Inhibition of Human Topoisomerase II by N,N,N-Trimethylethanammonium Iodide Alkylcarbazole Derivatives

Article abstract of DOI:10.1002/cmdc.201800546

Chemotherapy is used for the treatment of all stages of breast cancer, including the metastatic stage of the disease. Treatment regimens are generally tailored for each patient′s particular situation. However, chemotherapeutic agents are the leading cause of serious drug-related adverse effects; moreover, drug resistance often occurs. In this study, we designed and synthesized a new series of N-alkylcarbazoles derived from ellipticine, an alkaloid with a carbazole skeleton initially used in the treatment of metastatic breast cancer and later dismissed because of poor aqueous solubility and severe side effects. After evaluating the binding modes of our class of newly synthesized compounds with human topoisomerase II (hTopo II), we performed hTopo II decatenation assays, identifying compound 4 f (2-(4-((3-chloro-9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) as a good inhibitor. Moreover, 4 f and 4 g (2-(4-((3-chloro-9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N,N-trimethylethanammonium iodide) showed a good anti-proliferative activity toward breast cancer cells, causing apoptosis by activation of the caspase pathway. Interestingly, the activity of these two compounds on triple-negative MDA-MB-231 cells, which tend to be highly metastatic and aggressive, is strictly connected to the observed inhibition of hTopo II.

Full text of DOI:10.1002/cmdc.201800546

Accepted Article
Title: Inhibition of human Topoisomerase II by new N,N,N-
trimethylethanammonium iodide alkylcarbazole derivatives
Authors: Carmela Saturnino, Anna Caruso, domenico iacopetta,
Camillo Rosano, Jessica Ceramella, Noemi Muià, Annaluisa
Mariconda, Maria Grazia Bonomo, Marco Ponassi, Giuseppe
Rosace, Maria Stefania Sinicropi, and Pasquale Longo
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Inhibition of human Topoisomerase II by new N,N,N-
trimethylethanammonium iodide alkylcarbazole derivatives
Carmela Saturnino†[a], Anna Caruso†[b], Domenico Iacopetta* , Camillo Rosano* , Jessica Ceramella ,
[b]
[c]
[b]
[
b]
[d]
[a]
[c]
[d]
Noemi Muià , Annaluisa Mariconda , Maria Grazia Bonomo , Marco Ponassi , Giuseppe Rosace ,
Maria Stefania Sinicropi‡[b], Pasquale Longo‡[e]
†
[
a]
Prof. C. Saturnino , Dr. M.G. Bonomo
Department of Science, University of Basilicata, Viale dell'Ateneo Lucano 10, Potenza 85100, Italy
†
‡
[
b]
Dr. A. Caruso , Dr. D. Iacopetta, Dr. J. Ceramella, Dr. N. Muià, Prof. M.S. Sinicropi
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Via Pietro Bucci 87036, Arcavacata di Rende (Italy)
E-mail: domenico.iacopetta@unical.it
[c]
Dr. C. Rosano, Dr. M. Ponassi
Biopolymers and Proteomics IRCCS Ospedale Policlinico San Martino-IST, Genova, Italy
E-mail: camillo.rosano@gmail.com
[
[
d]
e]
Dr. G.Rosace
University of Bergamo, Department of Engineering and Applied Sciences, Viale Marconi 5, 24044 Dalmine (Bg) Italy
Dr. A. Mariconda, Prof. P. Longo
‡
Department of Biology and Chemistry, University of Salerno, Via Giovanni Paolo II, 132, Fisciano 84084, Italy
[
[
†]
‡]
Considered co-first authors
Considered co-senior authors.
Abstract: Chemotherapy is used for treatment of all stages of breast
cancer, including the metastatic disease and tends to be tailored for
each person situation. However, chemotherapeutic agents are the
leading cause of serious drug-related adverse effects and, often,
resistance occurs. In this paper, we designed and synthesized a new
series of N-alkylcarbazole derived by Ellipticine, an alcaloid with
carbazole structure initially used in the treatment of metastatic breast
cancer and then dismissed because of poor solubility in water and
dramatic side effects. After the evaluation of the binding modes of our
class of newly synthesized compounds with human Topoisomerase II
compounds represent a remarkable type of anticancer drug
candidates.
Carbazoles are an important class of indole-containing
[
5]
heterocycles with antitumor activities but, as well, antibacterial
,
anti-inflammatory, psychotropic, and anti-histamine properties [6].
Some derivatives showed interesting pharmaceutical properties
interfering, as well, with DNA-dependent enzymes, as
topoisomerases I/II and telomerase, or with other targets such as
cyclin-dependent kinases and estrogen receptors and, for these
reasons, they occupied an important place as antitumoral tools in
(
hTopo II) we performed hTopo II decatenation assay, obtaining that
preclinical and clinical trials [7-13]
.
compound 4f is a good inhibitor. Moreover, 4f and 4g showed a good
anti-proliferative activity on breast cancer cells, causing apoptosis by
activation of the caspases pathway. Interesting is the activity of these
two compounds on triple negative MDA-MB-231 cells, which tend to
be highly metastatic and aggressive, strictly connected with the
observed inhibition of hTopo II.
Of particular interest the interaction with DNA or its metabolizing
enzymes that induce the programmed cell death, namely
apoptosis [
14-18]
.
In particular, several analogues of carbazole derivatives
structurally correlated to the Ellipticine, alcaloid from Ochrosia
elliptica labil [ introduced in the treatment of metastatic breast
19]
cancer [
6, 20-23]
and then dismissed because of poor solubility in
water and dramatic side effects [24-25], were synthesized [26-30].
Trimethoxyphenylurea 1,4-dimethylcarbazoles
[31],
N-
Introduction
thioalkylcarbazoles , 1,4 dimethy-N-alkylcarbazoles [32], N-(1,4-
[15]
dimethyl-9H-carbazol-3-yl)-N’-alkylguanidines [ , benzofuro [2, 3
33]
In the last few decades cancer,
a multifactorial disease
f]quinazolin-1 (2H)-ones [34] and hydrazine-carbazole derivatives
-
[20]
characterized by uncontrolled growth and invasion of abnormal
cells, has emerged as one of the most alarming disease in the
world. Particularly, breast cancer has become the second leading
life-threatening disease for women accounting for high mortality
rates after lung cancer [ . It is a kind of malignant tumor that
affects, with an overwhelming incidence, young women with a
high mortality rate due, in several cases, to the development of
resistance against chemotherapeutics [2]. In fact, breast cancer in
younger women is often characterized by aggressive tumor
subtypes that are less likely to be amenable to treatment at the
time of diagnosis and have poorer survival outcomes than older
women [3-4]. Therefore, the poor response to chemotherapy and
the adverse effects of treatments currently used drives the search
towards the development of new chemopreventive agents able to
eradicate the cancer and with reduced undesirable side effects.
Among candidates for treating breast cancer, heterocyclic
were also prepared and some of them showed an interesting
anti-proliferative activity against breast cancer cells without
affecting non-tumoral cell lines viability.
Several studies have reported that carbazole derivatives, as
Ellipticine and Celiptium (Figure 1), are inhibitors of human
1]
Topoisomerases I and II [35-37]
.
1
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Figure 2. General structure of N-alkylcarbazole derivatives and their quaternary
ammonium iodide salts synthetized.
A
Solubility of synthesized compounds was improved through the
formation of salts, as in the case of 9-hydroxyellipticine
(
Celiptium) which possesses a higher DNA affinity than Ellipticine
and lacks of toxicity at therapeutic doses [49-51].
B
Some of these compounds have showed a noticeable cytotoxic
activity against the breast tumoral cells, due to the inhibition of
human Topoisomerase II and the activation of the apoptotic cell
death. Overall, these compounds may be considered new and
important tools for the exploration of the signaling pathways
involved in breast cancer growth and progression.
Results and Discussion
Docking studies
Figure 1. Structures of Ellipticine (A) and Celiptium (B).
hTopo I and II are essential enzymes, which play a crucial role
during DNA transcription and replication and are over-expressed
in tumor cells representing thus a valid target for the development
of new molecules for the treatment of a wide spectrum of tumors
First, in order to evaluate the binding modes and affinities
between our class of newly synthesized compounds and hTopo
II-Alpha, we performed molecular docking simulations using as
ligands all the moieties synthesized. Their binding modes are
reported in Figure 3. Using a “blind-docking approach” (no “a priori”
information about the binding site was provided to the system) we
discovered that all the compounds bind the ATPase domain, in
proximity to the ATP binding site. Particularly, compounds bind to
the same site of hTopo IIa positioning their carbazole moieties
within a mainly hydrophobic pocket composed by residues Ile 125,
Val 132, Leu 140, Ile 141, Thr 147, and forming a π-stacking with
the aromatic ring of Tyr 151. Aminoacids involved in hydrogen
bonds are Ser 149, Asp 152, Asp 154 and Glu 155. The
compounds are posed in an almost identical orientation, with the
exception of compound 4g, which carbazole group is displaced of
about 7Å towards the protein domain core occupying, therefore,
the ATP catalytic site and interacting with residues Asn 91, Ala 92,
Asp 94, Asn 95, Ile 141, Phe 142, Ser 148, Ala 167, Thr215 and
Ile 217. The binding modes of all the other compounds tested in
silico, however, do not seem to occupy directly the ATP binding
cleft; the inhibitory activity of these molecules may be due to
preventing the formation of the hTopo IIa homodimer by binding
to an area that is usually occupied by the N-terminal helices of the
ATPase domain (Figure 3, panels I and J) in the functional
complex. Through our “in silico” screening, we identified
compounds 3c, 4c, 3d, 4d, 3f, 4f, 3g and 4g as good candidates
to be tested in vitro, because they present good binding energies
and a favorable clusterization of the simulation results. The lack
of a significant in vitro inhibitory activity of 3c, 4c, 3d and 4d,
however, may be due to the absence of the chlorine moiety, which
is probably useful in stabilizing the interaction between the
compounds and the protein. Interestingly, the carbazole moiety of
[
38-39]
.
In particular, the principal mechanism of Ellipticine’s
cytotoxicity is related to the inhibition of topoisomerase II,
[
40]
however it may bind to proteins
, induce free radicals
generation [41], inhibit cytochrome P4501A1 [42], uncouple
oxidative phosphorylation , activate the transcription function of
mutant p53 [44] and induce endoplasmic reticulum stress [45]
[
43]
.
Recently some N-substituted carbazole derivatives, correlated to
Ellipticine, have been prepared and investigated for their cytotoxic
activity against different tumoral cell lines, revealing a remarkable
induction of cell cycle arrest and apoptosis [46-48]
In this study, a new serie of N-alkylcarbazole derivatives and their
.
quaternary ammonium iodide salts were designed, synthesized
(
Figure 2) and studied for their ability to inhibit human
topoisomerase II by in silico and in vitro studies. Moreover, their
antiproliferative activity was evaluated on two breast cancer cells
lines (MCF-7 and MDA-MB-231).
R
R
N
N
(
CH2)n
(
CH2)n
N
N
N
N
3
f and 4f are positioned within the hydrophobic cleft described
above, in different orientations being the two rings rotated of about
80 degrees. In this way, the chlorine atom in 4f points towards
H3C
CH3
N
N
H3C
1
CH3
I
H3C
the ATP binding cleft in hydrophobic contact with protein residues
Ile125, His130, Val137, Leu140 and Ile141 and maintaining the
polar bonds with Ser 149, Asp 152 and Glu 155. In the case of 3f,
the chlorine moiety is pointing outside the protein, towards the
R= H; n= 3,4,5,6,7
R= Cl; n= 5,6
2
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A
B
C
D
E
F
G
3
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H
I
J
K
L
M
Figure 3. Panel A) The three dimensional structure of the dimeric human Topoisomerase IIa is reported. Subunit A, drawn in green, adopt a different C-terminal
conformation according to the presence (or absence) of the ligand in its binding site. When in the dimeric conformation, the C-terminal alpha-helix (light blue) is
positioned inside a cleft of the b subunit, close to the ATP binding site, interacting with different residues, thus conferring stability to the dimeric structure. When the
ligand is bound, the C-term helix is pointing out (green helix) and the formation of the oligomer is prevented. Panel B) represents a model of the human
Topoisomerase IIa, bound to a DNA fragment. On the left of the figure, the ATP binding domain. Panel C) different molecules of this series in their binding mode as
forecasted by docking simulations. The crystallographic position of an ADP molecule is reported as reference. Panel D-K binding modes of the N-alkylcarbazole
derivatives as determined by docking simulations. Panel D) molecule 3c, Panel E) 4c, Panel F) 3d, Panel G) 4d, Panel H) 3f, Panel I) 4f, Panel J) 3g, Panel K) 4g,
panel L) 3g, Panel M) 4g (with the carbazole occupying the Adenine binding site).
4
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solvent, where the carbazole is in stacking with Tyr 151 and forms Biology
hydrophobic contacts with Val132, Leu140 and Thr 147.
Compound 4f, therefore, is supposed to have a better inhibitory
activity than 3f on hTopo IIa in vitro due to its minor exposition to
the solvent molecules.
Antitumor properties of N-alkylcarbazole derivatives
Once established, by in silico studies, that 4f is the best hTopo II
inhibitor out of our N-alkylcarbazole derivatives series (and with
the knowledge that they could act not only as hTopo II inhibitors
but, as well, they could interfere with other cellular metabolic
pathways [6]), we screened the entire series to determine their
antitumor properties in two models of breast cancer (namely the
human ERα positive MCF-7 and the triple negative MDA-MB-231
cells). As evidenced in table 1, reporting the IC50 values calculated
as described in the experimental section, six compounds (i.e. 3a,
4a, 3b, 4b, 3e and 4e) did not elicit inhibitory effects, whereas the
four compounds 3c, 4c, 3d and 4d displayed a very poor
antitumor activity on both the two breast cancer cell lines. A
noticeable improvement has been recorded using compounds 3f,
Chemistry
The N-alkylcarbazole derivatives (3a-g and 4a-g) were achieved
from the carbazole (1a) for 3a-e and 4a-e and from the 3-chloro-
9
H-carbazole (1b), commercially available, for 3f-g and 4f-g, in
two steps of reaction for 3a-g and in three steps for 4a-g as shown
in Scheme 1.
4
f, 3g and 4g. Particularly, compounds 4f and 4g exhibited the
best antitumor properties, and between the two models of breast
cancer we used in our experiments, the MDA-MB-231 cells
resulted more sensitive to the treatment. We suppose that this
peculiarity may be correlated with the higher duplication rate of
MDA-MB-231 (a cell line that is aggressive and metastatic) with
respect to MCF-7 cells. For this reason MDA-MB-231 cells need
a faster DNA synthesis to proceed through mitosis, thus they must
possess a very effective duplication machinery, including high
levels of topoisomerases. Therefore, it is reasonable that, being
the compound 4f the best hTopo II inhibitor out of the series, it
displays the antitumor activity mostly against MDA-MB-231 cells.
Finally, it should be noted that the active compounds do not
hamper the viability of the normal breast cells (MCF-10A),
whereas Ellipticine, used as reference molecule, exerted dramatic
effects.
Table 1. IC50 values of carbazole derivatives (3a-g, 4a-g) and Ellipticine,
expressed in micromolar (μM).
MCF-7
MDA-MB-231
MCF-10A°
Ellipticine
1.35 ± 0.30
1.75 ± 0.15
1.50 ± 0.15
3
4
3
4
3
a
a
b
b
c
>500
>500
>500
>500
>500
>500
>500
>500
> 500
> 500
> 500
> 500
>500
>500
> 500
> 500
>500
> 500
>500
>500
Scheme 1. Synthetic route for the preparation of 3a-g and 4a-g compounds.
The first step was the N-alkylation of the carbazole 1a or 1b with
>500
>500
357.0± 1.20
314.2± 0.90
381.0± 1.12
388.8± 1.27
>500
296.5± 1.10
270.3± 1.25
257.2± 1.35
221.9± 0.95
>500
4c
1,3-dibromopropane
or
1,4-dibromobutane
or
1,5-
3d
4d
3e
dibromopentane or 1,6-dibromohexane or 1,7-dibromoheptane to
obtain the intermediates 2a-g respectively. These N-alkylated
derivatives, in the second step were reacted to 1-[2-
(dimethylamino) ethyl]piperazine to give the (9H-carbazol-9-
4e
>500
>500
yl)piperazin-1-yl)-N,N-dimethylethanamine derivatives (3a-g).
Lastly, the compounds 3a-g, in the third step were reacted with
excess methyl iodide in acetone, the quaternary ammonium
iodide derivatives 4a-g were formed. All synthesized compounds
were characterized by mass spectrometry, NMR analysis and the
elemental analysis carried out on them, are in agreement with the
proposed formulations.
3
f
f
35.9± 0.55
10.2± 0.45
77.4± 0.35
20.4± 0.22
12.4± 0.75
4.4± 0.20
17.2± 0.25
5.8± 0.28
4
3g
4g
Topoisomerase II inhibition assay and cells death by
apoptosis induced by 4f.
5
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Figure 4. Human topoisomerase II decatenation assay. hTopo II has been
incubated in absence (control) or presence of the compounds to test at 10µM
for 1 h at 37 °C. The image is representative of three separated experiments.
Panels A, B and C: D: decatenated DNA marker, K: Kinetoplast DNA; C: Control
(DMSO); 3a-g and 4a-g assayed compounds, E, Ellipticine at 50µM.
Topoisomerase II (TopoII) is a nuclear enzyme that catalyzes
DNA topological reactions via
a DNA breakage/reunion
mechanism and it essential to resolve topological problems that
occur during DNA transcription, replication and chromosome
segregation[52]. For these reasons, TopoII has become one of the
most promising target in the anticancer drugs discovery field.
Several studies demonstrated that carbazole derivatives show a
It is well know that the biological functions of human
topoisomerases are fundamental for insuring genomic integrity,
thus a blockade of their activity results in DNA damage and,
consequently, in cell death. This strategy has held the interest of
researchers in cancer chemotherapy because many clinically
active drugs that generate enzyme-mediated DNA damage as, for
instance, topoisomerases targeting agents, were demonstrated to
be effective, minimizing the risk of secondary malignancy and with
low toxicity on the normal cells [ . Moreover, DNA damage is one
good inhibitor TopoII activity[
17, 53-54]
. Given that the in silico studies
results suggested interesting interactions with the hTopoII, we
performed, in parallel, a direct enzymatic assay on all the
synthetized series in order to verify the former hypotheses and
establish whether our compounds are human topoisomerase II
inhibitors or not. In particular, at a dose of 10µM, as indicated in
figure 4 (panels A and B) the ten compounds 3a, 4a, 3b, 4b, 3c,
57]
4
c, 3d, 4d, 3e and 4e were unable to block hTopo II enzymatic
activity given that the enzyme binds its substrate (kDNA) and
releases intact monomeric rings visible at the bottom of gel as two
bands, representing the nicked open circular minicircles and fully
closed circular rings (decatenation products). This happens,
clearly, in the control experiment where the vehicle (DMSO) was
added instead of the testing compounds (fig. 4, lane C) and hTopo
II retains is full activity. Conversely, compound 3g (10µM, fig.4,
panel B) was able to partially inhibits hTopo II activity; indeed, two
faint DNA bands are visible at the bottom of the gel but one band
appears at the top of agarose gel, indicating that the enzyme is
unable to fully cut the kDNA, which big size prevents the migration
on agarose gel. hTopo II inhibition increases in the case of
compounds 4g and 3f, (10µM, fig.4, panel B), which differs from
compound 3g for the presence of the quaternary ammonium
iodide and the length of the alkyl chain (connecting carbazole and
piperazine moieties) respectively. Finally, a total blockade of
hTopo II activity was achieved exposing the enzyme to compound
the stimuli that induce cell death by apoptosis, including nutrient
depletion, heat shock and spindle disruption [
11, 58]
, and is strictly
related to the activation of one of the apoptosis pathways, i.e. the
extrinsic and the intrinsic pathways [
.
59]
Thus, we first performed a TUNEL assay with the aim to
demonstrate that the observed hTopo II inhibition exerted by 4f
could induce MDA-MB-231 cells death by apoptosis. As
evidenced in figure 5, the exposition of cancer cells for 24 h to a
dose of 10 µM of 4f is able to induce genomic DNA fragmentation,
indicated with a green fluorescence (fig. 5, panel B, 4f) that
perfectly superposes with the blue fluorescence (fig. 5, panel C,
4f) indicating cell nuclei. A control experiment, performed with the
vehicle alone (DMSO) in the same conditions, did not produce the
green fluorescence (panel B, CTRL), confirming that no DNA
damage has occurred.
A
B
C
4f (10µM, fig. 4, panel B), which is the quaternary ammonium
iodide derivative of compound 3f; its inhibitory activity is
evidenced by the disappearance of the bottom DNA bands and
the presence of a clear kDNA band at the top of gel. We used, as
control molecule, Ellipticine, which inhibitory activity on hTopoII is
CTRL
[
15, 31, 34, 55-56]
well established
and, as expected, it is able to
completely block hTopoII activity (panel C, lane E) but at a
concentration five-fold higher with respect to the most active
compound, 4f. Overall, the obtained results well correlate with
docking studies previously discussed, and indicate compound 4f
as the best candidate for poisoning hTopo II.
4
f
Figure 5. TUNEL assay. Apoptotic effect of 4f used at 10 μM or vehicle (CTRL)
on MDA-MB-231 cells determined after 24 hours of treatment. Panels A: DAPI
(
excitation/emission wavelength 350 nm/460nm), used to locate the nuclei of
the cells; Panels B: CFTM488 A (excitation/emission wavelength 490 nm/515nm),
the green fluorescence indicates nuclei of cells undergoing apoptosis; Panels
C: Shows the overlay channel.
Successively, we investigated which caspases pathways was
involved using a cell-based luminescent assay. Particularly, we
treated MDA-MB-231 cells for 24 hours with 4f (or vehicle) at a
concentration of 10 µM and then we evaluated the variation of
caspases levels. Specifically, we measured caspase 8 and 9,
which are involved respectively in the extrinsic and intrinsic
pathway, and 3 and 7 (effectors or executioner caspases) that
share the upstream activators and show some overlapping
substrate specificity. Effectively, we noticed an increase of
caspases 9 and 3/7 activity under 4f treatment (fig. 6) with respect
6
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to the control reaction, where cancer cells were added by the
vehicle only (DMSO).
and affinity maps calculated. The search was carried out with
Lamarckian Genetic Algorithm: a population of 156 individuals with a
mutation rate of 0.02 were evolved for 100 generations. Evaluation of the
results was performed by listing the different ligand poses accordingly to
the predicted binding energy. A cluster analysis based on root mean
squares deviation values from the starting geometry was performed. The
lowest energetic conformation of the most populated cluster was consider
as the best candidate. When clusters are almost equipopulated and their
energy distribution is spread, their corresponding molecules were
considered as bad ligands [60-62].
a
Chemistry
All reagents and solvents were purchased from Sigma Aldrich s.r.l. (Milan,
Italy). The reactions were monitored by thin-layer chromatography (TLC),
using silica gel aluminum plates Whatman K6F and Allumina (Merck), with
fluorescence indicators and opportune solvents. A >95% purity could be
inferred from the 1H-NMR spectra. Melting points were taken on a
Gallenkamp melting point apparatus and were uncorrected. The organic
extracts were dried over sodium sulphate dry (Merck). The elemental
analyses for C, H, N, were performed according to standard
microanalytical procedures. Molecular weights were determined by a
JEOL JMS GCMate spectrometer at ionising potential of 70 eV (EI) or by
ESI mass spectrometry. ESI-MS analysis in positive and negative ion
mode, were made using a Finnigan LCQ ion trap instrument, manufactured
by Thermo Finnigan (San Jose, CA, USA), equipped with the Excalibur
software for processing the data acquired. The sample was dissolved in a
mixture of acetonitrile and methanol (50/50) and injected directly into the
electrospray source, using a syringe pump, which maintains constant flow
Figure 6. Caspases activity assay
at the concentration of 10 μM for 24h caused the activation of caspases 3/7 and
(% over the CTRL, vehicle-treated cells). Columns mean, bars SD, *p<0.001.
. The treatment of MDA-MB-231 cells with 4f
9
Conclusions
A
new series of N,N,N-trimethylethanammonium iodide
at 5 ml/min. The temperature of the capillary was set at 220 °C.
1
H NMR
and 13C NMR spectra were recorded at 298 K on a Bruker Avance 300
alkylcarbazole derivatives has been designed and synthetized
with the aim to test its hypothetical inhibitory effect on the human
Topoisomerase II (hTopo II), first by in silico and then by in vitro
studies. Amongst the whole series, four compound exhibited a
noticeable inhibitory activity, being compound 4f the most potent,
even more that the reference molecule, Ellipticine. Moreover, the
entire series has been screened against two models of breast
cancer, finding out that the two compounds 3f and 3g possess a
modest anticancer activity, whereas we noticed an improvement
using 4g and, mostly 4f. These effects are more evident against
MDA-MB-231 cells, which are more aggressive and metastatic
and, they do not affect the proliferation of normal breast cells
3
MHz spectrometer, using CDCl and referred to internal tetramethylsilane
[63-64].
Synthesis of N-alkylcarbazoles (2a-g)
TBEA (benzyltriethylamonium bromide) (2.73 mmol), was dissolved in an
aqueous solution of NaOH 50% p/p (15 ml) and toluene (20 ml) and stirred
for 15 min. After was added a solution of a suitable carbazole (1a) or 3-
chloro-9H-carbazole (1b) (5.45 mmol) in toluene (20 ml). To this mixture
was added 1,3-dibromopropane or 1,4-dibromobutane or 1,5-
dibromopentane or 1,6-dibromohexane or 1,7-dibromoheptane (16.3
mmol) and was stirred at reflux for 48 h. After the reaction, the solvent was
evaporated, poured into water and extracted with chloroform (2 x 50 ml).
(MCF-10A). These observations strengthens our data regarding
4
The organic layer was dried over anhydrous MgSO , filtered and the
solvent removed by evaporation. The resulting residue was purified on a
silica gel column chromatography in petroleum ether/ethyl acetate (1/9) to
the inhibition of hTopo II, which blockade diminishes the
uncontrolled duplication of these cells.
give as white/yellow solid (67% yield) (2a-g)[47, 51, 65]
General method for the synthesis of compounds 3a-g
2 3
-[2-(Dimethylamino)ethyl]piperazine (l.35 mmol) and Na CO (2.7 mmol)
were dissolved in DMF (10 ml). A solution of N-bromoalkyl-substituted
carbazole (2a-g) (1.35 mmol) in DMF (10 ml) was added at room
temperature. The mixture was stirred for 20 h. After the reaction, the
solvent was evaporated, poured into water and extracted with chloroform
.
Experimental Section
Docking simulations
1
To preliminary screen the potential activity of these compounds against
hTopo IIa “in silico”, we performed docking simulations using the program
Autodock (Goodsell et al., 1996) and its graphical interface ADT (Sanner
et al, 1999). A single monomer of the dimeric crystallographic structure
determined by X-ray crystallography of hTopo IIa in complex with AMP-
PNP (Schmidt at al, 2012; PDB code 4GFH) was used as a target for the
docking simulations. All the molecular structures of the ligands screened
were built using the program Marvin (ChemAxon, www.chemaxon.com).
The protein and the ligands were prepared using ADT. Polar hydrogens
were added to the protein Kollman charged assigned and solvatation
parameters added. The protein was considered as a rigid object while the
ligands as fully flexible. A searching grid was extended all over the protein
(
2 x 50 ml). The organic layer was dried over anhydrous MgSO
and the solvent removed by evaporation. Final product (3a-g) was a solid
Scheme 1).
4
, filtered
(
2-(4-((9H-carbazol-9-yl)propyl)piperazin-1-yl)-N,N-dimethylethanamine
1
(3a): White powder (68 % yield). Mp = 108 °C. H NMR (300 MHz, CDCl
3
):
δ 8.34 (d, 1H, Ar); 8.00 (d, 1H, Ar); 7,70 (d, 1H, Ar); 7.60 (d, 1H, Ar); 7.46-
7.27 (m, 4H, Ar); 2.55-2.50 (m, 4H, 2 CH ); 2.49-2.45 (m, 8H, CH
Pyperazine); 2.35 (t, 2H, CH N); 2.34-2.29 (m, 4H, 2 CH ); 2.26 (s, 6H, 2
CH ): 139.48, 125.65, 123.32, 121.35, 119.32,
). 13C NMR (75 MHz, CDCl
2
2
-
2
2
3
3
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800546
FULL PAPER
+
1
3
08.52, 53.54, 52.07, 51.66, 51.57, 50.04, 46.45, 43.34, 29.89. MS (ESI ):
Excess methyl iodide (5 mL) was added to a mixture of 3 (1 mmol) in
acetone (10 mL), the reaction mixture was heated at 30 °C for 1 h. After
evaporation of all the solvent, the solid residue was recrystallized from
methanol to give 4a-g.
+
32 4
65 (M +1). Anal. Calcd for C23H N : C, 75.78; H, 8.85; N, 15.37. Found:
C, 75.76; H, 8.88; N, 15.40.
2
-(4-((9H-carbazol-9-yl)butyl)piperazin-1-yl)-N,N-dimethylethanamine
1
(
3b): White powder (20 % yield). Mp = 103 °C. H NMR (300 MHz, CDCl
3
):
2-(4-((9H-carbazol-9-yl)propyl)piperazin-1-yl)-N,N,N-
δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar); 7,70 (d, 1H, Ar); 7.65 (d, 1H, Ar); 7.45-
.24 (m, 4H, Ar); 2.56-2.50 (m, 4H, 2 CH ); 2.49-2.46 (m, 8H, CH
Pyperazine); 2.39 (t, 2H, CH N); 2.38-2.30 (m, 6H, 3 CH ); 2.27 (s, 6H, 2
CH ): 132.45, 123.68, 121.43, 119.08, 118.98,
). 13C NMR (75 MHz, CDCl
10.92, 61.50, 56.65, 53.90, 53.64, 52.09, 50.85, 45.34, 28.32, 27.10. MS
trimethylethanammonium iodide (4a): White solid (70% yield). Mp = 112 °C.
7
2
2
-
1H NMR (300 MHz, CDCl
3
): δ 8.34 (d, 1H, Ar); 8.00 (d, 1H, Ar); 7.70 (d,
1H, Ar); 7,60 (d, 1H, Ar); 7.46-7.27 (m, 4H, Ar); 2.55-2.50 (m, 4H, 2 CH );
2.49-2.45 (m, 8H, CH -Pyperazine); 2.35 (t, 2H, CH N); 2.34-2.29 (m, 4H,
2 CH ); 2.28 (s, 9H, 3 CH ): δ 132.98, 121.98,
). 13C NMR (75 MHz, CDCl
2
2
2
3
3
2
2
1
2
3
3
+
+
(
ESI ): 379 (M +1). Anal. Calcd for C24
H
34
N
4
: C, 76.15; H, 9.05; N, 14.80.
120.17, 119.76, 119.43, 111.21, 65.00, 60.32, 54.45, 53.89, 53.74, 52.12,
46.36, 28.10, 26.32. MS (EI) m/z (%): 379 (M+ - I ). Anal. Calcd for
-
Found: C, 76.18; H, 9.08; N, 14.76.
24 4
C H35IN : C, 56.92; H, 6.97; N, 11.06. Found: C, 56.94; H, 6.99; N, 11.03.
2
-(4-((9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N-dimethylethanamine
1
(
3c): White powder (33 % yield). Mp = 109 °C. H NMR (300 MHz, CDCl
3
):
2-(4-((9H-carbazol-9-yl)butyl)piperazin-1-yl)-N,N,N-
δ 8.36 (d, 1H, Ar); 7.70 (d, 1H, Ar); 7,45 (d, 1H, Ar); 7.39 (d, 1H, Ar); 7.38-
.28 (m, 4H, Ar); 3.85-3.75 (m, 8H, 4 CH ); 2.53-2.49 (m, 8H, CH
Pyperazine); 2.48-2.46 (m, 4H, 2 CH ); 2.35 (t, 2H, CH N); 2.27 (s, 6H, 2
CH ): 134.65, 123.78, 121.54, 120.18, 119.10,
). 13C NMR (75 MHz, CDCl
11.93, 61.76, 56.69, 54.60, 53.80, 52.34, 50.70, 45.66, 30.45, 28.34,
trimethylethanammonium iodide (4b): White solid (75 % yield). Mp =
7
2
2
-
131 °C. 1H NMR (300 MHz, CDCl
3
): δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar);
2
2
7,70 (d, 1H, Ar); 7.65 (d, 1H, Ar); 7.45-7.24 (m, 4H, Ar); 3.34 (t, 2H, CH N);
2
3
3
3.30 (s, 9H, 3 CH
Pyperazine); 2.36-1.78 (m, 6H, 3 CH
3
); 2.56-2.50 (m, 4H, 2 CH
2
); 2.49-2.46 (m, 8H, CH
2
-
1
2
2
). 13C NMR (75 MHz, CDCl
3
): δ
+
+
5.09. MS (ESI ): 393 (M +1). Anal. Calcd for C25
36
H N
4
: C, 76.49; H, 9.24;
133.76, 122.28, 120.12, 119.32, 119.08, 111.12, 64.50, 60.65, 54.40,
53.74, 53.54, 52.75, 46.34, 28.12, 26.10. MS (EI) m/z (%): 393 (M+ - I-).
N, 14.27. Found: C, 76.51; H, 9.26; N, 14.24.
4
Anal. Calcd for C25H37IN : C, 57.69; H, 7.17; N, 10.76. Found: C, 57.71; H,
7
.15; N, 10.74.
2
-(4-((9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N-dimethylethanamine
1
(
3d): White powder (60 % yield). Mp = 121 °C. H NMR (300 MHz, CDCl
3
):
δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar); 7,70 (d, 1H, Ar); 7.45-7.28 (m, 4H, Ar);
2-(4-((9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N,N-
7
2
1
1
3
.27 (d, 1H, Ar); 3.85 (t, 2H, CH
.39-2.36 (m, 4H, 2 CH ); 2.35 (t, 2H, CH
.29 (m, 8H, 4 CH
). 13C NMR (75 MHz, CDCl
21.00, 119.19, 112.45, 61.89, 56.72, 54.65, 53.98, 52.36, 50.89, 45.98,
0.32, 28.45, 27.87, 25.65. MS (ESI ): 407 (M +1). Anal. Calcd for
: C, 76.80; H, 9.42; N, 13.78. Found: C, 76.82; H, 9.44; N, 13.75.
2
N); 2.46-2.40 (m, 8H, CH
N); 2.27 (s, 6H, 2 CH
): 133.67, 122.98, 121.79,
2
-Pyperazine);
trimethylethanammonium iodide (4c): Yellow solid (55% yield). Mp =
145 °C. 1H NMR (300 MHz, CDCl
): δ 8.36 (d, 1H, Ar); 7.70 (d, 1H, Ar);
7,45 (d, 1H, Ar); 7.39 (d, 1H, Ar); 7.38-7.28 (m, 4H, Ar); 3.85- 3.75 (m, 8H,
4 CH ); 2.50-2.46 (m, 4H, 2 CH ); 2.45-2.35 (m, 8H, CH -Pyperazine); 2.34
(t, 2H, CH N); 2.29 (s, 9H, 3 CH ): δ 133.86,
). 13C NMR (75 MHz, CDCl
122.33, 120.14, 119.43, 119.21, 112.12, 64.76, 61.67, 54.50, 53.87, 53.23,
2
2
3
); 1.77-
3
2
3
2
2
2
+
+
2
3
3
26 38 4
C H N
+
-
5
2.89, 46.31, 28.12, 26.15. MS (EI) m/z (%): 407 (M - I ). Anal. Calcd for
: C, 58.42; H, 7.35; N, 10.48. Found: C, 58.43; H, 7.33; N, 10.51.
26 4
C H39IN
2
-(4-((9H-carbazol-9-yl)heptyl)piperazin-1-yl)-N,N-dimethylethanamine
1
(
3e): White powder (24 % yield). Mp = 118 °C. H NMR (300 MHz, CDCl
3
):
δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar); 7,70 (d, 1H, Ar); 7.65 (d, 1H, Ar); 7.39-
2-(4-((9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N,N-
7
2
.24 (m, 4H, Ar); 3.85 (t, 2H, CH
.39-2.36 (m, 4H, 2 CH ); 2.30 (s, 6H, 2 CH
C NMR (75 MHz, CDCl ): 139.53, 125.00, 122.83, 120.54, 118.19,
08.49, 59.89, 54.86, 52.65, 51.68, 50.65, 50.00, 46.87, 30.98, 27.99,
2
N); 2.46-2.42 (m, 8H, CH
2
-Pyperazine);
trimethylethanammonium iodide (4d): White solid (78 % yield). Mp =
120 °C. 1H NMR (300 MHz, CDCl
): δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar);
7,70 (d, 1H, Ar); 7.39 (d, 1H, Ar); 7.45-7.24 (m, 4H, Ar); 3.85 (t, 2H, CH N);
2.46-2.40 (m, 8H, CH -Pyperazine); 2.39-2.36 (m, 4H, 2 CH ); 2.35 (t, 2H,
CH N); 2.30 (s, 9H, 3 CH ); 1.77-1.29 (m, 8H, 4 CH
). 13C NMR (75 MHz,
CDCl ): δ 140.09, 126.76, 123.79, 120.97, 119.00, 109.67, 65.76, 54.65,
3.65, 51.78, 50.36, 50.21, 49.96, 30.87, 28.87, 28.09, 27.65, 27.00. MS
2
3
); 1.77-1.38 (m, 12H, 6 CH
2
).
3
13
3
2
1
2
C
2
2
+
+
7.87, 27.65, 26.15, 25.95. MS (ESI ): 421 (M +1). Anal. Calcd for
: C, 77.10; H, 9.58; N, 13.32. Found: C, 77.13; H, 9.55; N, 13.29.
2
3
2
27
H
40
N
4
3
5
+
-
(
EI) m/z (%): 421 (M - I ). Anal. Calcd for C27
4
H41IN : C, 59.12; H, 7.53; N,
2
-(4-((3-chloro-9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N-
dimethylethanamine (3f): White powder (66 % yield). Mp = 112 °C. 1H
NMR (300 MHz, CDCl ): δ 8.17-8.10 (m, 4H, Ar); 7.46 (d, 1H, Ar); 7,44 (d,
H, Ar); 7.33 (d, 1H, Ar); 4.20 (t, 2H, CH N) 2.48-2.30 (m, 8H, CH
Pyperazine); 2.28-2.25 (m, 4H, 2 CH ); 2.24 (s, 6H, 2 CH ); 1.94-1.92 (m,
). 13C NMR (75 MHz, CDCl
): 140.12, 128.38, 126.98, 125.18,
10.21. Found: C, 59.15; H, 7.55; N, 10.23.
3
1
2
2
-
2-(4-((9H-carbazol-9-yl)heptyl)piperazin-1-yl)-N,N,N-
trimethylethanammonium iodide (4e): Yellow solid (68 % yield). Mp =
151 °C. 1H NMR (300 MHz, CDCl
): δ 8.36 (d, 1H, Ar); 8.00 (d, 1H, Ar);
7,70 (d, 1H, Ar); 7.65 (d, 1H, Ar); 7.39-7.24 (m, 4H, Ar); 3.85 (t, 2H, CH N);
2
3
8
1
5
+
7
H, 4 CH
2
3
3
22.54, 121.93, 121.54, 120.12, 113.21, 111.10, 104.21, 61.32, 58.69,
8.20, 57.76, 56.34, 53.30, 47.10, 30.21, 28.21, 25.02. MS (ESI ): 427 (M+
2
+
3.29 (s, 9H, 3 CH
3
); 2.46-2.42 (m, 8H, CH
2
-Pyperazine); 2.40-2.36 (m, 4H,
1). Anal. Calcd for C25H35ClN
4
: C, 70.32; H, 8.26; N, 13.12. Found: C,
2 CH ); 1.77-1.38 (m, 12H, 6 CH
2
2
). 13C NMR (75 MHz, CDCl
3
): δ 139.87,
0.30; H, 8.24; N, 13.14.
127.13, 124.00, 121.65, 120.12, 110.68, 66.46, 55.69, 54.25, 52.43, 50.56,
5
-
0.28, 50.12, 31.43, 29.86, 28.34, 27.87, 26.98. MS (EI) m/z (%): 435 (M+
I-). Anal. Calcd for C28
: C, 59.78; H, 7.70; N, 9.96. Found: C, 59.80;
H43IN
4
2
-(4-((3-chloro-9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N-
dimethylethanamine (3g): White powder (33 % yield). Mp = 110 °C. 1H
H, 7.68; N, 9.98.
NMR (300 MHz, CDCl
3
): δ 8.34 (d, 1H, Ar); 7.70-7.68 (m, 2H, Ar); 7,46 (d,
H, Ar); 7.41 (d, 1H, Ar); 7.18-7.14 (m, 2H, Ar); 4.873 (t, 2H, CH N); 2.70-
.66 (m, 8H, CH -Pyperazine); 2.38-2.35 (m, 4H, 2 CH ); 2.21 (s, 6H, 2
); 1.98 (t, 2H, CH N); 1.28-1.12 (m, 8H, 4 CH
). 13C NMR (75 MHz,
CDCl ): 140.12, 129.32, 125.79, 125.18, 122.59, 121.15, 121.09, 119.54,
12.32, 110.21, 103.54, 61.37, 58.65, 57.98, 57.21, 53.10, 46.76, 29.54,
1
2
CH
2
2-(4-((3-Chloro-9H-carbazol-9-yl)pentyl)piperazin-1-yl)-N,N,N-
trimethylethanammonium iodide (4f): White solid (56% yield). Mp = 115 °C.
2
2
1H NMR (300 MHz, CDCl
): δ 8.13-8.10 (m, 4H, Ar); 7.46 (d, 1H, Ar); 7,44
(d, 1H, Ar); 7.33 (d, 1H, Ar); 4.20 (t, 2H, CH N); 2.48-2.30 (m, 8H, CH
Pyperazine); 2.29 (s, 9H, 3 CH ); 2.28-2.25 (m, 4H, 2 CH ); 1.94-1.92 (m,
8H, 4 CH ): δ 139.67, 138.01, 135.65, 135.2,
). 13C NMR (75 MHz, CDCl
131.23, 128.34, 125.89, 123.11, 121.87, 117.21, 112.67, 112.45, 64.32,
0.65, 57.32, 56.87, 55.87, 54.60, 52.76, 46.87, 29.65, 27.87, 25.11. MS
3
2
2
3
3
2
2
-
1
2
7
3
2
8.65, 27.96. MS (ESI ): 441 (M+ +1). Anal. Calcd for C26
+
4
H37ClN : C,
2
3
0.80; H, 8.46; N, 12.70. Found: C, 70.82; H, 8.49; N, 12.68.
6
+
-
(
EI) m/z (%): 441 (M - I ). Anal. Calcd for C26
4
H38ClIN : C, 54.89; H, 6.73;
General method for the synthesis of compounds 4a-g
N, 9.85. Found: C, 54.91; H, 6.70; N, 9.87.
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201800546
FULL PAPER
2
-(4-((3-Chloro-9H-carbazol-9-yl)hexyl)piperazin-1-yl)-N,N,N-
additional washes with cold PBS, one drop of mounting solution was added,
then they were observed and imaged under a fluorescence microscope
(Leica DM 6000) (20X magnification) with excitation/emission wavelength
maxima of 490 nm/515 nm (CFTM488A) or 350 nm/460 nm (DAPI).
Representative fields were shown. The experiments were repeated three
times.
trimethylethanammonium iodide (4g): White solid (78 % yield). Mp =
1
7
1
CH
CH
13 °C. 1H NMR (300 MHz, CDCl
,69 (d, 1H, Ar); 7.46 (d, 1H, Ar); 7.41 (d, 1H, Ar); 7.18 (d, 1H, Ar); 7.14 (d,
H, Ar); 4.73 (t, 2H, CH N); 2.71 (s, 8H, CH -Pyperazine); 2.37 (s, 4H, 2
); 1.60-1.57 (m, 2H, CH ); 1.28-1.25 (m, 6H, 3
); 1.18-1.08 (m, 2H, CH ): δ 140.01, 128.71,
). 13C NMR (75 MHz, CDCl
3
): δ 8.34 (d, 1H, Ar); 7.70 (d, 1H, Ar);
2
2
2
); 2.27 (s, 9H, 3 CH
3
2
2
2
3
1
5
4
25.34, 125.12, 121.23, 121.11, 119.87, 112.65, 109.65, 103.38, 63.76,
Caspase Assay
8.21, 57.91, 56.87, 54.37, 48.09, 29.43, 28.30, 27.32. MS (EI) m/z (%):
-
55 (M+ - I ). Anal. Calcd for C27
4
H40ClIN : C, 55.63; H, 6.92; N, 9.61.
Caspases -3/7, -8 and -9 activities were measured with the Caspase-Glo
Assay, according to the guidelines of manufacturer (Caspase-GloR 3/7, 8
and 9 Assay Systems, Promega Corporation, Madison, WI, USA). Cells
were grown in white-walled 96-well plates and, after treatment, 100μL of
different caspases reagents were added to each well, as previously
described in Iacopetta et al [67]. Results were plotted as percentage (%)
over the vehicle-treated cells (CTRL), after background readings
subtraction.
Found: C, 55.65; H, 6.89; N, 9.63.
Biology
Cell culture and cell proliferation assay
Human estrogen receptor (ER)-positive MCF-7 and triple negative MDA-
MB-231 breast cancer cells were cultured in DMEM-F12 medium
containing 2 mmol/L L-glutamine, 1mg/ml penicillin-streptomycin and 5%
Newborn Calf Serum (NCS) or 5% Fetal Bovine Serum (FBS), respectively.
MCF-10A human mammary epithelial cells, were cultured in DMEM/F12
medium, supplemented with 5% horse serum (HS, Thermo Fisher
Scientific, Milan, Italy), 100 U mL-1 penicillin/streptomycin, 0.5 mg mL-1
hydrocortisone, 20 ng mL-1 human epidermal growth factor (hEGF), 10
mg mL-1 insulin, and 0.1 mg mL-1 cholera enterotoxin (Sigma–Aldrich,
Milan, Italy). The cell lines used in these experiments were obtained from
American Type Culture Collection (ATCC, Manassas, VA), were
Acknowledgements
This work was supported by MIUR (to M.S.S.)
Keywords: topoisomerase II; caspases; N-alkylcarbazoles;
apoptosis; docking simulation.
References:
maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO
2
and periodically screened for contamination. Cells were seeded in 48-well
and grown in complete medium, then starved in serum free medium for
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[
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hTopo II decatenation assays were performed in a final volume of 20 μL
as reported by Iacopetta et al, using 0.3 μg of kinetoplast DNA (kDNA)
M. S. Shaikh, R. Karpoormath, N. Thapliyal, R. A. Rane, M. B. Palkar, A.
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EB (0.5 μg/mL) has been used to stain agarose gel and after wash with
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0
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ChemMedChem
10.1002/cmdc.201800546
FULL PAPER
C. Saturnino, A. Caruso, D. Iacopetta*,
C. Rosano*, J. Ceramella, N. Muià, A.
Mariconda, M. G. Bonomo, M. Ponassi,
G. Rosace, M. S. Sinicropi, P. Longo.
New N,N,N-trimethylethanammonium
iodide alkylcarbazole derivatives. The
synthesized compounds have been
tested as hTopo II inhibitors by in silico
and in vitro studies. Compound 4f is the
most potent inhibitor and is able to
trigger the intrinsic apoptotic pathway in
MDA-MB-231 breast cancer cells
4
f
Inhibition of human Topoisomerase II by
new N,N,N-trimethylethanammonium iodide
alkylcarbazole derivatives
1
1
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