11a
0.13 2.60
15.39 0.91
Cisplatin
P valuea
1
l
M
1.92 1.78
26.30 2.35
NS
<0.01
10
lM
a
P values were calculated with the unpaired t-test. NS: not significantly different.
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⁄, ⁄⁄
(upper panel) or 48 h (lower panel).
and
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Stereoselective synthesis of spiro and condensed pyrazolines of steroidal
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The authors thank the Hungarian Scientific Research Fund
(OTKA K101659) and TÁMOP-4.2.2./B-10/1-2010-0012 for finan-
cial support. Special thanks should be given to Reinhard Machinek
(University of Göttingen) for his useful recommendations.

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