Synthesis and in Vitro antiproliferative evaluation of C-13 epimers of triazolyl-D-secoestrone alcohols: The first potent 13α-D-secoestrone derivative

Article abstract of DOI:10.3390/molecules21050611

The syntheses of C-13 epimeric 3-[(1-benzyl-1,2,3-triazol-4-yl)methoxy]-D-secoestrones are reported. Triazoles were prepared from 3-(prop-2-inyloxy)-D-secoalcohols and p-substituted benzyl azides via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The antiproliferative activities of the products and their precursors were determined in vitro against a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780) cell lines by means of MTT assays. The orientation of the angular methyl group and the substitution pattern of the benzyl group of the azide greatly influenced the cell growth-inhibitory potential of the compounds. The 13β derivatives generally proved to be more potent than their 13α counterparts. Introduction of a benzyltriazolylmethyl group onto the 3-OH position seemed to be advantageous. One 13α compound containing an unsubstituted benzyltriazolyl function displayed outstanding antiproliferative activities against three cell lines.

Full text of DOI:10.3390/molecules21050611

molecules
Article
Synthesis and in Vitro Antiproliferative Evaluation of
C-13 Epimers of Triazolyl-D-Secoestrone Alcohols:
The First Potent 13α-D-Secoestrone Derivative
Johanna Szabó 1, Nóra Jerkovics 1, Gyula Schneider 1, János Wölfling 1, Noémi Bózsity 2,
Renáta Minorics 2, István Zupkó 2 and Erzsébet Mernyák 1,
*
1
Department of Organic Chemistry, University of Szeged, Dóm tér 8, Szeged H-6720, Hungary;
johanna.szab@gmail.com (J.S.); jerkovicsnora@gmail.com (N.J.); schneider@chem.u-szeged.hu (G.S.);
wolfling@chem.u-szeged.hu (J.W.)
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, Szeged H-6720,
Hungary; bozsity.noemi@pharm.u-szeged.hu (N.B.); minorics@pharm.u-szeged.hu (R.M.);
zupko@pharm.u-szeged.hu (I.Z.)
2
*
Correspondence: bobe@chem.u-szeged.hu; Tel.: +36-62-544-277
Academic Editor: Maria Emília de Sousa
Received: 31 March 2016; Accepted: 29 April 2016; Published: 12 May 2016
Abstract: The syntheses of C-13 epimeric 3-[(1-benzyl-1,2,3-triazol-4-yl)methoxy]-D-secoestrones are
reported. Triazoles were prepared from 3-(prop-2-inyloxy)-D-secoalcohols and p-substituted benzyl
azides via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The antiproliferative activities of the
products and their precursors were determined in vitro against a panel of human adherent cervical
(HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231, MDA-MB-361 and T47D) and ovarian (A2780)
cell lines by means of MTT assays. The orientation of the angular methyl group and the substitution
pattern of the benzyl group of the azide greatly influenced the cell growth-inhibitory potential of the
compounds. The 13
Introduction of a benzyltriazolylmethyl group onto the 3-OH position seemed to be advantageous.
One 13 compound containing an unsubstituted benzyltriazolyl function displayed outstanding
β derivatives generally proved to be more potent than their 13α counterparts.
α
antiproliferative activities against three cell lines.
Keywords: antiproliferative effect; azide-alkyne cycloaddition; D-secoestrone; triazole
1. Introduction
Anticancer drug design based on synthetic modifications of naturally occurring biomolecules
may lead to nontoxic drug candidates with selective antitumoral potencies [
anticancer agents are already utilized in therapy, but one of the most important requirements of these
drugs is a lack of original hormonal activity [ ]. The literature provides evidence that inversion
of the configuration at C-13 or the opening of ring D of the estrane core may lead to the loss of
estrogenic activity [ ]. We recently reported that 3-benzyl ethers of D-secoestrone alcohol or
oxime (compounds and , Figure 1.) exert substantial in vitro cell growth-inhibitory action against
a number of cancer cell lines, with IC50 values in the low micromolar or submicromolar range [10 11].
Compounds and were diversified at several sites in the molecule, including different modifications
(etherifications, esterifications or debenzylations) at C-3 and/or C-17 and epimerization at C-13 (only
in the case of ). It was concluded that the nature of the 3- and 17-functional groups exerts a great
1,2]. Estrone-based
3,4
5–9
1
2
,
1
2
2
impact on the antiproliferative behavior of the compounds. 3-Ethers proved to be more potent than
their 3-OH counterparts. Derivatives containing a 17-oxime function displayed more pronounced
cytostatic properties than those of 17-hydroxymethyl derivatives. 3-Hydroxy-D-secooxime
2, but not
the D-secoalcohol , was further derivatized by introducing a terminal alkyne function onto the 3-OH
1
Molecules 2016, 21, 611; doi:10.3390/molecules21050611
www.mdpi.com/journal/molecules

Molecules 2016, 21, 611
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group by using propargyl bromide, and the resulting steroid alkyne was subjected to Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) with different substituted benzyl azides. The triazole moiety is
frequently used as a linker in bioconjugates, because it is a good mimic of peptide bonding with high
proteolytic and metabolic stability [12
–14]. Introduction of a triazolyl function into the oxime 2 led to
improved antiproliferative properties as compared with the 3-benzyl ether. The benzyltriazolylmethoxy
D-secooxime
cell lines.
3
(Figure 1) exerted substantial cell growth-inhibitory effects against several human cancer
17
12
CH2OH
11
CH=N-OH
R2
13
14
H
9
16
1
H
10
2
8
7
15
H
16a
A
H
A
H
H
BnO
3
5
R1O
6
4
1
2: R1 = Bn, R2 = CH=CH2
3: R1 = (1-benzyl-1,2,3-triazol-4-yl)methoxy;
R
2 = CH2-CH3
Figure 1. Structures of the potent antiproliferative D-secoestrones 1 and 2, 3.
The abovementioned outstanding results suggested that it might be useful to introduce the
benzyltriazolyl function onto other positions of the D-secoestrone scaffold. In a continuation
of our earlier work, we focused herein on the synthesis of steroidal alkynes bearing a terminal
alkyne function on the opened ring D. The D-secoestrone 17-carboxylic acids were reacted with
propargylamine using peptide coupling reagents [15]. The reactions were carried out in both the 13
α-
and the 13 -estrone series in order to obtain more compounds for structure-activity determinations.
β
The resulting N-propargyl 17-carboxamides were reacted with small molecule azides, such as
substituted benzyl azides, and the resulting triazoles were evaluated for their antiproliferative activities
against several human reproductive cancer cell lines. The activities of the compounds depended
greatly on the substitution pattern of the aromatic ring of the benzyl azide moiety. 3-Benzyl ethers
of 13
A2780 cells.
There are already a number of literature examples of the synthesis of antiproliferative steroidal
β-(p-alkylbenzyl)triazoles displayed outstanding selective antiproliferative potential against
triazoles formed mainly from steroid azides and small molecule alkynes, such as phenylacetylenes.
It has been found that the cytostatic activity of the resulting triazoles depends considerably on the
substitution pattern of the phenyl group of the acetylene [16–22].
In view of the abovementioned recent results, we aimed to introduce the benzyltriazolylmethoxy
moiety onto the 3-OH group of the C-13 epimeric D-secoalcohols. The synthesis of the D-secoestrone
alkynes were first planned at position C-3, followed by the CuAAC reactions of the secosteroidal
alkynes with substituted benzyl azides. The next goal was to perform comparative investigations of
the in vitro antiproliferative activities of the products and their precursors by means of MTT assays
against a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231,
MDA-MB-361 and T47D) and ovarian (A2780) cell lines. Our main objective was to establish some
structure-activity relationship, focusing particularly on the C-13 epimeric character of the compounds.
2. Results
We first synthesized steroidal alkynes in both the 13
After removal of the benzyl protecting group of compounds
propargyl bromide was added in the presence of K2CO3, leading to the 3-(prop-2-inyloxy)-17-alcohols
or 10. The terminal alkynes or 10 were reacted with benzyl azide (11a) or its substituted derivatives
11b under recently published CuAAC reaction conditions [20], furnishing the desired triazolyl
compounds 12a or 13a in high yields. The structures of the triazoles were established from the
corresponding 1H- and 13C-NMR spectra.
α
- and the 13
β-D-secoestrone series (Scheme 1).
1
or
6 by hydrogenolysis, an excess of
9
9
–
e
–
e
–e

Molecules 2016, 21, 611
3 of 13
O
17
CH2OH
13
CH2OH
H
ii
H
H
i
H
A
A
H
H
H
H
H
H
BnO
BnO 3
HO
4, 5
7, 8
1, 6
iii
13β-Me: 1, 4, 7, 9, 12
13α-Me: 5, 6, 8, 10, 13
CH2OH
H
H
H
C
O
9, 10
N3
HC
iv
R
11
CH2OH
11-13 R
H
a
H
H
H
b
c
Me
iPr
tBu
O
12a-e, 13a-e
N
N
N
d
e
NO2
R
Scheme 1. Synthesis of D-secoestrone derivatives (12a–e and 13a–e). Reagents and conditions: (i) 5 equiv.
KBH4, CH2Cl2:MeOH = 1:1, 0 ˝C—r.t., 0.5 h;˝(ii) H2 (20 bar), Pd/C, EtOAc, 3 h; (iii) propargyl bromide
(1.5 equiv.), K2CO3 (7 equiv.), acetone, 70 C, 24 h; (iv) CuI (0.05 equiv.), Ph3P (0.1 equiv.), DIPEA
(3 equiv.), toluene, reflux, 2 h.
The cell growth-inhibitory activities of the triazoles and their precursors were determined in vitro
against a panel of human adherent cervical (HeLa, SiHa and C33A), breast (MCF-7, MDA-MB-231,
MDA-MB-361 and T47D) and ovarian (A2780) cell lines by means of MTT assays (Table 1). The 3-benzyl
ether of the 13
>80% at 10 M. Its C-13 epimer
The 3-OH derivatives and exerted low antiproliferative potentials at both concentrations (even
at 30 M). Among the propargyl derivatives and 10, only the 13 epimer inhibited the growth
of nearly all the cell lines by >50% at 30 M. As concerns the triazoles, 12a displayed the highest
antiproliferative activity against A2780, HeLa and C33A, with inhibition values >80% at 10 M.
β-D-secoalcohol
1
selectively inhibited the growth of MCF-7 cells, with inhibition values
µ
6
at 10 M displayed <50% inhibition on all the examined cell lines.
µ
7
8
µ
9
β
9
µ
µ
The p-methyl derivative 12b displayed somewhat lower potential against the abovementioned cell
lines. Compounds 12c and 12d inhibited the growth of only two cell lines (A2780 and C33A) by
>80% at 30 µM. The 13α-epimeric triazole 13a displayed substantial antiproliferative potential against
A2780, Hela and C33A. 13b inhibited the growth of nearly all cell lines by >50% at 30
13α-epimeric triazoles 13c–e did not influence the growth of the cell lines effectively.
µM. The other
Compound 12a displayed limited growth inhibition against noncancerous human fibroblast cell
line (MRC-5) with values <30% even at 30 µM.
3. Discussion
The benzyl protecting groups of the D-secoalcohols
series were removed by hydrogenolysis, using Pd/C as a catalyst. The saturation of the
side-chain occurred simultaneously. The resulting 3,17-diols and were selectively alkylated
at their 3-OH functions, taking advantage of the more acidic behavior of the phenolic over the
alcoholic OH groups. The propargylations led to the desired terminal alkynes or 10 in high yields.
1,
6
in both the 13
α
- and the 13β-D-secoestrone
δ-alkenyl
7
8
9
The steroidal alkynes were subjected to azide-alkyne cycloadditions under the earlier published
reaction conditions [20], using a catalytic amount of CuI and PPh3 as an accelerating ligand.

Molecules 2016, 21, 611
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Table 1. Antiproliferative properties of the synthesized compounds. Mean value from two independent determinations with five parallel wells; standard
deviation <15%.
1
Inhibition (%) ˘ SEM (Calculated IC50
)
Comp.
Conc. (µM)
A2780
Hela
SiHa
C33A
MCF-7
T47D
MDA-MB-231 MDA-MB-361
2
10
30
42.3 ˘ 0.9
97.5 ˘ 0.1
31.4 ˘ 1.6
97.9 ˘ 0.3
39.2 ˘ 0.6
86.9 ˘ 0.7
81.3 ˘ 0.7
97.4 ˘ 0.3
(6.4)
26.1 ˘ 2.0
87.2 ˘ 0.9
-
28.2 ˘ 0.4
87.6 ˘ 0.4
-
1
84.3 ˘ 0.9
84.5 ˘ 0.9
10
30
43.6 ˘ 2.4
20.1 ˘ 1.8
-
40.4 ˘ 2.1
-
-
-
-
6
7
55.4 ˘ 2.5
60.3 ˘ 1.4
49.7 ˘ 1.4
53.8 ˘ 1.1
36.4 ˘ 1.2
59.6 ˘ 0.8
49.3 ˘ 1.6
77.2 ˘ 1.3
10
30
-
-
-
-
40.5 ˘ 0.8
42.3 ˘ 1.8
24.3 ˘ 2.6
-
-
-
-
-
-
35.1 ˘ 0.8
40.7 ˘ 1.7
51.6 ˘ 2.9
10
30
-
-
23.7 ˘ 0.9
-
-
-
-
-
-
-
-
-
-
-
8
64.5 ˘ 1.1
23.0 ˘ 1.7
10
30
22.4 ˘ 1.0
21.5 ˘ 0.8
89.3 ˘ 1.9
-
30.0 ˘ 1.1
-
-
36.8 ˘ 2.7
-
9
70.7 ˘ 0.4
84.5 ˘ 0.5
70.1 ˘ 0.7
52.5 ˘ 1.0
37.7 ˘ 1.3
81.0 ˘ 1.1
59.0 ˘ 2.9
10
30
-
-
-
-
-
-
-
-
-
-
10
29.7 ˘ 1.9
36.9 ˘ 1.8
39.9 ˘ 1.1
23.3 ˘ 0.6
45.5 ˘ 0.6
28.2 ˘ 2.2
10
30
81.5 ˘ 1.1
88.0 ˘ 0.1
(0.9)
85.4 ˘ 0.3
91.7 ˘ 0.3
(1.1) [23]
21.2 ˘ 1.1
34.5 ˘ 1.2
90.0 ˘ 0.3
95.1 ˘ 0.2
(1.8) [23]
66.3 ˘ 0.3
74.5 ˘ 1.7
(1.5)
51.0 ˘ 1.1
54.4 ˘ 1.8
53.5 ˘ 1.2
59.6 ˘ 1.8
59.3 ˘ 1.4
45.2 ˘ 1.1
12a
12b
10
30
96.8 ˘ 0.2
97.4 ˘ 0.1
(3.8)
52.6 ˘ 0.9
65.4 ˘ 0.9
48.1 ˘ 0.8
64.3 ˘ 1.0
86.8 ˘ 0.8
93.9 ˘ 0.9
(5.0)
71.6 ˘ 1.0
73.9 ˘ 1.0
(5.0)
65.7 ˘ 1.4
66.4 ˘ 1.2
58.3 ˘ 0.7
86.1 ˘ 0.3
(8.3)
87.2 ˘ 0.5
89.3 ˘ 1.1
(4.4)
10
30
83.3 ˘ 0.5
93.4 ˘ 0.1
(5.4)
27.1 ˘ 1.7
66.0 ˘ 2.4
-
57.5 ˘ 1.8
84.6 ˘ 0.9
(8.3)
-
-
33.6 ˘ 0.8
48.9 ˘ 0.7
47.1 ˘ 2.9
45.6 ˘ 0.6
12c
12d
12e
35.6 ˘ 0.3
66.1 ˘ 1.7
53.0 ˘ 1.6
10
30
20.5 ˘ 1.2
30.9 ˘ 3.0
-
-
25.3 ˘ 1.5
-
-
-
-
-
29.0 ˘ 2.0
45.9 ˘ 1.1
64.0 ˘ 1.8
29.5 ˘ 2.7
61.1 ˘ 1.6
63.4 ˘ 0.9
(6.6)
28.9 ˘ 0.9
41.5 ˘ 2.0
49.7 ˘ 2.5
45.5 ˘ 1.2
47.5 ˘ 0.7
46.7 ˘ 1.0
10
30
86.4 ˘ 0.3
89.6 ˘ 0.3
(4.6)
46.3 ˘ 2.5
72.5 ˘ 1.1
25.5 ˘ 2.0
23.2 ˘ 1.2
81.4 ˘ 1.9
88.9 ˘ 0.6
(5.4)
49.8 ˘ 0.7
48.8 ˘ 0.9

Molecules 2016, 21, 611
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Table 1. Cont.
1
Inhibition (%) ˘ SEM (Calculated IC50
)
Comp.
Conc. (µM)
A2780
Hela
SiHa
C33A
MCF-7
T47D
MDA-MB-231 MDA-MB-361
10
30
73.4 ˘ 0.9
83.8 ˘ 0.8
(3.0)
70.0 ˘ 0.7
90.9 ˘ 0.3
(5.3)
41.8 ˘ 1.7
37.1 ˘ 1.0
80.0 ˘ 0.5
93.9 ˘ 0.1
(4.4)
39.4 ˘ 1.2
73.8 ˘ 1.1
36.6 ˘ 0.7
69.1 ˘ 0.6
38.2 ˘ 2.2
59.7 ˘ 1.6
65.8 ˘ 1.0
48.6 ˘ 1.4
13a
13b
10
30
54.3 ˘ 0.8
81.0 ˘ 0.1
(9.8)
33.9 ˘ 1.1
74.4 ˘ 0.9
-
26.6 ˘ 1.8
86.1 ˘ 0.6
-
43.2 ˘ 0.3
89.3 ˘ 0.8
36.7 ˘ 1.8
56.8 ˘ 1.2
-
49.4 ˘ 0.8
65.6 ˘ 1.9
48.7 ˘ 1.0
10
30
-
21.3 ˘ 2.3
-
-
-
-
-
-
-
13c
13d
13e
52.7 ˘ 1.9
40.6 ˘ 0.9
41.7 ˘ 1.2
20.1 ˘ 0.8
39.4 ˘ 1.7
28.1 ˘ 0.7
25.7 ˘ 2.8
10
30
20.0 ˘ 1.9
24.4 ˘ 1.3
36.9 ˘ 0.8
-
26.1 ˘ 0.9
-
-
-
-
44.8 ˘ 1.1
23.0 ˘ 1.2
66.3 ˘ 0.6
30.8 ˘ 1.7
44.4 ˘ 1.7
37.5 ˘ 1.6
40.3 ˘ 2.1
10
30
-
-
-
-
-
-
-
-
-
-
-
-
29.1 ˘ 2.3
83.6 ˘ 1.2
95.0 ˘ 0.3
(1.3)
48.3 ˘ 2.0
42.6 ˘ 2.3
99.9 ˘ 0.3
(12.4)
34.2 ˘ 1.3
83.8 ˘ 0.8
94.0 ˘ 0.6
(3.7)
22.8 ˘ 1.9
51.0 ˘ 2.0
57.9 ˘ 1.5
(9.8)
10
30
88.6 ˘ 0.5
90.2 ˘ 1.8
(7.8)
66.9 ˘ 1.8
96.8 ˘ 0.4
(5.8)
-
67.5 ˘ 1.0
87.8 ˘ 1.1
(3.7)
Cisplatin
71.5 ˘ 1.2
(19.1)
1: Mean value from two independent determinations with five parallel wells; standard deviation <15%; 2: Inhibition values <20% are not presented.

Molecules 2016, 21, 611
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All the CuAACs furnished the triazoles 12a
–e
or 13a
–
e
in excellent yields. The orientation of the
angular methyl group and the substitution pattern of the N-benzyl ring did not influence the yields of
the reactions, as it was expected.
From the comparison of the 1H-NMR spectra of the 3-benzyl ethers
1
and
, the absence of the proton signals of the benzylic protecting group from
the aromatic region, and the presence of the singlet at around 9 ppm clearly indicates the successful
removal of the protecting group. In the 1H-NMR spectra of
and 10, the introduction of the propargyl
6 with those of their
phenolic counterparts
7 and 8
9
group onto the 3-O is supported by the singlet at 2.5 ppm, which relates to the terminal alkyne function,
and by the singlet of double intensity (the OCH2 group of the ether function). In the 13C-NMR spectra
of the triazoles 12a–e and 13a–e, the two OCH2 and the NCH2 carbon signals appear in the 55–70 ppm
range, indicating the presence of the N-benzyltriazolylmethoxy moiety on C-3. There are additional
quaternary carbon signals in the aromatic region of the spectra of the triazoles, belonging to the newly
introduced moiety. As concerns the epimeric character of the triazoles, C-18 in the 13
appears at higher chemical shift (~25 ppm) than that in the 13α counterparts 13a–e (~16 ppm).
The results of the MTT assays of the 3-OH and or 3-ether compounds and 10 revealed
their substantially lower inhibitory properties than those of some triazoles (Table 1). 3-OH derivatives
exerted the lowest growth inhibition, thus the presence of the phenolic OH function in the
β epimers 12a–e
7
8
1, 6, 9
7, 8
D-secoestrone seems to be disadvantageous. A bulky apolar benzyl or a smaller propargyl ether
protecting group on C-3 improved the antiproliferative behavior, leading to values >80% or >50% being
attained at 30
protecting group seemed to be beneficial in both C-13 epimer series. As concerns the triazoles 12a
and 13a , the 13 epimers 12a displayed overall higher inhibitions than their 13 counterparts
13a . In both series, the most potent derivative 12a or 13a was that bearing an unsubstituted N-benzyl
group, as observed earlier in the case of the D-secooxime 11]. The characterization of the mechanism
µM. Introduction of a triazolylmethyl linker between the oxygen on C-3 and the benzyl
–e
–
e
β
–
e
α
–
e
3
[
of the antiproliferative action of 12a on the three cervical cancer cell lines in under publication [23].
However, 12a displayed unusual behavior against the panel of breast cancer cell lines, with inhibition
levels <75% even at 30
µM. It can be stated that the presence of an electron-donating p-alkyl or
an electron-withdrawing p-nitro substituent on the N-benzyl ring of the triazoles 12c
–
e
and 13c
–e
usually proved to be detrimental for biological activity. The inhibitory effects decreased as the size
of the p-alkyl moiety increased. It may be noted that to date there have been no reports of the
13α-D-secoestrone derivatives with high antitumor activity.
The majority of cervical carcinomas originate from high-risk human papillomavirus (HPV)
infections of the epithelial layer of the cervix, including HPV-16, -18, -31 and -35 among others [24].
HeLa is known to be an HPV-18-positive cell line [25]. SiHa and C33A differ in HPV-16 status, since
only SiHa is infected by it. This pathological difference may cause a difference in antiproliferative
action of the compounds against these cell lines. Our test compounds did not significantly influence
the proliferation of SiHa cells, except in the cases of two ethers of the 13
>80% at 30 M. However, several triazoles were similarly potent against HeLa or C33A, independently
of the HPV status of the cell lines.
Substantial differences in the growth-inhibitory potential of compound
β epimer 1 or 9, with values of
µ
1
were determined against
a panel of breast cancer cell lines differing in receptor status [26]. These cell lines included T47D
(expressing the estrogen, progesterone and androgen receptors), MDA-MB-361 (expressing the estrogen
receptor and HER2) and a triple-negative cell line, MDA-MB-231. Compound
1 proved to be selective
against cell line MCF-7. Since the other test compounds displayed similar activities against this cell
line panel, the receptor status of the cells seems irrelevant, as we earlier observed for certain recently
published D-homoestrones [27].
The cancer selectivity of one of the most promising compounds 12a was tested by means of the
same MTT assay, using non-cancerous human fibroblast cells MRC5. Compound 12a elicited growth
inhibition of 24.9%
˘
4.9% (mean
˘
SEM) when applied at a final concentration of 30 µM. The reference
agent cisplatin at the same concentration caused a more substantial inhibition (70.7% ˘ 1.3%).

Molecules 2016, 21, 611
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On the basis of these results, it could be concluded that the selected compound displays limited
growth-inhibitory action against these non-cancerous cells, indicating some selective toxicity towards
fast growing cancer cells.
4. Materials and Methods
4.1. General Information
Melting points (mp) were determined with a Kofler hot-stage apparatus and are uncorrected.
Elemental analyses were performed with a CHN analyzer model 2400 (Perkin-Elmer, Waltham, MA,
USA). Thin-layer chromatography: silica gel 60 F254; layer thickness 0.2 mm (Merck, New York, NY,
USA); eluents: (A) 2% ethyl acetate/98% dichloromethane, (B) 10% ethyl acetate/90% dichloromethane;
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
µm
1
(Merck). H-NMR spectra were recorded in CDCl3 solution (if not otherwise stated) with a DRX-500
instrument (Bruker, Billerica, MA, USA) at 500 MHz, with Me4Si as internal standard. 13C-NMR
spectra were recorded with the same instrument at 125 MHz under the same conditions.
4.2. Chemistry
4.2.1. General Procedure for the Synthesis of 3-Benzyloxy-D-secoalcohols 1, 6
D-secoaldehyde 4 or 5 [10] (374 mg, 1.00 mmol) was dissolved in a 1:1 mixture of dichloromethane
and methanol (10 mL) in an ice-water bath and potassium borohydride (270 mg, 5.00 mmol) was added
in small portions. The mixture was allowed to stand at room temperature for 0.5 h, then diluted with
water and extracted with dichloromethane. The combined organic phases were washed with water
until neutral and dried over sodium sulfate. The crude product was subjected to flash chromatography
with dichloromethane as eluent.
3-Benzyloxy-13
Section 4.2.1, D-secoaldehyde
(270 mg, 5.00 mmol). Compound
oil, Rf = 0.53 (A). H-NMR
α
-hydroxymethyl-14
β
-(prop-2-enyl)-des-D-estra-1,3,5(10)-triene (
(374 mg, 1.00 mmol) was reacted with potassium borohydride
is identical with the compound described in the literature [10]:
m, 2 1H,
1). As described in
4
1
1
δ
ppm 0.80 (s, 3H, 18-H3), 2.85 (m, 2H, 6-H2), 3.30 and 3.61 (2
ˆ
ˆ
17-H2), 5.03 (m, 2H, 16a-H2), 5.04 (s, 2H, OCH2), 5.93 (m, 1H, 16-H), 6.73 (d, 1H, J = 2.3 Hz, 4-H), 6.79
(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H), 7.32 (t, 1H, J = 7.3 Hz, 41-H), 7.38 (t,
1
1
1
1
2H, J = 7.3 Hz, 3 -H and 5 -H), 7.43 (d, 2H, J = 7.3 Hz, 2 -H and 6 -H).
3-Benzyloxy-13 -hydroxymethyl-14 -(prop-2-enyl)-des-D-estra-1,3,5(10)-triene (
Section 4.2.1, D-secoaldehyde (374 mg, 1.00 mmol) was reacted with potassium borohydride (270 mg,
5.00 mmol). Compound was obtained as a white solid. Yield: 347 mg (92%). Mp 50–52 C,
Rf = 0.47 (A). Anal. Calcd. for C26H32O2: C, 82.94; H, 8.57. Found: C, 83.05; H, 8.66. 1H-NMR
ppm 1.06 (s, 3H, 18-H3); 2.82 (m, 2H, 6-H2); 3.53 and 3.72 (2 d, 2 1H, J = 10.8 Hz, 17-H2); 4.96–5.07
(overlapping multiplets, 4H, 16a-H2, OCH2); 5.87 (m, 1H, 16-H); 6.71 (s, 1H, 4-H); 6.79 (d, 1H, J = 8.3 Hz
2-H); 7.21 (d, 1H, J = 8.3 Hz, 1-H); 7.32 (t, 1H, J = 6.9 Hz, 41-H); 7.38 (t, 2H, J = 7.1 Hz, 31-H and 51-H);
β
β
6). As described in
5
˝
6
δ
ˆ
ˆ
,
7.42 (d, 2H, J = 6.7 Hz, 2 -H and 6 -H), 13C-NMR
43.7; 50.7; 64.5 (C-17); 69.9 (OCH2); 112.4 (C-2); 114.5 (C-4); 114.6 (C-16a); 126.3 (C-1); 127.4 (2C:C-3 ,5 );
127.8; 128.5 (2C:C-2 ,6 ); 133.0 (C-4 ); 137.3 (C-10); 137.9 (C-5); 140.2 (C-16); 156.8 (C-3).
δ ppm 25.3 (C-18); 26.5; 27.8; 30.3; 32.4; 35.6; 38.8; 41.2;
1
1
1
1
1
1
1
4.2.2. General Procedure for the Synthesis of 3-Hydroxy-D-secoestrones 7, 8
A suspension of 1 or 6 (376 mg, 1.00 mmol) and Pd/C (0.30 g, 10%) in ethyl acetate (20 mL) was
subjected to 20 bar of hydrogen pressure 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
product was subjected to flash chromatography with dichloromethane as eluent.

Molecules 2016, 21, 611
8 of 13
3-Hydroxy-13
compound
compound described in the literature [10]: Mp 60–62 ˝C, Rf = 0.17 (A). 1H-NMR
18-H3), 2.82 (m, 2H, 6-H2), 3.35 and 3.52 (2 d, 2
6.63 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.16 (d, 1H, J = 8.5 Hz, 1-H).
α
-hydroxymethyl-14
β
-propyl-des-D-estra-1,3,5(10)-triene (
7
). As described in Section 4.2.2,
1
(376 mg, 1.00 mmol) was subjected to hydrogenolysis. Compound
7
is identical with
ppm 0.77 (s, 3H,
δ
ˆ
ˆ
1H, J = 10.9 Hz, 17-H2), 6.56 (d, 1H, J = 2.3 Hz, 4-H),
3-Hydroxy-13 -hydroxymethyl-14 -propyl-des-D-estra-1,3,5(10)-triene (
compound (376 mg, 1.00 mmol) was subjected to hydrogenolysis. The chromatographic purification
of the crude product yielded
for C19H28O2: C, 79.12; H, 9.78. Found: C, 79.25; H, 9.96. DMSO-d6 1H-NMR
J = 6.7 Hz, 16a-H3); 1.17 (s, 3H, 18-H3); 2.69 (m, 2H, 6-H2); 3.19 and 3.48 (2 m, 2
1H, 17-OH); 6.41 (s, 1H, 4-H); 6.50 (dd, 1H, J = 1.76 Hz, J = 8.2 Hz, 2-H); 7.04 (d, 1H, J = 8.5 Hz, 1-H);
8.96 (s, 1H, 3-OH), 13C-NMR
ppm 14.4 (C-16a); 25.0; 25.2 (C-18); 26.3; 27.4; 29.8; 30.3; 35.1; 38.0; 41.3;
β
β
8). As described in Section 4.2.2,
6
˝
8
as a white solid (268 mg, 93%). Mp 50-52 C, Rf = 0.27 (A). Anal. Calcd.
ppm 0.89 (t, 3H,
1H, 17-H2); 4.18 (s,
δ
ˆ
ˆ
δ
43.2; 50.8; 61.5 (C-17); 112.7 (C-2); 114.5 (C-4); 126.0 (C-1); 130.6 (C-10); 136.9 (C-5); 154.8 (C-3).
4.2.3. General Procedure for the Synthesis of 3-(Prop-2-inyloxy)-D-secoestrones 9, 10
3-Hydroxy-D-secoalcohol
7 or 8 (288 mg, 1.00 mmol) was dissolved in acetone (10 mL), propargyl
bromide (0.17 mL (80 wt % in toluene), 1.50 mmol) and potassium carbonate (968 mg, 7.00 mmol) were
added. The reaction mixture was stirred at 70 ˝C for 24 h, the solvent was evaporated off, and the
residue was subjected to flash chromatography with 2% ethyl acetate/98% dichloromethane as eluent.
3-(Prop-2-ynyloxy)-13
Section 4.2.3, 3-hydroxy-D-secoalcohol
(0.17 mL (80 wt % in toluene), 1.50 mmol). Compound
Mp 41-43 ˝C, Rf = 0.40 (A). Anal. Calcd. for C22H30O2: C, 80.94; H, 9.26. Found: C, 81.02; H, 9.35.
1H-NMR
ppm 0.78 (s, 3H, 18-H3); 0.92 (t, 3H, J = 6.9 Hz, 16a-H3); 2.50 (s, 1H, C CH); 2.86 (m, 2H,
6-H2); 3.34 and 3.52 (2 d, 2 1H, J = 10.9 Hz, 17-H2); 4.66 (s, 2H, OCH2); 6.70 (d, 1H, J = 2.3 Hz, 4-H);
6.79 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.24 (d, 1H, J = 8.5 Hz, 1-H), 13C-NMR
ppm 14.7 (C-16a);
15.9 (C-18); 25.0; 26.4; 27.5; 30.7; 31.2; 35.6; 38.7; 41.7; 43.5; 45.2; 55.7 and 71.3 (2
OCH2); 74.9 (C”CH);
78.5 (C”CH); 112.4 (C-2); 114.5 (C-4); 126.6 (C-1); 133.8 (C-10); 138.1 (C-5); 155.4 (C-3).
α
-hydroxymethyl-14
β
-propyl-des-D-estra-1,3,5(10)-triene (
(288 mg, 1.00 mmol) was reacted with propargyl bromide
was obtained as a white solid (280 mg, 86%).
9). As described in
7
9
δ
”
ˆ
ˆ
δ
ˆ
3-(Prop-2-ynyloxy)-13
β
-hydroxymethyl-14
β
-propyl-des-D-estra-1,3,5(10)-triene (10). As described in
Section 4.2.3, 3-hydroxy-D-secoalcohol
8
(288 mg, 1.00 mmol) was reacted with propargylbromide
(0.17 mL (80 wt % in toluene), 1.50 mmol). Compound 10 was obtained as a white solid (271 mg, 83%).
Mp 41–43 ˝C, Rf = 0.50 (A). Anal. Calcd. for C22H30O2: C, 80.94; H, 9.26. Found: C, 80.87; H, 9.42.
1H-NMR
6-H2); 3.47 and 3.73 (2
6.78 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.22 (d, 1H, J = 8.5 Hz, 1-H), 13C-NMR
24.9 (C-18); 25.5; 26.6; 27.7; 30.6; 31.0; 35.2; 38.6; 41.7; 43.7; 51.4; 55.7 and 64.1 (2
δ
ppm 0.92 (t, 3H, J = 6.9 Hz, 16a-H3); 1.03 (s, 3H, 18-H3); 2.50 (s, 1H, C
d, 2 1H, J = 10.9 Hz, 17-H2); 4.65 (s, 2H, OCH2); 6.68(d, 1H, J = 2.3 Hz, 4-H);
ppm 14.6 (C-16a);
OCH2); 75.2 (C”CH);
”
CH); 2.84 (m, 2H,
ˆ
ˆ
δ
ˆ
78.9 (C”CH); 112.4 (C-2); 114.5 (C-4); 126.5 (C-1); 133.8 (C-10); 137.9 (C-5); 155.4 (C-3).
4.2.4. General Procedure for the “Click” Reaction
To a stirred solution of 3-(prop-2-inlyoxy)-D-secoalcohol
(5 mL), PPh3 (52 mg, 0.20 mmol), CuI (19 mg, 0.10 mmol), DIPEA (0.52 ml, 3.00 mmol) and the
appropriate benzyl azide 11 (1 equiv., see [28 31] for their preparation) were added. The reaction
mixture was refluxed for 2 h, allowed to cool and evaporated in vacuo. The residue 12a 13a was
purified by flash chromatography with 10% ethyl acetate/90% dichloromethane as eluent.
9 or 10 (326 mg, 1.00 mmol) in toluene
–
–e
,
–e
3-[{1-Benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13 -hydroxymethyl-14 -propyl-des-D-estra-1,3,5(10)-triene (12a).
α
β
As described in Section 4.2.4, alkyne
9 (326 mg, 1.00 mmol) was reacted with benzyl azide 11a (133 mg,
1.0 mmol). Yield: 428 mg (93%). Mp 41-43 ˝C, Rf = 0.46 (B). Anal. Calcd. for C29H37N3O2: C, 75.78;
1
H, 8.11. Found: C, 75.94; H, 8.25. H-NMR
δ
ppm 0.77 (s, 3H, 18-H3); 0.92 (t, 3H, J = 6.9 Hz, 16a-H3);

Molecules 2016, 21, 611
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2.84 (m, 2H, 6-H2); 3.34 and 3.52 (2
ˆ
d, 2
ˆ
1H, J = 10.9 Hz, 17-H2); 5.16 (s, 2H, OCH2); 5.53 (s, 2H,
NCH2); 6.69 (d, 1H, J = 2.3 Hz, 4-H); 6.77 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.21 (d, 1H, J = 8.5 Hz,
1-H); 17.28 (dd, 2H, J = 8.6 Hz, J = 2.9 Hz, 21-H and 61-H), 7.38 (overlapping multiplets, 3H, 31-H, 41-H
and 5 -H); 7.52 (s, 1H, C=CH), 13C-NMR
δ ppm 14.6 (C-18); 16.0 (C-16a); 25.0; 26.4; 27.4; 30.6; 31.2; 35.6;
38.7; 41.7; 43.5; 45.2; 54.2 (NCH2); 62.1 (OCH2); 71.3 (C-17); 112.4 (C-2); 114.4 (C-4); 122.5 (C=CH); 126.6
(C-1); 128.1 (2C: C-31,51); 128.8 (C-41); 129.1 (2C: C-21,61); 133.5 (C-10); 134.4 (C-11); 138.1 (C-5); 144.9
(C=CH); 156.0 (C-3).
3-[{1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13
α
-hydroxymethyl-14
β-propyl-des-D-estra-1,3,5
(10)-triene (12b). As described in Section 4.2.4, alkyne
4-methylbenzyl azide 11b (147 mg, 1.00 mmol). Yield: 436 mg (92%). Mp 50-52 C, Rf = 0.26 (B).
Anal. Calcd. for C30H39N3O2: C, 76.07; H, 8.30. Found: C, 75.92; H, 8.54. H-NMR
9
(326 mg, 1.00 mmol) was reacted with
˝
1
δ
ppm 0.77 (s, 3H,
18-H3); 0.91 (t, 3H, J = 6.9 Hz, 16a-H3); 2.35 (s, 3H, tolyl-CH3); 2.83 (m, 2H, 6-H2); 3.34 and 3.52 (2ˆ d,
2ˆ 1H
,
J = 10.9 Hz, 17-H2); 5.15 (s, 2H, OCH2); 5.48 (s, 2H, NCH2); 6.68 (d, 1H, J = 2.3 1Hz, 41-H); 6.76
1
(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.17-7.20 (overlapping multiplets, 6H, 1-H, C=CH, 2 -H, 3 -H, 5 -H
and 61-H), 13C-NMR
δ ppm 14.6 (C-18); 15.9 (C-16a); 21.1 (tolyl-CH3); 24.9; 26.4; 27.4; 30.6; 31.2; 35.6;
38.7; 41.7; 43.5; 45.3; 54.2 (NCH2); 62.1 (OCH2); 71.3 (C-17); 112.4 (C-2); 114.4 (C-4); 122.4 (C=CH); 126.5
(C-1); 128.2 (2C: C-31,51); 129.7 (2C: C-21,61); 131.3 and 133.5 (C-10 and C-41); 138.1 (C-5); 138.7 (C-11);
144.7 (C=CH); 156.0 (C-3).
3-[{1-(4-[Prop-2-yl]benzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-hydroxymethyl-14β-propyl-des-D-estra-1,3,5
(10)-triene (12c). As described in Section 4.2.4, alkyne
9 (326 mg, 1.00 mmol) was reacted with
4-(prop-2-yl)-benzyl azide 11c (175 mg, 1.00 mmol). Yield: 452 mg (90%). Mp 41-43 ˝C, Rf = 0.30 (B).
1
Anal. Calcd. for C32H43N3O2: C, 76.61; H, 8.64. Found: C, 76.85; H, 8.76. H-NMR
δ
ppm 0.77 (s, 3H,
18-H3); 0.91 (t, 3H, J = 6.9 Hz, 16a-H3); 1.24 (d, 6H, 2
(2 d, 2 1H, J = 10.9 Hz, 17-H2); 5.19 (s, 2H, OCH2); 5.50 (s, 2H, NCH2); 6.68 (d, 1H, J = 2.3 Hz, 4-H);
6.75 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.22-7.24 (overlapping multiplets, 5H, 1-H, 21-H, 31-H, 51-H
and 61-H); 7.55 (s, 1H, C=CH), 13C-NMR
ppm 14.6 and 15.9 (C-18 and C-16a); 23.8 (2C: CH( H3)2);
24.9; 26.4; 27.4; 30.6; 31.2; 33.8 (
ˆ
prop-2-yl-CH3); 2.83 (m, 2H, 6-H2); 3.33 and 3.52
ˆ
ˆ
δ
C
C
H(CH3)2); 35.6; 38.7; 41.7; 43.5; 45.2; 54.0 (NCH2); 62.0 (OCH2); 71.2
(C-117); 112.3 (C-2); 114.3 (C-4); 122.5 (C=
C
1 H); 126.5 (C-1); 127.1 (2C: C-31,51); 128.2 (2C: C-21,61); 131.7
(C-1 ); 133.5 (C-10); 138.1 (C-5); 144.7 (C-4 ); 149.6 (C=CH); 156.1 (C-3).
3-[{1-(4-tert-Butylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-hydroxymethyl-14β-propyl-des-D-estra-1,3,5
(10)-triene (12d). As described in Section 4.2.4, alkyne
9 (326 mg, 1.00 mmol) was reacted with
4-tert-butylbenzyl azide 11d (189 mg, 1.00 mmol). Yield: 475 mg (92%). Mp 58-60 ˝C, Rf = 0.32 (B).
1
Anal. Calcd. for C33H45N3O2: C, 76.85; H, 8.79. Found: C, 75.98; H, 8.95. H-NMR
δ
ppm 0.77 (s, 3H,
18-H3); 0.91 (t, 3H, J = 6.9 Hz, 16a-H3); 1.31 (s, 9H, 3
ˆ
tBu-CH3); 2.83 (m, 2H, 6-H2); 3.33 and 3.52 (2ˆ d
,
2
ˆ
1H, J = 10.9 Hz, 17-H2); 5.18 (s, 2H, OCH2); 5.50 (s, 2H, NCH2); 6.68 (d, 1H,1J = 2.3 Hz, 4-H); 6.77
1
(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.20-7.24 (overlapping multiplets, 3H, 1-H, 2 -H and 6 -H); 7.39 (d,
2H, J = 8.1 Hz, 31-H and 51-H); 7.54 (s, 1H, C=CH), 13C-NMR
24.9; 26.4; 27.4; 30.7; 31.1 (3C: C( H3)3); 31.2; 34.6 (
62.1 (OCH2); 71.3 (C-17); 112.4 (C-2); 114.4 (C-4); 122.5 (C=
δ ppm 14.7 and 15.9 (C-18 and C-16a);
C
C(CH3)3); 35.6; 38.7; 41.7; 43.5; 45.2; 54.0 (NCH2);
C
1H); 126.0 (2C: C-31,51); 126.6 (C-1); 127.9
1
1
1
(2C: C-2 ,6 ); 131.3 (C-1 ); 133.5 (C-10); 138.1 (C-5); 144.8 (C-4 ); 151.9 (C=CH); 156.0 (C-3).
3-[{1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13α-hydroxymethyl-14β-propyl-des-D-estra-1,3,5(10)
-triene (12e). As described in Section 4.2.4, alkyne 9 (326 mg, 1.00 mmol) was reacted with 4-nitrobenzyl
azide 11e (178 mg, 1.00 mmol). Yield: 475 mg, 94%). Mp 65-67 ˝C, Rf = 0.20 (B). Anal. Calcd. for
1
C29H36N4O4: C, 69.02; H, 7.19. Found: C, 69.15; H, 7.02. H-NMR
δ
ppm 0.77 (s, 3H, 18-H3); 0.91 (t,
3H, J = 6.9 Hz, 16a-H3); 2.83 (m, 2H, 6-H2); 3.34 and 3.52 (2
ˆ
d, 2
ˆ
1H, J = 10.9 Hz, 17-H2); 5.19 (s, 2H,
OCH2); 5.64 (s, 2H, NCH2); 6.67 (d, 1H, J = 2.3 Hz, 4-H); 6.76 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H); 7.21
(d, 1H, J = 8.5 Hz, 1-H); 7.40 (d, 2H, J = 8.6 Hz, 21-H, 61-H); 7.62 (s, 1H, C=CH); 8.22 (d, J = 8.6 Hz, 2H,
31-H, 51-H), 13C-NMR
δ ppm 14.6 (C-18); 15.9 (C-16a); 24.9; 26.4; 27.4; 30.7; 31.2; 35.6; 38.7; 41.7; 43.5;

Molecules 2016, 21, 611
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45.2; 53.1 (NCH2); 62.0 (OCH2); 71.2 (C-17); 112.3 (C-2); 114.3 (C-4); 122.8 (
126.6 (C-1); 128.6 (2C: C-2 ,6 ); 133.7 (C-10); 138.2 (C-5); 141.5 and 145.5 (C-1 and C=CH); 148.1 (C-4 );
155.9 (C-3).
C
1 H=C); 124.3 (2C: C-31,511);
1
1
3-[{1-Benzyl-1H-1,2,3-triazol-4-yl}methoxy]-13
β-hydroxymethyl-14β-propyl-des-D-estra-1,3,5(10)-triene
(13a). As described in Section 4.2.4, alkyne 10 (326 mg, 1.00 mmol) was reacted with benzyl azide 11a
(133 mg, 1.00 mmol). Yield: 437 mg (95%). Oil, Rf = 0.19 (B). Anal. Calcd. for C29H37N3O2: C, 75.78; H,
1
8.11. Found: C, 75.93; H, 8.02. H-NMR
δ
ppm 0.89 (t, 3H, J = 7.2 Hz, 16a-H3); 1.00 (s, 3H, 18-H3); 2.80
(m, 2H, 6-H2); 3.44 and 3.70 (2
ˆ
d, 2
ˆ
1H, J = 10.9 Hz, 17-H2); 5.13 (s, 2H, OCH2); 5.50 (s, 2H, NCH2);
6.66 (d, 1H, J = 2.4 Hz, 4-H); 6.73 (dd, 1H, J = 8.5 Hz, J = 2.1 Hz, 2-H); 7.16 (d, 1H, J = 8.5 Hz, 1-H);
7.24 (d, 2H, J = 7.6 Hz, 21-H and 61-H), 7.35 (overlapping multiplets, 3H, 31-H, 41-H and 51-H); 7.51 (s,
1H, C=CH), 13C-NMR
δ
ppm 14.5 (C-16a); 24.9 (C-18); 25.5; 26.6; 27.7; 30.5; 30.9; 35.2; 38.5; 41.6; 43.7;
51.4; 54.2 (NCH2); 62.0 and 64.0 (OCH2 and C-17); 112.3 (C-2); 114.3 (C-4); 122.5 (C= H); 126.5 (C-1);
=CH);
C
1
1
1
1
1
1
128.1 (2C: C-3 ,5 ); 128.8 (C-4 ); 129.1 (2C: C-2 ,6 ); 133.4 (C-10); 134.4 (C-1 ); 137.9 (C-5); 145.0 (
C
156.0 (C-3).
3-[{1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13β-hydroxymethyl-14β-propyl-des-D-estra-1,3,5
(10)-triene (13b). As described in Section 4.2.4, alkyne 10 (326 mg, 1.00 mmol) was reacted with
˝
4-methylbenzyl azide 11b (147 mg, 1.00 mmol). Yield: 431 mg (91%). Mp 49-51 C, Rf = 0.16 (B).
1
Anal. Calcd. for C30H39N3O2: C, 76.07; H, 8.30. Found: C, 75.94; H, 8.22. H-NMR
δ ppm 0.90 (t,
3H, J = 7.5 Hz, 16a-H3); 1.02 (s, 3H, 18-H3); 2.36 (s, 3H, tolyl-CH3); 2.82 (m, 2H, 6-H2); 3.47 and 3.73
2ˆ d, 2 1H, J = 10.9 Hz, 17-H2); 5.14 (s, 2H, OCH2); 5.48 (s, 2H, NCH2); 6.68 (s, 1H, 4-H); 6.76 (d, 1H,
J = 7.8 Hz, 2-H); 7.17 (overlapping multiplets, 6H, 1-H, C=CH, 21,31,51,61-H), 13C-NMR
ppm 14.6
(C-16a); 21.1 (tolyl-CH3); 24.9 (C-18); 25.5; 26.6; 27.7; 30.5; 30.9; 35.2; 38.6; 41.7; 43.7; 51.4; 54.2 (NCH2);
(
ˆ
δ
62.0 and 64.1 (O1CH2 and C-17); 112.4 (C-2); 114.3 (C-4); 122.4 (C=1
C
H); 126.5 (C-1); 128.2 (2C: C-31,51);
129.8 (2C: C-2 ,6 ); 131.3 (C-4 ); 133.4 (C-10); 137.9 (C-5); 138.8 (C-1 ); 144.7 (C=CH); 156.0 (C-3).
1
1
3-[{1-(4-[Prop-2-yl]benzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13β-hydroxymethyl-14β-propyl-des-D-estra-1,3,5
(10)-triene (13c). As described in Section 4.2.4, alkyne 10 (326 mg, 1.00 mmol) was reacted with
4-(prop-2-yl)-benzyl azide 11c (175 mg, 1.00 mmol). Yield: 462 mg (92%). Mp 47-49 ˝C, Rf = 0.19 (B).
1
Anal. Calcd. for C32H43N3O2: C, 76.61; H, 8.64. Found: C, 76.85; H, 8.53. H-NMR
δ
ppm 0.91 (t, 3H,
J = 6.8 Hz, 16a-H3); 1.02 (s, 3H, 18-H3); 1.24 (d, 2
6-H2); 2.90 (m, 1H, prop-2-yl-CH); 3.47 and 3.73 (2
ˆ
3H, J = 11.4 Hz, 2
ˆ
prop-2-yl-CH3); 2.82 (m, 2H,
ˆ
d, 2 1H, J = 10.8 Hz, 17-H2); 5.15 (s, 2H, OCH2);
ˆ
5.49 (s, 2H, NCH2); 6.68 (d, 1H, J = 2.21Hz, 4H); 6.76 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H); 7.19–7.23
δ ppm 14.5 (C-16a); 23.8
1
1
1
(overlapping multiplets, 5H, 1-H, 2 ,3 ,5 ,6 -H); 7.57 (s, 1H, C=CH), 13C-NMR
(2C: CH(
CH3)2); 24.8 (C-18); 25.5; 26.6; 27.7; 30.6; 31.0; 33.8 (CH(CH3)2); 35.2; 38.6; 41.7; 43.7; 51.4; 54.2
1 1
H); 126.5 (C-1); 127.2 (2C: C-3 ,5 );
(NCH2); 62.1 (OCH2); 64.1 (C-17); 112.4 (C-2); 114.4 (C-4); 122.6 (C=
128.2 (2C: C-2 ,6 ); 131.7 (C-1 ); 133.4 (C-10); 137.9 (C-5); 149.7 (C-4 ); 150.1 (C=CH); 156.0 (C-3).
C
1
1
1
1
3-[{1-(4-tert-Butylbenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13 -hydroxymethyl-14 -propyl-des-D-estra-1,3,5
β
β
(10)-triene (13d). As described in Section 4.2.4, alkyne 10 (326 mg, 1.00 mmol) was reacted with
4-tert-butylbenzyl azide 11d (189 mg, 1.00 mmol). Yield: 470 mg (91%). Mp 58-60 ˝C, Rf = 0.32 (B).
1
Anal. Calcd. for C33H45N3O2: C, 76.85; H, 8.79. Found: C, 76.72; H, 8.90. H NMR
δ
ppm 0.91 (t, 3H,
J = 7.2 Hz, 16a-H3); 1.02 (s, 3H, 18-H3); 1.32 (s, 3
(2 d, 2 1H, J = 10.9 Hz, 17-H2); 5.16 (s, 2H, OCH2); 5.49 (s, 2H, NCH2); 6.67 (d, 1H, J = 2.2 Hz, 4H);
6.76 (dd, 1H, J = 8.6 Hz, J = 2.2 Hz, 2-H); 7.18 (d, 1H, J = 8.6 Hz, 1-H); 7.21 (d, 2H, J = 8.2 Hz, 21, 61-H);
7.39 (d, 2H, J = 8.2 Hz, 31, 51-H); 7.54 (s, 1H, C=CH), 13C-NMR
ppm 14.5 (C-16a); 24.9 (C-18); 25.5;
26.6; 27.7; 30.5; 30.9; 31.2 (3C: C( H3)3); 34.6 (
(OCH2); 64.1 (C-17); 112.4 (C-2); 114.3 (C-4); 122.6 (C=
ˆ
3H, 3
ˆ
tBu-CH3); 2.82 (m, 2H, 6-H2); 3.47 and 3.73
ˆ
ˆ
δ
C
C(CH3)3); 35.2; 38.6; 41.7; 43.7; 51.4; 54.0 (NCH2); 62.1
C
1H); 126.0 (2C: C-31,51); 126.5 (C-1); 127.9 (2C:
1
1
1
C-2 ,6 ); 131.3 (C-1 ); 133.4 (C-10); 137.9 (C-5); 144.7 (C-4 ); 151.9 (C=CH); 156.0 (C-3).
3-[{1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl}methoxy]-13β-hydroxymethyl-14β-propyl-des-D-estra-1,3,5(10)
-triene (13e). As described in Section 4.2.4, alkyne 10 (326 mg, 1.00 mmol) was reacted with
4-nitrobenzyl azide 11e (178 mg, 1.00 mmol). Yield: 475 mg, 94%). Mp 50-52 ˝C, Rf = 0.12 (B). Anal.

Molecules 2016, 21, 611
11 of 13
Calcd. for C29H36N4O4: C, 69.02; H, 7.19. Found: C, 69.21; H, 7.05. 1H-NMR
δ
ppm 0.91 (t, 3H,
J = 6.9 Hz, 16a-H3); 1.02 (s, 3H, 18-H3); 2.82 (m, 2H, 6-H2); 3.47 and 3.73 (2
ˆ
d, 2
ˆ
1H, J = 10.9 Hz,
17-H2); 5.19 (s, 2H, OCH2); 5.65 (s, 2H, NCH2); 6.68 (s, 1H, 4-H); 6.76 (d, 1H, J = 8.6 Hz, 2-H); 7.210 (d,
1
1
1
1H, J = 8.6 Hz, 1-H); 7.40 (d, 2H, J = 8.3 Hz, 2 ,6 -H); 7.64 (s, 1H, C=CH); 8.23 (d, 2H, J = 8.5 Hz, 3 ,5 -H),
13C-NMR
δ
ppm 14.5 (C-16a); 24.9 (C-18); 25.5; 26.6; 27.7; 30.6; 31.0; 35.12; 38.6; 41.7; 43.7; 51.4; 53.3
1
(NCH2); 62.0 (OCH2); 64.1 (C-17); 112.3 (C-2); 114.4 (C-4); 124.3 (2C: C-3 ,5 ); 124.8 (C=CH); 126.6 (C-1);
1
1
1
1
128.6 (2C: C-2 ,6 ); 133.6 (C-10); 138.0 (C-5); 141.4 (C-1 ); 144.0 (C=CH); 144.8 (C-4 ); 155.9 (C-3).
4.3. Determination of Antiproliferative Activities
The antiproliferative properties of the prepared triazoles 12a–e or 13a–e and compounds 1, 6–10
were determined on a panel of human adherent gynecological cancer cell lines. MCF-7, MDA-MB-231,
MDA-MB-361 and T47D were isolated from breast cancers differing in biochemical background,
while HeLa, SiHa and C33A cells were from cervical cancers of various pathological histories, and
A2780 cells were isolated from ovarian cancer. Non-cancerous human fibroblast cells (MRC-5) was
additionally used to assess the cancer selectivity of the most effective compound. All cell lines were
purchased from European Collection of Cell Cultures (ECCAC, Salisbury, UK) except for SiHa and
C33A, which were obtained from LGC Standards GmbH (Wesel, Germany). Cells were cultivated
in minimal essential medium supplemented with 10% fetal bovine serum, 1% non-essential amino
acids and an antibiotic-antimycotic mixture. All media and supplements were obtained from Lonza
Group Ltd., (Basel, Switzerland). Near-confluent cancer cells were seeded onto a 96-well microplate
(5000 cells/well except for C33A and MDA-MB-361, which were seeded at 10,000/well) and, after
overnight standing, 200
added. After incubation for 72 h at 37 ˝C in humidified air containing 5% CO2, the living cells were
assayed by the addition of 20 L of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
µL new medium, containing the tested compounds at 10 and 30 µM, was
µ
bromide (MTT) solution. MTT was converted by intact mitochondrial reductase and precipitated as
purple crystals during a 4-h contact period. The medium was next removed and the precipitated
˝
formazan crystals were dissolved in 100 µL of DMSO during a 60-min period of shaking at 37 C.
Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with untreated
cells served as control [32]. In the case of the most active compounds (i.e., higher than 70% growth
inhibition at 30 µM), the assays were repeated with a set of dilutions, sigmoidal dose-response curves
were fitted to the determined data and the IC50 values (the concentration at which the extent of cell
proliferation was half that of the untreated control) were calculated by means of GraphPad Prism
4.0 (GraphPad Software, San Diego, CA, USA). All in vitro experiments were carried out on two
microplates with at least five parallel wells. Stock solutions of the tested substances (10 mM) were
prepared in DMSO. The highest DMSO content of the medium (0.3%) did not have any substantial
effect on the cell proliferation. Cisplatin (Ebewe Pharma GmbH, Unterach, Austria) was used as
positive control.
5. Conclusions
Novel antiproliferative triazolyl D-secoestrone derivatives were synthesized by introducing the
triazolylmethyl linker between the 3-OH and the benzyl or p-substituted benzyl protecting group.
The “clicking” of benzyl azides to the 3-propargyl-D-secoestrones led to potent antiproliferative
compounds. The synthesized derivatives differed at two sites of the molecules: in the orientation
of the angular methyl function and in the nature of the substituent present on the 3-OH group.
It can be stated that both variables substantially influenced the antiproliferative behavior. The 3-OH
derivatives displayed the lowest growth-inhibitory action. Etherification of the phenolic OH
group improved the cytostatic properties moderately, but the incorporation of the triazolylmethyl
linker between the protecting group and the 3-O nevertheless increased the inhibitory values of
the compounds substantially. 13β-Methyl derivatives proved to be more potent than their 13α
counterparts overall. As concerns the p-substituent on the N-benzyl ring, neither the presence of the
electron-withdrawing nor that of the electron-donating group appeared to be advantageous, in contrast

Molecules 2016, 21, 611
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with the unsubstituted derivative. It can be concluded that the combination of the 13
β-methyl and
the 3-(N-benzyltriazolylmethoxy) group on the D-secoestrone scaffold intensifies the antiproliferative
potential. These compounds are the most potent cell-growth-inhibitor D-secoestrones reported to date.
Antiproliferative potential of 13α-D-secoestrone derivatives is a novel finding.
Acknowledgments: The authors are grateful for financial support from the Hungarian Scientific Research Fund
(OTKA K113150 and K109293). The work of Noémi Bózsity and Renáta Minorics was supported by a PhD
Fellowship of the Talentum Fund of Richter Gedeon Plc. (Budapest) and a János Bolyai Research Scholarship of
the Hungarian Academy of Sciences, respectively.
Author Contributions: Johanna Szabó, Nóra Jerkovics and Noémi Bózsity performed the experiments;
János Wölfling and Gyula Schneider contributed reagents, materials and analysis tools; Erzsébet Mernyák,
Renáta Minorics and István Zupkó conceived and designed the experiments; Erzsébet Mernyák and
Renáta Minorics and István Zupkó analyzed the data; Erzsébet Mernyák and István Zupkó wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).