New thiophene analogues of kenpaullone: Synthesis and biological evaluation in breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2005.02.010

Thieno analogues of kenpaullone have been synthesized using an established method. We investigated the effect of five structural analogues of kenpaullone on vincristine sensitive and resistant MCF7 (human mammary adenocarcinoma) cells. One analogue, 8-Bro

Full text of DOI:10.1016/j.ejmech.2005.02.010

European Journal of Medicinal Chemistry 40 (2005) 757–763
www.elsevier.com/locate/ejmech
Original article
New thiophene analogues of kenpaullone:
synthesis and biological evaluation in breast cancer cells
a
a,b
a
a
Laurent Brault , Evelyne Migianu , Adrien Néguesque , Eric Battaglia ,
a,
a
Denyse Bagrel *, Gilbert Kirsch
a
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, UFR SciFA, Université de Metz, Campus Bridoux, rue du Général Delestraint,
7070 Metz, France
5
b
Present address: Laboratoire de Chimie Structurale et Spectroscopie Biomoléculaire, UMR CNRS 7033, 74, rue Marcel Cachin, 93017 Bobigny, France
Received 19 October 2004; received in revised form 31 December 2004; accepted 3 February 2005
Available online 12 April 2005
Abstract
Thieno analogues of kenpaullone have been synthesized using an established method. We investigated the effect of five structural ana-
logues of kenpaullone on vincristine sensitive and resistant MCF7 (human mammary adenocarcinoma) cells. One analogue, 8-Bromo-6,11-
dihydro-thieno-[3′,2′:2,3]azepino[4,5-b]indol-5(4H)-one (3a), showed an antiproliferative activity in the drug sensitive cell line that led to cell
accumulation in G2/M phase. In addition, repression of cdk1, a G2/M transition key regulator, as well as induction of p21 were observed at the
mRNA level. Programmed cell death (apoptosis) was induced in early time treatments and was accompanied by p53 mRNA induction. The
antiproliferative and proapoptotic properties of 3a make this CDK inhibitor an interesting candidate for further investigations.
©
2005 Elsevier SAS. All rights reserved.
Keywords: Paullone analogues; Cytotoxicity; Cell cycle; Apoptosis; Breast cancer
1
. Introduction
direct partners play a key role in cell proliferation and they
are also implicated in other physiological functions such as
RNA transcription and processing, cell death and neuronal
functions [5,6]. For these reasons, they are attractive targets
in numerous diseases as different as cancer, neurodegenera-
tive or cardiovascular disorders and viral infections. There-
fore, multiple CDK pharmacological inhibitors have been
identified to date, all being competitive inhibitors of these
kinases, mostly by interacting with the ATP binding site [7].
They belong to various chemical classes, including flavones
(flavopiridol), purine analogues (e.g. olomoucine, pur-
vanalol, roscovitine), pyrimidine analogues, oxindoles and
paullones [8]. Members of the latter class were identified as
CDK inhibitors using the COMPARE analysis of a database
of compounds screened in cancer cell lines [9]. Using this
method, kenpaullone was identified as inhibiting ATP bind-
ing and, since then, numerous paullone derivatives have been
synthesized, such as alsterpaullone and gwennpaullone
[10–12].
Over the last few years, comprehension of eukaryotic cell
division mechanisms has largely improved. The progression
of the cell cycle through its four distinct phases (G1, S, G2 and
M) is dependent on the integration of extra- and intracellular
signals among which cyclin-dependent kinases (CDKs) play
a central role [1,2]. These enzymes are a family of serine/
threonine kinases which become active when associated with
regulatory proteins, their sequential activation ensuring the
correct crossing of checkpoints separating each cell cycle
phase. If numerous CDKs and cyclin-box-containing pro-
teins have been isolated, only about 10 cyclin/CDKs com-
plexes are identified as involved in the regulation of cell cycle
progression [3]. While cyclin D is required for entering the
cycle, the progression from G1 phase into and through S phase
depends on the catalytically active complex cyclin E/CDK2
and cyclin A/CDK2. The entry into mitosis is under the con-
trol of the cyclin B/CDK1 complex [4]. Thus, CDKs and their
Using an established synthesis procedure [13], we pre-
pared four new thiophene derivatives of kenpaullone (3b–3e)
(Fig. 1). We synthesized original compounds by replacing
*
Corresponding author. Tel.: +33 3 87 37 84 04; fax: +33 3 87 37 84 23.
E-mail address: bagrel@sciences.univ-metz.fr (D. Bagrel).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.02.010

758
L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
3
.1. Antiproliferative activity
Cell viability was first evaluated in our screening condi-
tions (vide supra).As shown in Fig. 2A, compound 3a induced
in MCF7 an important decrease of cell viability, the number
of living cells being about 8% of control after 72 h of expo-
sure. Except for analogue 3b which displayed a slight effect
Fig. 1. General structure of paullones.
(
64% of viable cells as compared to control), the other com-
the aromatic cycleA by a variously substituted thiophene ring.
The aim of this study was to characterize the antiproliferative
action of these analogues of paullone in breast cancer cells,
i.e. the adenocarcinoma cell line MCF7 and its counterpart
resistant to vincristine, Vcr-R [14]. Cell cycle repartition as
well as cell death mechanisms implicated were also evalu-
ated for cells exposed to these compounds.
pounds were inactive. In the resistant cell line Vcr-R, only
compound 3a showed a significant activity with 72% of liv-
ing cells as compared to control. As referred to its activity
toward MCF7 cells, 3a was also tested at lower doses for
2
4–72 h-treatments (Fig. 2B). Our results indicate that this
analogue exerts a dose- and time-dependent decrease of cell
viability. Thus, a 25 µM concentration induced a significant
effect for a 24 h-treatment. It lowered to 10 µM for a 72 h-
exposure.
2. Chemistry
Having developed a method to access variously substi-
3.2. Cell cycle progression of MCF7 cells
tuted 3-aminothiophenes [15], we used an established strat-
egy to synthesize new analogs of paullones among them 3a
recently described by Kunick et al. [13]. The analogues 3 were
prepared by applying the Fischer indole synthesis to 6,7-
dihydro-4H-thieno[3,4-b]azepine-5,8 diones 2 (Table 1). The
latter were prepared from the corresponding 3-amino-2-
carbethoxy substituted thiophenes 1. The synthesis of these
thienoazepine-diones has been fully described earlier [15].
The Fischer indole synthesis was made by reacting the cor-
responding dione with the appropriate phenylhydrazine in ace-
tic acid followed by cyclization at 70 °C for 1 h [13]. The
yields of the isolated paullone analogues ranged from 9% to
Given the antiproliferative activity of these kenpaullone
analogues, cell cycle distribution was assessed by flow cytom-
etry. As shown in Fig. 3A, no significant modification was
observed for the resistant cells whatever the compound. In
contrast, a markedly change in MCF7 cell cycle repartition
was induced only for compound 3a for a 48 and 72 h-
treatment (data not shown and Fig. 3A). Cell accumulation in
G2/M phase, caused by 3a, was accompanied by a diminu-
tion of cell proportion in G0/G1 phase. For this compound,
the cell cycle blockage was observed as soon as 24 h. It per-
sisted for longer treatments (Fig. 3B, C) and was dose-
dependent for concentrations above 25 µM. This cell accu-
mulation was accompanied by a dose-dependent repression
of mRNA expression of cdk1 implicated in G2/M transition
(Fig. 4). A dose-dependent induction was also observed for
p21 mRNA, implicated in cell cycle blockage by inhibiting
CDK activity but also in apoptosis induction.
49%.
3. Biological evaluation
Breast cancer cell lines used in this study were the wild-
type MCF7, sensitive to miscellaneous drugs, and its coun-
terpart Vcr-R, resistant to vincristine. Paullone analogues
3.3. Induction of apoptosis
(compounds 3a to 3e) were screened by treating cells with
100 µM for 48 and 72 h, before using additional incubation
As shown in Fig. 4, the level of p53 mRNA was increased
times and concentrations for active molecules.
after a 24 h-treatment by compound 3a. p53 is well-known as
Table 1
Synthesis and structure of kenpaullone thieno analogues
Compound
R
R1
H
R2
H
3
3
3
3
3
a
b
c
d
e
Br
H
tBu
tBu
Ph
Ph
H
Br
H
H
CH3
CH3
Br
(
i) ClCO(CH ) CO CH , K CO , toluene, reflux; (ii) KH, toluene,DMF,80 °C; (iii) H O, DMSO, 140 °C; (iv) RPhNHNH , AcOH,70 °C.
2 2 2 3 2 3 2 2

L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
759
Fig. 2. Antiproliferative effect of thieno analogues of kenpaullone. (A) Cell viability (trypan-blue exclusion) of MCF7 and Vcr-R incubated with kenpaullone
analogues at 100 µM for 72 h (M live cells, n dead cells), ** significantly different from control P < 0.01. (B) Percent of live MCF7 cells after 24, 48 and 72 h
treatment with compound 3a. Each result represents the mean of three independent experiments. Results are significantly different from control (P < 0.05)
except for 24 and 48 h-treatment at 10 µM.
a key regulator of apoptosis and is also implicated in positive
regulation of the expression of p21.
increase of the proportion of apoptotic cells (Fig. 6). Thus,
3a is able to induce apoptosis and then apoptotic cells and
bodies enter secondary necrosis in less than 24 h. The appar-
ent discrepancy between TUNEL andAnnexin-V results could
be linked to the fact that the flow cytometric analysis reveals
only early apoptosis, whereas the immunostaining technique
allows to show late apoptosis. The only extensive study of
apoptosis induction triggered by paullone derivatives was per-
formed by Lahusen et al. [11]. They showed that, in Jurkat
cells, the apoptosis induced by alsterpaullone was observed
within early time treatment, which is in good agreement with
our results. It is worth noting that cell death is not due to the
induction of oxidative stress since treatments were not accom-
panied by a significant modification in reactive oxygen spe-
cies level as assessed by the dichlorofluorescein-diacetate
(DCF-DA) flow cytometric assay (data not shown).
Therefore, in order to determine if programmed cell death
was induced together with the observed cell cycle arrest,
Annexin-V binding was assessed by flow cytometry in both
cell types. In our screening conditions, no significant cell death
was induced for resistant cells, and was only detected in sen-
sitive MCF7 cells following exposure to 3a (Fig. 5A). It is
worth noting that these results are consistent with those
obtained with the cell viability test. Furthermore, cell death
was significantly induced by a 24 h-treatment with 100 µM
(Fig. 5B, C). For longer time treatments, a significant effect
was observed with lower concentration (50 µM) of com-
pound 3a (Fig. 5C). However, apoptosis seems to be only
slightly increased with this treatment (Fig. 5B). Thus, the pro-
portion of early apoptotic cells, detected by flow cytometry,
varied from 1.9 ± 0.6% for control to 4.8 ± 0.6% for a treat-
ment with 100 µM of compound 3a.
Given these results and taking into account the induction
of the mRNA level of p53, we investigated if apoptosis is an
early phenomenon by taking advantage of the terminal deoxy-
nucleotidyl transferase-mediated dUTP nick end-labeling
4. Conclusions
Analogues of paullones occupy, as CDK inhibitors, an
attractive position in the current generation of antitumor drugs.
The ability to prepare variously substituted thiophenes per-
mitted us to synthesize new analogues of paullone. These
structural modifications allowed to get some informations
about the structure–activity behavior of these compounds.
Among them, 3a, containing a bromo substitution (Table 1),
(TUNEL) method. This assay revealed a significant induc-
tion of apoptosis for a 24 h-treatment at 50 µM with a large
amount of necrotic cells which are in a large part in second-
ary necrosis (i.e. necrotic lysis of floating apoptotic bodies).
When used at 100 µM compound 3a led to an eightfold

760
L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
Fig. 3
.
Modulation of cell cycle progression. (A) MCF7 andVcr-R cells were treated with paullone analogues (100 µM) for 72 h and analyzed by flow cytometry
(
M G /G , n G /M). (B) DNA content of MCF7 cells exposed (dashed line) or not (full line) to compound 3a (100 µM) for 24, 48 and 72 h. (C) Dose-dependent
0 1 2
modulation of cell cycle distribution of MCF7 cells after 24–72 h incubation with compound 3a (M G0/G1, n G2/M). Each column and vertical bar represent
mean ± S.E.M. (n = 3), * and ** significantly different from control P < 0.05 and P < 0.01, respectively.
presents an interesting antiproliferative activity in MCF7
breast cancer cells. Furthermore, introduction of substitu-
tions on the thiophene ring (3c and 3e) was detrimental to the
biological activity of this compound. The inhibitory effect of
3a has been recently reported for purified CDK1 enzyme with
an IC50 of 0.6 µM [16] which is in good agreement with our
results on cultured cells. Our work was focused on the cellu-
lar effects of the thieno analogues of kenpaullone and we
showed an antiproliferative activity of this compound char-
acterized by the repression of the expression of ckd1 gene
accompanied by a cell accumulation in G2/M phase. The
decrease in mRNA level of cdk1 could be a direct conse-
quence of inhibition of the activity of the kinase. Indeed,
cdk1 expression is dependent on its own activity through a
positive feedback loop. Compound 3a was also able to
increase p21 mRNA level which is implicated in cell cycle
arrest, through inhibition of CDK activity especially CDK1,
but also in apoptosis regulation. It also increased the expres-
Fig. 4. RT-PCR analysis of the mRNA level of genes implicated in cell cycle
and apoptosis regulation in MCF7 cells incubated with compound 3a. Ethi-
dium bromide stained gel showing amplicons corresponding to cdk1, p21,
p53 and b-actin mRNA following exposure to compound 3a at 0, 25, 50 and
100 µM for 24 h.

L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
761
Fig. 5
. Cell death pathway induced by the kenpaullone analogues. (A) Percent of dead cells of MCF7 and Vcr-R treated with the compounds (100 µM) for 48 h
(M live cells, n dead cells) assessed by Annexin-V staining. (B) Dose–response assessment of cell death upon exposure to compound 3a during 24 h Liv.: live
cells; Apo.: early apoptotic cells; Nec.: late apoptotic and necrotic cells. (C) Dose–response assessment of cell death for MCF7 cells exposed to compound 3a
for 24, 48 and 72 h (M live cells, ♦ dead cells). Each column/symbol and vertical bar represent mean ± S.E.M. (n = 3), * and ** significantly different from
control P < 0.05 and P < 0.01, respectively.
sion of p53, another well-known cycle regulator and apopto-
sis inducer. For MCF7 cells, this modulation of mRNAs lev-
els went together with a rapidly induced apoptosis which
occurred within 24 h as demonstrated by TUNEL analysis.
While it displayed a very slight effect in vincristine resistant
cells, the antiproliferative and proapoptotic properties of com-
pound 3a on MCF7 breast cancer cells highlight the potential
interest of this molecule among the CDK inhibitors. Future
studies of its pharmacological properties and in vivo activity
would be of interest for therapeutic strategies against cancer.

762
L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
Fig. 6
.
TUNEL detection of apoptotic MCF7 cells. (A) Control cells. (B) 24 h-exposure to compound 3a (100 µM). DAB labeling led to brown staining of
apoptotic cell nuclei (examples: arrows). Harris’ hematoxylin was used for nuclear counter-staining (blue). (C) Apoptotic indexes, evaluated as the percentage
of apoptotic cells and bodies by reference to the total cellular population determined after a 24 h-treatment with compound 3a at 0, 25, 50 and 100 µM. Columns
and vertical bars represent mean ± S.E.M. for three independent experiments. A minimum of 600 cells were counted in each test. * and ** significantly different
from control P < 0.05 and P < 0.01, respectively.
1
5. Experimental section
dec. > 300 °C; Anal. C H N OS (C, H, N); H-NMR
1
8
18
2
(
250 MHz, DMSO-d ): 1.39 (s, 9H, C(CH ) ), 3.52 (s, 2H,
6 3 3
5.1. Chemistry
CH ), 6.73 (s, 1H, H ), 7.03–7.16 (m, 2H, 2H indole), 7.36
2
3
(
d, 1H, J = 7.7 Hz, H indole), 7.60 (d, 1H, J = 7.7 Hz, H
Thieno[b]azepinediones 1 were prepared according the
indole), 10.23 (bs, 1H, NH, D O exchangeable H), 11.45 (bs,
1
2
procedure we described previously [15]. Phenylhydrazines 2
were purchased fromAcros Organics (France). Melting points
were determined on a Stuart SMP3 melting point apparatus
and are uncorrected. Microanalysis (C, H, N) were
H, NH, D O exchangeable H). Anal. C H N OS (C, H,
2 18 18 2
N): C: 69.65, H: 5.84, N: 9.02; Found: C: 69.49, H: 6.03, N:
.95.
8
1
within ± 0.4% of the theoretical values. H-NMR spectra were
8-Bromo-2-tert-butyl-6,11-dihydro-thieno-[3′,2′:2,3]aze-
recorded on a AC Bruker 250 MHz spectrometer in DMSO-
pino[4,5-b]indol-5(4H)-one (3c). Pale brown solid (14%);
1
d .
m.p. dec. > 300 °C;Anal. C H BrN OS (C, H, N); H-NMR
6
18 17
2
General procedure for the synthesis of thienoazepino-indol-
(250 MHz, DMSO-d ): 1.39 (s, 9H, C(CH ) ), 3.53 (s, 2H,
6 3 3
5-ones 3 [13]. 0.12 ml (1.2 mmol) of adequate phenylhydra-
CH ), 6.73 (s, 1H, H ), 7.22 (d, 1H, J = 8.6 Hz, H indole),
2
3
zine in 3 ml of acetic acid was added dropwise to 1 mmol of
thieno[b]azepinedione 1 dissolved in 5 ml of acetic acid. The
reaction mixture was stirred at 70 °C for 1 h and then cooled
at 0 °C. Next, 0.1 ml of concentrated sulfuric acid was added
and the reaction mixture was stirred again at 70 °C for 1 h.
After incubation at room temperature, the mixture was poured
into 20 ml of ice-cold 10% aqueous sodium acetate solution.
The obtained precipitate was filtered, washed with water and
dried. The crude product was purified by trituration in hot
methanol and filtrated to give the expected thienoazepino-
indol-5-one 3.
7
1
.31 (d, 1H, J = 8.6 Hz, H indole), 7.84 (s, 1H, H indole),
0.28 (bs, 1H, NH, D O exchangeable H), 11.68 (bs, 1H,
2
NH, D O exchangeable H). Anal. C H BrN OS (C, H, N):
2
18 17
2
C: 55.53, H: 4.40, N: 7.20; Found: C: 55.37, H: 4.59, N: 7.13.
-Methyl-2-phenyl-6,11-dihydro-thieno-[3′,2′:2,3]aze-
pino[4,5-b]indol-5(4H)-one (3d). Pale brown solid (49%);
3
1
m.p. dec. > 300 °C; Anal. C H N OS (C, H, N); H-NMR
21 16
2
(250 MHz, DMSO-d ): 2.29 (s, 3H, CH ), 3.54 (s, 2H, CH ),
6 3 2
7.10–7.21 (m, 2H, 2H indole), 7.40–7.47 (m, 2H, 1H
phenyl + 1H indole), 7.52–7.54 (m, 4H, 4H phenyl), 7.67 (d,
1H, J = 7.9 Hz, H indole), 9.91 (bs, 1H, NH, D O exchange-
able H), 11.62 (bs, 1H, NH, D O exchangeable H). Anal.
C H N OS (C, H, N): C: 73.23, H: 4.68, N: 8.13; Found:
8
-Bromo-6,11-dihydro-thieno-[3′,2′:2,3]azepino[4,5-
b]indol-5(4H)-one (3a) (9%). See Ref. [13].
-tert-Butyl-6,11-dihydro-thieno-[3′,2′:2,3]azepino[4,5-
b]indol-5(4H)-one (3b). Pale brown solid (34.5%); m.p.
2
2
2
2
1 16 2
C: 72.97, H: 4.61, N: 7.91.

L. Brault et al. / European Journal of Medicinal Chemistry 40 (2005) 757–763
763
8
-Bromo-3-methyl-2-phenyl-6,11-dihydro-thieno-
The cell suspension was incubated in the dark for 15 min
with 5 µl of Annexin-V FITC and 5 µl of propidium iodide.
Analysis was performed on a FACScan andAnnexin-V FITC
binding was detected in the FL-1 (green) and PI in the FL-2
(red). In some instances, parallels samples were assessed by
the TUNEL in situ staining. TUNEL was performed accord-
ing to an optimized protocol previously described [17]. Cells
in suspension in the culture medium were collected together
with the adherent cells, fixed and studied as cytospots. Per-
oxidase labeling was detected using diaminobenzidine (DAB)
color reaction (brown). Samples were counterstained with
Harris’ hematoxylin, photographied and quantified using a
Eclipse 80i microscope (Nikon, France) and the Lucia
4.8 software (Nikon, France).
[
3′,2′:2,3]azepino[4,5-b]indol-5(4H)-one (3e). Green solid
24%); m.p. dec. > 300 °C; Anal. C H BrN OS (C, H, N);
H-NMR (250 MHz, DMSO-d ): 2.28 (s, 3H, CH ), 3.56 (s,
H, CH ), 7.28 (d, 1H, J = 8.5 Hz, H indole), 7.38 (d, 1H,
J = 8.5 Hz, H indole), 7.43–7.48 (m, 1H, H phenyl), 7.52–
.55 (m, 4H, 4H phenyl), 7.92 (s, 1H, H indole), 9.95 (bs,
H, NH, D O exchangeable H), 11.85 (bs, 1H, NH, D O
exchangeable H). Anal. C H BrN OS (C, H, N): C: 59.58,
H: 3.57, N: 6.62; Found: C: 59.29, H: 3.50, N: 6.22.
(
21
15
2
1
6
3
2
2
7
1
2
2
21
15
2
5
5
.2. Biology
.2.1. Cell culture and viability test
Cells were grown in RPMI-1640 medium, containing 10%
4
fetal calf serum, antibiotics and antimycotics [10 UI/ml peni-
cillin G, 10 mg/ml streptomycin, 25 µg/ml amphotericin B]
Acknowledgements
(Eurobio, France) at 37 °C in a humidified atmosphere con-
taining 5% CO . Cells in suspension in the culture medium
The authors are very grateful to M. Ghavam for TUNEL
experiments. This study was supported by grants from the
Ligue contre le Cancer (Comités de Moselle, de Meurthe &
Moselle et de Meuse) and the Conseil Régional de Lorraine.
L.B. and E.M. were recipients of a fellowship provided by
the Ligue contre le Cancer (Comité de Moselle).
2
were collected together with the adherent cells. Trypan-blue
staining was used to test cellular viability: cells were sus-
pended in 1% Trypan-blue in PBS (phosphate buffered saline),
incubated for 5 min, and counted for viability using visible-
light microscopy. Live cells remained white and light-
refracting, while dead cells were blue-stained.
5.2.2. Cell cycle analysis
References
At the indicated times, total cells were trypsin-harvested,
washed with PBS, fixed with 80% cold ethanol, stained with
propidium iodide solution (50 µg/ml PI, 20 µg/ml of Rnase
A, 0.1% Triton X-100) for 15 min, and analyzed by flow
cytometry (FACScan, Becton-Dickinson, USA).
[1] M. Malumbres, M. Barbacid, Nat. Rev. Cancer 1 (2001) 222–231.
[
[
2] E.G. Nabel, Nat. Rev. Drug Discov. 1 (2002) 587–598.
3] K. Vermeulen, D.R. Van Bockstaele, Z.N. Berneman, Cell Prolif. 36
(
2003) 131–149.
4] V.A. Smits, R.H. Medema, Biochim. Biophys. Acta 1519 (2001)
–12.
[5] M. Knockaert, P. Greengard, L. Meijer, Trends Pharmacol. Sci. 23
2002) 417–425.
[
1
5
.2.3. RNA isolation and RT-PCR
Total cellular RNA was isolated from cultured cells using
(
[
[
6] Y. Dia, S. Grant, Curr. Opin. Pharmacol. 3 (2003) 362–370.
7] P.M. Fischer, J. Endicott, L. Meijer, Prog. Cell Cycle Res. 5 (2003)
the ExtractAll (Eurobio, France) according the manufactur-
er’s instructions. The primer sequences for b-actin (an invari-
ant housekeeping gene control), p21, p53 and cdk1 were as
follows: b-actin (5′-ggacgacatggagaaaatctgg-3′, 5′-tggatagca-
acgtacatggctg-3′), p21 (5′-aggaggcgccatgtcagaac-3′, 5′-agg-
actgcaggcttcctgtg-3′), p53 (5′-attctgggacagccaagtct-3′, 5′-
ggagtcttccagtgtgatga-3′), cdk1 (5′-ggagttgtgtataagggtag-3′,
2
35–248.
[8] A. Huwe, R. Mazitschek, A. Giannis, Angew. Chem. Int. Ed. Engl. 42
(2003) 2122–2138.
[
9] D.W. Zaharevitz, R. Gussio, M. Leost, A.M. Senderowicz,
T. Lahusen, C. Kunick, et al., Cancer Res. 59 (1999) 2566–2569.
10] R. Gussio, D.W. Zaharevitz, C.F. McGrath, N. Pattabiraman,
G.E. Kellogg, C. Schultz, et al., Anticancer Drug Des. 15 (2000)
53–66.
[
5
′-gagtgttacctcatgtg-3′). Total RNA (5 µg) was transcribed
using MMLV-reverse transcriptase according to the manufac-
turer’s instructions and PCR was performed at an annealing
temperature of 60 °C and 32 cycles.
[11] T. Lahusen, A. De Siervi, C. Kunick, A.M. Senderowicz, Mol. Car-
cinog. 36 (2003) 183–194.
[
12] M. Knockaert, K. Wieking, S. Schmitt, M. Leost, K.M. Grant,
J.C. Mottram, et al., J. Biol. Chem. 277 (2002) 25493–25501.
13] C. Kunick, K. Lauenroth, K. Wieking, X. Xie, C. Schultz, R. Gussio,
et al., J. Med. Chem. 47 (2004) 22–36.
[
5
.2.4. Detection of apoptosis by Annexin-V and TUNEL
staining
After exposure of the cells to the compounds, Annexin-V
[14] R.D. Whelan, C.J. Waring, C.R. Wolf, J.D. Hayes, L.K. Hosking,
B.T. Hill, Int. J. Cancer 52 (1992) 241–246.
[
[
15] E. Migianu, G. Kirsch, Synthesis (Mass.) (2002) 1096–1100.
16] C. Kunick, K. Lauenroth, M. Leost, L. Meijer, T. Lemcke, Bioorg.
Med. Chem. Lett. 14 (2004) 413–416.
FITC staining was performed as instructed by the manufac-
turer (BD Pharmingen, USA). Briefly, the MCF7 cells were
trypsinized, harvested by centrifugation together with sus-
pension cells and resuspended in 100 µl of binding buffer.
[
17] A. Negoescu, P. Lorimier, F. Labat-Moleur, C. Drouet, C. Robert,
C. Guillermet, et al., J. Histochem. Cytochem. 44 (1996) 959–968.