Synthesis and biological evaluation of triazolyl 13α-estrone-nucleoside bioconjugates

Article abstract of DOI:10.3390/molecules21091212

2' -Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne-azide click reaction (CuAAC). For the introduction of the azido group the 5? -position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3? -hydroxy groups of the nucleosides were protected by acetyl groups and the 5? -hydroxy groups were modified by the tosyl-azide exchange method. The commonly used conditions for click reaction between the protected-5? -azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC₅₀ value of 9 μM). The thymidine conjugate displayed considerable 17β-HSD1 inhibitory activity (IC₅₀ = 19 μM).

Full text of DOI:10.3390/molecules21091212

molecules
Article
Synthesis and Biological Evaluation of Triazolyl
13α-Estrone–Nucleoside Bioconjugates
Brigitta Bodnár 1, Erzsébet Mernyák 2, János Wölfling 2, Gyula Schneider 2,
Bianka Edina Herman 3, Mihály Szécsi 3, Izabella Sinka 4, István Zupkó 4, Zoltán Kupihár 1,
*
and Lajos Kovács 1,
*
1
Department of Medicinal Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary;
bodnar.brigitta@med.u-szeged.hu
2
3
4
Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary;
bobe@chem.u-szeged.hu (E.M.); wolfling@chem.u-szeged.hu (J.W.); schneider@chem.u-szeged.hu (G.S.)
1st Department of Medicine, University of Szeged, Korányi fasor 8-10, H-6720 Szeged, Hungary;
herman.bius@gmail.com (B.E.H.); szecsi.mihaly@med.u-szeged.hu (M.S.)
Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6,
H-6720 Szeged, Hungary; sinkaiza@gmail.com (I.S.); zupko@pharm.u-szeged.hu (I.Z.)
Correspondence: kupihar.zoltan@med.u-szeged.hu (Z.K.), kovacs.lajos@med.u-szeged.hu (L.K.);
Tel.: +36-62-545145 (L.K. & Z.K.); Fax: +36-62-545971 (L.K. & Z.K.)
*
Academic Editor: Gyorgy M. Keseru
Received: 28 July 2016; Accepted: 6 September 2016; Published: 10 September 2016
Abstract: 20-Deoxynucleoside conjugates of 13
α-estrone were synthesized by applying the
copper-catalyzed alkyne–azide click reaction (CuAAC). For the introduction of the azido group
the 50-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of
13α
-estrone were chosen. The best yields were realized in our hands when the 30-hydroxy groups
of the nucleosides were protected by acetyl groups and the 50-hydroxy groups were modified by
the tosyl–azide exchange method. The commonly used conditions for click reaction between the
protected-50-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent
of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for
antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and
the potential inhibitory activity of the new conjugates on human 17
1 (17 -HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates
displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines
β-hydroxysteroid dehydrogenase
β
(the protected cytidine conjugate proved to be the most potent with IC50 value of 9 µM). The thymidine
conjugate displayed considerable 17β-HSD1 inhibitory activity (IC50 = 19 µM).
Keywords: nucleosides; 13
antiproliferative; 17β-HSD1
α-estrone; copper-catalyzed alkyne–azide click reaction; triazoles;
1. Introduction
Estrogens are synthesized biochemically in a multistep process from cholesterol in human body [1],
and are responsible for the development of secondary sexual characteristics in females and maintenance
of central nervous system, cardiovascular system and bones. Since they play a crucial role in the cell
proliferation, overproduction of estrogens may lead to enhanced proliferation of hormone sensitive
cells, resulting in hormone dependent cancers: ovarian, uterine, breast, prostate and endometrial [
Estrone-based anticancer drugs have been developed as antiproliferative/antihormonal or cytotoxic
agents acting on non-hormonal targets [ ]. Antiproliferative estrogens exert their activity as enzyme
2].
3
inhibitors or as antiestrogens (acting through their receptors). Although the proliferation process
is rather complex, one of potential enzymes which plays crucial role in the proliferation process of
Molecules 2016, 21, 1212; doi:10.3390/molecules21091212
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1212
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some cancer cell types is the human 17β-hydroxysteroid dehydrogenase 1 (17
β-HSD1). It catalyzes
the reduction of estrone to 17
High activity of this isozyme can be detected in female reproductive tissues, e.g., in the ovaries
and in the placenta [ ]. 17 -HSD1 has been found to be responsible for the local overproduction of
17 -estradiol in various breast cancers and ovarian cancers [ ]. The inhibition of 17 -HSD1 with
suitable pharmacons decreases synthesis of 17 -estradiol and causes significant estrogen deprivation
and antitumor effect in hormone dependent cancers, therefore 17 -HSD1 inhibitors could be promising
candidates of anti-estrogen therapy [ ]. Regardless of the mechanism of antiproliferative action, there
is a general requirement in the development of all estrone-based anticancer drugs: the lack of estrogenic
activity. Chemical modifications of estrone may lead to compounds lacking hormonal behavior [ 10].
Substitution at C-2, opening of ring D or inversion at C-13 of the estrane skeleton usually leads to
the loss of estrogenic activity [11 16]. 13 -Estrone may be an excellent scaffold for the design of
hormonally inactive agents having antiproliferative activity and fortunately it is readily available
from native 13 -estrone by the method of Yaremenko and Khvat, using 1,2-phenylenediamine and
acetic acid [17]. We recently published 13 -estrone, 1,2,3-triazolyl 13 - and D-secoestrone derivatives
possessing substantial cytostatic and/or 17 -HSD1 inhibitory properties [18 22]. 13 -Estrone itself
exerted outstanding 17 -HSD1 inhibitory activity with an IC50 value comparable to that of the
β-estradiol, which enhances the proliferation of certain cancer cells [4].
5
β
β
6
β
β
β
7,8
3,9,
–
α
β
α
α
β
–
α
β
reference estrone [22]. Concerning the triazoles, the heterocyclic ring was introduced to C-3 or
to C-16 directly or via a short linker. The cell growth-inhibitory potential depended on the position
of the triazolyl moiety and on the nature of the functional group at C-3. 3-Hydroxy or 3-ether
derivatives displayed lower cytostatic potentials than their 3-O-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]
counterparts. The latter triazoles displayed one order of magnitude higher activities (submicromolar
IC50 values) than the earlier described potent 16-triazolyl 3-O-benzyl ethers [18]. It can be stated
that concerning the antiproliferative behaviour of triazolyl 13
over C-16 seems more preferential. One of the most potent antiproliferative compounds was
3-O-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-13 -estrone bearing intact ring D ( , Figure 1). Based
on its remarkable cytostatic potential (IC50 = 0.3–0.9 M, [20], this 13 -estrone triazole conjugate
) may be used as a model compound for further derivatization with dual aim: to improve its cell
α-estrones, functionalization at C-3
α
1
µ
α
(1
growth-inhibitory potential to nanomolar scale, and to enhance its tumor selectivity. A potential route
to enhance this selectivity is to prepare bioconjugates. Our recent results suggest that the triazole ring
at 3-O should remain to retain the biological activity, but the azide counterpart used in the CuAAC
reaction may be another biomolecule, such as a nucleoside.
Figure 1. Structure of 3-O-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-13α-estrone.
The advantage of using a nucleoside azide could be either the higher activity on targeted
enzymes overrepresented in cancer cells or an enhanced cellular uptake of the bioconjugates in
these cells compared to the healthy ones. As the cancer cells require higher amount of nucleoside
building blocks for their proliferation, they have significantly higher uptake of nucleosides by the
different nucleoside transporters [23
synthesized so far for anticancer purposes contain the nucleoside units as the bioactive components,
like 20-deoxy-5-fluorouridine or coenzyme mimics in bisubstrate inhibitors [26
27]. However, to our
–25]. The nucleoside–steroid bioconjugates, which have been
,
knowledge nucleosides, which can selectively enhance the transport into the cancer cells, are not used
as targeting carriers.

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Therefore our aim was to test this possibility in case of an estrone derivative with established
antiproliferative activity. If the coupled nucleoside unit increases the active transport of the steroid
molecule without effecting the antiproliferative activity of the steroid part, one should see an increased
inhibition of cell growth.
The connection types applied in the nucleoside–steroid conjugates prepared so far are generally
ester bonds [26
the CuAAC method is a widely used alternative [32
,
28
–
31]. On the other hand, in case of conjugates of polyfunctional biomolecules,
33] which forms a stable 1,2,3-triazole ring.
,
1,2,3-Triazoles (for sake of simplicity hereinafter referred to as “triazoles”; systematic compound
names are given in the Materials and Methods section) are extensively used linkers in synthetic
bioactive conjugates because of their stability against metabolic degradation and their favourable
hydrogen-bonding properties. Incorporation of a triazole ring into the estrane skeleton has additional
advantages: it may enhance the water solubility, bioavailability and as mentioned above this structural
moiety enhanced the antiproliferative activity of an estrone derivative [18]. The CuAAC is a highly
selective coupling method [34] which requires an azide and an alkyne function on the biomolecules
and copper(I) ion as a catalyst.
In our case two potential approaches can be considered for the introduction of alkyne and azide
moieties suitable for CuAAC reaction: (1) the nucleosides contain the azido group and the steroid has
a terminal alkyne function or (2) the nucleosides have the terminal alkyne functional group and the
steroid contains the azide. The formation of an alkynyl ether function on the phenolic OH group of an
estrone derivative is not problematic, but the derivatization of nucleosides is not as straightforward
as it seems. Regarding the first possibility, the preparation of 50-azido-50-deoxy nucleosides in the
commonly used two-step method (50-O-activation by introduction of tosylate [35
–39], mesylate [38],
halogen [35,39–46] or other leaving group [47,48] followed by azide substitution is hampered by
several side-reactions and variable yields. The most relevant side-reactions are 30,50-bis-O-tosylation,
30,50-diazide formation [35], 40,50-elimination and 2,30/50-anhydronucleoside formation (in the case of
pyrimidine nucleosides) [43
on the identity of nucleobase (cytidine and especially guanine are troublesome) [37
configuration of sugar moiety [35 49], protecting group pattern [36] of the nucleobase and the sugar,
steric congestion [40 44] and the actual method used [39 46 48]. Hence, the overall yields of these
reactions are usually not very high, generally around 40%–60% or even lower (Scheme 1, path a).
Alternatively, Mitsunobu reaction [50 52] with hydrogen azide, trimethylsilyl azide or zinc
,47]. As a rule, the yields of these transformations are highly dependent
,
41–43,45,47],
,
,
, ,
–
azide-pyridine complex [53] is also a possible alternative to prepare azides from nucleosides, provided
that the senstive sugar moieties survive these conditions (Scheme 1, path d).
The second possibility (alkynylation of nucleosides) is also problematic: low yields [54–58],
concomintant O,N-dialkylation hamper the effective derivatization of nucleosides [57,58]; it was also
observed that cation chelation and solvents greatly influence the outcome of the reactions [59].
Here we chose the above mentioned 13
synthesizing nucleoside bioconjugates and we planned to investigate the antiproliferative properties
and potential 17 -HSD1 inhibitory effect of these conjugates. Based on our earlier experience in the
preparation and in vitro biological assays of triazolyl 13 -estrone derivatives with anticancer activity,
-estrone for the introduction of the terminal alkyne
α-estrone as a starting compound with the aim of
β
α
we have opted for the 3-hydroxy group of 13
α
0
function. On the nucleosides the 5 -hydroxy seems to be the best choice because its chemical reactivity
is higher than that of 30, and also the better biocompatibility, as the nucleosides in the cells are mainly
derivatized on their 50-end. Hence, it is more likely that the recognition of the nucleosides on the
receptor, enzyme and transporter proteins are favoured in case the nucleosides are only modified on
their 50-end and the rest of the nucleosides are freely available for the proteins.

Molecules 2016, 21, 1212
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0
0
0
Scheme 1. Synthesis of 5 -azido-2 ,5 -dideoxynucleosides.
2. Results and Discussion
2.1. Preparation of 50-Azido-20,50-dideoxynucleosides
For the preparation of 50-azido-20,50-dideoxynucleosides, first we have followed the tosyl–azide
replacement method based on the literature but the isolated yields were significantly lower in our
hands (Scheme 1, path a) compared to those described in the literature [35–39] therefore we decided
to protect the 30-hydroxy groups (Scheme 1, path b). Although the 30-protection requires two more
steps (30-O-acylation and 50-O-deprotection when the starting material is a 50-O-(4,40-dimethoxytrityl
(DMTr))-N-acyl-protected nucleoside), it helps avoid the bis-30,50-O-tosylation and increases the
solubility of the nucleosides which might also be a reason of the low yields. We have chosen
the acetyl-protection of the 30-hydroxy because it was considered to be compatible with the
final deprotection of the steroid–nucleoside conjugates. The crude acetylated material was
used for the 50-O-DMTr deprotection without chromatographic purification. As a commonly
used 3% trichloroacetic acid/dichloromethane deprotection resulted in a considerable amount
of depurination side-products (mainly in the case of 20-deoxyadenosine) therefore we have
changed the reagent to a deprotection mixture containing the Lewis acid boron trifluoride in a
1,1,1,3,3,3-hexafluoroisopropanol–nitromethane solution [60] (Scheme 1, path b).
Using the 50-OH containing, 30-O-acetyl-protected nucleosides (8a
–d) we have carried out the
50-O-tosylation in pyridine at room temperature and after purification the tosyl–azide exchange
reaction in DMF at 50 ◦C (Scheme 1, path c). We have obtained better yields compared to the ones
without 30-O-protection but the yields were still not too high, around 50%. Moreover, in case of
0
2 -deoxguanosine, the tosylation reaction gave a very low yield (<10%), probably due to the very poor
solubility. Therefore we stopped our attempts to obtain the tosylate 9d and its further derivatization
was also abandoned.
As we were not satisfied with the isolated yields of azidonucleosides we attempted to improve
them by applying Mitsunobu reaction to obtain the 50-azides directly in a one-step reaction from
the 30-O-acetyl-protected nucleosides (Scheme 1, path d). The Mitsunobu reaction requires an

Molecules 2016, 21, 1212
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acid component which should be HN3 in our case but we did not want to use a Brønsted acid
to avoid the potential glycoside bond cleavage therefore, instead of the protic acid, we have
tried two Lewis acids, Zn(N3)2 2 py or trimethylsilyl azide which were also used for Mitsunobu
reactions [50
–
53]. Although we have tried to optimize the re0action conditions by varying the starting
0
0
materials (2 -deoxyadenosine, thymidine, 2 -deoxycitidine, 2 -deoxyguanosine,), the azodicarboxylate
reagents (diethyl or diisopropyl esters), the azide-containing acids and also applying different
temperatures (0 C and room temperature), we were unable to detect a considerable amount of
50-azido-20,50-dideoxynucleosides. Only in case of 20-deoxyadenosine we have got a 20% of the
◦
◦
desired product, by using trimethylsilyl azide reagent at 0 C in a 1 h reaction time. In all other
cases only the 50-O-trimethylsilylated nucleoside side-products were found by mass spectrometry
analyses of the newly appearing TLC spots. These side-products have decomposed during the work
up procedure giving back the starting nucleosides. As the Mitsunobu reaction failed to produce the
desired 50-azido-20,50-dideoxynucleosides, we eventually have used the 30-O-protected–tosyl–azide
exchange route to prepare the required amounts of 50-azido-20,50-dideoxynucleosides 10a
conjugation reactions from crude tosylates 9a–c (Scheme 1, path c).
–c for the
2.2. Optimization of the Click Reaction between 50-Azido-nucleoside Derivatives and
3-O-Propargyl-13α-estrone
The 50-azido-20,50-dideoxynucleosides 10a
11) [20] in a CuAAC reaction (Scheme 2). The solvent of the click-reaction was toluene (thymidine
–c were connected to 3-O-propargyl-13α-estrone
(
and adenosine) or anhydrous tetrahydrofuran (cytidine) due to solubility problems. Initially, we have
followed the commonly used literature method (catalytic amounts of copper(II) salts in the presence
of sodium ascorbate, aq. tert-butanol at room temperature) [32] but to no avail (Table 1). Alternative
methods [36,38,61], using 0.01-0.2 equivalent of copper(I) iodide catalyst along with 0.2 equivalent
of triphenylphosphane and DIPEA have also been tested but the reaction did not proceed well
even if higher temperature, different solvents and prolonged reaction times (3 days) were applied.
The highest reaction temperature was limited to as high as 50 ◦C, to avoid the potential side-reactions
on the nucleoside part. We supposed that the reason of the very low yields (<30% according to TLC
monitoring) could be the high complex-forming affinity of the N-acyl-protected nucleosides which
trapped the Cu(I) ion catalysts [54]. Application of inert argone athmosphere did not improve the
yield. Therefore we increased the amount of the Cu(I) catalyst and DIPEA to 1.5 equivalent and
eliminated triphenylphosphane from the reaction mixture. With this modified method all conjugation
◦
reactions were complete in one day at 50 C according to TLC and the final isolated yields of protected
conjugates 12a–c were acceptable (Table 1).
O
H
O
X
H
H
N3
+
O
X =
O
AcO
10a-c
11
toluene
or THF
61–76%
CuI
DIPEA
NHZ
N
NHZ
N
O
N
N
NH
H
N
O
N
O
N
H
H
O
O
X
12a
Z = Bz, R = Ac
13a
12b
R = Ac
13b
12c
Z = Bz, R = Ac
13c
N
N
N
OR
12a-c
13a-c
NH3/MeOH
81–89%
Z = R = H
R = H
Z = R = H
Scheme 2. CuAAC conjugation reaction of 30-O-acetyl-50-azido-20,50-dideoxynucleosides and
3-O-propargyl-13α-estrone.

Molecules 2016, 21, 1212
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Table 1. Optimization of CuAAC conjugation reaction of 30-O-acetyl-50-azido-20,50-dideoxy-
nucleosides and 3-O-propargyl-13α-estrone.
Conditions and Yields of CuAAC Reaction
Refs. [36,38,61] 2
Ref. [32] 1
This work 3
Product
Code
Product
Yield (%)
Recovered
Product
Yield (%)
Recovered
Product
Yield (%)
Recovered
Nucleoside (%) 4
Nucleoside (%) 4
Nucleoside (%) 5
12a
12b
12c
0
0
0
93
92
>95
18–22
23–28
8–11
63–68
64–70
78–82
68
76
61
-
-
-
1
1 mol % CuSO4, 5% (m/v) aq. sodium ascorbate, water: tert-butanol 2:1 (v/v), r.t., 8 h; 2 0.01–0.2 equiv. CuI,
3
0.2 equiv. Ph3P, 0.2 equiv. DIPEA, toluene (12a
,
12b) or THF (12c), 50 ◦C, 16–72 h; 1.5 equiv. CuI, 3 equiv.
DIPEA, toluene (12a,
12b) or THF (12c), 50 ◦C, 16 h; 4 Determined using TLC densitometry of the UV-active
spots; 5 Not determined.
2.3. Optimization of the Deprotection of Conjugates (Nucleobase N-Benzoyl and 20-Deoxy-D-ribose-30-O-
acetyl Deprotection)
0
The synthesized bioconjugates contained the 3 -O-acetyl and N-benzoyl protecting groups on the
nucleoside moiety which helped increase the solubility in the synthetic reactions but for the biological
experiments we needed the unprotected nucleoside conjugates. Aqueous ammonia is commonly
used for the deprotection of these protecting groups in the nucleic acid chemistry but the protected
conjugates were not soluble in the aqueous media therefore this deprotection method failed. We have
tested the Zemplén deacetylation protocol using 0.1 M of sodium methylate in methanol but only the
acetyl group was removed from the 30-hydroxy group. Finally, 4 M ammonia solution in methanol
◦
was used at 50 C which removed all the acyl protecting groups of the conjugates in 16 h to yield
derivatives 13a–c.
2.4. Antiproliferative Activities
The antiproliferative properties of the newly synthesized nucleoside conjugates were
characterized in vitro on a panel of human adherent cancer cell lines (HeLa, MCF-7 and A2780) by
means of MTT assays. Structurally similar estrone analogs earlier exhibited growth inhibitory activity
against these cells [
moiety on the cytostatic properties was investigated and the results are shown in Table 2. The protected
cytidine conjugate 12c proved to be the most potent with IC50 values in the range 9.0–10.4 M, although
this value is an order of magnitude higher than the value of the 13 -estrone triazole 20]. The removal
of the benzoyl and/or acetyl protecting groups from the nucleoside–13 -estrone conjugates 12a
resulted in unprotected conjugates 13a with generally reduced cytostatic properties. Although we
do not know the mechanism of action of our conjugates, but compared to the reference steroid
9,18–21]. The influence of the nature of the protected or unprotected nucleoside
µ
α
1 [
α
–c
–c
1,
the non-polar benzyl group was replaced by the polar deoxynucleoside units, therefore the lower
activity could be due to the more pronounced steric effect and polar properties of the nucleoside
units. Interestingly, the conjugates containing less polar but larger protected nucleosides gave higher
antiproliferative activity than the unprotected, more polar but smaller nucleoside-containing ones.
This fact highlights the importance of both the limited size and non-polar characteristics of the group at
C-3 triazolyl moiety of 13α-estrone. On the other hand, this finding also suggests that the hypothesized,
potentially selective increase in the uptake of nucleoside conjugates of the estrone derivative might not
be operative. The reason of the lower overall uptake could be the decrease of the passive transport of
the more polar, nucleoside–estrone derivative through the cell membrane. If the passive transport has
much higher contribution to the overall uptake than the nucleoside transporter mediated routes, then
the loss of antiproliferative activity of the more polar, unprotected nucleoside–13
can be explained by lower concentration of the conjugates inside the cell. Either explanation is true,
unfortunately the triazolyl-deoxynucleoside modification of 13 -estrone on C-3 position did not help
improving the antiproliferative activity of the model compound 1.
α-estrone conjugates
α

Molecules 2016, 21, 1212
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Table 2. Antiproliferative properties of the synthesized compounds. Mean value from two independent determinations with five parallel wells; standard
deviation <15%.
Inhibition (%) ± SEM [Calculated IC50, µM] 1
Compd Code or Name [ref.]
12a
Conc. (µM)
Structure
A2780
HeLa
MCF-7
O
2
2
NHBz
10
30
39.4 ± 2.4
70.2 ± 1.6
[10.9]
-
-
H
N
N
N
H
H
55.5 ± 0.6
49.6 ± 1.4
N
O
O
N
N
[>30] 3
N
[16.3]
OAc
O
O
N
10
30
29.8 ± 0.2
35.1 ± 2.7
[>30]
-
-
29.2 ± 2.9
26.7 ± 2.0
[>30]
H
HN
H
H
O
12b
12c
O
O
N
N
N
[>30]
OAc
O
NHBz
10
30
63.2 ± 1.5
66.6 ± 1.6
[9.0]
53.5 ± 1.0
61.9 ± 1.2
[9.0]
47.4 ± 2.4
57.3 ± 1.8
[10.4]
H
N
H
H
O
N
O
O
N
N
N
OAc
O
NH2
10
30
-
26.6 ± 1.8
60.1 ± 0.7
[23.5]
-
H
N
N
N
H
H
41.9 ± 1.7
36.6 ± 1.0
N
13a
O
O
N
N
N
[>30]
[>30]
OH
O
O
N
10
30
-
25.9 ± 0.8
38.2 ± 2.1
[>30]
-
-
H
HN
H
H
O
32.4 ± 2.0
13b
O
O
N
N
N
[>30]
[>30]
OH
O
NH2
N
10
30
31.8 ± 2.5
31.6 ± 3.6
[>30]
41.4 ± 1.5
46.1 ± 2.6
[>30]
-
H
N
H
H
O
26.4 ± 2.2
13c
O
O
N
N
N
[>30]
OH
O
10
30
77.5 ± 0.4
78.4 ± 0.9
[0.5]
90.9 ± 0.3
93.3 ± 0.2
[0.9]
85.8 ± 1.3
85.0 ± 0.2
[0.6]
H
1 [20]
cisplatin
H
H
O
N
N
N
10
30
83.6 ± 1.2
95.0 ± 0.3
[1.3]
42.6 ± 2.3
99.9 ± 0.3
[12.4]
66.9 ± 1.8
96.8 ± 0.4
[5.8]
1
2
3
Mean value from two independent determinations with five parallel wells; standard deviation <15%; Inhibition values <20% are not presented. IC50 values > 30
not calculated.
µM are

Molecules 2016, 21, 1212
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2.5. Inhibition of 17β-HSD1
The newly synthesized nucleoside conjugates 12a
–
c
and 13a
–c
of 13α
-estrone were tested against
the human 17β-HSD1 in vitro. The results in Table 3 show that only the unprotected thymidine
conjugate 13b exerted any substantial inhibition, with an IC50 value of 19
displayed weak inhibition. Compared to our previous results on 13
µ
M. Other tested conjugates
-estrone and its 3-methyl
α
ether, these conjugates showed lower inhibitory effect, similar to those of the 3-benzyl ether of
13 -estrone [22]. The decrease in the inhibitory potential of nucleoside conjugates is probably caused
by the steric effect of the larger nucleoside–triazolyl unit on the C-3 position.
α
Table 3. Inhibition results on 17β-HSD1. Relative conversions (control incubation with no inhibition is
100%) measured in the presence of 10
µM of the compound tested. IC50: The inhibitor concentration
that decreases the enzyme activity to 50%. SD: standard deviation (for relative conversion n = 3).
Relative conversion ± SD at 10 µM (%)
Structure
Compd Code
[IC50 ± SD (µM) NADPH]
12a
90 ± 13
12b
12c
13a
13b
89 ± 7
98 ± 14
O
NHBz
N
H
N
H
H
O
O
O
N
N
N
OAc
O
NH2
H
N
N
N
H
H
113 ± 6
N
O
O
N
N
N
OH
O
O
H
HN
H
H
65 ± 3 [IC50 = 19 ± 10]
O
N
O
O
N
N
N
OH
O
NH2
H
N
H
H
13c
91 ± 4
O
N
O
O
N
N
N
OH

Molecules 2016, 21, 1212
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3. Materials and Methods
3.1. Chemistry
Melting points (mp) were determined on an IA8103 apparatus (Electrothermal, Stone, UK) and
are uncorrected. The reactions were monitored by TLC on Kieselgel-G (Si 254 F, Merck, Darmstadt,
Germany) layers (0.25 mm thick); solvent systems (ss): (A) EtOAc, (B) EtOAc/methanol (9:1 v/v), (C)
toluene/isopropanol (1:1 v/v). The conjugates were detected by spraying with 5% phosphomolybdic
acid in 50% aqueous phosphoric acid. For the identification of azides the TLC was placed in a 10%
solution of triphenylphosphane in CH2Cl2 for 2 min, then sprayed with ninhydrine solution (0.5%
ninhydrine and 5 mM NaOH in ethanol/water 3:1 v/v).The Rf values were determined for the spots
observed by illumination at 254 nm. Flash chromatography: Merck silica gel 60, 40–63 µm. All solvents
were distilled prior to use. Reagents and materials were obtained from commercial suppliers and were
used without purification. Elementary analysis data were determined with a CHN analyzer model
2400 (Perkin Elmer, Waltham, MA, USA). NMR spectra were obtained at room temperature with a
DRX 500 instrument (Bruker, Rheinstetten, Germany). Chemical shifts are reported in ppm (δ scale),
and coupling constants (J) in Hz. For the determination of multiplicities the J-modulated spin-echo
pulse sequence was used. Mass spectra were recorded on a MAT TSQ 7000 instrument (Finnigan,
Waltham, MA, USA) equipped with an electrospray ion source, using the following parameters:
◦
positive ionization mode, nebulizing gas N2 (3.45 bar), capillary temperature: 200 C, capillary voltage:
4500 V and an acetonitrile–water 1:1 (v/v) mixture containing 0.1% trifluoroacetic acid as eluent. The
samples were dissolved in acetonitrile and injected directly. The base peaks were usually the [M + H]+
signals along with far less abundant [2M + H]+ signals.
3.1.1. General Procedure for Synthesis of 30-O-Acetyl-N-acyl-protected-20-deoxynucleosides 8a–d
10 mmol of 50-O-DMTr-N-acyl-protected-20-deoxynucleoside 6a
pyridine (50 mL) and Ac2O (10 mmol, 945
L) was added. The reaction mixture was stirred at 0 ◦C
for 4 h, evaporated in vacuo, redissolved in EtOAc (100 mL) and extracted with water (2 100 mL).
, 6b, 6c or 6d was dissolved in
µ
×
The combined organic layers were dried over Na2SO4 and evaporated in vacuo. The resulting crude
product 7a, 7b, 7c or 7d was directly used for the next step without further purifications.
0
0
0
0
10 mmol of 5 -O-(4,4 -dimethoxytrityl)-3 -O-acetyl-N-acyl-protected-2 -deoxynucleoside 7a
or 7d was dissolved in nitromethane (20 mL) and a mixture of 1,1,1,3,3,3-hexafluoroisopropanol (20 mL,
190 mmol), boron trifluoride diethyl etherate (247 L, 2 mmol) and triethylsilane (6 mL, 38 mmol) was
, 7b, 7c
µ
added. The reaction mixture was stirred at room temperature overnight, then 10% aqueous NaHCO3
solution was added and evaporated in vacuo. The resulting crude product was purified by column
chromatography with EtOAc as eluent to give the corresponding nucleoside 8a, 8b, 8c or 8d.
3.1.2. General Procedure for Synthesis of
30-O-Acetyl-50-azido-N-acyl-protected-20,50-dideoxy-nucleosides 10a–c
30-O-Acetyl-N-acyl-protected-20-deoxynucleoside 8a
, 8b, 8c or 8d (1 mmol) was dissolved in
pyridine (15 mL) and p-toluenesulfonyl chloride (285 mg, 1.5 mmol) was added. The reaction mixture
was stirred at room temperature overnight, evaporated in vacuo, redissolved in EtOAc (100 mL)
and extracted with 1% aqueous KHSO4 solution (2
dried over Na2SO4 and evaporated in vacuo. The resulting crude product was purified by column
×
100 mL). The combined organic layers were
chromatography with EtOAc as eluent to afford the tosylated nucleoside 9a, 9b, 9c or 9d.
30-O-Acetyl-N-acyl-protected-20-deoxy-50-O-tosylnucleoside 9a
, 9b or 9c (1 mmol) was dissolved
in dry DMF (20 mL) then sodium azide (195 mg, 3 mmol) and lithium bromide (261 mg, 3 mmol) were
added. The reaction mixture was stirred at 50 ◦C for 6 h. The reaction mixture was evaporated in
vacuo, redissolved in EtOAc and extracted with water (2
were dried over Na2SO4 and evaporated in vacuo. The resulting crude product was recrystallized
×
100 mL). The combined organic layers
from EtOAc to yield azidonucleoside 10a, 10b or 10c.

Molecules 2016, 21, 1212
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30-O-Acetyl-50-azido-6-N-benzoyl-20,50-dideoxyadenosine (10a
):
After purification, 10a was obtained as
◦
1
a white 0solid (346 mg, 82%), m.p. 135–136 C, Rf = 0.209 (ss A); H-NMR (CDCl3);
δ [ppm] = 2.11
(s, 3H, 3 -OAc), 2.63 (d, 1H, J = 8.0 Hz) and 3.27 (m, 1H): 2 -H2, 3.61 (d, 1H, J = 11.5 Hz) and 3.76 (d, 1H,
0
0
0
0
J = 12.0 Hz): 5 -H2, 4.27 (s, 1H), 5.39 (s, 1H), 6.54 (m, 1H): 1 -, 3 -, 4 -H, 7.55 (t, 2H, J = 2
(d, 1H, J = 7.5 Hz), 8.06 (d, 2H, J = 7 Hz): benzoyl protons, 8.74 (s, 1H) and 8.79 (s, 1H): 2-H and 8-H,
11.23 (s, 1H, 6-NH); 13C-NMR (CDCl3); [ppm] = 20.7 (30-OAc), 35.0 (C-20), 51.5 (C-50), 74.6, 82.9, 83.8,
×
7.0 Hz), 7.64
δ
125.9 (C-5), 128.3 (2C), 128.4 (2C), 132.4: benzoyl CHs, 133.2: benzoyl Cq, 143.3 (C-8), 150.4 (C-4), 151.9
(C-6), 152.3 (C-2), 165.5 (Bz-CO), 170.0 (Ac-CO); ESI-MS: 423 [M + H]+; Anal. Calcd for C19H18N8O4:
C, 54.03; H, 4.30; N, 26.53. Found: C, 53.96; H, 4.47; N, 26.95.
30-O-Acetyl-50-azido-50-deoxythymidine (10b): After purification, 10b was obtained as a white solid
◦
(263 mg, 85%), m.p. 115–116 C, Rf = 0.65 (ss A); 1H-NMR (CDCl3);
δ [ppm] = 1.57 (s, 3H, 5-CH3), 1.83
(s, 3H, 30-OAc), 2.02 and 2.27 (2
×
m, 2
×
1H, 200-H2), 3.40 (m, 2H), 3.86 (d, 1H, J = 2.0 Hz), 4.91 (d, 1H,
0
0
0
J = 3.5 Hz), 5.95 (t, 1H, J = 10.0 Hz, J = 5.0 Hz): 1 -, 3 -, 4 -H, 5 -H2, 7.33 (s, 1H, 6-H), 11.16 (s, 1H, 3-NH);
13C-NMR (CDCl3);
δ
[ppm] = 12.0 (5-CH3), 20.7 (30-OAc), 35.0 (C-20), 51.6 (C-50), 74.1, 81.9, 83.9, 109.9
(C-5), 135.9 (C-6), 150.4, 163.6, 170.0 (Ac-CO); ESI-MS: 310 [M + H]+; Anal. Calcd for C12H15N5O5:
C, 46.60; H, 4.89; N, 22.64. Found: C, 46.82; H, 5.02; N, 22.83.
30-O-Acetyl-50-azido-4-N-benzoyl-20,50-dideoxycytidine (10c): After purification, 10c was obtained as a
white solid (302 mg, 76%), m.p. 146–147 ◦C, Rf = 0.32 (ss A); 1H-NMR (CDCl3);
δ [ppm] = 2.11 (s, 3H,
30-OAc), 2.53 (m, 2H, 20-H2), 3.77 (dd, 2H, J = 9.5 Hz, J = 12.0 Hz, 50-H2), 4.25 (s, 1H), 5.21 (s, 1H), 6.24
(d, 1H, J = 4.5 Hz): 10-, 30-, 40-H, 7.42 (s, 1H, 50-H), 7.54 (d, 2H, J = 5.5 Hz), 7.65 (d, 1H, J = 5.5 Hz), 8.04
(m, 2H): benzoyl protons, 8.24 (s, 1H, 6’-H), 11.34 (s, 1H, 40-NH); 13C-NMR (CDCl3); δ [ppm] = 20.7
(30-OAc), 36.6 (C-20), 51.6 (C-50), 74.3, 82.7, 86.5, 96.6 (C-5), 128.3 (2C), 128.4 (2C), 132.7: benzoyl CH-s,
133.1: benzoyl Cq, 145.2 (C-6), 154.1 (C-2), 163.6 (C-4), 165.1 (Bz-CO), 170.0 (Ac-CO); ESI-MS: 399
[M + H]+; Anal. Calcd for C18H18N6O5: C, 54.27; H, 4.55; N, 21.10. Found: C, 54.18; H, 4.63; N, 19.85.
3.1.3. General Procedure for Click Reactions: Preparation of 12a–c
Azidonucleoside 10a
(0.225 mmol, 42.75 mg, 1.5 equiv.), DIPEA (78
,
10b or 10c (0.15 mmol) was dissolved in toluene (10 mL) or THF (10mL), CuI
L, 0.45 mmol, 3 equiv.) and 3-O-propargyl-13 -estrone
20] (0.165 mmol, 1.1 equiv.) were added to the reaction mixture and it was stirred for overnight at
µ
α
11 ◦
[
50 C. Then the mixture was evaporated in vacuo, the resulting crude product was purified by column
chromatography with EtOAc as eluent to yield protected conjugate 12a, 12b or 12c.
3-{[1-(30-O-Acetyl-6-N-benzoyl-20,50-dideoxyadenosine-50-yl)-1H-1,2,3-triazole-4-yl]methyl-oxy}-13
1,3,5(10)-trien-17-one (12a): After purification, protected conjugate 12a was obtained as a white solid
α-estra-
◦
(78 mg, 68%), m.p. 141–143 C, Rf = 0.55 (ss B); 1H-NMR (CDCl3);
δ [ppm] = 1.00 (s, 3H, 18-CH3), 1.89
(s, 3H, 30-OAc), 2.27 (dd, 2H, J = 10.5 Hz, J = 12.5 Hz, 20-H2), 2.54 (d, 1H, J = 11.0 Hz) and 2.82 (d, 1H,
0
0
J = 11.5 Hz): 6-H2, 2.55 (m, 1H), 4.45 (m, 1H), 4.84 (dd, 2H, J = 13.5 Hz, J = 14.0 Hz), 5.54 (s, 1H): 1 -, 3 -,
40-H, 50-H2, 5.04 (s, 2H, OCH2), 6.42 (s, 1H, 4-H), 6.61 (d, 1H, J = 6.0 Hz, 2-H) 6.69 (d, 1H, J = 8.5 Hz,
1-H), 7.06 (s, 1H), 7.46 (s, 3H), 7.55 (s, 1H), 8.00 (s, 2H): benzoyl protons, 2”-, 8”-H, 7.59 (s, 1H, HC=C),
8.72 (s, 1H, 6”0-NH); 13C-NMR (CDCl3); δ [ppm] = 20.7 (C-18), 20.8, 20.9 (30-OAc), 24.9 (2C), 30.2, 31.9,
0
33.3, 36.1 (C-2 ), 41.2 (2C, C-8 and C-9), 49.1 (C-14), 50.0 (C-13), 51.4 (C-5 ), 61.6 (OCH2), 74.4, 83.1, 85.3,
112.3 (C-2), 114.3 (C-4), 125.3 (HC=C), 123.8 (C-5”), 126.7 (C-1), 128.1 (2C). 128.6 (2C), 132.8: benzoyl
CH-s, 132.4 (C-10), 135.7: benzoyl Cq, 138.0 (C-5), 139.5 (C-8”), 144.1 (HC=C), 147.1, 151.4, 155.9 (C-3),
153.4, 165.2 (Bz-CO), 170.3 (Ac-CO), 221.7 (C-17); ESI-MS: 731 [M + H]+; Anal. Calcd for C40H42N8O6:
C, 65.74; H, 5.79; N, 15.33. Found: C, 65.67; H, 5.92; N, 15.46.
0
0
0
3-{[1-(3 -O-Acetyl-5 -deoxythymidine-5 -yl)-1H-1,2,3-triazole-4-yl]methyloxy}-13α-estra-1,3,5(10)-trien-17-one
(
12b): After purification, protected conjugate 12b was obtained as a white solid (74 mg, 76%), m.p.
189–191 ◦C, Rf = 0.5 (ss A); 1H-NMR (CDCl3);
δ
[ppm] = 1.03 (s, 3H, 18-CH3), 1.24 (s, 2H), 1.87 (s, 3H,

Molecules 2016, 21, 1212
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0
0
5”-CH3), 2.09 (s, 3H, 3 -OAc), 2.20 (d, 2H, J = 11.0 Hz, 2 -H2), 2.78 (m, 2H, 6-H2), 4.29 (m, 1H), 4.84 (dd,
0
0
0
0
2H, J = 9.5 Hz, J = 11.5 Hz), 5.29 (s, 1H), 6.16 (m, 1H): 1 -, 3 -, 4 -H, 5 -H2, 5.17 (s, 2H, OCH2), 6.64 (s, 1H,
4-H), 6.72 (d, 1H, J = 6.0 Hz, 2-H), 6.90 (s, 1H, 6”-H), 7.14 (d, 1H, J = 8.5 Hz, 1-H), 7.79 (s, 1H, HC=C),
9.46 (s, 1H, 3”-NH); 13C-NMR (CDCl3);
δ
[ppm] =12.4 (5”-CH3), 20.8 (30-OAc), 21.0, 205.0 (C-18), 28.2
0
(2C), 30.3, 32.0, 33.4, 36.0 (C-2 ), 41.3 (2C, C-8 and C-9), 49.2 (C-14), 50.1 (C-13), 51.6 (C-5 ), 61.6 (OCH2),
74.3, 82.0, 85.3, 111.8 (C-5”), 112.4 (C-2), 114.4 (C-4), 124.8 (H
(C-6”), 138.2 (C-5), 144.2 (HC= ), 150.3, 163.6, 155.8 (C-3), 170.6 (Ac-CO), 218.8 (C-17); ESI-MS: 618 [M
+ H]+; Anal. Calcd for C33H39N5O7: C, 64.17; H, 6.37; N, 11.34. Found: C, 64.25; H, 6.52; N, 11.49.
C=C), 126.9 (C-1), 132.7 (C-10), 135.6
C
3-{[1-(30-O-Acetyl-4-N-benzoyl-20,50-dideoxycytidine-50-yl)-1H-1,2,3-triazole-4-yl]methyl-oxy}-13
α-estra-
1,3,5(10)-trien-17-one (1◦2c): After purification, protected conjugate 12c was obtained as a white solid
(67 mg, 61%), m.p. 170 C (dec.), Rf = 0.47 (ss B); 1H-NMR (CDCl3);
δ [ppm] = 1.04 (s, 3H, 18-CH3), 2.13
(s, 3H, 30-OAc), 2.63 (d, 2H, J = 9.0 Hz, 20-H2), 2.78 (m, 2H, 6-H2), 4.43 (m, 1H), 4.82 (m, 2H), 5.24–5.26
(overlapping m, 2H): 10-, 30-, 40-H, 50-H2, 5.17 (s, 2H, OCH2), 6.68 (s, 1H, 4-H), 6.76 (d, 1H, J = 6.0 Hz,
2-H) 7.15 (d, 1H, J = 8.5 Hz, 1-H), 7.53 (t, 3H, J = 7.0 Hz), 7.64 (t, 2H J = 14.5 Hz): benzoyl protons,
7.73 (s, 1H, HC=C), 7.94 (s, 1H, 6”-H), 8.04 (s, 1H, 4”-NH); 13C-NMR (CDCl3);
δ [ppm] = 20.8 (C-18),
21.0, 25.1 (30-OCH3), 28.2 (2C), 29.6, 30.3, 32.0, 33.4, 37.5 (C-20), 41.4 (2C, C-8 and C-9), 49.2 (C-14), 50.1
(C-13), 51.4 (C-50), 56.3, 62.8 (OCH2), 74.1, 84.0, 97.3 (C-5”), 112.5 (C-2), 114.5 (C-4), 123.9 (C-1), 127.0
(HC=C), 129.2 (2C). 129.5 (2C), 132.6: benzoyl CH-s, 132.9 (C-10), 137.6: benzoyl Cq, 138.3 (C-5), 143.5
(HC=
C
), 156.0 (C-3), 157.5 (C-2”), 162.7 (C-4”), 170.6 (Bz-CO), 171.2 (Ac-CO), 220.5 (C-17); ESI-MS: 707
[M + H]+; Anal. Calcd for C39H42N6O7: C, 66.27; H, 5.99; N, 11.89. Found: C, 66.32; H, 6.13; N, 12.10.
3.1.4. General Procedure for Deprotection of Conjugates 13a–c
Protected conjugate 12a, 12b or 12c (0.1 mmol) was deacylated by dissolving in 4 M NH3
in MeOH (5 mL), stirring the mixture overnight at room temperature and then the mixture was
evaporated in vacuo and the resulting crude product was purified by column chromatography with
EtOAc/acetonitrile (9:1) as eluent to afford unprotected conjugate 13a, 13b or 13c.
0
0
0
3-{[1-(2 ,5 -Dideoxyadenosine-5 -yl)-1H-1,2,3-triazole-4-yl]methyloxy}-13
α
-estra-1,3,5(10)-trien-17-one (13a):
◦
After purification, conjugate 13a was obtained as a white solid (50 mg, 87%), m.p. 160 C (dec.),
Rf = 0.33 (ss B); 1H-NMR (DMSO-d6);
δ
[ppm] = 0.96 (s, 3H, 18-CH3), 2.71 (m, 2H, 20-H2), 2.83 (m, 2H,
6-H2), 4.21 (s, 1H), 4.51 (s, 1H), 4.68-4.77 (overlapping m, 2H), 4.99 (dd, 2H J = 10.0 Hz, J = 12.0 Hz),
5.60 (s, 1H): 10-, 30-, 40-H, 50-H2, OCH2, 6.63 (s, 1H, 4-H), 6.70 (d, 1H, J = 8.0 Hz, 2-H) 7.12 (d, 1H,
J = 8.5 Hz, 1-H), 7.47 (s, 2H, 6”-NH), 7.47 (s, 1H, HC=C), 8.21 (s, 1H), 8.32 (s, 1H): 2”-H and 8”-H;
13C-NMR (DMSO-d6);
δ
[ppm] = 20.5, 24.60 (C-18), 27.7, 28.0, 29.7, 31.6, 32.9, 38.0 (C-20), 40.8 (2C, C-8
and C-9), 48.5 (C-14), 49.4 (C-13), 51.5 (C-5 ), 60.8 (OCH2), 71.1, 83.8, 84.9, 112.4 (C-2), 114.1 (C-4), 119.1,
125.0 (HC=C), 126.7 (C-1), 132.0 (C-10), 137.9 (C-5), 140.1, 142.8 (HC=C), 150.4, 151.9, 155.6 (C-6”), 155.8
(C-3), 220.7 (C-17); ESI-MS: 585 [M + H]+; Anal. Calcd for C31H36N8O5: C, 63.68; H, 6.21; N, 19.17.
Found: C, 63.84; H, 6.35; N, 19.38.
3-{[1-(50-Deoxythymidine-50-yl)-1H-1,2,3-triazole-4-yl]methyloxy}-13
α
-estra-1,3,5(10)-trien-17-one (13b):
◦
After purification, conjugate 13b was obtained as a white solid (51 mg, 89%), m.p. 211–212 C,
Rf = 0.63 (ss B); 1H-NMR (DMSO-d6);
and 2.30 (m, 1H): 2 -H2, 2.74 (d, 2H, J = 3.0 Hz, 6-H2), 4.28 (m, 1H), 4.63 (m, 1H) and 4.71 (m, 1H): 5 -H2,
δ
[ppm] = 0.97 (s, 3H, 18-CH3), 1.77 (s, 3H, 5”-CH3), 1.88 (m, 1H)
0
0
0
0
0
5.01 (s, 2H, OCH2), 4.09 (d, 1H, J = 3.0 Hz), 5.50 (s, 1H), 6.16 (t, 1H, J = 5.0 Hz, J = 6.0 Hz): 1 -, 3 -, 4 -H,
6.69 (s, 1H, 4-H), 6.76 (d, 1H, J = 8.5 Hz, 2-H), 7.16 (d, 1H, J = 7.5 Hz, 1-H), 7.33 (s, 1H, HC=C), 8.16
(s, 1H, 3”-NH); 13C-NMR (DMSO-d6);
δ
[ppm] = 12.0 (5”-CH3), 20.4, 24.5 (C-18), 27.6, 27.9, 29.7, 31.5,
32.5, 37.8 (C-20), 40.7 (2C, C-8 and C-9), 48.4 (C-14), 49.4 (C-13), 51.1 (C-50), 60.8 (OCH2), 70.7 (C-30),
83.8, 83.9, 109.8 (C-5”), 112.3 (C-2), 114.1 (C-4), 125.0 (H =C), 126.6 (C-1), 131.9 (C-10), 135.9 (C-6”),
C

Molecules 2016, 21, 1212
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137.8 (C-5), 142.9 (HC=
C
), 150.3, 155.7 (C-3), 163.5, 220.5 (C-17); ESI-MS: 576 [M + H]+; Anal. Calcd for
C31H37N5O6: C, 64.68; H, 6.48; N, 12.17. Found: C, 64.53; H, 6.62; N, 12.35.
3-{[1-(20,50-Dideoxycytidine-50-yl)-1H-1,2,3-triazole-4-yl]methyloxy}-13
α
-estra-1,3,5(10)-trien-17-one (13c):
After purification, conjugate 13c was obtained as a white solid (45 mg, 81%), m.p. 170 ◦C (dec.), Rf
= 0.41 (ss C); 1H-NMR (DMSO-d6);
δ
[ppm] = 0.96 (s, 3H, 18-CH3),02.29 (m, 02H, 20-H2), 2.74 (m, 2H,
0
0
6-H2), 3.75 (m, 1H), 4.21 (m, 2H), 4.62-4.72 (overlapping m, 2H): 1 -, 3 -, 4 -H, 5 -H2, 5.06 (s, 2H, OCH2),
6.70 (s, 1H, 4-H), 6.75 (d, 1H, J = 6.5 Hz, 2-H) 7.15 (d, 1H, J = 8.0 Hz, 1-H), 7.29 (s, 1H, HC=C), 8.04
(d, 1H, J = 6.5 Hz, 6”-H), 8.19 (s, 1H, 4”-NH2); 13C-NMR (DMSO-d6); δ [ppm] = 24.6 (C-18), 27.8, 28.1,
0
0
29.8, 31.7, 33.0, 36.1 (C-2 ), 40.8 (2C, C-8 and C-9), 43.4, 48.6 (C-14), 50.5 (C-13), 51.5 (C-5 ), 61.3 (OCH2),
84.1, 84.9, 87.3, 94.5 (C-5”), 112.5 (C-2), 114.2 (C-4), 125.2 (C-1), 126.8 (H =C), 132.1 (C-10), 137.9 (C-5),
141.1 (C-6”), 142.9 (HC=
), 155.0 (C-3), 155.2 (C-2”), 165.6 (C-4”), 220.7 (C-17); ESI-MS: 561 [M + H]+;
C
C
Anal. Calcd for C30H36N6O5: C, 64.27; H, 6.47; N, 14.99. Found: C, 64.42; H, 6.34; N, 15.18.
3.2. Cell Cultures and Antiproliferative Assays
The growth-inhibitory effects of the conjugates 12a–c and 13a–c were tested in vitro by means
of the MTT assay on a panel of malignant human cell lines of gynecological origin including HeLa,
MCF-7 and A2780 cells, isolated from cervical, breast and ovarian cancers. The cell lines were obtained
from the European Collection of Cell Cultures (Salisbury, UK). The cells were maintained in minimal
essential medium supplemented with 10% fetal bovine serum (FBS), 1% nonessential aminoacids and
an antibiotic-antimycotic mixture (AAM). 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 were grown in a humidified atmosphere of 5% CO2 at
◦
37 C. For pharmacological investigations, 10 mM stock solutions of the tested conjugates 12a
–
c
and 13a–c and cisplatin controls were prepared in 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, then the medium containing the
tested compounds (12a
increasing concentrations (0.03
viability was determined by the addition of 20
–
c
and 13a
–
c
and cisplatin controls) were added. After a 72 h incubation with
M) of the compounds (12a and 13a and cisplatin controls),
L MTT solution (5 mg/mL) for 4 h. The precipitated
−
30
µ
–
c
–c
µ
formazan crystals were solubilized in DMSO and the absorbance values were determined at 545 nm
with an ELISA reader [62] utilizing untreated cells as controls. Two independent experiments were
performed with 5 parallel wells and cisplatin, a clinically applied anticancer agent, was used as
reference compound. For the most effective conjugates (12a, 12c and 13a) 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).
3.3. Determination of 17β-HSD1 Inhibition
The inhibitory effects exerted on 17
determined via an in vitro radiosubstrate incubation method described in detail earlier [22
Conversion of estrone to 17 -estradiol was measured in the presence of NADPH cofactor and
microsomes of human termed placenta served as enzyme source. Relative conversions in the presence
of 10 M test compounds were measured. IC50 value (the inhibitor concentration that decreases the
β-HSD1 activity by the newly synthesized conjugates were
,
63].
β
µ
enzyme activity to 50%) was determined for 12b. A reference IC50 value was determined earlier for
unlabelled estrone and that was 0.63 ± 0.11 µM.
4. Conclusions
Triazolyl nucleoside conjugates of 13
to investigate their antiproliferative and 17
α
-estrone have been synthesized succesfully in order
-HSD1 enzyme inhibitory activity. The CuAAC
β

Molecules 2016, 21, 1212
13 of 16
reaction conditions have been optimized for the coupling of the protected azidonucleosides
with 3-O-propargyl-13 -estrone. The biological assays showed lower antiproliferative and
17 -HSD1 enzyme inhibitory activities for the conjugates compared to our earlier synthesized
3-O-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]-13 -estrone ( ) which is probably due to the steric effect
and the polar behavior of the inserted nucleoside units. The protected, cytidine–13 -estrone conjugate
12c showed the best antiproliferative activity with IC50 values of 9.0–10.4 M and the unprotected,
thymidine–13 -estrone conjugate 13b displayed the best enzyme inhibitory potential (IC50 = 19 M).
13 -Estrone lost its 17 -HSD1 inhibitory activity upon attaching the nucleoside moiety to its 3-OH
group by a triazolylmethyl linker. The antiproliferative activity was also decreased by changing the
benzyl group for the nucleoside unit in our potent model compound . Concerning that the most
active 17 -HSD1 inhibitor was different from the best cell growth-inhibitor in this series, it can be
α
β
α
1
α
µ
α
µ
α
β
1
β
stated, that the two biological effects should be attributed to other mechanisms. From the biological
results, it seems that neither the improvement in the activity nor the improvement in the selectivity
was demonstrated for the synthesized nucleoside–13α-estrone conjugates.
Acknowledgments: The authors are grateful for financial support from the Hungarian Scientific Research Fund
(grants OTKA K113150 and K109293).
Author Contributions: Brigitta Bodnár, Edina Bianka Herman and Izabella Sinka performed the experiments;
János Wölfling and Gyula Schneider contributed reagents, materials and analysis tools; Zoltán Kupihár,
Erzsébet Mernyák, Mihály Szécsi and István Zupkó conceived and designed the experiments; Zoltán Kupihár,
Erzsébet Mernyák and István Zupkó analyzed the data; Zoltán Kupihár, Erzsébet Mernyák, Mihály Szécsi,
István Zupkó and Lajos Kovács wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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