A facile 'click' approach to novel 15β-triazolyl-5α-androstane derivatives, and an evaluation of their antiproliferative activities in vitro

Article abstract of DOI:10.1016/j.bmc.2012.01.008

Intermolecular Cu(I)-catalyzed azide-alkyne cycloadditions of 15β-azido-17β-hydroxy-5α-androstan-3β-yl acetate with different terminal alkynes under optimized reaction conditions were carried out to furnish 15β-triazolyl derivatives in good yields. Subseq

Full text of DOI:10.1016/j.bmc.2012.01.008

Bioorganic & Medicinal Chemistry 20 (2012) 1396–1402
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A facile ‘click’ approach to novel 15b-triazolyl-5
a
-androstane derivatives,
and an evaluation of their antiproliferative activities in vitro
Zalán Kádár a, , Judit Molnár b, , Gyula Schneider a, István Zupkó b, , Éva Frank a,
⇑
⇑
a Department of Organic Chemistry, University of Szeged, Dóm tér 8., Szeged H-6720, Hungary
b Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6., Szeged H-6720, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Intermolecular Cu(I)-catalyzed azide-alkyne cycloadditions of 15b-azido-17b-hydroxy-5a-androstan-
Received 25 November 2011
Revised 3 January 2012
Accepted 4 January 2012
Available online 12 January 2012
3b-yl acetate with different terminal alkynes under optimized reaction conditions were carried out to
furnish 15b-triazolyl derivatives in good yields. Subsequent oxidation of the ‘click’ products with the
Jones reagent afforded the corresponding 17-ketones. All the synthetized compounds were tested on
three malignant human cell lines (HeLa, MCF7 and A431) in order to investigate their antiproliferative
activities in vitro. Evidence of cell cycle blockade and apoptosis induction was obtained for the most
effective five selected compounds by means of flow cytometry and microscopic techniques. The
Keywords:
Steroidal azides
Cu(I) catalysis
1,3-Dipolar cycloaddition
Triazoles
15b-triazolyl-5
a-androstane framework may be considered an appropriate base for the design of
steroidal antiproliferative agents.
Ó 2012 Elsevier Ltd. All rights reserved.
Antiproliferative activity
1. Introduction
The synthetic strategies for the formation of five-membered
heterocyclic ring systems include 1,3-dipolar cycloaddition
reactions, which have enjoyed undiminished popularity for several
decades in view of the large numbers of potential dipoles and
dipolarophiles.3 As one such possibility, Cu(I)-catalyzed azide–
alkyne cycloaddition (CuAAC)4 has received considerable attention
in recent years as it meets all of the criteria for ‘click’ chemistry
introduced by Sharpless in 2001 (e.g., regioselectivity, lack of
side-reactions, increased reaction rates, high conversions, and
tolerability to a variety of common reaction parameters).5 The
1,4-disubstituted 1,2,3-triazole formed during the reaction can
mimic the atom placement and electronic properties of a peptide
bond; however, it is essentially chemically inert against oxidation,
reduction and hydrolytic conditions, and possesses a much stron-
ger dipole moment than that of an amide bond.6 Although a num-
ber of diverse triazolyl derivatives have been reported to exhibit
varied biological activities, including anti-HIV,7 antibacterial,8 anti-
histamine9 or cytostatic effects,10 steroids containing this kind of
structural moiety have received less attention from both synthetic
and pharmacological aspects.11 Banday et al. recently reported the
syntheses of 21-triazolyl derivatives of pregnenolone as potential
anticancer agents through use of the ‘click’ chemistry approach,12
but without any suggestion as to their mode of action.
Sexual hormones and their derivatives are widely used in the
medication of different diseases, though undesirable side-effects
may appear because of their primary hormonal activities. Currently
the main driving force toward the preparation of steroidal com-
pounds is the development of novel analogs with a biological tar-
get other than a hormone receptor, and therefore the reduction
or elimination of unwanted hormonal effects. One of the synthetic
tools for modifying biological activity is the design of heterocyclic
derivatives that are not recognized by the hormone receptor pro-
tein in consequence of their specific structure or the fact that their
geometry differs from that of the natural hormones. Thus, a variety
of steroids with unusual and interesting structures have been syn-
thetized and evaluated for their anti-tumor activities.1 Experimen-
tal results during the past few years have revealed that a number of
natural or synthetic steroidal heterocycles play important roles in
complex signal transduction mechanisms, and therefore affect the
proliferation of human cancer cells without influencing the divi-
sion of intact cells.2 Consequently, the application of heterocyclic
steroidal compounds in the treatment of cancer offers an excellent
possibility for targeted therapy.
The most frequent synthetic modifications of the original ster-
ane framework are performed at the positions adjacent to the
existing C-3, C-17 or C-20 functional groups, where substitution
is facilitated. Substitution at other positions of the skeleton (e.g.,
the introduction of an azido group) has proved to be more difficult,
⇑
Corresponding authors. Tel.: +36 62 544275; fax: +36 62 544200.
E-mail addresses: zupko@pharm.u-szeged.hu (I. Zupkó), frank@chem.u-szeged.
hu (Éva Frank).
Authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.008

Z. Kádár et al. / Bioorg. Med. Chem. 20 (2012) 1396–1402
1397
necessitating several reaction steps, and is therefore rarely applied.
In this regard, we recently reported the stereoselective synthesis of
1a-azidoandrostanes and their CuAAC with terminal alkynes to
hol 3 was then reacted with phenylacetylene (5a) under various
‘click’ reaction conditions in order to determine the parameters
(catalyst, additives, solvent and temperature) needed for the opti-
mal yields of the desired product 6. The results are presented in Ta-
ble 1.
furnish novel 1a-triazolyl derivatives with noteworthy in vitro
cytostatic effects.13
The foregoing results led us to set out to introduce an azido
group at the unconventional position 15 of the sterane skeleton,
and ultimately to synthetize novel 15-exo-triazolyl derivatives
via CuAAC. To the best of our knowledge, only a few 15-substituted
steroidal derivatives have been synthetized to date; they include
15b-methylated estrone 3-methyl ether, which exerts in vitro cyto-
toxic activity against human gastric cancer cell line MGC-803.14
Since some steroid triazoles are also known to exhibit antiprolifer-
ative activity,11c–13 we decided to screen all the synthetized com-
pounds in vitro for their activities against a panel of three human
cancer cell lines (HeLa, MCF7 and A431). The most effective com-
pounds were subjected to additional in vitro experiments on HeLa
cells, including flow cytometric cell cycle analysis and fluorescence
microscopy, in order to characterize the mechanism of their action.
The most common technique, involving the in situ generation of
the Cu(I) species by the reduction of CuSO4ꢀ5H2O with sodium
ascorbate, resulted in the corresponding cycloadduct 6 in a yield
of only 11%, with a major amount of unreacted starting material
3 (entry 1). A similar result was obtained on the use of CuI catalyst
in acetonitrile at room temperature, although both an amine base
additive and a complexing ligand were used for formation of the
Cu–acetylide complex and to protect the Cu(I) from oxidation (en-
try 2). Nevertheless, better yields were achieved when toluene as
solvent was applied instead of acetonitrile (entries 3 and 4). Tri-
phenylphosphane is presumed to accelerate the rate of the reaction
by complexing to Cu(I), since lower conversions were attained
without its addition to the reaction mixture. Since CuI-catalyzed
cycloadditions are often favored by elevated temperature,17 the
further experiments were carried out under reflux in order to im-
prove the yields of the heterocyclic product 6 and to accelerate the
reaction. The best conversion was found on the use of a catalytic
amount of CuI with the simultaneous addition of triphenylphos-
pane as stabilizing ligand and excess N,N-diisopropylethylamine
(DIPEA) as amine base; these conditions furnished 6 in a yield of
72% on refluxing in toluene for 4 h (entry 6). The reaction of 5a
with the acetylated azidoalcohol 4 proceeded similarly as for 3,
indicating that the functional group on C-17 does not have a signif-
icant effect on the ring-closure (entry 7). However, full conversion
of the starting materials 3 or 4 could not be achieved even at ele-
vated temperature; this may be attributed to the OH or OAc group
on C-17, which is cis and therefore spatially close to the azide di-
pole, presumably causing a crowded transition state in the Cu(I)-
catalyzed process. Similar steric hindrance was observed earlier
2. Results and discussion
2.1. Synthesis
Preliminary ring-closure experiments on model compound 3,
synthetized from 3-methoxy-1,3,5(10),15-estratetraen-17-one
(1), were first carried out in order to optimize the reaction condi-
tions (Scheme 1). The azido group was introduced stereoselectively
onto position 15b of the sterane skeleton of 1 by the 1,4-Michael
addition of azoimide,15 generated in situ from sodium azide and
acetic acid, to afford azidoketone 2 in a yield of 75% after purifica-
tion by flash chromatography. Since b-substituted ketones such as
2 are often susceptible to elimination and undergo facile transfor-
mation to the corresponding enone,16 2 was reduced with KBH4 so
as to avoid this adverse side-reaction. The resultant cis-azidoalco-
during the synthesis of 1
After determination of the optimal conditions, an azidoalcohol
(9) in the 5 -androstane series, readily available from dehydroepi-
a
-triazolyl derivatives.13
a
androsterone in a multistep pathway,15 was subjected to similar
cycloadditions with different aryl-substituted acetylenes (5a–d).
This resulted in steroidal 15b-exo-triazolyl derivatives 10a–d in
yields of ꢁ70%, independently of the substituent on the alkyne
dipolarophile (Table 2). Subsequent Jones oxidation of triazolyl
alcohols 6 and 10a–d furnished the corresponding 17-keto analogs
8 and 11a–d in good yields.
The structures of all synthetized compounds were confirmed by
1H and 13C NMR measurements. The 1H NMR spectra of triazoles
containing an aromatic moiety connected to the hetero ring (6–8,
10a–d, 11a–d) revealed the signals of the incorporated aryl groups
at 7.1–7.8 ppm as compared with the spectra of the starting azides
(3, 4 and 9). The 50-H singlet of the newly formed heterocycles was
identified at 7.7–8.9 ppm.
O
O
NaN3/AcOH
THF
H
H
15
H
H
H
N3
MeO
MeO
2
1
KBH4 MeOH
OR2
OR2
Ph C CH
5a
H
H
Cu(I)
H
H
H
N
N
N3
N
2.2. Pharmacology
6 R2 = H
Ph
3 R2 = H
4 R2 = Ac
Ac2O
py
7 R2 = Ac
With the exception of the 17-acetate in the estrone series (7),
the novel triazolyl derivatives (6, 8, 10a–d and 11a–d) were sub-
jected to in vitro pharmacological studies of their antiproliferative
effects (Table 3). The activities were determined by using three
malignant adherent cell lines in the microplate-based MTT colori-
metric assay,18 in comparison with cisplatin as positive control.
Although there is no generally accepted threshold for efficacy, a
substance exhibiting less than 50% inhibition of cell growth at
(for 6) Jones ox.
O
H
H
H
N
N
MeO
30
l
M can not be considered a promising lead compound.
N
17b-Triazolyl estrones 6 and 8, similarly to the reference agent
cisplatin, exhibited marked antiproliferative effects against A431
cells, while the other two cell lines seemed less sensitive. Since
8
Ph
Scheme 1. Synthesis of 15b-triazolylestrones (6–8).

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Z. Kádár et al. / Bioorg. Med. Chem. 20 (2012) 1396–1402
Table 1
CuAAC of model compounds 3 and 4 with phenylacetylene 5a under different reaction conditions
Entry
Azide
5a (equiv)
Catalyst (equiv)
Base (equiv)
Ligand (equiv)
Solvent
T (°C)
t (h)
Yielda (%)
1
2
3
4
5
6
7
3
3
3
3
3
3
4
(1.0)
(1.0)
(1.0)
(1.0)
(1.1)
(1.1)
(1.1)
CuSO4 (0.02) Na ascorbate (0.1)
—
—
tBuOH/H2O = 1:1
CH3CN
Toluene
Toluene
Toluene
25
25
25
96
96
96
96
4
11
9
CuI (0.1)
CuI (0.1)
CuI (0.1)
CuI (0.1)
CuI (0.1)
CuI (0.1)
Et3N (1.0)
Et3N (1.0)
Et3N (1.0)
—
DIPEA (3.0)
DIPEA (3.0)
Ph3P (0.2)
—
Ph3P (0.2)
Ph3P (0.2)
Ph3P (0.2)
Ph3P (0.2)
18
40
52
72
70
25
111
111
111
Toluene
Toluene
4
4
a
Yields of purified isolated products.
Table 2
Treatment of HeLa cells with 3 and 10 lM of the selected agents
CuAAC of the cis-azidoalcohol
9
with terminal alkynes 5a–d under optimized
for 24 or 48 h was followed by flow cytometric cell cycle analysis.
The 24-h treatment with each of these compounds resulted in a
concentration-dependent decrease in the number of cells in the
G1 phase, and also in an accumulation of the G2/M population
(Fig. 1). Compound 10c did not exert any effect on the G2/M phase,
but increased the proportion of cells in the synthetic (S) phase.
10a–c and 11a also resulted in modest but statistically significant
increases in the number of hypodiploid (subG1) cells, which are
generally regarded as an apoptotic population.19 This apoptotic
proportion became more pronounced after incubation for 48 h
(Fig. 2).
The programmed cell death-inducing capacities of the tested
agents were confirmed by detection of the cell morphology and
membrane integrity. Separate pictures were taken, illustrating
Hoechst 33258 and propidium iodide (PI) fluorescence as morpho-
logical markers. After incubation for 24 h, concentration-depen-
dent increases in nuclear condensation and cell membrane
permeability were generally detected, indicated by blue and red
fluorescence, respectively (Fig. 3). This finding suggests that, after
conditions
OH
OH
R C CH
5a−d
17
H
CuI (0.1 equiv.)
H
H
H
N3 Ph3P (0.2 equiv.)
DIPEA (3 equiv.)
toluene
N
AcO
N
H
9
N
R
111 °C, 16 h
10a−d
Jones ox.
(93-96%)
11a−d (C17=O)
Entry
Alkyne
5a
R
Triazole
Yielda (%)
1
2
3
4
10a
10b
10c
10d
73
75
72
72
5b
treatment with these 5a-androstane derivatives, the cells can not
complete the S-G2/M phases of the cell cycle and this blockade
may initiate the apoptotic machinery.
5c
5d
F
An anticancer agent is generally expected to cause the death of
the tumor cells, but the manner of cell death can be crucially rele-
vant. While apoptosis involves a precisely regulated self-decompo-
sition of the cells in question, protecting the surrounding intact
cells, necrosis involves the prompt release of the intracellular con-
tent, which results in the deterioration of neighboring cells.20 The
biochemical and morphological results indicate that the initiation
of apoptosis predominates in the antiproliferative action of the
present compounds and their structure can therefore be consid-
ered a reasonable skeleton for the design and synthesis of further
anticancer lead candidates.
a
Yields of purified isolated products.
Table 3
Calculated IC50 values of the synthetized triazole derivatives
IC50 values (l
M)*
HeLa cells
MCF-7 cells
A431 cells
6
>30
>30
1.70
4.77
20.69
22.43
>30
>30
9.69
10.66
>30
16.03
2.84
8
14.88
7.70
9.40
6.52
>30
11.35
19.24
10.28
>30
10a
10b
10c
10d
11a
11b
11c
11d
Cisplatin
3. Conclusions
>30
9.16
1.69
2.68
8.40
3.39
9.63
In summary, the CuAAC of steroidal 15b,17b-azidoalcohols with
different terminal alkynes under optimized reaction conditions
gave 15b-exo-triazolyl derivatives in good yields. The synthetized
10.27
15.01
10.96
12.43
5a-androstane derivatives are of interest from a pharmacological
*
aspect, since several of the analogs proved to exert marked
in vitro antiproliferative activity. As apoptosis initiation is involved
in their mode of action, these compounds may be considered for
the design of further anticancer lead candidates.
Mean values from two independent determinations with five parallel wells;
standard deviation less than 15%.
substantial differences were not detected between the IC50 values
of the 5
a
-androstane counterparts, it could be concluded that the
4. Experimental
4.1. General
substituent on the triazolyl ring does not have a crucial impact
on the anticancer capacities of this skeleton. Although not conse-
quently, the 17-keto function seemed to be favored over the 17-hy-
droxy group. The results of the MTT assays led to the selection of
10a–c, 11a and 11b for additional experiments in an attempt to
elucidate the mechanism of their action.
Melting points (mp) were determined on a Kofler block and are
uncorrected. The reactions were monitored by TLC on Kieselgel-G
(Merck Si 254 F) layers (0.25 mm thick); solvent systems (ss): (A)

Z. Kádár et al. / Bioorg. Med. Chem. 20 (2012) 1396–1402
1399
Figure 1. Effects of 10a–c and 11a–b on HeLa cell cycle distribution after incubation for 24 h. The data are mean values from three determinations. ꢂ and ꢂꢂ indicate p <0.05
and p <0.01, respectively, as compared with the control cells.
Figure 2. Effects of 10a–c and 11a–b on HeLa cell cycle distribution after incubation for 48 h. The data are mean values from three determinations. ꢂ and ꢂꢂ indicate p <0.05
and p <0.01, respectively, as compared with the control cells.
CH2Cl2, (B) CH2Cl2/EtOAc (98:2 v/v), (C) CH2Cl2/EtOAc (95:5 v/v),
(D) CH2Cl2/EtOAc (80:20 v/v), (F) CH2Cl2/EtOAc (50:50 v/v), (G)
CH2Cl2/hexane (80:20 v/v). The spots were detected by spraying
with 5% phosphomolybdic acid in 50% aqueous phosphoric acid.

1400
Z. Kádár et al. / Bioorg. Med. Chem. 20 (2012) 1396–1402
Figure 3. Fluorescence microscopy images of Hoechst 33258—PI double staining. Separate pictures from the same field were recorded for the two markers. HeLa cells were
treated with vehicle (control), or with 10a–c or 11a–b at 3, 10 and 30 M. The blue fluorescence indicates Hoeschs 33258, and the red coloration is a result of cellular PI
accumulation. The bar in the Hoeschs 33258 control picture indicates 100 m.
l
l
The Rf values were determined for the spots observed by illumina-
tion at 254 and 365 nm. Flash chromatography: Merck silica gel 60,
40–63 lm. All solvents were distilled prior to use. Reagents and
then refluxed for 10 h, allowed to cool and evaporated in vacuo.
The resulting crude product was purified by column
chromatography.
materials were obtained from commercial suppliers and were used
without purification. Elementary analysis data were determined
with a PerkinElmer CHN analyzer model 2400. NMR spectra were
obtained at room temperature with a Bruker DRX 500 instrument.
Chemical shifts are reported in ppm (d scale), and coupling con-
stants (J) in Hz. For the determination of multiplicities, the J-
MOD pulse sequence was used. Automated flow injection analyses
were performed by using an HPLC/MSD system. The system com-
prised an Agilent 1100 micro vacuum degasser, a quaternary
pump, a micro-well plate autoinjector and a 1946A MSD equipped
with an electrospray ion source (ESI) operated in positive ion
mode. The ESI parameters were: nebulizing gas N2, at 35 psi; dry-
ing gas N2, at 350 °C and 12 L/min; capillary voltage (VCap) 3000 V;
fragmentor voltage 70 V. The MSD was operated in scan mode with
4.2.1. 3b-Acetoxy-15b-[40-phenyl-10H-10,20,30-triazol-10-yl]-5
androstan-17b-ol (10a)
Alkyne: phenylacetylene (5a, 0.09 mL). After purification with
CH2Cl2/EtOAc (80:20) as eluent, 10a was obtained as a white solid
(279 mg, 73%), mp 243–245 °C, Rf = 0.23 (ss D); 1H NMR (500 MHz,
CDCl3): dH = 0.81 (s, 3H, 19-H3), 1.15 (s, 3H, 18-H3), 1.99 (s, 3H, Ac-
a-
H3), 2.80 (dt, 1H, J = 14.1 Hz, J = 8.5 Hz, 16-H ), 3.75 (t, 1H,
a
J = 8.6 Hz, 17-H), 4.64 (m, 1H, 3-H), 4.96 (m, 1H, 15-H), 7.32 (t,
1H, J = 7.6 Hz, 400-H), 7.42 (t, 2H, J = 7.6 Hz, 300-H and 500-H), 7.78
(s, 1H, 50-H), 7.82 (d, 2H, J = 7.6 Hz, 200-H and 600-H); 13C NMR
(125 MHz, CDCl3): dC = 12.3 (C-19), 14.3 (C-18), 20.6 (CH2), 21.4
(Ac-CH3), 27.4 (CH2), 28.1 (CH2), 30.8 (CH2), 33.1 (CH), 33.8
(CH2), 35.7 (C-10), 36.7 (CH2), 38.9 (CH2), 40.8 (CH2), 42.9 (C-13),
44.8 (CH), 55.1 (CH), 55.6 (CH), 58.5 (C-15), 73.4 (C-3), 80.3 (C-
17), 120.1 (C-50), 125.6 (2C, C-200 and C-600), 128.1 (C-400), 128.8
(2C, C-300 and C-500), 130.6 (C-100), 147.0 (C-40), 170.7 (Ac-C); ESI-
MS: 478 [M+H]+; Anal. Calcd for C29H39N3O3 C, 72.92; H, 8.23; N,
8.80. Found: C, 73.08; H, 8.41; N, 8.67.
a mass range of m/z 60–620. Samples (0.2 lL) with automated nee-
dle wash were injected directly into the solvent flow (0.3 mL/min)
of CH3CN/H2O 70:30 (v/v) supplemented with 0.1% formic acid.
The system was controlled by Agilent LC/MSD Chemstation
software.
4.2. General procedure for the preparation of triazoles 10a–d
4.3. General procedure for the preparation of triazoles 11a–d
To a solution of 15b-azido-17b-hydroxy-5
a-androstan-3b-yl
Compound 10a–d (200 mg) was dissolved in acetone (10 mL)
and Jones reagent (0.5 mL) was dropped into the solution, which
was then stirred at room temperature for 20 min., and diluted with
water. The precipitate that formed was filtered off and dried, and
the crude product was purified by column chromatography.
acetate (9)15 (300 mg, 0.80 mmol) in toluene (10 mL) were added
Ph3P (41 mg, 0.16 mmol), CuI (15 mg, 0.08 mmol) and DIPEA
(0.40 mL, 2.4 mmol). Finally, the appropriate terminal alkyne
(5a–d, 1.1 equiv) was added to the reaction mixture, which was

Z. Kádár et al. / Bioorg. Med. Chem. 20 (2012) 1396–1402
1401
4.3.1. 3b-Acetoxy-15b-[40-phenyl-10H-10,20,30-triazol-10-yl]-5
a-
for 1 h at 37 °C and were then photographed by means of a Ni-
androstan-17-one (11a)
kon Eclipse microscope equipped with an epifluorescence
Substrate: 10a (0.42 mmol); product: 11a (189 mg, 95%), mp
217–218 °C, Rf = 0.33 (ss C); 1H NMR (500 MHz, CDCl3): dH = 0.80
(s, 3H, 19-H3), 0.86 (s, 3H, 18-H3), 2.01 (s, 3H, Ac-H3), 2.89 (dd,
attachment containing the appropriate optical blocks and a
QCapture CCD camera. The staining allowed the identification
of live, early-apoptotic, late-apoptotic and necrotic cells. Hoechst
33258 permeates all the cells and makes the nuclei appear blue.
Apoptosis was revealed by nuclear changes such as chromatin
condensation and nuclear fragmentation. The necrotic and the
late-apoptotic cells were identified as cells which displayed PI
uptake, which indicates the loss of membrane integrity, the cell
nuclei being stained red.
1H, J = 19.5 Hz, J = 7.5 Hz, 16-Hb), 3.38 (d, 1H, J = 19.5 Hz, 16-H ),
a
4.69 (m, 1H, 3-H), 5.15 (m, 1H, 15-H), 7.34 (t, 1H, J = 7.6 Hz, 400-
H), 7.42 (t, 2H, J = 7.6 Hz, 300-H and 500-H), 7.81 (d, 2H, J = 7.6 Hz,
200-H and 600-H), 7.83 (s, 1H, 50-H); 13C NMR (125 MHz, CDCl3):
dC = 12.2 (C-19), 16.3 (C-18), 20.3 (CH2), 21.4 (Ac-CH3), 27.3
(CH2), 28.0 (CH2), 31.1 (CH2), 33.2 (CH), 33.8 (CH2), 34.3 (CH2),
35.9 (C-10), 36.6 (CH2), 43.6 (CH2), 44.9 (CH), 46.3 (C-13), 55.4
(CH), 56.3 (CH), 56.4 (CH), 73.3 (C-3), 119.8 (C-50), 125.6 (2C, C-
200 and C-600), 128.3 (C-400), 128.9 (2C, C-300 and C-500), 130.2 (C-100),
147.6 (C-40), 170.7 (Ac-C), 216.7 (C-17); ESI-MS: 476 [M+H]+; Anal.
Calcd for C29H37N3O3 C, 73.23; H, 7.84; N, 8.83. Found: C, 73.41; H,
7.68; N, 9.09.
Acknowledgments
This work was supported by the New Hungary Development
Plan (TÁMOP 4.2.1/B-09/1/KONV-2010-0005) and the Hungarian
Scientific Research Fund (OTKA K101659). The project ‘TÁMOP-
4.2.1/B-09/1/KONV-2010-0005—Creating the Center of Excellence
at the University of Szeged’ is supported by the European Union
and co-financed by the European Regional Fund. The authors thank
Mrs. Irén Forgó and Ms. Judit Czinkota (University of Szeged,
Hungary) for technical support.
4.4. Cell cultures and antiproliferative assays
Human cancer cell lines (HeLa, MCF-7 and A431, isolated from
cervical adenocarcinoma, breast adenocarcinoma and skin epider-
moid carcinoma, respectively) were maintained in minimal essen-
tial medium supplemented with 10% fetal bovine serum, and 1%
non-essential amino acids and an antibiotic-antimycotic mixture.
All cell lines were purchased from the European Collection of Cell
Cultures, Salisbury, UK. The cells were grown in a humidified
atmosphere of 5% CO2 at 37 °C. Cells were seeded onto 96-well
plates at a density of 5000 cells/well and allowed to stand over-
night, after which the medium containing the tested compound
was added. After a 72-h incubation period, viability was deter-
Supplementary data
Supplementary data (experimental procedures for the
preparation and NMR spectral data of all synthetized compounds)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2012.01.008.
References and notes
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