N-thioalkylcarbazoles derivatives as new anti-proliferative agents: synthesis, characterisation and molecular mechanism evaluation

Article abstract of DOI:10.1080/14756366.2017.1419216

Synthetic or natural carbazole derivatives constitute an interesting class of heterocycles, which showed several pharmaceutical properties and occupied a promising place as antitumour tools in preclinical studies. They target several cellular key-points, e.g. DNA and Topoisomerases I and II. The most studied representative, i.e. Ellipticine, was introduced in the treatment of metastatic breast cancer. However, because of the onset of dramatic side effects, its use was almost dismissed. Many efforts were made in order to design and synthesise new carbazole derivatives with good activity and reduced side effects. The major goal of the present study was to synthesise a series of new N-thioalkylcarbazole derivatives with anti-proliferative effects. Two compounds, 5a and 5c, possess an interesting anti-proliferative activity against breast and uterine cancer cell lines without affecting non-tumoural cell lines viability. The most active compound (5c) induces cancer cells death triggering the intrinsic apoptotic pathway by inhibition of Topoisomerase II.

Full text of DOI:10.1080/14756366.2017.1419216

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
N-thioalkylcarbazoles derivatives as new anti-
proliferative agents: synthesis, characterisation
and molecular mechanism evaluation
Maria Stefania Sinicropi, Domenico Iacopetta, Camillo Rosano, Rosario
Randino, Anna Caruso, Carmela Saturnino, Noemi Muià, Jessica Ceramella,
Francesco Puoci, Manuela Rodriquez, Pasquale Longo & Maria Rosaria
Plutino
To cite this article: Maria Stefania Sinicropi, Domenico Iacopetta, Camillo Rosano, Rosario
Randino, Anna Caruso, Carmela Saturnino, Noemi Muià, Jessica Ceramella, Francesco Puoci,
Manuela Rodriquez, Pasquale Longo & Maria Rosaria Plutino (2018) N-thioalkylcarbazoles
derivatives as new anti-proliferative agents: synthesis, characterisation and molecular mechanism
evaluation, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 434-444, DOI:
10.1080/14756366.2017.1419216
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 434–444
https://doi.org/10.1080/14756366.2017.1419216
RESEARCH PAPER
N-thioalkylcarbazoles derivatives as new anti-proliferative agents: synthesis,
characterisation and molecular mechanism evaluation
a
a
b
c
a
ꢀ
ꢀ
Maria Stefania Sinicropi , Domenico Iacopetta , Camillo Rosano , Rosario Randino , Anna Caruso ,
d
a
a
c
ꢀa
Carmela Saturnino , Noemi Muia , Jessica Ceramella , Francesco Puoci , Manuela Rodriquez ,
Pasquale Longoe† and Maria Rosaria Plutinof†
b
aDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, Italy; Biopolymers and Proteomics
c
d
IRCCS Policlinico San Martino-IST, Genova, Italy; Department of Pharmacy, University of Salerno, Fisciano, Italy; Department of Science,
e
f
University of Basilicata, Potenza, Italy; Department of Chemistry and Biology, University of Salerno, Fisciano, Italy; Institute for the Study of
Nanostructured Materials, ISMN-CNR, Palermo, c/o Department of ChiBioFarAm, University of Messina, Messina, Italy
ABSTRACT
ARTICLE HISTORY
Received 16 August 2017
Revised 12 December 2017
Accepted 13 December 2017
Synthetic or natural carbazole derivatives constitute an interesting class of heterocycles, which showed sev-
eral pharmaceutical properties and occupied a promising place as antitumour tools in preclinical studies.
They target several cellular key-points, e.g. DNA and Topoisomerases I and II. The most studied representa-
tive, i.e. Ellipticine, was introduced in the treatment of metastatic breast cancer. However, because of the
onset of dramatic side effects, its use was almost dismissed. Many efforts were made in order to design
and synthesise new carbazole derivatives with good activity and reduced side effects. The major goal of
the present study was to synthesise a series of new N-thioalkylcarbazole derivatives with anti-proliferative
effects. Two compounds, 5a and 5c, possess an interesting anti-proliferative activity against breast and
uterine cancer cell lines without affecting non-tumoural cell lines viability. The most active compound (5c)
induces cancer cells death triggering the intrinsic apoptotic pathway by inhibition of Topoisomerase II.
KEYWORDS
Carbazole derivatives;
apoptosis; topoisomerase II;
immunofluorescence;
caspases
Introduction
some of them were also used as antifungal, photoconductive,
antioxidant, antibacterial, anti-malaria, anti-tuberculosis, anti-HIV
agents and in the treatment of obesity5–7. Regarding their antitu-
mour activity, several key-points, namely telomerases, topoisom-
erase I and II, tubulin were identified8–11. The most studied
representative of this class of compounds, i.e. Ellipticine
(Figure 1) was introduced in the treatment of metastatic breast
cancer1,12–15, then dismissed because of poor solubility in water
Recently, one of the principal targets of the pharmaceutical
research is represented by the design of new antitumoural drugs
with higher selectivity on neoplastic cells, few side effects and
able to overcome resistance onset1. In this scenario, carbazoles
were widely studied since they possess not only antitumour
activity but also antimicrobic, antiepileptic, antihistaminic, antioxi-
dant, anti-inflammatory, analgesic and neuroprotective proper-
ties2–4. In recent studies, N-substituted carbazoles were reported
and dramatic side effects16,17
. An improved solubility was
as neuroprotective agents with strong anti-oxidant activity and achieved through the formation of salts as, for instance, in the
CONTACT Maria Stefania Sinicropi
s.sinicropi@unical.it
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende,
Department of Science, University of Basilicata, Potenza, Italy
Italy; Carmela Saturnino
carmela.saturnino@unibas.it
ꢀ
These authors contributed equally to this work.
†These authors contributed equally to this work.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
435
Figure 1. Structures of Ellipticine (A) and Celiptium (B).
case of 9-hydroxyellipticine (Celiptium, Figure 1), which was nanoAquity, Waters, Milford, MA) in positive mode, and either pro-
tonated molecular ions [M þ H]þ were used for empirical formula
widely used, alone or in combination with other chemotherapeu-
confirmation, unless otherwise stated. The 9-(bromoalkyl)-9H-car-
tics, possesses a higher DNA affinity than Ellipticine and lacks of
toxicity at therapeutic doses18–20
.
bazoles (2, 3, 4 a–c) intermediates were prepared as described in
the literature28 by the corresponding 1a–c carbazoles31–34
.
In other previous studies, the carbazole scaffold was variously
functionalised obtaining 6-aryl, 3-acyl (or 3-aroyl) and 9-alkyl-1,4-
9-(7-Bromoheptyl)-9H-carbazole (2a). Colourless oil (145 mg,
1
dimethyl-9H-carbazoles21–26
,
with improved pharmacokinetic
71%). H NMR (400 MHz, CDCl3) d 8.17 (d, J ¼ 7.7 Hz, 2H), 7.54–7.51
(m, 2H), 7.45 (d, J ¼ 8.1 Hz, 2H), 7.31–7.28 (m, 2H), 4.35–4.33
(m, 2H), 3.42–3.40 (m, 2H), 1.95–1.87 (m, 2H), 1.85–1.82 (m, 2H),
1.47–1.36 (m, 6H) ppm. HRMS (ESI-Q-TOF) m/z: Calcd. for
C19H22BrN 343.0936, found 344.0939 [M þ Hþ]þ.
properties. Furthermore, benzofuroquinazolinones derivatives were
synthesised as carbazole bioisosters27 and they showed interesting
antitumour properties at sub-micromolar concentrations28. In this
paper, we report the synthesis of a new series of N-thioalkylcarba-
zoles differently substituted at the 1, 4, 6 and 9 positions of the
carbazole core. In these molecules, the carbazole nitrogen bears
alkyl chains with seven, eight and nine methylene groups, linked
to a thiol terminal group.
9-(8-Bromooctyl)-9H-carbazole (3a). Colourless oil (129 mg,
1
61%). H NMR (400 MHz, CDCl3) d: 7.51–7.37 (m, 2H), 7.36–7.22 (m,
2H), 7.17–7.12 (m, 2H), 7.11–7.06 (m, 2H), 4.10–4.06 (m, 4H),
3.37–3.33 (m, 2H), 1.86–1.82 (m, 2H), 1.68–1.64 (m, 2H), 1.40–1.29
(m, 6H) ppm. HRMS (ESI-Q-TOF) m/z: Calcd. for C20H24BrN
357.1092, found 359.1095 [M þ 2]þ.
Two of these molecules have demonstrated an interesting anti-
proliferative activity against two breast cancer cell lines, MCF-7
and MDA-MB-231, and, to a greater extent, against cervical HeLa
and endometrial ISHIKAWA cells. Moreover, the assayed com-
pounds did not show effects on the proliferation of non-tumoural
MCF-10A and 3T3-L1 cells. The antitumour activity resides in the
ability to trigger the apoptotic intrinsic pathway, also known as
mitochondrial pathway, and to inhibit the human Topoisomerase
II (hTopo II) activity, as shown by direct enzyme assays and dock-
ing simulations. hTopoII is an essential enzymes playing a crucial
role during transcription and replication and is over-expressed in
tumour cells, thus it is a valid target for the development of new
9-(9-Bromononyl)-9H-carbazole (4a). Colourless oil (144 mg,
1
65%). H NMR (400 MHz, CDCl3) d 8.16 (d, J ¼ 7.7 Hz, 2H), 7.54–7.50
(m, 2H), 7.45 (d, J ¼ 8.1 Hz, 2H), 7.30–7.27 (m, 2H), 4.35–4.32 (m,
2H), 3.44–3.42 (m, 2H), 1.94–1.87 (m, 2H), 1.85–1.83 (m, 2H),
1.44–1.30 (m, 10H) ppm. HRMS (ESI-Q-TOF) m/z: Calcd. for
C21H26BrN 371.1249, found 373.1245 [M þ 2]þ.
9-(7-Bromoheptyl)-1,4,6-trimethyl-9H-carbazole
(2b).
1
Colourless oil (75 mg, 81%). H NMR 400 MHz, CDCl3) d 8.00 (s, 1H),
7.35–7.28 (m, 2H), 7.09 (d, J ¼ 7.3 Hz, 1H), 6.90 (d, J ¼ 7.3 Hz, 1H),
4.53–4.50 (m, 2H), 3.45–3.40 (m, 2H), 2.88 (s, 3H), 2.81 (s, 3H), 2.59
(s, 3H), 1.92–1.81 (m, 4H), 1.51–1.35 (m, 6H) ppm. HRMS (ESI-Q-
TOF) m/z: Calcd. for C22H28BrN 385.1405, found 387.1402 [M þ 2]þ.
molecules for the treatment of a wide spectrum of tumours29,30
.
Our outcomes set the compound 5c as a valid lead compound to
be useful for cancer treatment.
9-(8-Bromooctyl)-1,4,6-trimethyl-9H-carbazole
(3b).
Colourless oil (70 mg, 73%). 1H NMR (400 MHz, CDCl3) d 7.96 (s,
1H), 7.28–7.24 (m, 2H), 7.03 (d, J ¼ 7.3 Hz, 1H), 6.85 (d, J ¼ 7.3 Hz,
1H), 4.44–4.41 (m, 2H), 3.36–3.34 (m, 2H), 2.82 (s, 3H), 2.74 (s, 3H),
2.53 (s, 3H), 1.81–1.72 (m, 4H), 1.40–1.25 (m, 8H) ppm. HRMS (ESI-
Q-TOF) m/z: Calcd. for C23H30BrN 399.1562, found 401.1565
[M þ 2]þ.
Materials and methods
Synthesis
Commercial reagents were purchased from Sigma-Aldrich (St.
Louis, MO) and used without additional purification. Silica gel 60
(300 ꢁ 400 mesh, Merck, Kenilworth, NJ) was used for flash chro-
matography. Preparative TLC was performed on 20 ꢂ 20 cm glass
plates coated with a 2 mm layer of silica gel PF254 Merk. 1HNMR
and 13CNMR spectra were recorded on a Bruker Avance 400 spec-
trometer (Bruker Corporation, Billerica, MA). Chemical shifts are
expressed in parts per million downfield from tetramethylsilane as
an internal standard. Multiplicities are represented by s (singlet), d
(doublet), t (triplet), q (quartet) and m (multiplet). Coupling con-
stants (J) are reported in Hertz (Hz). Mass spectra were obtained
using an ESI mass spectrometer: LC-ADVANTAGE MAX (ESI). High-
resolution mass spectra (HRMS) were recorded on a high-reso-
lution mass spectrometer equipped by electrospray (ESI) and
nanospray sources, and a quadrupole-time of flight hybrid ana-
9-(9-Bromononyl)-1,4,6-trimethyl-9H-carbazole
(4b).
1
Colourless oil (64 mg, 64%). H NMR (400 MHz, CDCl3) d 8.01 (s, 1H),
7.34–7.28 (m, 2H), 7.09 (d, J ¼ 7.3 Hz, 1H), 6.90 (d, J ¼ 7.2 Hz, 1H),
4.52–4.49 (m, 2H), 3.44–3.41 (m, 2H), 2.87 (s, 3H), 2.80 (s, 3H), 2.58
(s, 3H), 1.89–1.78 (m, 4H), 1.58–1.28 (m, 10H) ppm. HRMS (ESI-Q-
TOF) m/z: Calcd. for C24H32BrN 413.1718; found 415.1721 [M þ 2]þ.
6-Bromo-9-(7-bromoheptyl)-1,4-dimethyl-9H-carbazole (2c).
Colourless oil (102 mg, 41%). 1H NMR (400 MHz, CDCl3) d 8.13 (s,
1H), 7.40–7.37 (m, 1H), 7.13 (d, J ¼ 8.9 Hz, 1H), 6.96 (d, J ¼ 7.3 Hz,
1H), 6.77 (d, J ¼ 7.3 Hz, 1H), 4.35–4.31 (m, 2H), 3.25–3.22 (m, 2H),
2.67 (s, 3H), 2.63 (s, 3H), 1.70–1.61 (m, 4H), 1.30–1.18 (m, 6H) ppm.
HRMS (ESI-Q-TOF) m/z: Calcd. for C21H25Br2N 449.0354; found
451.0355 [M þ 2]þ.
6-Bromo-9-(8-bromooctyl)-1,4-dimethyl-9H-carbazole
(3c).
lyser, coupled with capillary UPLC system (Q-TOF Premier/ Colourless oil (95 mg, 37%). 1H NMR (400 MHz, CDCl3) d 8.13

436
M. S. SINICROPI ET AL.
(s, 1H), 7.39–7.36 (m, 1H), 7.13–7.11 (m, 1H), 6.96 (d, J ¼ 7.3 Hz, 1H),
8-(1,4,6-Trimethyl-9H-carbazol-9-yl)-octane-1-thiol
(6b).
6.76 (d, J ¼ 7.3 Hz, 1H), 4.32–4.28 (m, 2H), 3.29–3.23 (m, 4H), 2.66 Yellow oil (14 mg, 31%). 1H NMR (400 MHz, CDCl3) d: 8.07 (s, 1H,
(s, 3H), 2.62 (s, 3H), 1.75–1.63 (m, 4H), 1.29–1.17 (m, 6H). HRMS
Ar), 7.36–7.34 (m, 2H, Ar), 7.14 (d, J ¼ 7.2 Hz, 1H, Ar), 6.95 (d,
(ESI-Q-TOF) m/z: Calcd. for C22H27Br2N 463.0510; found 465.0513
J ¼ 7.2 Hz, 1H, Ar), 4.54–4.50 (m, 2H, NCH2), 2.93 (s, 3H, CH3), 2.84
[M þ 2]þ.
(s, 3H, CH3), 2.75–2.72 (m, 2H CH2S), 2.64 (s, 3H, CH3), 1.85–1.82
6-Bromo-9-(9-bromononyl)-1,4-dimethyl-9H-carbazole (4c).
Colourless oil (105 mg, 61%). 1H NMR (400 MHz, CDCl3) d 8.13 (s,
1H), 7.38 (d, J ¼ 8.6 Hz, 1H), 7.13 (d, J ¼ 8.8 Hz, 1H), 6.96 (d,
J ¼ 7.3 Hz, 1H), 6.77 (d, J ¼ 7.2 Hz, 1H), 4.33–4.30 (m, 2H), 3.25 (t,
J ¼ 6.8 Hz, 2H), 2.67 (s, 3H), 2.63 (s, 3H), 1.73–1.60 (m, 4H),
1.26–1.12 (m, 10H). HRMS (ESI-Q-TOF) m/z: Calcd. for C23H29Br2N
477,0667; found 479.1599 [M þ 2]þ.
(m, 2H, CH2), 1.75–1.71 (m, 2H, CH2), 1.65–1.33 (m, 9H, 4 CH2, SH)
ppm. 13C NMR (100 MHz, CDCl3) d: 139.01, 136.81, 131.45, 130.73,
127.92, 127.11, 125.67, 123.58, 123.13, 122.63, 120.23, 111.57,
48.35, 33.25, 28.96, 28.61, 27.95, 27.55, 25.38, 21.21, 20.58,
18.02 ppm. MS (ESI): C23H31NS (354.56) [M þ Hþ]þ. HRMS (ESI-Q-
TOF) m/z: [M þ H]þ Calcd. for C23H31NS 353.2177, found 354.2178
[M þ Hþ]þ.
General procedure for the synthesis of N-thioalkylcarbazole
derivatives (5, 6, 7a–c). To a mixture of the 9-(bromoalkyl)-9H-
carbazoles 2, 3, 4a–c (0.21 mmol) in i-PrOH (13 ml) thiourea
(0.84 mmol) was added and the solution was refluxed for 12 h
under nitrogen atmosphere35. At the residue obtained, after
removal of the solvent, was added 6 N NaOH (27 ml), the resulting
mixture was refluxed for 5 h, then it was neutralised by dropwise
addition of 3 N HCl. Finally, by extraction with CH2Cl2 (3 ꢂ 100 ml),
we obtained the crude compounds, purified by preparative thin
layer chromatography (PTLC) (n-hexane/ethyl acetate 97:3) to give
pure compounds.
7-(9H-Carbazol-9-yl)heptane-1-thiol (5a). Yellow oil (17 mg,
25%). 1H NMR (400 MHz, CDCl3) d: 8.14 (d, J ¼ 7.6 Hz, 2H, Ar),
7.51–7.48 (m, 2H, Ar), 7.43 (d, J ¼ 8.1 Hz, 2H, Ar), 7.27 (m, 2H, Ar),
4.33–4.31 (m, 2H, NCH2), 2.66–2.63 (m, 2H, CH2S), 1.92–1.86 (m, 2H,
NCH2CH2), 1.66–1.64 (m, 2H, HSCH2CH2), 1.41–1.37 (m, 7H, 3CH2,
SH) ppm. 13C NMR (100 MHz, CDCl3) d: 140.73, 126.45, 124.15,
121.68, 120.48, 109.63, 47.43, 33.25, 28.96, 28.61, 27.95, 27.55,
25.38 ppm. MS(ESI): C19H23NS (298.46) [M þ Hþ]þ. HRMS (ESI-Q-
TOF) m/z: Calcd. for C19H23NS 297.1551, found 298.1549
[M þ Hþ]þ.
9-(1,4,6-Trimethyl-9H-carbazol-9-yl)-nonane-1-thiol (7b).
Yellow oil (18 mg, 51%). 1H NMR (400 MHz, CDCl3) d: 8.03 (s, 1H,
Ar), 7.36–7.32 (m, 2H, Ar), 7.11 (d, J ¼ 7.2 Hz, 1H, Ar), 6.92 (d,
J ¼ 7.2 Hz, 1H, Ar), 4.53–4.49 (m, 2H, NCH2), 2.90 (s, 3H, CH3), 2.82 (s,
3H, CH3), 2.73–2.70 (m, 2H, CH2S), 2.60 (s, 3H, CH3), 1.83–1.77 (m,
2H, CH2), 1.72–1.62 (m, 2H, CH2), 1.43–1.31 (m, 11H, 5CH2, SH) ppm.
13C NMR (100 MHz, CDCl3) d: 139.01, 136.81, 131.45, 130.73, 127.92,
127.11, 125.67, 123.58, 123.13, 122.63, 120.23, 111.57, 48.35, 33.25,
28.96, 28.61, 27.95, 27.55, 25.38, 21.21, 20.58, 18.02 ppm. MS (ESI):
C24H33NS (368.59) [M þ Hþ]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for
C24H33NS 367.2334, found 368.2336 [M þ Hþ]þ.
7-(6-Bromo-1, 4-dimethyl-9H-carbazol-9-yl)-heptane-1-thiol
(5c). Yellow oil (22 mg, 48%). 1H NMR (400 MHz, CDCl3) d 8.28 (s,
1H, Ar), 7.54–7.51 (m, 1H, Ar), 7.29–7.26 (m, 1H, Ar), 7.12–7.10 (m,
1H, Ar), 6.91 (d, J ¼ 7.3 Hz, 1H, Ar), 4.49–4.46 (m, 2H, NCH2), 2.81 (s,
3H, CH3), 2.77 (s, 3H, CH3), 2.53–2.47 (m, 2H, CH2S), 1.81–1.77 (m,
2H, CH2), 1.60–1.55 (m, 2H, CH2), 1.41–1.25 (m, 7H, 3CH2, SH) ppm.
13C NMR (100 MHz, CDCl3) d: 142.37, 139.01, 130.73, 128.14, 127.83,
127.11, 124.71, 123.13, 122.63, 120.23, 118.45, 112.31, 48.35, 33.25,
28.96, 28.61, 27.95, 27.55, 25.38, 20.58, 18.02 ppm. MS (ESI):
C21H26BrNS (405.41) [M þ 2]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for
C21H26BrNS 403.0969, found 405.4066 [M þ 2]þ.
8-(9H-Carbazol-9-yl)octane-1-thiol (6a). Yellow oil (14 mg,
23%).1H NMR (300 MHz, CDCl3) d: 8.10 (d, J ¼ 7.8 Hz, 2H, Ar),
7.49–7.32 (m, 4H, Ar), 7.24–7.19 (m, 2H, Ar), 4.33–4.24 (m, 2H,
NCH2), 2.64–2.59 (m, 2H, CH2S), 1.87–1.82 (m, 2H NCH2CH2),
1.63–1.55 (m, 2H, HSCH2CH2), 1.47–1.25 (m, 9H, 4CH2, SH) ppm.
13C NMR (100 MHz, CDCl3) d: 140.73, 126.45, 124.15, 121.68, 120.48,
109.63, 47.43, 33.25, 28.96, 28.61, 27.95, 27.55, 25.38 ppm. MS (ESI):
C20H25NS (312.48) [M þ Hþ]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for
C20H25NS 311.1708, found 312.4844 [M þ Hþ]þ.
8-(6-Bromo-1, 4-dimethyl-9H-carbazol-9-yl)-octane-1-thiol (6c).
Yellow oil (14 mg, 31%). 1H NMR (400 MHz, CDCl3) d 8.26 (s, 1H,
Ar), 7.53–7.51 (m, 1H, Ar), 7.28–7.26 (m, 1H, Ar), 7.11–7.09 (m, 1H,
Ar), 6.91–6.89 (m, 1H, Ar), 4.48–4.44 (m, 2H, NCH2), 2.81 (s, 3H,
CH3), 2.77 (s, 3H, CH3), 2.68–2.63 (m, 2H, CH2S), 1.80–1.76 (m, 2H,
CH2), 1.66–1.62 (m, 2H, CH2), 1.36–1.30 (m, 9H, 4CH2, SH) ppm. 13C
NMR (100 MHz, CDCl3) d: 142.37, 139.01, 130.73, 128.14, 127.83,
127.11, 124.71, 123.13, 122.63, 120.23, 118.45, 112.31, 48.35, 33.25,
28.96, 28.61, 27.95, 27.55, 25.38, 20.58, 18.02 ppm. MS (ESI):
C22H28BrNS (419.43) [M þ 2]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for
C22H28BrNS 417.1126, found 418.1129 [M þ Hþ]þ.
9-(9H-Carbazol-9-yl)nonane-1-thiol (7a). Yellow oil (13 mg,
40%). 1H NMR (300 MHz, CDCl3) d: 7.95 (d, J ¼ 7.7 Hz, 2H, Ar),
7.33–7.24 (m, 4H, Ar), 7.11–7.06 (m, 2H Ar), 4.16–4.12 (t, 2H, NCH2),
2.51–2.47 (m, 2H, CH2S), 1.73–1.67 (m, 2H, CH2), 1.53–1.44 (m, 2H,
9-(6-Bromo-1,4-dimethyl-9H-carbazol-9-yl)-nonane-1-thiol (7c).
CH2), 1.20–0.98 (m, 11H, 5CH2, SH) ppm. 13C NMR (100 MHz, CDCl3) Yellow oil (12 mg, 29%). 1H NMR (400 MHz, CDCl3) d 8.26 (s, 1H,
d 140.73, 126.45, 124.15, 121.68, 120.48, 109.63, 47.43, 33.25, 28.96, Ar), 7.53–7.51 (m, 1H, Ar), 7.28–7.26 (m, 1H, Ar), 7.11–7.09 (m, 1H,
Ar), 6.91–6.89 (m, 1H, Ar), 4.46–4.44 (m, 2H, NCH2), 2.81 (s, 3H,
CH3), 2.77 (s, 3H, CH3), 2.68–2.64 (m, 2H, CH2S), 1.81–1.74 (m, 2H,
CH2), 1.69–1.65 (m, 2H, CH2), 1.37–1.26 (m, 11H, 5CH2, SH) ppm.
13C NMR (100 MHz, CDCl3) d 142.37, 139.01, 130.73, 128.14, 127.83,
127.11, 124.71, 123.13, 122.63, 120.23, 118.45, 112.31, 48.35, 33.25,
28.96, 28.61, 27.95, 27.55, 25.38, 20.58, 18.02 ppm. MS (ESI):
C23H30BrNS (433.46) [M þ 2]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for
C23H30BrNS 431.1282, found 432.1129 [M þ Hþ]þ.
28.61, 27.95, 27.55, 25.38 ppm. MS (ESI): C21H27NS (326.51)
[M þ Hþ]þ. HRMS (ESI-Q-TOF) m/z: Calcd. for C21H27NS 325.1864,
found 326.1844 [M þ Hþ]þ.
7-(1,4,6-Trimethyl-9H-carbazol-9-yl)-heptane-1-thiol (5b).
Yellow oil (23 mg, 52%). 1H NMR (400 MHz, CDCl3) d: 8.05 (s, 1H,
Ar), 7.36–7.28 (m, 2H, Ar), 7.12 (d, J ¼ 7.2 Hz, 1H, Ar), 6.94 (d,
J ¼ 7.2 Hz, 1H, Ar), 4.57–4.45 (m, 2H, NCH2), 2.83 (s, 3H, CH3), 2.77
(s, 3H, CH3), 2.74–2.71 (m, 2H, CH2S), 2.60 (s, 3H, CH3), 1.83–1.77
(m, 2H, CH2), 1.76–1.74 (m, 2H, CH2), 150–1.27 (m, 7H, 3CH2, SH)
ppm. 13C NMR (100 MHz, CDCl3) d: 139.01, 136.81, 131.45, 130.73,
127.92, 127.11, 125.67, 123.58, 123.13, 122.63, 120.23, 111.57,
48.35, 33.25, 28.96, 28.61, 27.95, 27.55, 25.38, 21.21, 20.58,
18.02 ppm. MS (ESI): C22H29NS (340.54) [M þ Hþ]þ. HRMS (ESI-Q-
TOF) m/z: [M þ H]þ Calcd. for C22H29NS 339.2021, found 340.2017
[M þ Hþ]þ.
Biological assay
Cell cultures
The six cell lines used in this work were purchased from American
Type Culture Collection (ATCC, Manassas, VA). MCF-7 human breast
cancer cells, oestrogen receptor (ER) positives, were maintained in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
437
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham with TBST containing 5% milk for 1 h (blocking solution) and,
(DMEM/F12), supplemented with 5% foetal bovine serum (FBS) and then, incubated overnight at 4 ꢃC with the primary antibody
100 U/ml penicillin/streptomycin, as previously described36. MDA- raised against Parp-1(7150) or GADPH (25778), purchased from
Santa Cruz-Biotechnology Inc. (Santa Cruz, CA) and used at 1:500
and 1:2000 dilutions, respectively. After incubation with the
appropriate secondary antibody (VWR International PBI, MI, Italy)
for 1 h at room temperature, the proteins of interest were
detected on the membranes by using enhanced chemilumines-
cence (Amersham ECL Prime Western Blotting Detection Reagent,
GE Healthcare, Piscataway, NJ).
MB-231, human breast cancer cells, oestrogen receptor (ER) nega-
tive, were maintained in Dulbecco’s Modified Eagle’s Medium/
Nutrient Mixture F-12 Ham (DMEM/F12), supplemented with 5%
Newborn Calf Serum (NCS) and 100 U/ml penicillin/streptomycin.
HeLa human epithelial cervix carcinoma cells, oestrogen receptor
(ER) negative, and ISHIKAWA human endometrial adenocarcinoma
cell, oestrogen receptor positive, were maintained in minimum
essential medium (MEM), supplemented with 10% FBS, 100 U/ml
penicillin/streptomycin and 1% non-essential amino acid. 3T3-L1
cells line of murine fibroblasts of embryonic type, were maintained
in DMEM, supplemented with 10% NCS and 100 U/ml penicillin/
streptomycin. MCF-10A human mammary epithelial cells were cul-
tured in DMEM/F12 medium, supplemented with 5% horse serum
(HS) (Eurobio, Les Ullis, Cedex, France), 100 U/ml penicillin/strepto-
mycin, 0.5 mg/ml hydrocortisone, 20 ng/ml hEGF (human epidermal
growth factor), 10 lg/ml insulin and 0.1 mg/ml cholera enterotoxin
(Sigma-Aldrich, Milano, Italy). Cells were maintained at 37 ꢃC in a
humidified atmosphere of 95% air and 5% CO2 and periodically
TUNEL assay
Apoptosis was detected by the TUNEL assay, according to the
guidelines of the manufacturer (CFTM488A TUNEL Assay Apoptosis
Detection Kit, Biotium, Hayward, CA)8. Briefly, cells were grown on
glass coverslips and, after treatment, they were washed trice with
PBS, then methanol-fixed at ꢁ20 ꢃC for 15 min. Fixed cells were
washed trice with 0.01% (V/V) Triton X-100 in PBS and incubated
with 100 mL of TUNEL equilibration buffer for 5 min. After its
removal, 50 mL of TUNEL reaction mixture containing 1 mL of ter-
minal deoxynucleotidyl transferase (TdT) were added to each sam-
ple and incubated in a dark and humidified chamber for 2 h at
37 ꢃC. Samples were washed trice with ice-cold phosphate-buf-
fered saline (PBS) containing 0.1% Triton X-100 and 5 mg/mL
bovine serum albumin (BSA). 2-(4-Amidinophenyl)-6-indolecarba-
midine dihydrochloride (DAPI) (0.2 mg/mL) counterstain was per-
formed (10 min, 37 ꢃC, dark and humidified conditions). After three
additional washes with cold PBS, one drop of mounting solution
was added, then they were observed and imaged under a fluores-
cence microscope (Leica DM 6000, Leica, Frankfurt am Main,
Germany) (20ꢂ magnification) with excitation/emission wave-
length maxima of 490 nm/515 nm (CFTM488A) or 350 nm/460 nm
(DAPI). Representative fields were shown. The experiments were
repeated three times41.
screened for contamination37
.
Cell viability
MDA-MB-231, MCF-7, HeLa and ISHIKAWA cells were grown in
complete medium and, before being treated, they were serum
deprived for 24 h, to allow cell cycle synchronisation. Then cells
were grown in phenol red-free medium supplemented with 1%
DCC (destran-coated charcoal treated) FBS, then treated with
increasing concentrations (0.1, 1, 10, 20, 40, 100 mM) of each com-
pound for 72 h. Compounds were dissolved in dimethylsulphoxide
(DMSO) (Sigma, St. Louis, MO) and diluted in each cell medium.
Untreated cells, used as a control, were added with DMSO alone
(final concentration 0.1%). Cell viability was assessed using the 3-
[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reagent
(MTT), according to the protocol from manufacturer (Sigma-
Aldrich, Milan, Italy), as previously described37,38. For each sample,
the mean absorbance, measured at 570 nm, was expressed as a
percentage of the control and plotted versus drug concentration
to determine for each cell line the IC50 values (namely, drugs con-
centrations able to reduce cell viability of 50% with respect to the
control), using GraphPad Prism 5 Software (GraphPad Inc., San
Diego, CA)39. Each experiment was carried out in quadruplicate.
Standard deviations (SD) were shown.
Immunofluorescence
Cells were grown on glass coverslips in full media, then serum-
deprived for 24 h and exposed to compound to test, at the indi-
cated time. Then, they were PBS-washed, fixed with cold methanol
(15 min/ꢁ20 ꢃC) and washed three times (10 min/room tempera-
ture) with cold PBS containing 0.01% TritonX-100. After incubation
(30 min/room temperature) with blocking solution (PBS, 2% BSA),
they were incubated with primary antibody diluted in blocking
solution (4 ꢃC/overnight). The mouse anti-cytochrome c (556433)
was purchased from BD Biosciences (Franklin Lakes, NJ) and used
at 1:100 dilution. Coverslips were then washed three times with
PBS, then fixed cells were incubated with the secondary antibody
Cell proteins extraction and immunoblot analysis
Cells were serum deprived for 24 h, treated for 24, 48 and 72 h
with the compound to be tested. At the end, cells were rinsed
twice with ice cold PBS and immediately lysed in lysis buffer
(50 mM Tris-HCl, pH 7, 5–8; 150 mM NaCl, 1% Igepal CA-630;
0.1% SDS; 0.5% sodium deoxycholate) containing 1% sodium
orthovanadate and 1% phenylmethylsulphonyl fluoride (PMSF).
Cell lysates were cleared by centrifugation at 14,000 g for 10 min
at 4 ꢃC. Protein concentrations were determined using the
Bradford protein assay40 (Bio-Rad Laboratories, Milan, Italy)
according to the directions from the manufacturer. Equal
amounts of cell extracts (about 20 mg) were resolved under dena-
turing conditions by electrophoresis in 10% polyacrylamide gels
containing SDS (SDS-PAGE) and transferred to nitrocellulose
membranes by electroblotting (GE Healthcare, Piscataway, NJ).
Membranes were first stained with Ponceau S, washed with 1ꢂ
TBST (Tween-20 0,1%, Tris/HCl 10 mM e NaCl 100 mM), incubated
R
V
Alexa Fluor 568 conjugate goat-anti-mouse (1:500, Thermo Fisher
Scientific, Waltham, MA). Nuclei were stained using DAPI (Sigma-
Aldrich, Milan, Italy) for 10 min at a concentration of 0.2 mg/mL
then washed three times with PBS. Fluorescence was detected
using a fluorescence microscope (Leica DM 6000 Leica, Frankfurt
am Main, Germany). LASX software was used to acquire and pro-
cess all images42.
Caspase assay
Caspases-3/7, -8 and -9 activities were measured with the
Caspase-Glo Assay, according to the guidelines of manufacturer
R
(Caspase-GloV 3/7, 8 and 9 Assay Systems, Promega Corporation,
Madison, WI) as described in Iacopetta et al.43.

438
M. S. SINICROPI ET AL.
Statistical analysis
The obtained residue was purified by silica gel column chromatog-
raphy (Et2O/hexane, 2/3 as eluent) to give the pure compounds.
Data were analysed for statistical significance (p < .001) using one-
way ANOVA followed by Dunnett’s test performed by GraphPad
Prism 5 (GraphPad Software, La Jolla, CA). Standard deviations (SD)
are shown.
Antiproliferative activity
All synthesised carbazole derivatives (5a–c, 6a–c, 7a–c) were
examined to test their antiproliferative activity against two human
breast cancer cell lines, namely the MCF-7 cells expressing the oes-
trogen receptor a (ERa), and the MDA-MB-231 triple negative cells
(ER-, PR- and HER-2/Neu not amplified), and two cell lines of uter-
ine cancer, which are the Hela cells, from uterine cervix epithelium
(ERꢁ), and endometrial ISHIKAWA cells (ERþ). Cells were subjected
to 72 h of continuous exposure to the compounds to test and, at
the end, their viability was measured by MTT assay. Ellipticine,
which represents the main natural molecule containing the carba-
Human topoisomerase I relaxation assay: hTopoI relaxation
assays were performed as reported in Iacopetta et al.8.
Human topoisomerase II decatenation assay: hTopoII decatena-
tion assays were performed as reported in Iacopetta et al.8.
Computer modelling and docking simulations: The crystal struc-
ture of hTopoII in complex with a short DNA fragment and the
ATP-analogue AMPPNP [PDB Code 4GFH] has been used as a tem-
plate to build the complete three-dimensional models of hTopoII,
as previously described8. The atomic coordinates of the three-
dimensional model obtained were then used as a target for all the
following molecular docking simulations. The ligand structures zole moiety, was used as reference molecule for these studies. The
have been built and energy minimised using the program
MarvinSketch [ChemAxon ltd, Budapest, Hungry]. In order to inves-
tigate the possible binding modes of 5c to the protein, calcula-
tions were carried out with “Achilles” Blind Docking Server,
results, summarised in Table 1, clearly indicate that only two com-
pounds, 5a and 5c, possess an antitumour activity. In both breast
cancer cells, a moderate antitumour activity was noticed; indeed
the IC50 values for the compounds 5a and 5c in MCF-7 cells were
found to be, respectively, 50.4 0.96 mM and 54.9 1.00 mM. In
MDA-MB-231 cells, the IC50 values resulted lower than those deter-
mined for the MCF-7 cells and were 39.9 0.52 mM and
27.2 0.75 mM, respectively. However, a higher antitumour activity
was found against HeLa and ISHIKAWA cells, where compound 5a
showed IC50 values of 12.8 0.62 mM and 19.2 1.13 mM, respect-
ively. Compound 5c showed IC50 values of 11.3 0.63 mM (HeLa)
and 15.7 0.95 mM (ISHIKAWA). These data set the compound 5c
the most active and selective for uterine tumour cells. Moreover,
both compounds did not affect the proliferation of MCF-10A cells,
human non-malignant breast epithelial cells, and 3T3-L1 murine
fibroblast-like cells, at least at doses lower than 100 mM. Finally,
Ellipticine demonstrates a higher antitumour activity in all cell
lines used in these assays with respect to compounds 5a and 5c,
but together with dramatic cytotoxic effects on normal cells
(Table 1). Thus, a relevant antitumour activity was obtained for
compound 5c, which hold two methyl substituents at the position
1 and 4, a bromine at position 6 and a 7 terms chain on the car-
bazole ring nitrogen. The replacement of bromine at position 6
with a methyl group (5b)12,22,24,25,28 and the elongation of the
chain produced a loss of the antitumour activity (6c and 7c). With
respect to compound 5c, a lesser antitumour activity was noticed
available
at:
http://bio-hpc.eu/software/blind-docking-server/.
Using a “blind docking” approach, the docking of the small mol-
ecule to the targets is done without a priori knowledge of the
location of the binding site by the system44. Figures were drawn
using the program Chimera45.
Results and discussion
Chemistry
The N-thioalkylcarbazole derivatives (5, 6, 7a-c) were synthesised
by the reaction of appropriate 9-(bromoalkyl)-9H-carbazoles (2, 3,
4a-c) with thiourea in i-PrOH (at reflux for 12 h), under a nitrogen
atmosphere (Scheme 1). The obtained residue, after removal of
the solvent, was first treated with 6 N NaOH (reflux for 5 h), and
then with 3 N HCl (at room temperature, hereafter rt). The
obtained crude was purified by PTLC (n-hexane/ethyl acetate 97:3)
to give the pure compounds. The intermediates 9-(bromoalkyl)-9H-
carbazoles (2, 3, 4a–c) were prepared following general synthetic
methods28. In particular, carbazole derivatives (1a–c) and dry DMF
were stirred at rt until became clear. Then, NaH 60% oil dispersion
and, successively, the appropriate dibromoalkane were added at
0 ꢃC, and the mixture was stirred for 5 h at rt. Finally, water
was added and the resulting mixture was extracted with EtOAc. for compound 5a. The latter does not possess substituents on the
Scheme 1. Synthesis of N-9H-alkyltiocarbazoles 5–7a–c. Reagents and conditions: (i) 1, N-dibromoalkane, NaH 60%, DMF, rt; (ii) Thiourea, i-PrOH, reflux, 12 h; NaOH
6 N, reflux, 5 h; HCl 3 N, rt, 10 min.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
439
Table 1. Antiproliferative activity: IC50 values of carbazole derivatives (5a–c, 6a–c, 7a–c) and Ellipticine, expressed in micromolar (mM).
MCF-7
MDA-MB-231
ISHIKAWA
HeLa
MCF-10a
3T3-L1
Ellipticine
1.25 0.30
50.4 0.96
>100
54.9 1.00
>100
>100
>100
>100
>100
1.85 0.15
39.9 0.52
>100
27.2 0.75
>100
>100
>100
>100
>100
1.70 0.80
19.2 1.13
>100
15.70 0.95
>100
>100
>100
>100
>100
1.05 0.50
12.8 0.62
>100
11.3 0.63
>100
>100
>100
>100
>100
1.20 0.20
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.98 0.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
5a
5b
5c
6a
6b
6c
7a
7b
7c
>100
>100
>100
>100
(Figure 2, lane E) at the concentration of 50 mM to obtain a com-
plete inhibition27.
Docking simulations
The binding site of 5c to hTopoII was identified by our docking
simulation as coincident to the ATP-binding site (Figure 3, panel
A), with the docked moiety almost perfectly superposed to the
one of ANP-PNP (a non-hydrolysable ATP analogue), determined
experimentally through X-ray diffraction (Figure 3, panel B). The
binding site includes different amino acids from both chains of
hTopoII, which contribute to binding stability mostly through polar
and hydrophobic interactions (Figure 3, panel C). The best pose
for molecule in the most populated cluster has a binding energy
of ꢁ7.10 kcal/mol, as calculated by the server “Achilles”. A visual
inspection of the crystallographic structure of hTopoII in complex
with AMPPNP indicates that (1) the adenine moiety is hydrogen
bonded to residues Asn 67, Asn 92 and Thr 187; (2) the ribose
ring is stabilised by hydrogen bonds with Ser 120 and Ser 121; (3)
the phosphates tail interacts with the peptidic nitrogen of Gly 138
and with the side chains of Asn 63 Ser 120, Asn 122, Lys 140, Gln
348 and Lys 350 through hydrogen bonds. Our simulation poses
5c with the carbazole moiety almost superposed to the adenine
of the crystallographic AMPPNP, with the bromine interacting with
Asn 92 and Thr 187 through halogen bonds and the SH group
forming a weak hydrogen bond to Asn 122. Residues Asp 66, Ile
97, Ile 113, Phe 114, and Ala 139 further contribute to stabilise 5c
binding mode to hTopoII by hydrophobic interactions.
Figure 2. hTopoII inhibition assay. kDNA was incubated with human
Topoisomerase II in the absence (lane C, vehicle DMSO) or presence of compound
5c (lane 5c) or Ellipticine (lane E) at 10 and 50 mM, respectively. Lane K, kineto-
plast DNA (kDNA).
carbazolic scaffold and holds a 7 terms chain on the nitrogen at
position 9.
Compound 5c induces parp-1 cleavage
The observed inhibition of hTopoII pushed us to evaluate whether
cells were undergoing cell death by apoptosis. The Topoisomerase
II, indeed, plays an important role in DNA replication, transcription,
recombination, thus its inhibition provokes genotoxic and muta-
genic effects, leading to cell death. In response to DNA damage,
cells may utilise the poly-(ADP) ribose polymerase (Parp-1), a
nuclear enzyme that catalyses the transfer of the ADP-ribose por-
tion of NADþ into a specific subgroup of nuclear substrates49.
Parp-1 is an important regulator of the DNA base excision repair
Human topoisomerases I and II inhibition assays
Several studies reported that carbazole derivatives and Ellipticine
are inhibitors of human Topoisomerases I and II. In particular,
Ellipticine exhibits multi-modal mechanism of action with DNA
intercalation and hTopoII inhibition46–48. For these reasons, the
most active compound 5c, structurally correlated to Ellipticine,
was evaluated for its ability to inhibit DNA metabolising enzymes,
i.e. hTopoI and II direct in vitro assays. The latter evidenced no
inhibition of the hTopoI (data not shown) upon 5c exposure, at (BER) pathway and it is involved in the maintenance of genomic
least under the adopted experimental conditions. On the contrary, integrity and survival following genotoxic insults. Parp-1 is specific-
in hTopo II decatenation assays, the same compound was able to ally proteolysed by executioner caspases (caspases 3 and 7) to an
inhibit hTopoII activity at 10 mM concentration (Figure 2, lane 5c), 89 kDa catalytic fragment during the final steps of apoptosis50,51
.
preventing the hTopoII from cutting the catenated kinetoplast Indeed, Parp-1 cleavage promotes apoptosis by preventing DNA
DNA (KDNA), as instead happens in the experiment control (Figure repair-induced survival and across the block of energy depletion
2, lane C) where the enzyme releases the nicked open circular induced by necrosis52. With this in mind, we treated HeLa cells
minicircles and fully closed circular rings (decatenation products) with compound 5c at a concentration of 10 mM and at three differ-
visible at the bottom of gel as two distinct DNA bands. As well in ent times (24, 48 and 72 h). At the end of the treatment, the cells
these experiments, Ellipticine was used as reference molecule were lysed and the total cellular protein content was extracted

440
M. S. SINICROPI ET AL.
Figure 3. Panel (A) Ribbon representation of a dimeric hTopoII (chains A and B coloured in pink and olive green respectively) in complex with DNA (yellow ribbon and
blue lego). The area within the orange rectangle highlights ATP binding site. Panel (B) A close up of the ATP binding site showing the different binding modes of
AMP-PNP, a non-hydrolisable ATP analogue, (orange) and 5c (black sticks) as suggested by docking simulations. Panel (C) shows the protein residues involved in 5c
binding.
and used for western blot analyses. The obtained results, reported
in Figure 2, showed the presence of the cleaved form of Parp-1
(about 89 KDa) in cells treated with compound 5c, visible at 24 h
and increased at 48 and 72 h, together with a decrease of the
native protein, due to the cleavage. At 72 h, the only appreciable
form is the cleaved one, whereas, on the contrary, in vehicle-
treated cells only the whole form is present and not the cleaved
one. In Figure 4, the shown control is referred only to 72 h, but
same results were obtained at 24 and 48 h (data not shown).
In order to assess whether the observed cytotoxic effects could
depend on the induction of apoptosis under treatment, we
exposed Hela cells to 5c compound, at the concentration equal to
its IC50 value for 24 h. As in Figure 5, a green nuclear fluorescence
(5c, panel B) indicates DNA damage and that cells have under-
Figure 4. Parp-1 cleavage: time course of PARP-1 cleavage in uterine cancer Hela
cells treated with 5c, used at a concentration equal to its IC50 value. The control
gone the apoptotic process due to the exposure to the 5c com-
pound, whereas no fluorescence resulted in the vehicle-treated
shown is referred to 72 h of vehicle exposure, but same results was obtained for
cells used as experiment control (Figure 5, CTRL, panel B). These 24 and 48 h. GAPDH was used as loading normalisation.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
441
Figure 5. TUNEL assay of HeLa cells. Cells were treated for 24 h with compound 5c (at the concentration equal to its IC50) or with vehicle (CTRL). Then they were fixed,
subjected to TdT reaction, washed, dyed with DAPI and imaged under a fluorescence microscope (20ꢂ magnification, excitation/emission wavelength 490 nm/515 nm
for CFTM488A, panels B, and 350nm/460nm for DAPI, panels A). Panels C show the merge.
data confirm that the selected compound induced DNA damage
and led to cell death triggering the apoptotic mechanism.
Compound 5c induces apoptosis by the intrinsic pathway
Even though the mechanisms of apoptosis are complexes and
involve an energy-dependent cascade of molecular events, it is
noteworthy that there are at least two main pathways,53 not
necessarily independent from each other, i.e. the extrinsic or death
receptor pathway and the intrinsic or mitochondrial one54. In
order to establish which pathway was involved, we evaluate the
activity of initiators caspases, namely caspases 8 and 9, that are
involved, respectively, in the extrinsic and intrinsic pathway, and
effectors or executioner caspases 3 and 7, by means of a lumines-
cent assay. Caspases are usually expressed in an inactive form and
after activation they may initiate a proteolytic cascade,55 in which
one caspase may activate other caspases or cleave cell proteins,
included Parp-1, amplifying the apoptotic cell death53. Effectively,
we noticed a little, but significant, rise of caspase-9 activity in
HeLa cells treated with compound 5c for 24 h, whereas no
increase of caspase-8 activity was evidenced (Figure 6). Moreover,
Figure 6. Caspases activity. Activation of caspases 3/7 and 9 following to the
treatment of HeLa cells with the compound 5c at a concentration equal to its
ꢀ
we observed a clear increase of caspases 3 and 7 activity (Figure
6), which are cleaved by the initiator caspase-9 and that, in turn,
may activate other pro-apoptotic proteins and cleave Parp-1, as
we already observed (Figure 4). Summing up, the exposure of
HeLa cells to compound 5c induces the activation of caspase-9
that activates caspases 3/7, responsible of the observed Parp-1
cleavage that provokes the inability of DNA reparation processes
and leads to cell death by apoptosis.
Another important event of the intrinsic apoptotic pathway is
represented by the release of Cytochrome c from mitochondria to
the cytosol56. Thus, we wanted to evaluate the localisation of
Cytochrome c, by means of immunofluorescence assays in HeLa
cells treated with the compound 5c for 24 h at the concentration
equal to its IC50, and fixed (see experimental section for details).
These assays confirmed that compound 5c determines the release
of Cytochrome c from mitochondria to the cytosol (Figure 5).
IC50 value for 24 h. Columns mean, bars SD, p < .001.
delocalised throughout the cell cytoplasm, whereas in vehicle-
treated cells (CTRL), the fluorescence is strictly localised in well-
defined areas of the cell, given that Cytochrome c is normally
localised in the mitochondrial compartment. In Figure 5, it should
be evidenced how it is possible, as already reported57, to identify
the different stages of mitochondria disruption during apoptosis.
In fact, a punctiform fluorescence in the cytosol (white arrow 1)
indicates mitochondria fragmentation and partial release of
Cytochrome c into the cytosol, which occurs during the early
stages of apoptosis. Successively, the release of Cytochrome c is
complete (white arrow 2, Figure 7) and the fluorescence (red) is
greatly increased and spread throughout the cytosol. On the con-
Indeed, the fluorescence (red colour in Figure 5) is diffused and trary, in cells treated with the vehicle only, the fluorescence (red)

442
M. S. SINICROPI ET AL.
Figure 7. Cytochrome c release during apoptosis in Hela cells. In vehicle-treated cells Cytochrome c is localised within the mitochondria (panel B, CTRL). During the
early stages of apoptosis, induced by compound 5c treatment at the concentration equal to its IC50 for 24 h, mitochondria fragmentation occurs (as shown by the red
dots indicated by arrow 1 in panel B, 5c) and Cytochrome c is partially released. In the last apoptotic cell stage, a full release of Cytochrome c is observed (diffused
R
V
red fluorescence, indicated by the white arrow 2). Panel A, DAPI (excitation/emission wavelength 350nm/460nm), panel B, Alexa Fluor 568 (excitation/emission wave-
length 578nm/603nm), panel C, overlay. Images were acquired at 63ꢂ magnification and are representative of three separated experiments.
is defined and organised in an ordered and filamentous way, indi- caspase-9 activation, which targets several proteins, amongst them
cating that mitochondria are intact.
caspases 3 and 7, known as executioner caspases. The latter are
able to continue the cascade of proteolytic cleavages, as we
observed for Parp-1, one of the most important protein involved
in DNA damage repair. Since Parp-1 is cleaved, cells are unable to
repair their DNA, which increased damage, together with hTopoII
inhibition, is the major stimuli that direct cells to die by apoptosis.
Effectively, Hela cells treated with 5c are clearly undergoing apop-
tosis, as demonstrated by TUNEL assay and, particularly, through
the mitochondrial mechanism. Indeed, immunofluorescences stud-
ies evidenced as Cytochrome c is released from its natural site,
namely mitochondria, to the cytosol of treated Hela cells.
Considered the biological features of compound 5c, we are confi-
dent that this lead compound may be a useful and interesting
tool in the treatment of uterine cancer.
Conclusions
Cancer is one of the leading causes of death in economically
developed countries, probably due to population aging and life-
style choices, such as smoking, lack of physical activity and unbal-
anced diets58. Among the human cancers, breast and uterine
cancers represent the death cause of almost the 26% and 7% of
women, respectively59. Even though the earlier diagnosis, as result
of the publicity surrounding the disease, and its prevention
increased the survival, the availability of new and improved che-
motherapies facing the resistance occurrence and the toxic side
effects is still a major challenge. Under this point of view, the pre-
clinical research may offer a panel of new potential anticancer
molecules that may be useful as valid alternative to the classic
therapies or in co-treatment. Herein, we have studied a series of
new N-thioalkylcarbazole derivatives, structurally correlated to the
most known Ellipticine, which exert interesting antiproliferative
effects on two in vitro models of the most common cancer types
in women. Our outcomes indicates that two derivatives, 5a and
5c, possess a good antitumour activity against two models of
breast cancer cells and a higher selectivity toward the cervical and
endometrial cancer cells. Even though Ellipticine, the ancestor of
carbazoles, possess a higher, but non-selective, activity compared
with these derivatives, it should be evidenced how these proper-
ties come together with very high cytotoxic effects on non-tumou-
ral cells used in these studies. On the contrary, the two lead
molecules, did not affect the viability of non-tumoural cells, at
least at the doses lower than 100 mM. The underlying molecular
mechanism by which the most active compound exerts its antipro-
liferative activity is represented by the inhibition of hTopoII and
the activation of the mitochondrial or intrinsic apoptotic pathway.
Acknowledgements
C. R. would like to acknowledge networking contribution by the
COST Action CM1407 “Challenging organic syntheses inspired by
nature – from natural products chemistry to drug discovery”.
Disclosure statement
The authors report no conflicts of interest.
Funding
This work was supported by MIUR (to M. S. S. and C. S.) and by
Indeed, the treatment of HeLa cells with compound 5c induces CNR (to M. R. P.).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
443
derivative covalently linked to a thymidine nucleoside as a
potential targeted antitumoral agent. I. Chem Pharm Bull
(Tokyo) 2003;51:971–4.
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