Synthesis and biological evaluation of 13α-estrone derivatives as potential antiproliferative agents

Article abstract of DOI:10.1016/j.steroids.2016.05.010

13α-Estrone derivatives containing various substituents on C-3 and C-17 were synthesized, and evaluated by means of MTT assays for in vitro antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF-7, A2780 and A431). Compounds with N-benzyltriazolylmethoxy moieties on C-3 proved to be more potent than their 3-hydroxy or 3-ether counterparts. Some triazoles exerted substantial cytostatic effects against particular tumor cell lines, with submicromolar IC₅₀ values.

Full text of DOI:10.1016/j.steroids.2016.05.010

Accepted Manuscript
Synthesis and biological evaluation of 13α-estrone derivatives as potential an-
tiproliferative agents
Johanna Szabó, Zoltán Pataki, János Wölfling, Gyula Schneider, Noémi
Bózsity, Renáta Minorics, István Zupkó, Erzsébet Mernyák
PII:
S0039-128X(16)30054-X
http://dx.doi.org/10.1016/j.steroids.2016.05.010
STE 7981
DOI:
Reference:
To appear in:
Steroids
Received Date:
Revised Date:
Accepted Date:
20 December 2015
25 May 2016
31 May 2016
Please cite this article as: Szabó, J., Pataki, Z., Wölfling, J., Schneider, G., Bózsity, N., Minorics, R., Zupkó, I.,
Mernyák, E., Synthesis and biological evaluation of 13α-estrone derivatives as potential antiproliferative agents,
Steroids (2016), doi: http://dx.doi.org/10.1016/j.steroids.2016.05.010
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1
Synthesis and biological evaluation of 13α-estrone derivatives as potential
antiproliferative agents
1
1
1
1
2
Johanna Szabó, Zoltán Pataki, János Wölfling, Gyula Schneider, Noémi Bózsity,
2
2*
1*
Renáta Minorics, István Zupkó, Erzsébet Mernyák
1
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged,
Hungary
2
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6., H-
6
720 Szeged, Hungary
*Corresponding authors. Tel.: +36 62 544277; fax: +36 62 544200 (E. Mernyák).
E-mail addresses: bobe@chem.u-szeged.hu (E. Mernyák), zupko@pharm.u-szeged.hu (I.
Zupkó).

2
Abstract
3α-Estrone derivatives containing various substituents on C-3 and C-17 were synthesized,
1
and evaluated by means of MTT assays for in vitro antiproliferative activity against a panel of
human adherent cancer cell lines (HeLa, MCF-7, A2780 and A431). Compounds with N-
benzyltriazolylmethoxy moieties on C-3 proved to be more potent than their 3-hydroxy or 3-
ether counterparts. Some triazoles exerted substantial cytostatic effects against particular
tumor cell lines, with submicromolar IC50 values.
Keywords
1
3α-estrone, azide-alkyne cycloaddition, 17-deoxy-13α-estrone, antiproliferative effect.

3
1
. Introduction
Anticancer drugs administered for the treatment of estrogen-dependent cancers are often
analogs of natural estrogenic compounds. The development of estrone-based anticancer
agents [1‒6] lacking estrogenic activity is one of the major challenges in the medicinal
chemistry of steroids. The literature reveals that inversion of the configuration of C-13 in
estrone (E1) or 17α/β-estradiols leads to a complete loss of the estrogenic activity, which
results from the conformational changes [7‒9]. 13α-Estrone, which is readily available from
E1 by the method of Yaremenko and Khvat [10], is an appropriate scaffold for the design of
hormonally inactive agents with selective biological potency. The first reported
antiproliferative 13α-estrone derivative was the 3-benzyl ether of the 16-oxime propionate,
which proved to be potent against particular cancer cell lines, inducing apoptosis with high
tumorselectivity [11]. We recently published the synthesis and CuAAC (azide-alkyne click
reaction) of steroidal azidoalcohols with terminal alkynes [12‒16]. Some of the formed
triazoles displayed pronounced in vitro antiproliferative effects on certain human malignant
cell lines. On the basis of these outstanding results, we continued the design of potentially
antitumoral 13α-estrone derivatives by incorporating triazole rings, in order to enhance the
solubility, the bioavailability and the stability against metabolic degradation [17]. We
therefore synthesized two series of diastereomeric trans-16-phenyltriazolyl-17-estradiol 3-
benzyl ethers of 13α-estrone. It was confirmed that the 16β,17α isomers bearing p-alkyl
substituents on the triazolylphenyl ring displayed substantial growth-inhibitory properties
against 7 cancer cell lines with apoptosis induction via the intrinsic pathway [18]. The
diastereomeric 13α-estradiols have been reported to be moderate antiproliferative agents [7],
and incorporation of the 3-benzyl and 16β-triazolyl functions into the 13α,17α-estradiol core
significantly improved the growth-inhibitory activity. These results indicate that the
introduction of structural elements such as a triazole moiety at other positions of 13α-estrone
may lead to enhanced antitumor properties. Drasar et al. recently described the synthesis of 3-
O-propargylestrone [20], using the propargylation procedure of Skorobogatyi et al. [19]. The
CuAAC reaction of O-propargylestrone with 2,6-bis(azidomethyl)pyridine furnished a
bridged steroidal homodimer. The triazolyl conjugate was tested on a panel of steroid receptor
reporter cell lines to determine the capacity of the compound to modulate the transcriptional
activity of different steroid receptors. The conjugate displayed moderate potency in both the
estrogen receptor α (ERα) and the estrogen receptor β (ERβ) assays, these modifications of E1
therefore resulting in retained estrogenic activity. The compound exerted substantial cytostatic

4
activity against the CEM–DNR-BULK cell line, which is a daunorubicin-resistant derivative
of CCRF-CEM cells. These results indicate that introduction of the triazolyl function onto the
3
-OH of the estrane skeleton may lead to cytostatic properties, but complete loss of the
estrogenic activity of the antitumoral derivative would be of primary importance.
In view of the promising literature evidences, we decided to synthesize triazolyl derivatives of
1
3α-estrone through incorporation of the heterocyclic moiety onto the 3-hydroxy function.
We aimed to determine the in vitro antiproliferative activities of the newly synthetized
derivatives and their precursors by means of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) assays against a panel of human adherent cancer cell lines
(
HeLa, MCF-7, A431 and A2780).

5
2
2
2
. Methods and materials
.1. Chemical synthesis
.1.1. General methods
Compounds used as starting materials 2 [8], 3 [21] and 15 [22] were obtained by the literature
methods. Debenzylation [21] of 3 led to steroid 1 [23]. Melting points (mp) were determined
with a Kofler hot-stage apparatus and are uncorrected. Elemental analyses were performed
with a PerkinElmer CHN analyzer model 2400. Thin-layer chromatography: silica gel 60
F254; layer thickness 0.2 mm (Merck); eluents: (A) CH
Cl
2 2
, (B) EtOAc/CH
2
Cl = 2/98, (C)
2
EtOAc/CH Cl = 5/95, (D) EtOAc/CH Cl = 30/70, (E) CH
2
2
2
2
2
Cl /hexane = 10/90; detection
2
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,
1
4
0–63 mm (Merck). H NMR spectra were recorded in CDCl
3
solution (if not otherwise
1
3
4
stated) with a Bruker DRX-500 instrument at 500 MHz, with Me Si as internal standard. C
NMR spectra were recorded with the same instrument at 125 MHz under the same conditions.
Full-scan mass spectra of the compounds were acquired in the range 50–800 m/z with an
Agilent 500MS Ion trap mass spectrometer equipped with an electrospray ionization source.
Analyses were performed in positive ion mode, if not otherwise stated. Capillary and needle
voltages were 80 and 5000V, respectively. RF loading was set at 88%. Nebulizing gas (N
and drying gas (N ) pressures were maintained at 60 and 20 psi, respectively. Drying gas
temperature was held at 300°C. The spectra were collected by continuous infusion of the
2
)
2
-
1
steroid solution at a concentration of 10 ngµl in acetonitrile/5 mM ammonium formate 50/50
-
1
(
v/v %) at a flow rate of 15 µlmin . The analytical HPLC measurements were performed on
an Agilent 1260 Infinity HPLC equipped with a Micro Vacuum Degasser, Binary Pump,
Standard Autosampler, Thermostated Column Compartment, and Variable Wavelength
Detector. The chromatographic separation was achieved at 40 °C on a Gemini NX C-18
analytical column (3 mm, 150 x 2 mm) from Phenomenex, equipped with a C-18 guard
column, using gradient elution. Mobile phase A was water (Sigma–Aldrich Ltd., Budapest,
Hungary), while mobile phase B was acetonitrile (Merck Ltd., Budapest, Hungary). A linear
gradient was applied from 20% B to 100% B in 10 min (holding time: 5 min), the B content
was then lowered to 20% in 5 min, and finally the column was re-equilibrated for 5 min. The
flow rate was set to 0.2 ml/min.

6
2
.1.2. General procedure for the reduction of 17-ketones (1‒3)
To a stirred solution of 3-hydroxy-13α-estra-1,3,5(10)-trien-17-one (1) (270 mg, 1.0 mmol)
or 3-methoxy-13α-estra-1,3,5(10)-trien-17-one (2) (284 mg, 1.0 mmol) or 3-benzyloxy-13α-
estra-1,3,5(10)-trien-17-one (3) (360 mg, 1.0 mmol) in a 1:1 mixture of MeOH and CH
ml), NaBH (189 mg, 5.0 mmol) was added. The reaction mixture was stirred at room
temperature for 3 h, then diluted with water and extracted with CH Cl . The combined
organic phases were washed with water until neutral, dried over Na SO and evaporated in
2 2
Cl (5
4
2
2
2
4
vacuo. The crude product was a mixture of the 17β-hydroxy (4–6) and the 17α-hydroxy (7–9)
diastereomers in a ratio of nearly 1:1.
2
.1.2.1. 3-Hydroxy-13α-estra-1,3,5(10)-trien-17β-ol (4) and 3-hydroxy-13α-estra-1,3,5(10)-
trien-17α-ol (7)
As described in Section 2.1.2, ketone 1 (270 mg, 1.0 mmol) was reacted with NaBH
4
(189
mg, 5.0 mmol). Compounds 4 and 7 were identical with compounds described in the literature
1
[
7]. 4: Mp 185‒187 °C, R
f
= 0.62 (ss D); H NMR (DMSO-d
): δ
6 H
0.84(s, 3H, H-18); 2.64(m,
2
2
3
1
H, H-6); 3.62(m, 1H, H-17); 4.40 (s, 1H, OH); 6.40(s, 1H, H-4); 6.50(d, J = 8.5 Hz, 1H, H-
1
3
6 C
); 7.02(d, J = 8.5 Hz, 1H, H-1). C NMR (DMSO-d ): δ 26.0; 28.6(2C); 29.7(C-18); 29.8;
1.8; 32.7; 39.9; 41.3; 43.8(C-13); 51.1; 81.2(C-17); 112.9(C-2); 114.4(C-4); 126.9(C-1);
1
31.3(C-10); 137.4(C-5); 154.5(C-3). 7: Mp 192‒194 °C, R
OD): δ
.48(s, 1H, H-4); 6.58(d, J = 8.5 Hz, 1H, H-2); 7.15(d, J = 8.5 Hz, 1H, H-1). C NMR
OD): δ 22.2(C-18); 23.6; 26.6; 28.6; 28,7; 30.2; 32.7; 42.3; 42.8; 43.3(C-13); 50.3;
f
= 0.53 (ss D); H NMR
(
CD
3
H
0.94(s, 3H, H-18); 2.74(m, 2H, H-6); 4.20(m, 1H, H-17); 4.40 (s, 1H, OH);
13
6
(
CD
3
C
7
3.2(C-17); 112.5(C-2); 114.4(C-4); 126.5(C-1); 131.1(C-10); 137.8(C-5); 154.5(C-3).
2
.1.2.2. 3-Methoxy-13α-estra-1,3,5(10)-trien-17β-ol (5) and 3-methoxy-13α-estra-1,3,5(10)-
trien-17α-ol (8)
As described in Section 2.1.2, ketone 2 (284 mg, 1.0 mmol) was reacted with NaBH
4
(189
mg, 5.0 mmol). Compounds 5 and 8 were identical with compounds described in the literature
1
[
8]. 5: Mp 75‒77 °C, R
f
= 0.52 (ss C); H NMR (DMSO-d
6
): δ 0.85(s, 3H, H-18); 2.71(m,
H
2
H, H-6); 3.63(m, 1H, H-17); 3.68 (s, 3H, 3-OMe); 4.43 (s, 1H, OH); 6.57(s, 1H, H-4);

7
1
3
6
2
1
1
3
.66(d, J = 8.5 Hz, 1H, H-2); 7.14(d, J = 8.5 Hz, 1H, H-1). C NMR (DMSO-d
6
): δ 26.2;
C
8.6; 28.7; 29.8(18-Me); 30.1; 31.9; 32.9; 40.1; 41.3; 43.9(C-13); 51.2; 54.9(3-OMe); 81.3(C-
7); 111.8(C-2); 113.0(C-4); 127.2(C-1); 133.1(C-10); 137.7(C-5); 156.8(C-3). 8: Mp
1
05‒107 °C, R
f
= 0.41 (ss C); H NMR (DMSO-d
6
): δ
H
0.82(s, 3H, H-18); 2.71(m, 2H, H-6);
.68 (s, 3H, 3-OMe); 4.01(m, 1H, H-17); 4.35 (s, 1H, OH); 6.58(s, 1H, H-4); 6.66(d, J = 8.5
13
Hz, 1H, H-2); 7.18(d, J = 8.5 Hz, 1H, H-1). C NMR (DMSO-d
6
): δ
6.3; 28.1; 29.2; 29.9; 32.7; 41.8(2C); 42.9(C-13); 49.7(C-14); 54.7(3-OMe); 71.7(C-17);
11.5(C-2); 113.1(C-4); 126.6(C-1); 131.9(C-10); 137.7(C-5); 156.9(C-3).
C
22.9; 23.6(18-Me);
2
1
2
1
.1.2.3. 3-Benzyloxy-13α-estra-1,3,5(10)-trien-17β-ol (6) and 3-benzyloxy-13α-estra-
,3,5(10)-trien-17α-ol (9)
As described in Section 2.1.2, ketone 3 (360 mg, 1.0 mmol) was reacted with NaBH
4
(189
mg, 5.0 mmol). The crude product was purified by flash chromatography with EtOAc/
CH
2
Cl
2
= 2/98 as eluent. The first-eluted compound 6 was obtained as a white solid after
1
evaporation of the eluent (102 mg, 28%), mp 64‒66 °C, R
f
= 0.58 (ss B); H NMR: δ
H
0.95(s,
3
6
H, H-18); 2.79(m, 2H, H-6); 3.83(m, 1H, H-17α); 5.03(s, 2H, OCH
2
); 6.69(s, 1H, H-4);
.77(dd, J = 8.6 Hz, J = 2.3 Hz, 1H, H-2); 7.19(d, J = 8.6 Hz, 1H, H-1);7.31(t, J = 7.3 Hz, 1H,
1
3
H-4’); 7.37(t, J = 7.3 Hz, 2H, H-3’, H-5’); 7.42(d, J = 7.3 Hz, 2H, H-2’, H-6’). C NMR: δ
C
2
8
6.7; 28.5; 29.0; 29.8(C-18); 30.5; 31.4; 33.2; 40.1; 42.2; 44.4(C-13); 51.5; 69.9(OCH
2
);
3.5(C-17); 112.6(C-2); 114.4(C-4); 127.4(3C: C-1, C-3’, C-5’); 127.8(C-4’); 128.5(2C: C-2’,
C-6’); 133.7(C-10); 137.4(C-1’); 138.3(C-5); 156.5(C-3). ESI-MS m/z (%): 363 (100,
+
[
M+H] ), 91 (15, Bn). Anal. Calcd. for C25
H
30
O
2
: C, 82.83; H, 8.34. Found: C, 82.95; H, 8.26.
Purity from HPLC: 99.1%. Continued elution yielded a mixture of alcohols 6 and 9 (119 mg,
1
3
0
4
1
δ
7
1
3
3%) and finally compound 9 (120 mg, 33%), mp 80‒84 °C, R
.95(s, 3H, H-18); 2.79(m, 2H, H-6); 4.21(m, 1H, H-17β); 5.03(s, 2H, OCH
); 6.81(dd, J = 8.6 Hz, J = 2.3 Hz, 1H, H-2); 7.23(d, J = 8.6 Hz, 1H, H-1);7.31(t, J = 7.3 Hz,
f
= 0.45 (ss B); H NMR: δ
H
2
); 6.72(s, 1H, H-
13
H, H-4’); 7.39(t, J = 7.3 Hz, 2H, H-3’, H-5’); 7.43(d, J = 7.3 Hz, 2H, H-2’, H-6’). C NMR:
22.1(C-18); 23.6; 26.2; 28.2; 29.7; 30.1; 32.7; 41.9(2C); 43.1(C-13); 49.9; 69.6(OCH );
C
2
3.8(C-17); 112.1(C-2); 114.2(C-4); 126.5(C-1); 127.0(2C: C-3’, C-5’); 127.4(C-4’);
28.1(2C: C-2’, C-6’); 132.4(C-10); 136.9(C-1’); 137.9(C-5); 156.3(C-3). ESI-MS m/z (%):
+
+
63 (100, [M+H] ), 345 (30, [M+H] -H
2
O), 91 (21, Bn). Anal. Calcd. for C25
H
30
O : C, 82.83;
2
H, 8.34. Found: C, 82.90; H, 8.37. Purity from HPLC: 98.2%.

8
2
.1.3. Hydrogenolysis of 3-benzyloxy-13α-estra-1,3,5(10),16-tetraene (15)
A suspension of 15 (345 mg, 1.0 mmol) and Pd/C (0.35 g, 10%) in EtOAc (20 ml) was
subjected to 20 bar of H at room temperature for 3 h. The catalyst was then removed by
filtration through a short pad of silica gel. After evaporation of the solvent in vacuo, the crude
2
product was subjected to column chromatography with CH
obtained as a white solid (253 mg, 99%), mp 164‒165 °C, R
):δ
2
Cl
2
as eluent. Compound 16 was
1
f
= 0.48 (ss A); H NMR (DMSO-
d
6
H
0.92(s, 3H, H-18); 2.66(m, 2H, H-6); 6.42(s, 1H, H-4); 6.50(m, 1H, H-2); 7.07(m, 1H,
1
3
H-1). C NMR: δ
1.7; 51.1; 112.7(C-2); 114.6(C-4); 126.5(C-1); 130.3(C-10); 137.4(C-5); 154.7(C-3). ESI-
MS m/z (%): 260 (100). Anal. Calcd. for C18 24O: C, 84.32; H, 9.44. Found: C, 84.21; H,
C
20.4; 26.6; 27.2; 27.9; 29.7(18-Me); 29.8; 33.0; 35.5; 40.8; 41.4(C-13);
4
H
9
.58. Purity from HPLC: 96.9%.
2
.1.4. General procedure for the synthesis of 3-(prop-2-inyloxy)-13α-estra-1,3,5(10)-trienes
(10, 17)
3
1
-Hydroxy-13α-estra-1,3,5(10)-trien-17-one (1, 270 mg, 1.0 mmol) or 17-deoxy-3-hydroxy-
3α-estra-1,3,5(10)-triene (16; 256 mg, 1.0 mmol) was dissolved in acetone (10 ml), and
propargylbromide (0.17 ml (80 wt.% in toluene), 1.5 mmol) and K
were added. The reaction mixture was stirred at 70 °C for 24 h, the solvent was then
2
evaporated off, and the residue was purified by flash chromatography with EtOAc/ CH Cl =
2
CO (968 mg, 7 mmol)
3
2
2
2 2
/98 or CH Cl /hexane = 10/90 as eluent.
2
.1.4.1. 3-(Prop-2-inyloxy)-13α-estra-1,3,5(10)-trien-17-one (10)
As described in Section 2.1.4, 3-hydroxy-13α-estra-1,3,5(10)-trien-17-one (1) (270 mg, 1.0
mmol) was reacted with propargylbromide (0.17 ml, 1.5 mmol). Compound 10 was obtained
1
as a white solid (299 mg, 97%), mp 132‒134 °C, R
f
= 0.70 (ss B); H NMR: δ
H
1.06(s, 3H, H-
1
8
2
7
2
8); 2.49(s, 1H, C≡CH); 2.83(m, 2H, H-6); 4.65(s, 2H, OCH
2
); 6.68(s, 1H, H-4); 6.77(d, J =
13
.5 Hz, 1H, H-2); 7.19(d, J = 8.5 Hz, 1H, H-1). C NMR: δ
C
20.6; 24.7(18-Me); 27.8; 27.9;
9.9; 31.7; 33.1; 41.1(2C: C-8, C-9); 48.9(C-14); 49.7(C-13); 55.3(OCH ); 74.9(C≡CH);
2
8.5(C≡CH); 112.1(C-2); 114.3(C-4); 126.5(C-1); 132.6(C-10); 137.7(C-5); 155.1(C-3);
-
21.2(C-17). ESI-MS (negative ion mode) m/z (%): 307 (54, [M-H] ), 299 (36), 285 (100).
24 2
Anal. Calcd. for C21H O : C, 81.78; H, 7.84. Found: C, 81.92; H, 7.63. Purity from HPLC:
9
7.0%.

9
2
.1.4.2. 17-Deoxy-3-(prop-2-inyloxy)-13α-estra-1,3,5(10)-triene (17)
As described in Section 2.1.4, 17-deoxy-3-hydroxy-13α-estra-1,3,5(10)-triene 16 (256 mg, 1.0
mmol) was reacted with propargylbromide (0.17 ml, 1.5 mmol). Compound 17 was obtained
1
as a white solid (279 mg, 95%), mp 140‒143 °C, R
f
= 0.32 (ss E); H NMR: δ
H
0.96(s, 3H, H-
1
8
2
7
2
8); 2.50(s, 1H, C≡CH); 2.81(m, 2H, H-6); 4.65(s, 2H, OCH ); 6.69(s, 1H, H-4); 6.78(dd, J =
13
.5 Hz, J = 2.0 Hz, 1H, H-2); 7.25(d, J = 8.5 Hz, 1H, H-1). C NMR: δ
8.3; 29.9(18-Me); 30.7; 33.6; 35.8; 41.2; 41.9(C-13); 42.3; 51.7; 55.7(OCH
8.8(C≡CH); 112.4(C-2); 114.6(C-4); 126.9(C-1); 134.0(C-10); 138.5(C-5); 155.3(C-3). ESI-
C
20.9; 26.9; 27.8;
2
); 75.2(C≡CH);
+
+
MS m/z (%): 295 (24, [M+H] ), 255 (36, [M -(CH≡C-CH
2
)]. Anal. Calcd. for C21
H26O: C,
8
5.67; H, 8.90. Found: C, 85.85; H, 9.05. Purity from HPLC: 98.8%.
2
.1.5. General procedure for the preparation of triazoles 12a‒e and 18
To a stirred solution of 3-(prop-2-inyloxy)-13α-estra-1,3,5(10)-trien-17-one 10 (308 mg, 1.0
mmol) or 17-deoxy-3-(prop-2-inyloxy)-13α-estra-1,3,5(10)-triene 17 (294 mg, 1.0 mmol) in
toluene (5 ml), Ph P (26 mg, 0.1 mmol), CuI (9.5 mg, 0.05 mmol), DIPEA (0.52 ml, 3.0
3
mmol) and the appropriate benzylazide 11 (1 equiv. [24–27]) were added. The reaction
mixture was refluxed for 2 h, then allowed to cool and evaporated in vacuo. The residue was
2 2 2 2
purified by flash chromatography with EtOAc/CH Cl = 5/95 or EtOAc/CH Cl = 2/98 as
eluent.
2
.1.5.1. 3-[{1-Benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17-one (12a)
As described in Section 2.1.5, alkyne 10 (308 mg, 1.0 mmol) was reacted with benzyl azide
(
11a, 1.0 mmol). Compound 12a was obtained as a white solid (433 mg, 98%), mp 163‒165
1
°
C, R
f
= 0.35 (ss C); H NMR: δ
H
1.05(s, 3H, H-18); 2.80(m, 2H, H-6); 5.14(s, 2H, OCH
2
);
5
8
.51(s, 2H, NCH
2
); 6.67(s, 1H, H-4); 6.75(dd, J = 8.5 Hz, J = 2.0 Hz, 1H, H-2); 7.16(d, J =
.5 Hz, 1H, H-1); 7.27(m, 2H, H-2’, H-6’); 7.36(m, 3H, H-3’, H-4’, H-5’); 7.50(s, 1H,
1
3
C=CH). C NMR: δ
C
21.0; 25.1(18-Me); 28.2; 28.3; 30.3; 32.0; 33.4; 41.4(2C: C-8, C-9);
4
1
1
9.3(C-14); 50.1(C-13); 54.2(NCH ); 62.1(OCH ); 112.5(C-2); 114.4(C-4); 122.4(C=CH);
2
2
26.9(C-1); 128.1(2C: C-3’, C-5’); 128.8(C-4’); 129.1(2C: C-2’, C-6’); 132.6(C-10);
34.4(C-1’); 138.1(C-5); 144.9(C=CH); 156.1(C-3); 221.5(C-17). ESI-MS m/z (%): 442 (100,
+
[
M+H] ), 144 (22), 91 (14, Bn). Anal. Calcd. for C28
H
31
N
3
2
O : C, 76.16; H, 7.08. Found: C,
7
6.38; H, 6.91. Purity from HPLC: 98.3%.

1
0
2
.1.5.2. 3-[{1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17-
one (12b)
As described in Section 2.1.5, alkyne 10 (308 mg, 1.0 mmol) was reacted with p-tolyl azide
(
11b, 1.0 mmol). Compound 12b was obtained as a white solid (442 mg, 97%), mp 119‒121
1
°
C, R
.13(s, 2H, OCH
H-2); 7.15‒7.17(overlapping multiplets, 5H, 1-H, H-2’, H-3’, 5’-H, H-6’); 7.49(s, 1H,
f
= 0.26 (ss C); H NMR: δ
H
1.05(s, 3H, H-18); 2.35(s, 3H, 4’-CH
3
); 2.80(m, 2H, H-6);
5
2
); 5.47(s, 2H, NCH
2
); 6.66(s, 1H, H-4); 6.74(dd, J = 8.5 Hz, J = 2.0 Hz, 1H,
1
3
C=CH). C NMR: δ
C
21.0; 21.1(4’-Me); 25.1(18-Me); 28.3; 29.7; 30.3; 32.0; 33.4; 41.4(2C:
); 62.1(OCH ); 112.5(C-2); 114.4(C-4);
22.4(C=CH); 126.9(C-1); 128.1(2C: C-3’, C-5’); 129.8(2C: C-2’, C-6’); 131.4(C-10);
C-8, C-9); 49.3(C-14); 50.1(C-13); 54.1(NCH
2
2
1
1
32.6(C-4’); 138.1(C-5); 138.7(C-1’); 144.7(C=CH); 156.1(C-3); 221.5(C-17). ESI-MS m/z
+
(
%): 456 (100, [M+H] ), 158 (29), 105 (27, p-xylol). Anal. Calcd. for C29
H
33
N
3
2
O : C, 76.45;
H, 7.30. Found: C, 76.32; H, 7.58. Purity from HPLC: 98.5%.
2
.1.5.3.
3-[{1-(4-[Prop-2-yl]benzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-
trien-17-one (12c)
As described in Section 2.1.5, alkyne 10 (308 mg, 1.0 mmol) was reacted with 4-(prop-2-
yl)benzyl azide (11c, 1.0 mmol). Compound 12c was obtained as a white solid (465 mg,
1
9
6%), mp 102‒104 °C, R
CH(CH
NCH ); 6.68(s, 1H, H-4); 6.75(d, J = 8.5 Hz, 1H, H-2); 7.15‒7.22(overlapping multiplets, 5H,
f
= 0.30 (ss C); H NMR: δ
H
1.05(s, 3H, H-18); 1.24(m, 2x3H,
3
)
2
); 2.80(m, 2H, H-6); 2.91(m, 1H, CH(CH
3
)
2
); 5.13(s, 2H, OCH ); 5.48(s, 2H,
2
2
1
3
1
2
1
1
1
1
-H, H-2’, H-3’, H-5’, H-6’); 7.65(s, 1H, C=CH). C NMR: δ
4.7(18-Me); 27.8; 27.9; 29.3; 29.9; 31.7; 33.1; 33.4(CH(CH ); 41.1(2C: C-8, C-9); 48.9(C-
4); 49.7(C-13); 53.9(NCH ); 61.7(OCH ); 112.2(C-2); 114.1(C-4); 125.9(C=CH); 126.5(C-
C 3 2
20.6; 23.5(2C: CH(CH ) );
)
3 2
2
2
); 126.8(2C: C-3’, C-5’); 127.8(2C: C-2’, C-6’); 131.3(C-10); 132.2(C-4’); 137.7(C-5);
+
44.8(C=CH); 149.3(C-1’); 155.8(C-3); 221.1(C-17). ESI-MS m/z (%): 484 (100, [M+H] ),
37 3 2
33 (33, 1-methyl-4-(propan-2-yl)benzene). Anal. Calcd. for C31H N O : C, 76.98; H, 7.71.
Found: C, 77.04; H, 7.85. Purity from HPLC: 99.3%.
2
1
.1.5.4. 3-[{1-(4-tert-Butylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-
7-one (12d)
As described in Section 2.1.5, alkyne 10 (308 mg, 1.0 mmol) was reacted with 4-tert-
butylbenzyl azide (11d, 1.0 mmol). Compound 12d was obtained as a white solid (483 mg,

1
1
1
9
7%), mp 110‒112 °C, R
C(CH
.78(m, 1H, H-2); 7.16‒7.20(overlapping multiplets, 3H, H-1, H-2’, H-6’); 7.39(d, 2H, H-3’,
f
= 0.28 (ss C); H NMR: δ
H
1.05(s, 3H, H-18); 1.31(s, 3x3H,
3
)
3
); 2.81(m, 2H, H-6); 5.11(s, 2H, OCH
2
); 5.50(s, 2H, NCH ); 6.69(s, 1H, H-4);
2
6
1
3
H-5’); 7.55(s, 1H, C=CH). C NMR: δ
C(CH ); 32.0; 33.4; 34.6; 37.5(C(CH
2.1(OCH ); 112.5(C-2); 114.4(C-4); 122.8(C=CH); 125.9(2C: C-3’, C-5’); 126.9(C-1);
C
20.9; 25.1(18-Me); 28.2; 28.3; 30.3; 31.2(3C:
3
)
3
3
)
3
); 41.4(2C: C-8, C-9); 49.2(C-14); 50.1(NCH );
2
6
1
1
2
27.9(2C: C-2’, C-6’); 131.3(C-10); 132.6(C-1’); 138.1(C-5); 144.9(C=CH); 151.9(C-4’);
+
56.1(C-3); 221.5(C-17). ESI-MS m/z (%): 498 (100, [M+H] ), 147 (36, 1-tert-butyl-4-
methylbenzene). Anal. Calcd. for C32
Purity from HPLC: 99.1%.
H
39
N
3
2
O : C, 77.23; H, 7.90. Found: C, 77.43; H, 8.02.
2
.1.5.5. 3-[{1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17-
one (12e)
As described in Section 2.1.5, alkyne 10 (308 mg, 1.0 mmol) was reacted with 4-nitrobenzyl
azide (11e, 1.0 mmol). Compound 12e was obtained as a white solid (463 mg, 95%), mp
1
1
5
44‒148 °C, R
f
= 0.27 (ss C); H NMR (DMSO-d
6
): δ
H
0.96(s, 3H, H-18); 2.74(m, 2H, H-6);
.08(s, 2H, OCH
2
); 5.78(s, 2H, NCH
2
); 6.69(s, 1H, H-4); 6.75(dd, J = 8.5 Hz, J = 2.0 Hz, 1H,
H-2); 7.16(d, J = 8.5 Hz, 1H, H-1); 7.52(d, J = 8.6 Hz, 2H, H-2’, H-6’); 8.23(d, J = 8.6 Hz,
1
3
2
3
1
1
2
6
H, H-3’, H-5’); 8.31(s, 1H, C=CH). C NMR: δ
C
20.4; 24.5(18-Me); 27.6; 27.9; 29.7; 31.5;
); 60.9(OCH ); 112.4(C-2);
2.8; 40.7(2C: C-8, C-9); 48.5(C-14); 49.4(C-13); 51.8(NCH
2
2
14.1(C-4); 123.8(2C: C-3’, C-5’); 124.8(C=CH); 126.7(C-1); 128.9(2C: C-2’, C-6’);
31.9(C-10); 137.8(C-5); 143.3 and 143.4(C-1’ and C=CH); 147.2(C-4’); 155.7(C-3);
+
20.6(C-17). ESI-MS m/z (%): 487 (100, [M+H] ), 143 (18). Anal. Calcd. for C28
9.12; H, 6.21. Found: C, 68.81; H, 6.45. Purity from HPLC: 97.6%.
30 4 4
H N O : C,
2
.1.5.6. 3-[{1-Benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-triene (18)
As described in Section 2.1.5, alkyne 17 (294 mg, 1.0 mmol) was reacted with benzyl azide
(
11a, 1.0 mmol). Compound 18 was obtained as a white solid (424 mg, 99%), mp 147‒148
1
°C, R
f
= 0.41 (ss B); H NMR: δ
H
0.96(s, 3H, H-18); 2.78(m, 2H, H-6); 5.15(s, 2H, OCH
2
);
5
1
.53(s, 2H, NCH
); 7.27(overlapping multiplets, 3H, C=CH, H-2’, H-6’); 7.37(m, 3H, H-3’, H-4’, H-5’). C
20.9; 26.9; 27.8; 28.3; 29.9(C-18); 30.6; 33.6; 35.8; 41.2; 41.9(C-13); 42.3; 51.7;
); 62.1(OCH ); 112.4(C-2); 114.5(C-4); 125.9(C=CH); 126.9(C-1); 128.2(2C: C-
2
); 6.68(s, 1H, H-4); 6.77(d, J = 8.5 Hz, 1H, H-2); 7.22(d, J = 8.5 Hz, 1H, H-
13
NMR: δ
C
5
7.3(NCH
2
2

1
2
3
1
’, C-5’); 128.8(C-4’); 129.1(2C: C-2’, C-6’); 133.7(C-10); 134.4(C-1’); 138.5(C-5);
+
42.1(C=CH); 155.9(C-3). ESI-MS m/z (%): 428 (100, [M+H] ), 144 (24). Anal. Calcd. for
28 33 3
C H N O: C, 78.65; H, 7.78. Found: C, 78.42; H, 7.86. Purity from HPLC: 98.6%.
2
.1.6. General procedure for reduction of the triazoles (12a, 12e)
To a stirred solution of 3-[{1-benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-
trien-17-one (12a) (442 mg, 1.0 mmol) or 3-[{1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-
yl}methoxy]-13α-estra-1,3,5(10)-trien-17-one (12e) (487 mg, 1.0 mmol) in a 1:1 mixture of
MeOH and CH
stirred at room temperature for 3 h, then diluted with water and extracted with CH
combined organic phases were washed with water until neutral, dried over Na
evaporated in vacuo. The crude product was a mixture of the 17β-hydroxy (13a, 13b) and
2
Cl
2
(5 ml), NaBH
4
(189 mg, 5.0 mmol) was added. The reaction mixture was
Cl . The
SO and
2
2
2
4
1
7α-hydroxy (14a, 14b) diastereomers in a ratio of 3:2.
2
.1.6.1. 3-[{1-Benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17β-ol (13a)
and 3-[{1-benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17α-ol (14a)
As described in Section 2.1.5, triazole 12a (442 mg, 1.0 mmol) was reacted with NaBH
4
(189
mg, 5.0 mmol). The first-eluted compound 13a was obtained as a white solid after
1
evaporation of the eluent (210 mg, 47%), mp 163‒165 °C, R
DMSO-d
multiplets, 2H, H-8 and H-12); 1.52(m, 1H, 16-H); 1.60–1.65(overlapping multiplets, 2H, H-
f
= 0.63 (ss D); H NMR: δ
H
(
6
) 0.85(s, 3H, H-18); 1.10(m, 1H, H-7); 1.23(m, 1H, H-14); 1.34–1.38(overlapping
1
1
3
8
7
1
9
1
1 and H-15); 1.79(m, 1H, H-15); 1.94–2.00(overlapping multiplets, 3H, H-7 or H-11 and H-
2 and H-16); 2.16(overlapping multiplets, 2H, H-9 and H-7 or H-11); 2.70(m, 2H, H-6);
.36(m, 1H, H-17α); 5.06(s, 2H, OCH
2
); 5.60(s, 2H, NCH
2
); 6.67(s, 1H, H-4); 6.75(dd, J =
.6 Hz, J = 2.3 Hz, 1H, H-2); 7.14(d, J = 8.6 Hz, 1H, H-1); 7.30(m, 2H, H-2’, H-6’);
1
3
.37(overlapping multiplets, 3H, H-3’, H-4’, H-5’); 8.24(s, 1H, C=CH). C NMR: δ
5); 28.4 and 28.5(C-11 and C-17); 29.7(C-18); 29.9(C-6); 31.8(C-12); 32.7(C-16); 39.9(C-
); 41.1(C-8); 43.8(C-13); 51.1(C-14); 52.7(NCH ); 60.9(OCH ); 81.2(C-17); 112.4(C-2);
13.9(C-4); 124.4(C=CH); 127.1(C-1); 127.8(2C: C-3’, C-5’); 128.0(C-4’); 128.6(2C: C-2’,
C
26.0(C-
2
2
C-6’); 133.4(C-10); 135.9(C-1’); 137.6(C-5); 143.2(C=CH); 155.4(C-3). ESI-MS m/z (%):
+
4
7
66 (35), 444 (100, [M+H] ), 144 (18), 91 (8, Bn). Anal. Calcd. for C28
H
33
N
3
O : C, 75.81; H,
2
.50. Found: C, 75.68; H, 7.72. Purity from HPLC: 98.5%.Continued elution yielded a

1
3
mixture of alcohols 13a and 14a (80 mg, 18%) and finally compound 14a (136 mg, 31%), mp
1
1
46‒148 °C, R
H-17β); 5.15(s, 2H, OCH
H, H-1); 7.26(m, 2H, H-2’, H-6’); 7.36(m, 3H, H-3’, H-4’, H-5’); 7.51(s, 1H, C=CH). C
NMR: δ 22.1(18-Me); 23.6; 26.2; 28.1; 29.7; 30.1; 32.6; 41.8(2C: C-8, C-9); 43.1(C-13);
9.9(C-14); 53.9(NCH ); 61.8(OCH ); 73.8(C-17); 112.1(C-2); 114.1(C-4); 122.1(C=CH);
f
= 0.52 (ss D); H NMR: δ
H
0.93(s, 3H, H-18); 2.77(m, 2H, H-6); 4.19(m, 1H,
2
); 5.52(s, 2H, NCH
2
); 6.66(s, 1H, H-4); 6.75(m, 1H, H-2); 7.19(m,
1
3
1
C
4
1
1
2
2
26.6(C-1); 127.8(2C: C-3’, C-5’); 128.4(C-4’); 128.7(2C: C-2’, C-6’); 132.8(C-10);
34.0(C-1’); 138.0(C-5); 144.6(C=CH); 155.6(C-3). ESI-MS m/z (%): 466 (29), 444 (100,
+
[
M+H] ), 144 (19), 91 (10, Bn). Anal. Calcd. for C28
H
33
N
3
O
2
: C, 75.81; H, 7.50. Found: C,
7
5.72; H, 7.84. Purity from HPLC: 98.2%.
2
.1.6.2. 3-[{1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-trien-17β-
and 3-[{1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-estra-1,3,5(10)-
trien17α-ol (14b)
As described in Section 2.1.5, triazole 12e (487 mg, 1.0 mmol) was reacted with NaBH
ol (13b)
4
(189
mg, 5.0 mmol). The first-eluted compound 13b was obtained as a white solid after
1
evaporation of the eluent (182 mg, 37%), mp 135‒136 °C, R
.93(s, 3H, H-18); 2.75(m, 2H, H-6); 3.82(m, 1H, H-17α); 5.18(s, 2H, OCH
NCH ); 6.64(s, 1H, H-4); 6.74(dd, J = 8.6 Hz, J = 2.3 Hz, 1H, H-2); 7.17(d, J = 8.6 Hz, 1H,
H-1); 7.38(d, J = 8.4 Hz, 2H, H-2’, H-6’); 7.58(s, 1H, C=CH); 8.21(d, J = 8.4 Hz, 2H, H-3’,
f
= 0.50 (ss D); H NMR: δ
H
0
2
); 5.63(s, 2H,
2
1
3
H-5’). C NMR: δ
C
26.2; 28.5; 28.7; 29.4(18-Me); 30.1; 31.1; 32.8; 39.8; 41.7; 44.0(C-13);
5
1
1
1.1; 52.7(NCH
2
); 61.6(OCH ); 83.1(C-17); 112.1(C-2); 113.9(C-4); 122.4(C=CH);
2
23.9(2C: C-3’, C-5’); 127.2(C-1); 128.2(2C: C-2’, C-6’); 133.8(C-10); 138.1(C-5); 141.2(C-
+
’); 145.3(C=CH); 147.7(C-4’); 155.3(C-3). ESI-MS m/z (%):469 (65, [M+H] -H
2
O), 453
+
(
100), 350 (39, p-nitrotoluene), 489 (14, [M+H] ). Anal. Calcd. for C28
H
32
N
4
O
4
: C, 68.83; H,
6
.60. Found: C, 69.04; H, 6.43. Purity from HPLC: 98.8%. Continued elution yielded a
mixture of alcohols 13b and 14b (75 mg, 15%) and finally compound 14b (218 mg, 45%), mp
1
1
75‒177 °C, R
H-17β); 5.19(s, 2H, OCH
Hz, J = 2.3 Hz, 1H, H-2); 7.21(d, J = 8.6 Hz, 1H, H-1); 7.39(d, J = 8.5 Hz, 2H, H-2’, H-6’);
f
= 0.40 (ss D); H NMR: δ
H
0.93(s, 3H, H-18); 2.77(m, 2H, H-6); 4.19(m, 1H,
2
); 5.64(s, 2H, NCH
2
); 6.67(d, J = 2.3 Hz, 1H, H-4); 6.76(dd, J = 8.6
1
3
7
2
6
.59(s, 1H, C=CH); 8.22(d, J = 8.5 Hz, 2H, H-3’, H-5’). C NMR: δ
6.5; 28.5; 29.7; 30.0; 32.9; 42.2(2C: C-8, C-9); 43.4(C-13); 50.2(C-14); 53.2(NCH
2.0(OCH ); 74.2(C-17); 112.4(C-2); 114.5(C-4); 122.7(C=CH); 124.3(2C: C-3’, C-5’);
C
22.5(18-Me); 23.9;
2
);
2

1
4
1
1
27.0(C-1); 128.6(2C: C-2’, C-6’); 133.3(C-10); 138.5(C-5); 141.5(C-1’); 145.6(C=CH);
+
48.5(C-4’); 155.9(C-3). ESI-MS m/z (%): 489 (100, [M+H] ). Anal. Calcd. for C28
H
32
N
4
O
4
:
C, 68.83; H, 6.60. Found: C, 68.95 ; H, 6.48. Purity from HPLC: 98.4%.
2
.2. Cell cultures and antiproliferative assays
The growth-inhibitory effects of the compounds were tested in vitro by means of the MTT
assay on HeLa, MCF-7, A2780 and A431 cells, isolated from cervical, breast, ovarian and
skin cancer. The cell lines were obtained from the European Collection of Cell Cultures
(
Salisbury, UK). The cells were maintained in minimal essential medium supplemented with
0% fetal bovine serum (FBS), 1% nonessential aminoacids and an antibiotic-antimycotic
1
mixture (AAM). Human skin fibroblasts (Sigma–Aldrich, Budapest, Hungary) were
maintained in DMEM/F12 medium, supplemented with 10% FBS. All media and
supplements were obtained from Lonza Ltd., (Basel, Switzerland). All chemicals, if otherwise
not specified, were purchased from Sigma-Aldrich Ltd. (Budapest, Hungary). All cell lines
o
were grown in a humidified atmosphere of 5% CO
2
at 37 C. For pharmacological
investigations, 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
exert any substantial effect on the determined cellular functions. All cell types were seeded
into 96-well plates at a density of 5000 cells/well and allowed to stand overnight under cell-
culturing conditions, and the medium containing the tested compound was then added. After a
7
2-h incubation with increasing concentrations (0.03−30 µM) of the compounds, viability was
determined by the addition of 20 µl MTT solution (5 mg/ml) for 4 h. The precipitated
formazan crystals were solubilized in DMSO and the absorbance was determined at 545 nm
with an ELISA reader [28]; wells with untreated cells were utilized as controls. Two
independent experiments were performed with 5 parallel wells; cisplatin, an agent
administered clinically in the treatment of certain gynecological malignancies, was used as
positive control in the same concentration range as the test compounds. For the most effective
compounds, the assays were repeated with a set of dilutions, and sigmoidal dose-response
curves were fitted to the measured data in order to determine the IC50 values by means of
Graphpad Prism 4.0 (Graphpad Software; San Diego, CA, USA).

1
5
3
. Results and discussion
In view of the results reported by Drasar et al. [20], we based our present study on
functionalization of the hormonally inactive 13α-estrone core at 3-OH. Our initial aim was to
introduce a terminal alkyne function onto the phenolic OH group. 13α-Estrone 3-benzyl ether
(
(
(
3) was therefore subjected to hydrogenolysis in order to remove the benzyl protecting group
Scheme 1). The resulting compound (1) was transformed into 3-O-propargyl-13α-estrone
10) by a modified procedure of Drasar et al. [20], using an excess of propargylbromide in the
presence of K
2
CO . The steroidal alkyne (10) was subjected to azide-alkyne ‛click’ reactions
3
with substituted benzyl azides according to a recently published procedure, using a catalytic
amount of CuI [13]. The novel triazoles (12) were obtained in excellent yields (entries 1‒5,
Table 1). In order to establish whether the 17-keto function is necessary for the cytostatic
effect, it was subjected to hydride reduction or was removed. Reduction of the triazoles (12a
4
or 12e) with KBH yielded diastereomeric 17-hydroxy derivatives 13a,b and 14a,b in a ratio
of 3:2 (Table 2, entries 4 and 5). Compounds 1, 2 or 3 were also subjected to hydride
reduction, yielding 4‒7 and 6‒9 in a ratio of nearly 1:1 (Table 2, entries 1, 2 and 3). 17-
Deoxy-13α-estrone (16, Scheme 2) was synthetized by hydrogenation of the 3-benzyloxy-
1
6,17
∆
-derivative of 13α-estrone, which is readily available by a literature method [22]. The
propargylation was carried out as described above, and the resulting terminal alkyne 17 was
subjected to the azide-alkyne ‛click’ reaction with benzyl azide (Table 1, entry 6).
1
13
The structures of the new compounds were confirmed by H and C NMR measurements. As
concerns the C-17 epimeric character of the 17-hydroxy derivatives (4–9, 13, 14), the
1
chemical shifts of the 17β- and 17α-H are characteristic for the appropriate epimers. In the H
NMR spectra of 17β-epimers (4–6, 13), multiplets of 17α-H are shown around 3.6 and 3.8
ppm, but around 4.2 in the spectra of 17α epimers (7–9, 14). The signal of C-18 in the spectra
of the 17β-epimers (4–6, 13) is displayed at higher chemical shift (around 30 ppm) than that
in the 17α counterparts (7–9, 14, around 22 ppm). The β-orientation of 17-OH in compound
1
3a was deduced from the NOESY spectra, since a crosspeak was observed between the
1
signals of 17α-H and 13α-Me. In the H NMR spectra of 10 and 17, the introduction of the
propargyl group onto the 3-O is supported by the singlet at 2.5 ppm, which relates to the
2
terminal alkyne function. The appearance of singlets of NCH at 5.1 ppm, and of C=CH in the
range of 7.5–8.3 ppm (depending on the nature of the p-substituent of the N-benzylphenyl
ring) indicate the presence of the N-benzyltriazolylmethoxy moiety in the triazoles (12).

1
6
Table 1
Scheme 1
The antiproliferative properties of the triazoles (12 and 18) and the 3-hydroxy or ether
derivatives (1‒3, 10 and 17) were characterized in vitro on a panel of human adherent cancer
cell lines (HeLa, MCF-7, A431 and A2780) by means of MTT assays (Table 3). 13α-Estrone
(
1) and its ethers (2 and 3) exerted less than 50% cell-growth inhibition at 30 µM, 2 and 3
proving to be slightly more effective than 1. The 13α-estradiols (4 and 7) displayed a
substantial selective antiproliferative potential against HeLa cells. Comparison of the results
for 17-ketones (2 and 3) and their 17-hydroxy counterparts (5–9) reveals that the presence of
the 17-OH function generally improved the cytostatic properties, especially for the 17α
derivative (9), which at 30 µM exerted 83% growth inhibition on A2780 cells. Following
introduction of a propargyl function onto the 3-OH function of compound 1, the growth
inhibition of the HeLa cells improved, approaching or exceeding 70% at both concentrations.
Further activity enhancements were observed in the cases of 12. The antiproliferative
potencies of the triazoles depended greatly on the substitution pattern of the triazolyl phenyl
function. The most potent compound (12a) exhibited substantial activities, with IC50 values in
the range 0.3‒0.9 µM. The unsubstituted 12a and the compounds containing small p-alkyl
t
groups (12b,c) were more potent than the p-nitro- (12e) or the Bu-phenyltriazolyl derivative
(
[
12d). This differs from the results obtained for the 16-(substitutedphenyl)triazolyl derivatives
18], where all the p-alkylphenyltriazoles were potent, with IC50 values for the most effective
agents in the range 2.4‒3.4 µM. The diastereomeric 17-alcohols of the triazoles (13a and 14a)
displayed similar activities as the 17-ketone 12a. In the cases of the p-nitro derivatives (12e,
1
3b and 14b), substantial enhancements in growth-inhibitory potential were observed for the
7-alcohols (13b and 14b). The orientation of the 17-OH group did not have an appreciable
1
influence on the cytostatic properties.
The cancer selectivity of the most promising compounds (12a, 13a and 14a) was additionally
tested by means of the MTT assay, using non-cancerous human skin fibroblasts. 12a, 13a and
1
4a elicited growth inhibition of 25.9 ± 2.8%, 38.9 ± 1.0 and 35.3 ± 1.9%, respectively (mean
SEM), when applied at a final concentration of 30 µM. Since all of the selected test
±
compounds induced less than 50% growth inhibition at the highest concentration, no IC50
values were calculated. Reference agent ciplatin displayed 73.2 ± 1.7% growth inhibition at

1
7
3
1
0 µM (IC50=21.7 µM), indicating a considerable degree of cancer selectivity of these potent
3α-estrone analogs.
Table 2
Scheme 2
Each of the 17-deoxy derivatives (17, 18) displayed limited growth inhibition in comparison
with their 17-keto analogs (10, 12a, Table 3). It can be stated that the presence of a 17-keto or
1
7-hydroxy function in the 3-triazoles is necessary for the desired cytostatic activity. In
comparison with the low micromolar IC50 values reported earlier for 17α-hydroxy-16-triazolyl
-benzyl ethers [18], introduction of the triazolyl function onto the 3-OH group improves the
3
antiproliferative potency to the submicromolar range.
Table 3

1
8
Conclusions
Novel 13α-estrones derivatized at C-3 and/or C-17 were synthesized. Introduction of an N-
benzyltriazolylmethyl moiety onto 3-OH resulted in compounds with submicromolar in vitro
antiproliferative properties on various human reproductive tumor cell lines. The antitumor
potential was improved by one order of magnitude (submicromolar IC50 values) via
functionalization at C-3 relative to that at C-16, as the earlier described 17α-hydroxy-16β-
triazolyl 3-benzyl ethers displayed only low micromolar cell-growth inhibitory potentials
(
[18]). The presence of the 17-keto or 17-OH function was demonstrated to be necessary for
substantial activity.

1
9
Acknowledgments
The authors are grateful for financial support from the Hungarian Scientific Research Fund
OTKA K113150 and K109293) and Richter Gedeon Plc.. 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’.

2
0
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[
1
9
α-triazolyl-5α-androstane derivatives as potent antiproliferative agents, Org. Biomol. Chem.
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[
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279–87.
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[
1
[
(
[
11a, 11b)
25] Maycock C.D., Santos J.P., Duarte M.F., Frenandez M.T., Costa M.L. (2010) Study of
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[
[

2
3
Legends for Tables
Table 1. Synthesis of 13α derivatives (12 and 18)
Table 2. Synthesis of 13α derivatives (4–9 and 13, 14)
Table 3. Antiproliferative properties of the synthetized compounds
a
Mean value from two independent determinations with five parallel wells; standard deviation
<
15%.
b
Inhibition values <10% are not presented.

2
4
Table 1
Entry
Azide
11a
11b
11c
Alkyne
10
Product
12a
Yield (%)
1
2
3
4
5
6
98
97
96
97
95
99
10
12b
12c
10
11d
11e
10
12d
12e
10
11a
17
18
Table 2
Entry
17-Ketone Products
Yield (%)
45+44
1
2
3
4
5
1
4+7
2
5+8
46+45
3
6+9
47+47
12a
12e
13a+14a
58+38
13b+14b 58+39
Table 3
Inhibition (%) ± SEM
Conc.
a
Comp.
[calculated IC50]
(
µM)
HeLa
A2780
A431
MCF-7
b
1
2
3
10
–
–
–
–
–
–
–
–
–
3
0
16.8 ± 1.7
–
–
–
10
–
–
3
0
44.7 ± 2.3
34.0 ± 0.2
47.5 ± 1.3
25.6 ± 2.2
11.6 ± 1.9
24.4 ± 1.5
19.7 ± 2.3
–
10
3
0
18.1 ± 1.4
9
5.4 ± 0.2
8.6 ± 0.1
4
5
10
–
–
–
–
9
3
0
53.8 ± 0.9
22.2 ± 2.4
[
4.7]
10
13.3 ± 2.0
38.0 ± 1.3
10.6 ± 2.0
39.5 ± 1.1
–
–
–
3
0
13.9 ± 2.6

2
5
6
7
10
29.1 ± 0.6
33.8 ± 0.9
17.9 ± 1.5
48.1 ± 1.2
29.2 ± 1.5
37.9 ± 1.9
–
3
0
16.8 ± 2.9
8
9.2 ± 0.8
8.7 ± 0.1
10
18.7 ± 0.8
75.4 ± 0.4
–
12.2 ± 1.9
54.2 ± 0.8
9
3
0
89.4 ± 0.6
[
8.7]
8
9
10
17.0 ± 0.5
46.9 ± 1.1
29.4 ± 1.0
44.7 ± 1.8
69.5 ± 1.1
81.3 ±2.5
21.3 ± 1.2
46.0 ± 1.1
31.6 ± 2.7
44.5 ± 2.8
48.6 ± 2.0
65.3 ± 0.6
77.5 ± 0.4
78.4 ± 0.9
[0.5]
–
–
3
0
–
23.0 ± 2.5
23.3 ± 2.6
59.6 ± 2.0
29.3 ± 2.8
46.0 ± 1.5
85.8 ± 1.3
85.0 ± 0.2
[0.6]
10
15.3 ± 1.8
83.4 ± 1.1
29.5 ± 1.3
24.9 ± 0.5
95.7 ± 0.3
95.3 ± 0.1
[0.3]
3
0
1
0
10
3
0
9
0.9 ± 0.3
3.3 ± 0.2
1
2a
10
9
3
0
[
0.9]
6
3.0 ± 1.8
7.3 ± 0.9
67.8 ± 0.7
76.5 ± 0.8
[6.9]
83.0 ± 0.5
92.1 ± 0.5
[6.2]
55.2 ± 2.1
60.4 ± 1.1
[9.6]
1
2b
10
7
3
0
[
7.9]
6
7.5 ± 1.6
8.1 ± 0.5
78.9 ± 1.7
79.0 ± 0.7
[4.4]
86.4 ± 2.4
89.2 ± 2.3
[5.4]
54.3 ± 0.9
63.7 ± 2.1
[9.7]
1
2c
10
7
3
0
[
7.4]
1
2d
10
20.4 ± 0.9
35.0 ± 2.6
30.5 ± 1.6
33.6 ± 1.2
33.5 ± 0.6
29.8 ± 2.4
58.8 ± 0.9
58.5 ± 0.5
83.1 ± 0.3
94.8 ± 0.4
[1.2]
18.4 ± 0.6
19.6 ± 3.0
25.0 ± 1.5
25.1 ± 1.1
87.8 ± 0.8
89.5 ± 0.4
[0.8]
-
3
0
10.8 ± 2.1
42.2 ± 1.5
43.9 ± 1.6
61.3 ± 1.6
68.5 ± 2.0
[1.0]
1
2e
3a
10
3
0
8
6.7 ± 1.5
5.8 ± 0.2
1
10
8
3
0
[
2.5]
6
9.9 ± 1.5
0.1 ± 0.7
73.4 ± 0.9
84.7 ± 0.2
[7.7]
37.1 ± 2.2
82.1 ± 0.9
[12.9]
60.9 ± 2.3
70.2 ± 1.3
[11.1]
1
3b
10
8
3
0
[
16.4]
8
5.1 ± 0.3
5.4 ± 0.2
83.1 ± 0.1
86.7 ± 0.1
[0.6]
86.9 ± 0.2
86.2 ± 0.1
[0.5]
72.6 ± 0.5
75.2 ± 1.4
[0.5]
1
4a
10
8
3
0
[
1.6]

2
6
7
0.9 ± 0.9
1.7 ± 0.2
80.7 ± 0.5
88.4 ± 0.3
[4.1]
69.0 ± 0.5
87.7 ± 0.5
[5.9]
52.3 ± 1.3
77.3 ± 1.1
[6.9]
1
4b
10
9
3
0
[
6.2]
–
1
6
7
8
10
–
–
–
3
0
–
46.9 ± 1.4
–
28.1 ± 1.7
–
24.3 ± 1.1
–
1
10
–
3
0
33.9 ± 2.9
24.3 ± 1.2
37.2 ± 1.2
12.9 ± 2.-2
25.1 ± 2.6
68.7 ± 0.7
83.6 ± 1.2
95.0 ± 0.3
[1.3]
–
–
1
10
–
13.5 ± 2.0
42.4 ± 1.3
66.9 ± 1.8
96.8 ± 0.4
[5.8]
3
0
64.0 ± 0.8
93.5 ± 1.2
96.5 ± 1.3
[8.8]
4
2.6± 2.3
99.9 ± 0.3
12.4]
1
3
0
0
cisplatin
[

2
7
Legends for Schemes and Figures
Scheme 1. Synthesis of 13α-estrone derivatives (4–9, 10, 12–14). Reagents and conditions: (i)
propargylbromide (1.5 equiv.), K CO (7 equiv.), acetone, 70 °C, 24 h; (ii) NaBH (5 equiv.),
MeOH/CH Cl = 1/1, rt, 3 h; (iii) CuI (0.05 equiv.), Ph P (0.1 equiv.), DIPEA (3 equiv.),
reflux, 2 h.
2
3
4
2
2
3
Scheme 2. Synthesis of 13α-estrone derivatives (16–18). Reagents and conditions: (i) H
bar), Pd/C, EtOAc, 3 h; (ii) propargylbromide (1.5 equiv.), K CO (7 equiv.), acetone, 70 °C,
4 h; (iii) CuI (0.05 equiv.), Ph P (0.1 equiv.), DIPEA (3 equiv.), toluene, reflux, 2 h.
2
(20
2
3
2
3

2
8
Scheme 1
OH
OH
O
O
ii
H
i
H
+
H
H
7-9
H
H
H
H
4-6
R1O
R1
1
2
3
C
O
10
HC
1,
4
,
7
H
Me
Bn
2
,
5
,
8
9
iii
N3 H2C
R2
3, 6,
1
1
O
H
H
H
R2
1
1, 12
O
1
2
N
a
b
c
H
N
Me
N
prop-2-yl
tBu
ii
d
e
NO2
R2
OH
OH
14 R2
13,
+
a
H
b
NO2
H
H
14
13
Scheme 2
O
i
H
H
H
H
H
H
H
BnO
HO
3
15
16
ii
2
H C
C
O
HC
17
N
3
H
2
C
iii
11
H
H
H
2
H C
2
H C
O
N
1
8
N
N

3
0