Inhibition of human topoisomerase I and II and anti-proliferative effects on MCF-7 cells by new titanocene complexes

Article abstract of DOI:10.1016/j.bmc.2015.10.030

The antitumor activity shown by many platinum complexes has produced a strong interest in research of new organometallic compounds having anticancer action. Among the many metal compounds synthesized and tested, those based on titanium have received consi

Full text of DOI:10.1016/j.bmc.2015.10.030

Bioorganic & Medicinal Chemistry 23 (2015) 7302–7312
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of human topoisomerase I and II and anti-proliferative
effects on MCF-7 cells by new titanocene complexes
a
a
Adele Chimento a,y, Carmela Saturnino b, ,y, Domenico Iacopetta a, , Rosaria Mazzotta , Anna Caruso ,
⇑
⇑
Maria Rosaria Plutino c, Annaluisa Mariconda b, Anna Ramunno b, Maria Stefania Sinicropi a,
,
⇑
Vincenzo Pezzi a,à, Pasquale Longo d,à
a Department of Pharmacy, Health and Nutrition Sciences, University of Calabria, Arcavacata di Rende, Cosenza, Italy
b Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Fisciano (SA), Italy
c Department of Chemistry, University of Messina and Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Vill. S. Agata, Messina, Italy
d Department of Chemistry and Biology, University of Salerno, Fisciano (SA), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The antitumor activity shown by many platinum complexes has produced a strong interest in research of
new organometallic compounds having anticancer action. Among the many metal compounds
synthesized and tested, those based on titanium have received considerable attention because of their
cytotoxic activity against solid tumors. Particularly, new titanocene compounds containing aromatic
groups linked to the Cp (cyclopentadienyl ring, C5H5) have been synthetized, such as the titanocene Y
(bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride) that displayed promising medium–high
cytotoxic activity on breast cancer cell lines. Other titanocene complexes recently synthesized, obtained
by replacing the substituent methoxy-aryl of cyclopentadienes of titanocene Y with ethenyl-methoxide
or ethenyl-phenoxide, showed increased cytotoxic activity on breast cancer cell lines being more stable
compounds. In this paper, we report that new titanocene complexes holding lipophilic groups, for
instance a methyl group on benzyl carbon, exhibit improved antiproliferative effect on breast cancer cell
line MCF-7. Similar results have been obtained introducing a 5-methoxy naphthyl group to further
stabilize the titanocene complexes. These inhibitory effects on breast cancer cells have been ascribed
to human topoisomerase I and II inhibition as demonstrated by specific enzymatic assays.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 31 July 2015
Revised 15 October 2015
Accepted 22 October 2015
Available online 24 October 2015
Keywords:
Titanocene complexes
cis-Platin
MCF-7 cells
Topoisomerase I
Topoisomerase II
Cell death
1. Introduction
range of human tumors, the onset of toxic side effects and/or
chemoresistance represents the principal limitation to their thera-
Several platinum agents, such as cis-platin, which exert antipro-
liferative activity in breast cancer targeting DNA,1 have produced a
strong interest in research of new organometallic compounds as
pharmacological anticancer tools. Even though cis-platin and plat-
inum-based drugs exhibit high antitumor activities against a wide
peutic efficacy.2,3 It has been already reported that DNA is a major
target for cis-platin even though only a small fraction (5–10%) of
intracellular cis-platin binds DNA, whereas the major amount
(75–85%) has been found bound to other intracellular targets, such
as enzymes and RNA.4,5 This aspecific binding to non-DNA targets
could represent a valid explanation for the onset of resistance and,
as well, for its high toxicity. Moreover, several studies revealed that
cis-platin induces the formation of oxygen reactive species (ROS),
responsible for its toxic side effects, for example, nephrotoxicity,
hepatotoxicity and neurotoxicity.6
The antitumor properties of different metal complexes have
been evaluated and, amongst them, titanium complexes have
received considerable attention because of their cytotoxic activity
against solid tumors.7 It has been proposed that such complexes
may interact with DNA and inhibit cell cycle, although the
antitumor mechanism depends on the transport and delivery of
Abbreviations: DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; ADP, adenosine
diphosphate; ATP, adenosine triphosphate; DAPI, 40,6-diamidino-2-phenylindole;
DMEM/F-12, Dulbecco’s modified eagle medium: nutrient mixture F-12; DNA,
deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAD, nicotinamide
adenine dinucleotide; Parp-1, poly(ADP-ribose)polymerase 1; RNA, ribonucleic acid.
⇑
Corresponding authors. Tel.: +39 089 969602; fax: +39 089 969769 (S.C.);
tel.: +39 0984 493200; fax: +39 0984 493298 (I.D.).
E-mail addresses: saturnino@unisa.it (C. Saturnino), domenico.iacopetta@unical.
it (D. Iacopetta), s.sinicropi@unical.it (M.S. Sinicropi).
y
Considered co-first authors.
Senior authors.
à
http://dx.doi.org/10.1016/j.bmc.2015.10.030
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
7303
Ti species into cancer cells. Furthermore, the hydrolysis rate plays a
major role for the tumor-inhibiting potency, because it is corre-
lated to the interaction with nucleic acids, proteins and other
potential intracellular targets.8 As a matter of fact, budotitane
[cis-diethoxybis (1-phenylbutane-1,3-dionate)titanium(IV)] and
the titanocene dichloride (Cp2TiCl2, TDC) (Fig. 1), showed very
promising results in preclinical studies, but did not pass the phase
I and II clinical trials.9 It has been observed a high rate of hydrolysis
at neutral pH and polymeric hydrolysis products precipitation.10,11
Additionally, these compounds showed a modest efficacy in
patients with metastatic breast cancer.12,13
Literature data reported that the titanocene dichloride, differ-
ently from cis-platin, interacts with phosphate groups and with
N-sites of bases forming strong DNA adducts, which have been
detected in tumor cells, at pH values lower than 5 and weaker at
physiological pH values. Furthermore, titanium(IV) binds strongly
to protein transferrin, displacing iron(III), may mediate the uptake
of Ti into cancer cells.10
Thus, these outcomes encouraged the development of new tita-
nocene complexes having a higher hydrolytic stability and higher
cytotoxic activity introducing polar side chains attached to the
Cp ligands, for example, alkoxo, amino or carboxylic acid and
esters,14–20 and also other titanocene derivatives containing
aromatic groups linked to the Cp have been prepared.20
One of the most interesting of these compounds, namely the
titanocene Y (bis-[(p-methoxybenzyl)-cyclopentadienyl]-titanium-
dichloride), (Fig. 1), showed antiproliferative activity in numerous
human tumor cell lines14,21 and in explanted human tumors,
particularly in MCF-7 with a medium–high cytotoxic activity
(IC50 of 76
M).16,22–24
Titanocene complexes obtained by replacing the substituent
methoxy-aryl of cyclopentadienes of titanocene Y with ethenyl-
methoxide or ethenyl-phenoxide were recently synthesized and
tested.17,19
l
Particularly, the complexes bis-[methoxy-ethenyl-cyclopenta-
dienyl]-titanium-dichloride (T2), bis-[phenoxy-ethenyl-cyclopen-
tadienyl]-titanium-dichloride
(T4)
and
[methoxy-ethenyl-
cyclopentadienyl]-titanium trichloride (T1) (Fig. 1) showed
a
good cytotoxicity, very similar or better to cis-platin on MCF-7,
comparable to the ones reported for titanocene Y.14
It has been also verified the influence of leaving ligands on the
activity by substituting chlorine atoms with dimethylamide,
oxalate or aminoacid groups. The results of the hydrolysis of our
titanocenes showed unequivocally that the leaving groups (Cl, N
(CH3)2, C2O4 or glycine) significantly affect even the hydrolysis rate
of cyclopentadienyl groups, being chloride and oxalate more
stable.17
Complex T1 was the first example of half-titanocene complex
that showed interesting cytotoxic activity, and afterwards, other
analog complexes synthesized and tested as antiproliferative
agents on MCF-7 and SkBr-3 breast cancer cells.17 These data high-
lighted the importance of coordinating substituents on cyclopenta-
dienyl ligands for the cytotoxic activity.19
Although generalizations regarding structure–activity relation-
ships are not yet clear, it could be assumed that the neutral
nucleophilic substituents of cyclopentadienyl (aryl methoxy or
OCH3
Cl
OCH3
Ti
Cl
Cl
Ti
Cl
Cl
Ti
Cl
OCH3
Cl
TDC
HY
Y
H3CO
H3CO
O
OCH3
R
OCH3
O
Cl
Cl
Cl
Cl
R
Cl
Cl
Ti
Ti
Cl
Cl
Ti
Ti
R
OCH3
Cl
O
Cl
OCH3
T1 R = CH3
T3 R = Ph
T2 R = CH3
T4 R = Ph
HDMT
DMT
Cl
Cl
Cl
Ti
Cl
Ti
Cl
HTB
TDB
Figure 1. (a) Titanocene dichloride (TDC); [(p-methoxybenzyl)cyclopentadienyl]titanium trichloride (HY), Bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride (Y);
[methoxy-ethenyl-cyclopentadienyl]-titanium-trichloride (T1) bis-[methoxy-ethenyl-cyclopentadienyl]-titanium-dichloride (T2), [phenoxy-ethenyl-cyclopentadienyl]-tita-
nium trichloride (T3), bis-[phenoxy-ethenyl-cyclopentadienyl]-titanium-dichloride (T4), [(2,4-dimethoxybenzyl)cyclopentadienyl]titanium trichloride (HDMT), Bis-[(2,4-
dimethoxybenzyl)cyclopentadienyl]titanium dichloride (DMT); [cyclopentadienyl-benzyl]titanium trichloride (HTB), bis-[cyclopentadienyl-benzyl]titanium dichloride (TB).

7304
A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
OCH3
Cl
Cl
Cl
Cl
Cl
Ti
Cl
Ti
Ti
Cl
Cl
1
2
3
H3CO
OCH3
OCH3
OCH3
OCH3
Cl
Cl
Cl
Cl
Ti
Ti
4
Cl
OCH3
Ti
Cl
OCH3
Cl
H3CO
5
6
OCH3
OCH3
Cl
Ti
Cl
Cl
Cl
Ti
OCH3
Cl
7
8
Figure 2. [(Cyclopentadienyl-ethyl-1-benzene)]-titanium-trichloride [CpCH(CH3)Ph]TiCl3 (1), bis[(cyclopentadienyl-ethyl-1-benzene)]-titanium-dichloride [CpCH(CH3)
Ph]2TiCl2 (2), [(cyclopentadienyl-ethyl-1-(4-methoxy-benzene)]-titanium-trichloride [CpCH(CH3)(p-C6H4AOCH3)]TiCl3 (3), bis-[(cyclopentadienyl-ethyl-1-(4-methoxy-
benzene)]-titanium-dichloride [CpCH(CH3)(p-C6H4AOCH3)]2TiCl2 (4), [(cyclopentadienyl-ethyl-1-(2,4-dimethoxy-benzene)]-titanium-trichloride [CpCH(CH3)(C6H3
(OCH3)2)]TiCl3 (5), bis-[(cyclopentadienyl-ethyl-1-(2,4-dimethoxy-benzene)]-titanium-dichloride [CpCH(CH3)(C6H3(OCH3)2)]2TiCl2 (6), [(cyclopentadienyl)-methylene-
(5-methoxy-naphtalene)]-titanium-trichloride [CpCH2(C10H6AOCH3)]TiCl3 (7), and bis-[(cyclopentadienyl)-methylene-(5-methoxy-naphtalene)]-titanium-dichloride
[CpCH2(C10H6A OCH3)]2TiCl2 (8).
ethenyl-methoxy groups) could intramolecularly coordinate to the
titanium cation, thus preventing decomposition reactions. On the
other hand, this hypothesis was suggested for analogous com-
plexes able to give polymerization of propene or styrene having
microstructures strongly influenced by the possible coordination
of neutral substituent of cyclopentadienyl at the metal center.25–27
In this work, we report the synthesis, the characterization and
the evaluation of the hydrolytic stability of some new titanocene
and half-titanocene compounds having a methyl group on the car-
bon 6 and a methoxy-naphthyl group as substituent of the
cyclopentadienyl (Fig. 2). We also tested the antiproliferative
effects exerted by these new complexes on human breast cancer
cell line MCF-7, evidencing that they act as inhibitors of important
DNA-metabolizing enzymes, that is, topoisomerase I and II. The
outcomes suggest that these complexes could represent a valid
alternative to the most used cis-platin, being itself a topoiso-
merases inhibitor with a proven clinical efficacy, but affecting, as
well, the proliferation of normal breast cells.
lipophilicity with respect to the already reported complexes with
cyclopentadienyl-benzyl ligands. Complexes 7 and 8 were synthe-
sized because the group 5-methoxy naphthyl could further stabi-
lize the titanocene complexes and give greater coordinating
ability to the reactive sites of DNA than the compounds HY, Y,
HDMT and DMT, moreover they are more lipophilic than the latter.
In general, the synthesis of 1–6 was carried out with good yields by
reaction of the lithium salt of the suitable ligand, obtained by reac-
tion of acetophenone derivatives with cyclopentadiene, with the
stoichiometric amount of TiCl4ꢀ2THF (see Scheme 1).28
Complexes 7 and 8 were prepared according to the Scheme 2.
The synthesis of the 5-methoxy-naphthyl-fulvene was carried out
as already reported in the literature28 and its lithium salt was
obtained by reaction with Super Hydride (LiBEt3H) in dry diethyl
ether. Then, it was isolated and subsequently reacted with the
suitable amount of TiCl4ꢀ2THF in dry THF.
Hydrolytic stability of the titanocene complexes has been deter-
mined in aqueous solution, 90% DMSO by 1H NMR spectroscopy, in
order to correlate the chemical stability of these complexes with
their potential cytotoxic activity. Since we can expect that rapid
hydrolysis of the leaving group (Cl) and cyclopentadienyl ligands
could give way to biologically inactive species, whereas active spe-
cies could be generated if the Cp rings remain metal bound. The
hydrolysis of aromatic rings was evaluated by the integration of
two signals of protons of cyclopentadienyl bound to metal, com-
pared to newly formed multiplet of substituted cyclopentadiene.
The obtained results (Table 1) showed that, among all the
2. Results and discussion
2.1. Chemistry
The new titanocene complexes herein reported (1–8) have been
obtained through different methodological synthetic approaches
(Fig. 2). Complexes 1–6 were synthesized with an additional
methyl group on benzyl carbon, in order to increase their

A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
7305
R
R'
R
R'
R
O
H3C
H3C
CH3
Pyr
MeOH
LiBEt3H
Et2O; r.t.
+
R'
-
Li+
1
/
l 4
2
T
C
i
i
C
THF; reflux
T
l
4
THF; reflux
R'
R
R'
R'
Cl
Cl
Cl
R
Ti
Ti
Cl
Cl
R
1
3
5
R = H
R = H
R' = H
2
4
6
R = H
R = H
R' = H
R' = OCH3
R' = OCH3
R = OCH3 R' = OCH3
R = OCH3 R' = OCH3
Scheme 1. Synthetic route for the preparation of complexes 1–6.
OCH3
O
H
OCH3
Pyr
MeOH
+
LiBEt3H
Et2O; r.t.
-
Li+
H3CO
1
l 4
/
C
i
2
Ti
T
C
T
ux
l
H
l
f
4
F
e
r
;
;
r
e
F
fl
H
u
T
x
OCH3
OCH3
Cl
Cl
Cl
Cl
Ti
Ti
Cl
OCH3
Scheme 2. Synthetic route for the preparation of complexes 7 and 8.
2.2. Biological activity
Table 1
Hydrolysis results of complexes in DMSO/D2O solution at room temperature followed
The effects of all new synthesized titanocene complexes on
tumor cells viability have been evaluated against estrogen receptor
positive (ER+) human breast cancer cell line MCF-7 (Fig. 3A and B)
by 1H NMR
Compound
% Cp rings hydrolysis
5 min
4 h
8 h
24 h
at different concentrations (1, 5, 10, 20, 40 lM) using the (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT
assay.29–32 MCF-7 cells were treated for 72 h with each compounds
using cis-platin as anticancer reference molecule. As reported in
Figure 3A, among the tested compounds 1, 3, 5 and 7, only 7
displays antiproliferative activity in a dose-dependent manner.
Notably, as shown in Figure 3B, the complex 4 is the most active,
with an antiproliferative activity comparable to that of cis-platin
(Table 2). Compound 2 (Fig. 3B) determined a clear dose–response
inhibitory effect on cell growth, while 8 (Fig. 3B) also elicited an
1
2
3
4
5
6
7
8
15
14
<1
<1
<1
<1
<1
<1
30
31
<1
<1
<1
<1
<1
<1
55
52
<1
<1
<1
<1
<1
<1
55
55
<5
<5
<5
<5
<5
<5
inhibition, but only at higher concentrations (40
6 resulted in a slight but statistically significant reduction of cell
vitality, but only at 20 and 40 M. These considerations indicate
that the higher rate of hydrolysis of compound 2 clearly influence
lM). Compound
synthesized titanocene complexes, only two (compounds 1 and 2)
resulted more rapidly hydrolysable, probably because of the
absence of further coordinating groups (methoxy groups) on the
phenyl or naphthyl rings.
l

7306
A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
Figure 3. Effects of different doses of titanocene complexes on MCF-7 cell proliferation. Cells were treated for 72 h with the indicated concentrations of half-titanocenes (A)
or titanocenes (B). Cell viability was evaluated by MTT assay. Graphs represent mean + SE of three independent experiments each performed in triplicate. Statistically
significant differences are indicated (⁄, P <0.05 vs basal).
Table 2
lipophilicity of compound 5, respect to 6, completely abolishes
the activity (Table 2). Concerning the compounds 7 and 8 that have
one or two methoxy-naphthyl group(s) as substituent of the
cyclopentadienyl respectively, the behavior is different. Indeed,
the half-titanocene presents a higher activity than the correspond-
IC50 of cis-platin and titanocene complexes for MCF-7 cells
Compounds
IC50
(
l
M)
95% confidence intervals
1
2
3
4
5
6
7
8
505.6
85.26
273.7
49.16
664.6
129.8
63.13
171.8
33.67
372.1–686.8
80.13–90.71
221.8–337.8
46.00–52.54
499.6–884.0
122.0–138.0
57.70–69.08
153.8–191.9
31.94–35.49
ing titanocene (the IC50 values of compounds 7 and 8 are 63.13
and 171.8 M, respectively).
lM
l
In addition, a control experiment using non-malignant breast
epithelial cells MCF-10A has been performed. As shown in Figure 4,
the proliferative behavior of these cells is not affected by com-
pound 2 but changed by compounds 4 and 6 only at the high dose
cis-Platin
of 40
significant inhibitory effect at the doses of 5–10-20
cis-platin decreases cell growth at 20 and 40 M. Moreover, the
l
M; the compound
7
exerts
a
slight but statistically
lM, whereas
its anti-proliferative effect, lowering its efficacy of about 70%
l
respect to compound 4 (indeed, the IC50 values of compound 2
and 4 are, respectively, 85.26 and 49.16 lM, Table 2). Moreover,
the lack of activity of compound 1 could be ascribed to the lower
lipophilicity respect to compound 2. In a similar fashion, the higher
lipophilic character of compound 4, respect to the compound 3,
IC50 values of the most active compounds (i.e., 2, 4, 6 and 7, Table 3)
and of cis-platin have been calculated, evidencing that these new
complexes have a more specific inhibitory effect on MCF-7 breast
cancer cells, exhibiting a lesser antiproliferative activity than
cis-platin on non-tumoral MCF-10A cells.
resulted in a higher anti-proliferative effect (IC50 49.16
l
M of com-
In addition, a control experiment using non-malignant breast
epithelial cells MCF-10A has been performed. As shown in Figure 4,
the proliferative behavior of these cells is not affected by com-
pound 2 but changed by compounds 4 and 6 only at the high dose
pound 2 respect to 273.7 M compound 3, Table 2). In the com-
l
pound 6 (IC50 129.8 lM) the presence of another methoxy group
at 20 position of phenyl ring brings to a reduction in the antiprolif-
erative activity respect to compound 4 and, once again, the lower
of 40 lM; the compound 7 exerts a slight but statistically

A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
7307
Figure 4. Effects of different doses of titanocene complexes on MCF-10A cell proliferation. Cells were treated for 72 h with the indicated concentrations of compounds 7, 2, 4,
6 and cis-platin. Cell viability was evaluated by MTT assay. Graphs represent mean + SE of three independent experiments each performed in triplicate. Statistically significant
differences are indicated (⁄, P <0.05 vs basal).
permanent DNA damages and triggering cell death.42 Therefore, it
would be desirable to synthesize compounds able to inhibit both
Table 3
IC50 of cis-platin and some titanocene complexes for MCF-10A cells
the types, in order to minimize the onset of toxic side effects, due
to the combinations of topo I and II poisons use and, eventually, drug
resistance. The latter phenomenon is frequent when a selective inhi-
bitor is used, probably due to the rise of the other isoforms in the
cancer cellular context.43 In order to examine whether these com-
plexes, found to be able to exert antiproliferative effects, could act
as topoisomerases poisons and, eventually, if they possess a dual
activity on topoisomerases, we performed inhibition assays using
human topoisomerases I and II. As shown in Figure 6A, the most
active complexes (i.e., 2, 4, 6 and 7, lanes d, e, f, and g respectively)
have been found able to completely inhibit topoisomerase I activity,
Compounds
IC50
(
l
M)
95% confidence intervals
2
4
6
7
395.0
181.7
145.4
90.59
80.23
286.0–545.5
159.5–207.1
129.3–163.5
67.95–120.8
74.97–85.86
cis-Platin
significant inhibitory effect at the doses of 5–10-20
cis-platin decreases cell growth at 20 and 40 M. Moreover, the
lM, whereas
l
IC50 values of the most active compounds (i.e., 2, 4, 6 and 7, Table 3)
and of cis-platin have been calculated, evidencing that these new
complexes have a more specific inhibitory effect on MCF-7 breast
cancer cells, exhibiting a lesser antiproliferative activity than
cis-platin on non-tumoral MCF-10A cells.
as cis-platin did, at the dose of 50 lM. Indeed super coiled (SC) DNA
cannot be relaxed by the enzyme as, on the contrary, happens in the
control reaction. Moreover, the same complexes 2, 4, 6 and 7 (lanes
d, e, f, and g, respectively) are, as well, inhibitors of topoisomerase II
activity, as indicated in Figure 6B, and this produces a fail of intact
big-sized kinetoplast DNA (kDNA) migration on agarose gel. Similar
results have been obtained using cis-platin as reference molecule
(Fig. 6A, lane h), whereas in the control sample (vehicle) the enzyme
is able to bind kDNA and release intact monomeric rings. The latter
are visible at the bottom of gel as two DNA bands representing the
nicked open circular minicircles and fully closed circular rings
(decatenation products, Fig. 6B, lane c). In summary, the new syn-
thesized complexes clearly block human topoisomerases I and II
In order to verify if the decrease in cell growth was associated
with nuclear morphological changes, we performed DAPI staining.
This assay demonstrated that untreated MCF-7 cells had round
nuclei with regular contours and large. After treatment with
compound 4 cells showed nuclei with condensed DNA (Fig. 5A), a
feature often associated with the onset of apoptosis, DNA damage
but, as well, with other cellular processes.33,34 We also investigated
the status of the poly-(ADP)ribose polymerase (Parp-1),35 which is a
nuclear enzyme that catalyzes the transfer of the ADP-ribose moi-
ety of NAD+ to a specific subset of nuclear substrates in response
to DNA damage.36 It is an important regulator of the DNA base exci-
sion repair (BER) pathway and it is involved in the maintenance of
genomic integrity and survival following genotoxic insults.37–39
In this paper, we revealed that treatment of compound 4 deter-
mines Parp-1 cleavage in the same way to cis-platin (Fig. 5B).
Thus, we next performed TUNEL assay in order to establish
whether compound 4 was able to induce cell death by apoptosis;
however, no clear evidence that treated cells were going under
apoptosis have been found (data not shown).
Literature data reported that cis-platin is an inhibitors of DNA
topoisomerases, which are essential enzymes involved in the regu-
lation of the topological state of DNA during its replication, tran-
scription, recombination and, as well, chromatin remodeling and
are important for ensuing genomic integrity so that the possibility
to interfere with these enzymes represents an effective strategy
for cancer therapy.40,41 Concerning this, DNA topoisomerases I and
II are good targets of clinically significant classes of anticancer drugs
being able to interfere with the enzyme–DNA complexes producing
activity at the dose of 50 lM, as well as cis-platin. These results
may well correlate with the chromatin condensation and parp-1
activation observed and discussed before, according to other
published results.44–46
3. Conclusions
In this paper we reported the synthesis of new titanocene and
half-titanocene complexes with a higher chemical stability to
hydrolysis, low toxic effects on the proliferation of non-malignant
breast cells and interesting antiproliferative effects on ER(+) breast
cancer cells MCF-7, caused by a clear DNA topoisomerases inhibi-
tory activity. Nevertheless further studies are needed to better
define the molecular mechanism induced by these compounds that
determine the breast cancer cell death, the presented outcomes
open new interesting perspectives about the effectiveness in
therapy of metal-based drugs.

7308
A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
BF
DAPI
A
bs
20X
20X
4
20X
20X
40X
B
4
cis-platin
20 40
0
20
40
[µM]
116KDa
89KDa
Parp-1
cleaved form
GAPDH
Figure 5. Effects of compound 4 treatment on MCF7 cell nuclei morphology and Parp-1 activation. MCF-7 cells were treated with compound 4 and cis-platin (20, 40 lM) for
72 h (A) or 24 h (B). (A) After treatment MCF7 cells were fixed with paraformaldehyde, dyed with DAPI and observed under fluorescent microscope (objective 20x or 40x). BF,
brightfield; arrows indicate condensed nuclei. Images are from a representative experiment. (B) Western blot analyses of Parp-1 was performed on equal amounts of total
proteins. Blots are representative of three independent experiments with similar results. GAPDH was used as a loading control.

A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
7309
4.2. General synthesis
a
b
c
d
e
f
g
h
A
B
4.2.1. Synthesis of 6-methyl-6-aryl-fulvenes
The synthesis was carried out under nitrogen. Pyrrolidine
(30.0 mmol) was added to a solution of suitable acetophenone
derivatives or 6-methoxy-2-naphtaldehyde (20 mmol) and
cyclopentadiene (50.0 mmol) in 30 ml of methanol. After addition,
the color of the solution turned from colorless to red–orange. After
20 h, acetic acid (1.8 ml, 32.0 mmol) was added. The reaction mix-
ture was diluted with 20 ml of a mixture of diethyl ether and water
(1:1). The resultant organic layer was separated and the aqueous
layer was washed with diethyl ether (3 ꢁ 20 ml). The combined
organic extracts were washed with a saturated aqueous NaCl solu-
tion. The organic solution was dried over magnesium sulfate. The
crude product was purified by column chromatography over silica
gel and a mixture of n-hexane/ethyl acetate (9:1) as the eluent,
obtaining a yield of between 22% and 35%.
a
b
c
d
e
f
g
h
4.2.1.1. Spectral data of 6-methyl-6-aryl fulvenes. 6-Methyl- 6-
phenyl-fulvene [C5H4@CHA50-C10H6A(OCH3)]: 1H NMR (d ppm,
CDCl3): 7.39–7.29 (m, 5H, C6H5); 6.65–6.47 (m, 4H, C5H4); 2.54
(s, 3H, CH3).
6-Methyl-6-(40-methoxyphenyl)fulvene [C5H4@C(CH3)Ap-C6H4
AOCH3]: 1H NMR (d ppm, CDCl3): 7.38–6.91 (m, 4H, C6H4); 6.63–
6.23 (m, 4H, C5H4); 3.83 (s, 3H, OCH3); 2.52 (s, 3H, CH3).
6-Methyl-6-(20,40-dimethoxyphenyl)fulvene [C5H4@C(CH3)A2,
4AC6H3A(OCH3)2] 1H NMR (d ppm, CDCl3): 7.04–6.56 (m, 3H,
C6H3); 6.47–6.06 (m, 4H, C5H4); 3.85 (s, 3H, OCH3); 3.73 (s, 3H,
OCH3); 2.50 (s, 3H, CH3).
Figure 6. Evaluation of human topoisomerase I and II activity. (A): relaxation assay.
Supercoiled DNA was incubated without or with human topoisomerase I in the
absence or presence of the titanocene complexes or cis-platin at 50 lM: Lane a,
super coiled DNA, lane b, relaxed DNA marker, lane c, vehicle (DMSO), lanes d, e, f, g
and h, complexes 2, 4, 6, 7 and cis-platin, respectively. (B): decatenation assay.
kDNA was incubated without or with human topoisomerase II in the absence or
6-(50-Methoxynaphthyl)fulvene 1H NMR (d ppm, CDCl3):
7.97–7.14 (m, 6H, C10H6); 7.34 (s, 1H, C10H6ACHACp); 6.69–6.36
(m, 4H, C5H4); 3.95 (s, 3H, OCH3).
presence of the titanocene complexes or cis-platin at 50 lM: Lane a, kDNA, lane b,
decatenated DNA marker, lane c, linear kDNA, lane d, vehicle (DMSO), lanes d, e, f, g
and h, complexes 2, 4, 6, 7 and cis-platin, respectively.
4.2.2. Synthesis of half-titanocene complexes (1, 3, 5, 7)
LiBEt3H (10 ml of a 1.0 M solution in THF) was concentrated by
removal of the solvent by heating it to 60 °C under a vacuum of
10ꢂ2 mbar for 40 min and to 90 °C for other 20 min. The concen-
trated reagent was dissolved in diethyl ether (50 ml) and was
transferred to a solution of 6-methyl-6-aryl-fulvene derivatives
(5 mmol) in diethyl ether (60 ml). The solution was stirred (12 h),
during which time the lithium cyclopentadienide intermediate
precipitated from the solution and the color of the solution chan-
ged from red to yellow. After stirring, the precipitate was allowed
to settle and was filtered to remove the filtrate. The white lithium
salt was then collected on a frit and washed with diethyl ether
(25 ml), dried briefly in vacuo and transferred to a Schlenk flask
under nitrogen.
TiCl4ꢀ2THF (0.4 mmol) was added to 20 ml of dry THF. The solu-
tion turned immediately from colourless to pale yellow. 0.4 mmol
of the lithium cyclopenadienide intermediate were dissolved in
30 ml of THF dry and added dropwise to the solution containing
the TiCl4.The solution turned from yellow to dark red during addi-
tion. After this addition, the mixture was refluxed overnight and
then cooled. The solvent was removed under reduced pression.
The remaining residue was extracted with dichloromethane
(30 ml) and filtered twice through celite to remove the LiCl. The
black filtrate was washed twice with hexane (20 ml) and then
dried under reduced pression to give a solid.
4. Experimental section
4.1. Chemistry
The elemental analyses for C, H, N, were recorded on a
ThermoFinnigan Flash EA 1112 series and performed according
to standard microanalytical procedures. 1H NMR, homodecoupled
1H NMR, 1H COSY and 13C NMR spectra were recorded at 298 K
on a Bruker Avance 300 Spectrometer operating at 300 MHz (1H)
and 75 MHz (13C) and referred to internal tetramethylsilane.
Molecular weights were determined 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 dis-
solved in acetonitrile and injected directly into the electrospray
source, using a syringe pump, which maintains constant flow at
5 ll/min. The temperature of the capillary was set at 220 °C. All
manipulations were carried out under oxygen- and moisture-free
atmosphere in an MBraun MB 200 glove-box. All the solvents were
thoroughly deoxygenated and dehydrated under argon by reflux-
ing over suitable drying agents; while NMR deuterated solvents
(Euriso-Top products) were kept in the dark over molecular sieves.
TiCl4, Titanium(IV) chloride tetrahydrofuran complex, Super
Hydride (LiBEt3H, 1.0 M solution in THF), and all chemicals were
obtained from Aldrich chemical Co. and used without further
purification. Cyclopentadiene was obtained by freshly cracked
dicyclopentadiene. The four fulvenes and their relative lithium salt
were prepared by following slightly modified reported procedures.
4.2.2.1. Spectral data of half-titanocene complexes. [(Cyclopenta-
dienyl-ethyl-1-benzene)]-titanium-trichloride [C5H4-CH(CH3)-C6H5]
TiCl3 (1): 1H NMR (d ppm, THF): 7.23–7.17 (5H, m, C5H4ACH(CH3)A
C6H5); 6.43–6.35 (4H, m, C5H4ACH(CH3)AC6H5); 4.45 (1H, s, C5H4ACH
(CH3)AC6H5); 1.58 (3H, s, C5H4ACH(CH3)AC6H4(OCH3)). 13C NMR (d
ppm, THF): 23.9 (C5H4ACH(CH3)AC6H4(OCH3)), 43.6 (C5H4ACH(CH3)A
C6H5), 109.2, 114.8, 120.1, 127.5, 127.8, 138.8 (C5H4ACH(CH3)AC6H5).

7310
A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
Mass (E.I., 70 eV, m/z): 286 [L-Ti-Cl2]Å+. Anal. Calcd for C13H13Cl3Ti: C,
48.27; H, 4.05. Found: C, 48.10; H, 3.95.
115.7, 119.6, 120.3, 129.1, 138.3, 142.7, 158.7 [C5H4ACH(CH3)A
C6H4(OCH3)]. Mass (E.I., 70 eV, m/z): 479 [L2-Ti-Cl]Å+. Anal. Calcd
for C28H30Cl2O2Ti: C, 65.01; H, 5.85. Found: C, 65.32; H, 5.42.
Bis-[(cyclopentadienyl-ethyl-1-(2,4-dimethoxy-benzene)]-tita-
nium-dichloride [C5H4CH(CH3)(2,4-C6H3(OCH3)2]2TiCl2 (6): 1H
NMR (d ppm, C6D6): 6.88–6.28 (m, 6H, C5H4ACH(CH3)A
C6H3(OCH3)2]; 5.92–5.70 (m, 8H, C5H4ACH(CH3)AC6H3(OCH3)2);
5.07 (2H, C5H4ACH(CH3)AC6H3(OCH3)2); 3.32, 3.20 [2s, 12H,
C5H4ACH(CH3)AC6H3(OCH3)2]; 1.70 (s, 6H, C5H4ACH(CH3)A
C6H3(OCH3)2). 13C NMR (d ppm, C6D6): 23.3 [C5H4ACH(CH3)
AC6H3(OCH3)2], 38.8 [C5H4ACH(CH3)AC6H3(OCH3)2], 52.1, 53.2
[C5H4ACH(CH3)AC6H3(OCH3)2], 98.4, 103.5, 115.8, 121.3, 122.0,
136.3, 135.5, 153.0, 159.6 [C5H4ACH(CH3)AC6H3(OCH3)2]. Mass
(E.I., 70 eV, m/z): 504 [L2-Ti]Å+. Anal. Calcd for C30H34Cl2O4Ti: C,
62.41; H, 5.94. Found: C, 62.59; H, 5.59.
[(Cyclopentadienyl-ethyl-1-(4-methoxy-benzene)]-titanium-
trichloride [C5H4ACH(CH3)(p-C6H4AOCH3)]TiCl3 (3): 1H NMR (d
ppm, C6D6): 7.04–6.37 (m, 4H, C5H4ACH(CH3)AC6H4(OCH3));
5.75–5.45 (m, 4H, C5H4ACH(CH3)AC6H4(OCH3)); 4.63 (s, 1H,
C5H4ACH(CH3)AC6H4(OCH3)); 3.30 (s, 3H, C5H4ACH(CH3)A
C6H4(OCH3)); 1.58 (s, 3H, C5H4ACH(CH3)AC6H4(OCH3)). 13C NMR
(d ppm, C6D6): 23.2 [C5H4ACH(CH3)AC6H4(OCH3)], 40.6 [C5H4A
CH(CH3)AC6H4(OCH3)], 55.0 [C5H4ACH(CH3)AC6H4(OCH3)], 114.2,
114.5, 120.0, 120.8, 122.0, 123.3, 129.5, 138.8, 159.1 [C5H4ACH
(CH3)AC6H4(OCH3)]. Mass (E.I., 70 eV, m/z): 317 [L-Ti-Cl2]Å+. Anal.
Calcd for C14H15Cl3OTi: C, 47.57; H, 4.28. Found: C, 47.72; H, 3.98.
[(Cyclopentadienyl-ethyl-1-(2,4-dimethoxy-benzene)]-titanium-
trichloride [C5H4ACH(CH3)(2,4-C6H3A(OCH3)2)]TiCl3 (5): 1H NMR (d
ppm, C6D6): 6.91–5.92 (m, 7H, C5H4ACH(CH3)AC6H3(OCH3)2); 5.06
(s, 1H, C5H4ACH(CH3)AC6H3(OCH3)2); 3.22, 3.23 (2s, 6H, C5H4ACH
(CH3)AC6H3(OCH3)2); 1.70 (s, 3H, C5H4ACH(CH3)AC6H3(OCH3)2).
13C NMR (d ppm, C6D6): 23.7 [C5H4ACH(CH3)AC6H3(OCH3)2], 30.5
[C5H4ACH(CH3)AC6H3(OCH3)2], 54.3, 55.2 [C5H4ACH(CH3)AC6H3
(OCH3)2], 98.6, 103.7, 114.5, 120.8, 121.4, 131.4, 136.4, 150.2,
160.1 [C5H4ACH(CH3)AC6H3(OCH3)2]. Mass (E.I., 70 eV, m/z): 347
[L-Ti-Cl2]Å+. Anal. Calcd for C15H17Cl3O2Ti: C, 46.98; H, 4.47. Found:
C, 47.01; H, 4.26.
Bis-[(cyclopentadienyl)-methylene-(5-methoxy-naphtalene)]-
titanium-dichloride [C5H4ACH2A(C10H6AOCH3)]2TiCl2 (8): 1H
NMR (d ppm, CD2Cl2): 7.68–7.01 (m, 12H, C10H6); 6.38–6.35 (m,
8H, C5H4); 3.90 (s, 4H, C10H6ACH2ACp); 3.88 (s, 6H, OCH3). 13C
NMR (d ppm, CD2Cl2): 34 (C10H6ACH2ACp), 55.2 (OCH3), 105.4,
124.3, 125.2, 128.0, 128.8, 129.4, 132.8, 146.8, 156.1 (C5H4,
C
10H6). Mass (E.I., 70 eV, m/z): 516 [L2-Ti]Å+. Anal. Calcd for
C34H30Cl2O2Ti: C, 69.29; H, 5.13. Found: C, 69.58; H, 4.89.
[(Cyclopentadienyl)-methylene-(5-methoxy-naphtalene)]-tita-
nium-trichloride [C5H4CH2(C10H6AOCH3)]TiCl3 (7): 1H NMR (d
ppm, THF): 7.66–7.07 (m, 6H, C10H6); 6.46–6.38 (m, 4H, C5H4);
4.19 (s, 2H, C10H6ACH2ACp); 3.85 (s, 3H, OCH3). 13C NMR (d ppm
CD2Cl2): 35.1 (C10H6ACH2ACp), 55.9 (OCH3), 104.9, 124.1, 125.4,
128.1, 128.7, 129.3, 132.5, 147.0, 155.9 (C5H4, C10H6). Mass (E.I.,
70 eV, m/z): 353 [L-Ti-Cl2]Å+. Anal. Calcd for C17H15Cl3OTi: C,
52.42; H, 3.88. Found: C, 52.78; H, 3.65.
4.3. Cell cultures and treatments
MCF-7 breast cancer cells (an ER positive human breast cancer
cells, obtained from American Type Culture Collection (ATCC),
Manassas, VA, USA) were maintained as previously described.47
MCF-10A human mammary epithelial cells, obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA), were
maintained in DMEM-F12 supplemented with 10% horse serum
(HS), 1% glutamine, 1% penicillin/streptomycin, 0.5 mg/ml hydro-
cortisone, 20 ng/ml hEGF (human epidermal growth factor) and
0.1 mg/ml cholera enterotoxin (Sigma–Aldrich, Milano, Italy)
(complete medium). Cells were maintained at 37 °C in a humidi-
fied atmosphere of 95% air and 5% CO2 and were screened period-
ically for Mycoplasma contamination. All titanocene complexes
were dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, Mis-
souri, USA) at a concentration of 10 mM and diluted in DMEM/F12
medium supplemented with 1% DCC-FBS (dextran-coated char-
coal-treated newborn calf serum serum) to obtain the working
concentration.
4.2.3. Synthesis of titanocene complexes (2, 4, 6, 8)
The lithium intermediate was obtained as described before for
the synthesis of half-titanocenes.
TiCl4ꢀ2THF (0.28 mmol) was added to 20 ml of dry THF. The
solution turned immediately from colourless to pale yellow.
0.56 mmol of the lithium cyclopentadienide intermediate were
dissolved in 30 ml of THF dry and added dropwise to the solution
containing the TiCl4.The solution turned from yellow to dark red
during addition. After this addition, the mixture was refluxed over-
night and then cooled. The solvent was removed under reduced
pression. The remaining residue was extracted with dichloro-
methane (30 ml) and filtered twice through celite to remove the
LiCl. The black filtrate was washed twice with hexane (20 ml)
and then dried under reduced pression to give a dark red solid.
4.4. Assessment of cell viability
MCF-7 and MCF-10A cells were seeded on twenty-four well
plates (0.2 ꢁ 105 cells/well) and grown for 48 h in complete med-
ium. Before being treated, cells were starved in DMEM/F12 serum
free medium for 24 h to the purpose of cell cycle synchronization.
The effect of the different doses of different titanocene
complexes was measured using the MTT assay as previously
described.32,47–50 Seventy two hours after treatments, fresh MTT
(Sigma), re-suspended in PBS, was added to each well (final concen-
tration (0.33 mg/mL). After 2 h incubation, cells were lysed with
1 mL of DMSO. Each experiment was performed in triplicate and
the optical density was measured at 570 nm in a spectrophotome-
ter. Each experiment was performed in triplicate and the optical
density was measured at 570 nm in a spectrophotometer. Results
are represented as percent (%) of basal.
4.2.3.1. Spectral data of titanocene complexes. Bis[(cyclopenta-
dienyl-ethyl-1-benzene)]-titanium-dichloride [C5H4CH(CH3)C6H5]2
TiCl2 (2): 1H NMR (d ppm, THF): 7.23–7.16 (m, 10H, C5H4ACH
(CH3)AC6H5); 6.40–6.35 (m, 8H, C5H4ACH(CH3)AC6H5)); 4.44 (s,
2H, C5H4ACH(CH3)AC6H5); 1.58 (s, 6H, C5H4ACH(CH3)A
C6H4(OCH3)). 13C NMR (d ppm, THF): 22.9 [C5H4ACH(CH3)A
C6H4(OCH3)], 43.4 [C5H4ACH(CH3)AC6H5], 110.2, 115.1, 120.4,
127.0, 127.8, 139.1 [C5H4ACH(CH3)AC6H5]. Mass (E.I., 70 eV, m/z):
384 [L2-Ti]Å+. Anal. Calcd for C26H26Cl2Ti: C, 68.28; H, 5.73. Found:
C, 68.62; H, 5.32.
Bis-[(cyclopentadienyl-ethyl-1-(4-methoxy-benzene)]-titanium-
dichloride [C5H4CH(CH3)(pC6H4AOCH3)]2TiCl2 (4): 1H NMR (d ppm,
C6D6): 7.04–6.37 (m, 8H, C5H4ACH(CH3)AC6H4(OCH3)); 5.81–5.44
(m, 8H, C5H4ACH(CH3)AC6H4(OCH3)); 4.60 (s, 2H, C5H4ACH
(CH3)AC6H4(OCH3)); 3.28 (s, 6H, C5H4ACH(CH3)AC6H4(OCH3));
1.58 (s, 6H, C5H4ACH(CH3)AC6H4(OCH3)). 13C NMR (d ppm,
C6D6): 21.9 [C5H4ACH(CH3)AC6H4(OCH3)], 40.2 [C5H4ACH(CH3)A
C6H4(OCH3)], 54.6 [C5H4ACH(CH3)AC6H4(OCH3)], 113.8, 114.2,
4.5. Western Blot analysis
Twenty l
g of protein were subjected to western blot analysis.51
Blots were incubated overnight at 4 °C with antibodies against
Parp-1(from Santa Cruz Biotechnology, Santa Cruz CA, USA).

A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
7311
Membranes were incubated with horseradish peroxidase (HRP)-
conjugated secondary antibodies (Amersham Pharmacia Biotech,
Piscataway, NJ) and immunoreactive bands were visualized with
the ECL western blotting detection system (Amersham Pharmacia
Biotech, Piscataway, NJ). To assure equal loading of proteins, mem-
branes were stripped and incubated overnight with Glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz
Biotechnology).
analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.; La
Jolla, CA) software. Basal (untreated cells) and treated cells
were compared using the analysis of variance (ANOVA) with
Kruskal–Wallace test and post hoc Dunn’s Multiple Comparison
Test. Significance was defined as p <0.05.
Acknowledgments
This work was supported by the Programma Operativo
Nazionale (PON) Ricerca e Competitività per le Regioni della
Convergenza, 2007/2013-CCI: 2007IT161PO006.
4.6. Determination of nuclear morphological changes
Cells were grown on glass coverslips, and then treated in
FBS-DCC medium for 24 h. Cells were washed with PBS and fixed
in 4% formaldehyde for 10 min at room temperature. Fixed cells
were washed with PBS and incubated with 2-(4-amidinophenyl)-
References and notes
1. Petrelli, F.; Coinu, A.; Borgonovo, K.; Cabiddu, M.; Ghilardi, M.; Lonati, V.; Barni,
S. Breast Cancer Res. Treat. 2014, 144, 223.
2. Kater, L.; Claffey, J.; Hogan, M.; Jesse, P.; Kater, B.; Strauss, S.; Tacke, M.; Prokop,
A. Toxicol. in vitro 2012, 26, 119.
3. Hanif, M.; Babak, M. V.; Hartinger, C. G. Drug Discovery Today 2014, 19, 1640.
4. Gomez-Ruiz, S.; Maksimovic-Ivanic, D.; Mijatovic, S.; Kaluderovic, G. N.
Bioinorg. Chem. Appl. 2012.
5. Olszewski, U.; Deally, A.; Tacke, M.; Hamilton, G. Neoplasia 2012, 14, 813.
6. Ingawale, D. K.; Mandlik, S. K.; Naik, S. R. Environ. Toxicol. Pharmacol. 2014, 37,
118.
6-indolecarbamidine dihydrochloride (DAPI) (0.2 lg/mL) for
10 min in a humidified chamber, protected from light, at 37 °C.
Cells were then washed three times with cold PBS and one drop
of mounting solution was added. Cell nuclei were observed and
imaged under an inverted fluorescence microscope (200ꢁ magnifi-
cation) with excitation at 350 nm and emission at 460 nm.
7. Harding, M. M.; Mokdsi, G. Curr. Med. Chem. 2000, 7, 1289.
8. Kostova, I. Anti-Cancer Agent Me 2009, 9, 827.
4.7. Human topoisomerase I relaxation assay
9. Strohfeldt, K.; Tacke, M. Chem. Soc. Rev. 2008, 37, 1174.
10. Guo, M. L.; Sun, H. Z.; McArdle, H. J.; Gambling, L.; Sadler, P. J. Biochemistry-Us
2000, 39, 10023.
11. Lummen, G.; Sperling, H.; Luboldt, H.; Otto, T.; Rubben, H. Cancer Chemother.
Pharmacol. 1998, 42, 415.
12. Kroger, N.; Kleeberg, U. R.; Mross, K.; Edler, L.; Sass, G.; Hossfeld, D. K.; Onc, P. I.
I. S. G. A. M. Onkologie 2000, 23, 60.
13. Caruso, F.; Rossi, M. Met. Ions Biol. Syst. 2004, 42, 353.
14. Claffey, J.; Hogan, M.; Muller-Bunz, H.; Pampillon, C.; Tacke, M. ChemMedChem
2008, 3, 729.
15. Sweeney, N.; Gallagher, W. M.; Muller-Bunz, H.; Pampillon, C.; Strohfeldt, K.;
Tacke, M. J. Inorg. Biochem. 2006, 100, 1479.
16. Allen, O. R.; Knox, R. J.; McGowan, P. C. Dalton Trans. 2008, 5293.
17. Napoli, M.; Saturnino, C.; Sirignano, E.; Popolo, A.; Pinto, A.; Longo, P. Eur. J.
Med. Chem. 2011, 46, 122.
18. Saturnino, C.; Sirignano, E.; Botta, A.; Sinicropi, M. S.; Caruso, A.; Pisano, A.;
Lappano, R.; Maggiolini, M.; Longo, P. Bioorg. Med. Chem. Lett. 2014, 24,
136.
19. Sirignano, E.; Saturnino, C.; Botta, A.; Sinicropi, M. S.; Caruso, A.; Pisano, A.;
Lappano, R.; Maggiolini, M.; Longo, P. Bioorg. Med. Chem. Lett. 2013, 23, 3458.
20. Top, S.; Kaloun, E. B.; Vessieres, A.; Laios, I.; Leclercq, G.; Jaouen, G. J.
Organomet. Chem. 2002, 643, 350.
Relaxation assays have been performed in a final volume of
20 lL and assembled as follows. 0.25 lg of supercoiled pHOT1 in
TE buffer [TE: 10 mM Tris–HCl (pH 7.5), 1 mM EDTA] (TopoGEN)
has been added to a solution containing water (variable volume)
and 1ꢁ assay buffer (10 mM Tris–HCl (pH 7.9), 1 mM EDTA,
0.5 mM NaCl, 0.1% bovine serum albumin, 0.1 mM spermidine
and 5% glycerol). Next, the compounds to be tested have been
added and the reaction initiated by addition of recombinant
human topo I (2 U) (TopoGEN). The reactions were incubated at
37 °C for 30 min and terminated by the addition of 5 ꢁ stop buffer
(5% sarkosyl, 25% glycerol, 0.125% bromophenol blue). Samples
underwent to Proteinase K digestion (50 lg/mL) at 37 °C for
30 min, followed by extraction with an equal volume of chloro-
form/isoamylic alcohol (24:1), vortexed and centrifuged for 30 s.
The upper aqueous phase has been withdrawn, loaded onto a 1%
agarose gel containing 1X TAE buffer (diluted from 50ꢁ buffer con-
taining 242 g Tris base, 57.1 ml glacial acetic acid and 100 ml of
0.5 M EDTA) without ethidium bromide (EB). After the run, agarose
21. Hogan, M.; Tacke, M. Top Organomet. Chem. 2010, 32, 119.
22. Claffey, J.; Hogan, M.; Muller-Bunz, H.; Pampillon, C.; Tacke, M. J. Organomet.
Chem. 2008, 693, 526.
gel has been colored with 1ꢁ TAE buffer containing EB (0.5
lg/ml)
23. Lally, G.; Deally, A.; Hackenberg, F.; Quinn, S. J.; Tacke, M. Lett. Drug Des. Discov.
2013, 10, 675.
24. Schur, J.; Manna, C. M.; Deally, A.; Koster, R. W.; Tacke, M.; Tshuva, E. Y.; Ott, I.
Chem. Commun. 2013, 4785.
for 30 min and destained with distilled water for 15 min, then
visualized using a UV transilluminator.
25. Longo, P.; Amendola, A. G.; Fortunato, E.; Boccia, A. C.; Zambelli, A. Macromol.
Rapid Commun. 2001, 22, 339.
26. De Rosa, C.; Auriemma, F.; Fanelli, E.; Talarico, G.; Capitani, D. Macromolecules
2003, 36, 1850.
27. Napoli, M.; Grisi, F.; Longo, P. Macromolecules 2009, 42, 2516.
28. Sirignano, E.; Pisano, A.; Caruso, A.; Saturnino, C.; Sinicropi, M. S.; Lappano, R.;
Botta, A.; Iacopetta, D.; Maggiolini, M.; Longo, P. Anti-Cancer Agent Me 2015, 15,
468.
29. Sirianni, R.; Zolea, F.; Chimento, A.; Ruggiero, C.; Cerquetti, L.; Fallo, F.; Pilon, C.;
Arnaldi, G.; Carpinelli, G.; Stigliano, A.; Pezzi, V. J. Clin. Endocr. Metab. 2012, 97,
E2238.
30. Saturnino, C.; Sinicropi, M. S.; Parisi, O. I.; Iacopetta, D.; Popolo, A.; Marzocco,
S.; Autore, G.; Caruso, A.; Cappello, A. R.; Longo, P.; Puoci, F. Biomed. Res. Int.
2014.
31. Di Donna, L.; Iacopetta, D.; Cappello, A. R.; Gallucci, G.; Martello, E.; Fiorillo, M.;
Dolce, V.; Sindona, G. J. Funct. Foods 2014, 7, 558.
32. Sinicropi, M. S.; Caruso, A.; Conforti, F.; Marrelli, M.; El Kashef, H.; Lancelot, J. C.;
Rault, S.; Statti, G. A.; Menichini, F. J. Enzyme Inhib. Med. Chem. 2009, 24, 1148.
33. Widlak, P.; Palyvoda, O.; Kumala, S.; Garrard, W. T. J. Biol. Chem. 2002, 277,
21683.
4.8. Human Topoisomerase II decatenation assay
Decatenation assays have been performed in a final volume of
20 lL, as follows. 0.3 lg of kDNA (topoGEN) have been added to
a solution containing water, 1ꢁ assay buffer [which contains:
50 mM Tris–HCl, pH of 8, 150 mM NaCl, 10 mM MgCl2, 0.5 mM
Dithiothreitol (DTT) and 30 lg/mL bovine serum albumin (BSA)],
and 1 mM ATP. The compounds to be tested have been added
and the reaction started by the addition of 3 U of human topoiso-
merase II (topoGEN) and incubated at 37 °C for 30 min. At the
end, 5ꢁ stop buffer has been added and the samples have been
treated as described in the previous paragraph. The aqueous phase
has been loaded on a 1% agarose gel containing 1ꢁ TAE buffer with
EB (0.5 lg/ml) and visualized using an UV transilluminator.
34. Hendzel, M. J.; Nishioka, W. K.; Raymond, Y.; Allis, C. D.; Bazett-Jones, D. P.;
Th’ng, J. P. H. J. Biol. Chem. 1998, 273, 24470.
35. Soldani, C.; Scovassi, A. I. Apoptosis 2002, 7, 321.
4.9. Data analysis and statistical methods
36. D’Amours, D.; Desnoyers, S.; D’Silva, I.; Poirier, G. G. Biochem. J. 1999, 342, 249.
37. deMurcia, J. M.; Niedergang, C.; Trucco, C.; Ricoul, M.; Dutrillaux, B.; Mark, M.;
Oliver, F. J.; Masson, M.; Dierich, A.; LeMeur, M.; Walztinger, C.; Chambon, P.;
deMurcia, G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7303.
All experiments were performed at least three times. Data were
expressed as mean values standard deviation (SD), statistical
significance between control (basal) and treated samples were

7312
A. Chimento et al. / Bioorg. Med. Chem. 23 (2015) 7302–7312
38. Trucco, C.; Oliver, F. J.; de Murcia, G.; Menissier-de Murcia, J. Nucleic Acids Res.
1998, 26, 2644.
46. Montero, J.; Dutta, C.; van Bodegom, D.; Weinstock, D.; Letai, A. Cell Death
Differ. 2013, 20, 1465.
39. Vodenicharov, M. D.; Sallmann, F. R.; Satoh, M. S.; Poirier, G. G. Nucleic Acids
Res. 2000, 28, 3887.
47. Sirianni, R.; Capparelli, C.; Chimento, A.; Panza, S.; Catalano, S.; Lanzino, M.;
Pezzi, V.; Ando, S. Mol. Cell. Endocrinol. 2012, 363, 100.
40. Brill, S. J.; Dinardo, S.; Voelkelmeiman, K.; Sternglanz, R. Nature 1987, 326, 414.
41. Pommier, Y. Chem. Rev. 2009, 109, 2894.
42. Vicker, N.; Burgess, L.; Chuckowree, I. S.; Dodd, R.; Folkes, A. J.; Hardick, D. J.;
Hancox, T. C.; Miller, W.; Milton, J.; Sohal, S.; Wang, S. M.; Wren, S. P.; Charlton,
P. A.; Dangerfield, W.; Liddle, C.; Mistry, P.; Stewart, A. J.; Denny, W. A. J. Med.
Chem. 2002, 45, 721.
48. Sala, M.; Chimento, A.; Saturnino, C.; Gomez-Monterrey, I. M.; Musella, S.;
Bertamino, A.; Milite, C.; Sinicropi, M. S.; Caruso, A.; Sirianni, R.; Tortorella, P.;
Novellino, E.; Campiglia, P.; Pezzi, V. Bioorg. Med. Chem. Lett. 2013, 23, 4990.
49. Chimento, A.; Casaburi, I.; Rosano, C.; Avena, P.; De Luca, A.; Campana, C.;
Martire, E.; Santolla, M. F.; Maggiolini, M.; Pezzi, V.; Sirianni, R. Mol. Nutr. Food
Res. 2014, 58, 478.
43. Salerno, S.; Da Settimo, F.; Taliani, S.; Simorini, F.; La Motta, C.; Fornaciari, G.;
Marini, A. M. Curr. Med. Chem. 2010, 17, 4270.
44. Meyer-Ficca, M. L.; Lonchar, J. D.; Ihara, M.; Meistrich, M. L.; Austin, C. A.;
Meyer, R. G. Biol. Reprod. 2011, 84, 900.
45. Har-Vardi, I.; Mali, R.; Breietman, M.; Sonin, Y.; Albotiano, S.; Levitas, E.;
Potashnik, G.; Priel, E. Hum. Reprod. 2007, 22, 2183.
50. Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J. C.; Panno, A.; Pezzi, V.;
Saturnino, C.; Sinicropi, M. S.; Sirianni, R.; Rault, S. J. Enzyme Inhib. Med. Chem.
2012, 27, 609.
51. Sirianni, R.; Chimento, A.; Malivindi, R.; Mazzitelli, I.; Ando, S.; Pezzi, V. Cancer
Res. 2007, 67, 8368.