New titanocene derivatives with high antiproliferative activity against breast cancer cells

Article abstract of DOI:10.1016/j.bmcl.2013.11.058

The synthesis and characterization of some new titanocene-complexes, having a ethenyl-phenoxide or a benzyl group as substituents of the cyclopentadienyl rings, are reported. The synthesized compounds have been evaluated for their cytotoxic potential against two human breast cancer cell lines, that is: MCF7 and SkBr3. Most of these compounds have shown significant cytotoxic effects, compared to cisplatin, in MTT-based cell tests.

Full text of DOI:10.1016/j.bmcl.2013.11.058

Bioorganic & Medicinal Chemistry Letters 24 (2014) 136–140
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New titanocene derivatives with high antiproliferative activity
against breast cancer cells
Carmela Saturnino , Esther Sirignano , Antonio Botta , Maria Stefania Sinicropi , Anna Caruso b,
a,
a,
a
b,
⇑
b
b
b
c
Assunta Pisano , Rosamaria Lappano , Marcello Maggiolini , Pasquale Longo
a
Department of Pharmaceutical and Biomedical Sciences, University of Salerno, Italy
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Italy
Department of Chemistry and Biology, University of Salerno, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and characterization of some new titanocene-complexes, having a ethenyl-phenoxide or a
benzyl group as substituents of the cyclopentadienyl rings, are reported. The synthesized compounds
have been evaluated for their cytotoxic potential against two human breast cancer cell lines, that is:
MCF7 and SkBr3. Most of these compounds have shown significant cytotoxic effects, compared to
cisplatin, in MTT-based cell tests.
Received 16 September 2013
Revised 20 November 2013
Accepted 23 November 2013
Available online 3 December 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Metal-based anticancer drugs
Titanocene compounds
Breast cancer cells
The remarkable antitumor activity shown by cisplatin and other
interesting of this series, is bis-[(p-methoxybenzyl)cyclopentadi-
enyl]-titanium-dichloride (titanocene Y), shown in Figure 1a. Its
antiproliferative activity was studied in 36 human tumor cell
1
–6
platinum complexes
has meant that new metal-based antican-
cer drugs have become a noteworthy subject of research. Among
all synthesized compounds, a great deal of research has been
focused on titanium-based complexes, whose cytotoxic activity
against solid tumors is well known.
1
4
15–17
lines and in explanted human tumors.
In vitro and ex vivo
experiments show that prostate, cervix and kidney tumors are a
major target for this novel class of titanocene. Furthermore, titano-
cene Y has been tested on MCF-7 breast cancer cells, revealing a
promising medium–high cytotoxic activity with IC50 values of
Budotitane, [cis-diethoxybis(1-phenyl-1,3-butanedionato)-tita-
nium(IV)], proved to be very promising in the preclinical evalua-
tion phase, but did not pass phase I clinical trials, due to its rate
1
6
76
lM.
7
of hydrolysis.
The oxalate complex obtained from titanocene Y by a simple
Titanocene dichloride (Cp
2
TiCl
2
), (Fig. 1a), is much more resis-
anion exchange reaction was reported to have 13-fold increased
activity in relation to titanocene Y during in vitro studies against
the LLC-PK pig kidney cells.
tant in this respect, shows moderate antiproliferative activity
in vitro, but promising results in vivo,8 reaching phase II clinical
trials. Unfortunately, its efficiency in patients with metastatic renal
cell carcinoma or metastatic breast cancer was too low to be
,9
14
Recently, some of us have reported the synthesis and cytotoxic
activity of some titanocene complexes. We have also verified, by
substituting chlorine atoms, the influence of other leaving ligands
1
0,11
pursued.
As proposed by Sadler and co-workers, titanocene
1
8
dichloride is able to interact with DNA, binding to phosphate
groups instead of binding to nucleotides and nitrogen bases, as
on the activity of the complexes. Some of the synthesized com-
pounds showed a good cytotoxicity, in particular, the complexes
1
2
does cisplatin. Moreover, the titanium(IV) ion, by binding to spe-
cific iron(III), forms a strongly-bound complex with transferrin, a
protein of human plasma, and thus it is supplied to tumor cells
as this complex.1
bis-[methoxy-ethenyl-cyclopentadienyl]-titanium-dichloride (T
and [methoxy-ethenyl-cyclopentadienyl]-titanium trichloride (T
2
)
)
1
(Fig. 1a) gave IC50 values very similar to cisplatin on MCF-7, compa-
2
19,20
rable to the ones reported for titanocene Y.
A lot of analogues of titanocene containing aromatic groups
1
Complex T has been the first example of half-titanocene com-
linked to the Cp have been synthesized.13 One of the most
plex with interesting cytotoxic activity. Therefore, lately, we have
synthesized a series of novel half-titanocene complexes and tested
them for their regulatory effects on MCF-7 and SkBr3 breast cancer
⇑
Corresponding author. Fax: +39 0984 493298.
E-mail address: s.sinicropi@unical.it (M.S. Sinicropi).
These authors equally contributed to the manuscript.
1
9
cells. Some of these compounds elicited relevant repressive ef-
fects on both cell lines if compared to cisplatin. These data provide
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.058

C. Saturnino et al. / Bioorg. Med. Chem. Lett. 24 (2014) 136–140
137
Figure 1. (a) Titanocene dichloride (TDC); bis-[(p-methoxybenzyl)cyclopentadienyl]titanium dichloride (titanocene Y); bis-[methoxy-ethenyl-cyclopentadienyl]-titanium-
dichloride (T ) and [methoxy-ethenyl-cyclopentadienyl]-titanium trichloride (T ); (b) structures of synthesized complexes.
2
1
evidence that the presence of coordinating groups on cyclopentadi-
enyl ring (substituted aryl or even the phenyl itself) are important
for the biological effectiveness of these compounds.19
In order to understand if different ether groups can influence
the intramolecular coordination and consequently the stability of
the titanocene complexes and whether the ether group is essential
for their cytotoxic activity, we synthesized several new-titanocene
complexes (Fig. 1b), having as ligands the 2-cyclopentadienyl-eth-
2
[Cp-CH -Ph], and we evaluated their potential antiproliferative
effects on MCF7 and SkBr3 breast cancer cells.
In particular, the aim of this paper is to report the synthesis and
the characterization by nuclear magnetic resonance (NMR), mass
spectroscopy and elemental analysis of bis-[2(cyclopentadienyl-
ethoxy-benzene)]-titanium-dichloride (1) and of the homologous
complexes obtained by the substitution of chlorides with glycinate
(2) and oxalate (3) and of bis-cyclopentadienyl-benzyl-titanium-
dichloride (4). In addition we have also synthesized the
2 2
oxy-benzene [Cp-CH CH -O-Ph] or the cyclopentadienyl-benzyl
Scheme 1. Synthetic route for the preparation of ligands and complexes 1–3.

1
38
C. Saturnino et al. / Bioorg. Med. Chem. Lett. 24 (2014) 136–140
Table 1
Hydrolysis results of complexes in DMSO/D
2
O solution at rt followed by 1H NMR
Complex
% Cp ring hydrolysis
min 4 h
5
8 h
24 h
1
2
3
4
5
<1
<1
<1
<1
25
<1
<1
<1
<1
40
<1
<1
<1
13
48
<5
<5
<5
40
60
out by the reaction of 1 with two equivalents of glycine in methanol
containing 1% of water (Scheme 1).
Instead, the synthesis of (bis-2-cyclopentadienyl-ethoxy-
5 4 2 2 2 2 4
benzene)-titanium-oxalate [C H CH CH OPh] Ti(C O ) (3) was
performed by reaction of 1 with silver-oxalate in dry THF (see
Scheme 1).
Scheme 2. Synthetic route for the preparation of complex 4.
Complex 4, bis-cyclopentadienyl-benzyl-titanium-dichloride
(
(
1,2-diphenyl-1,2-dicyclopentadienyl-ethane)-titanium-dichloride
5) in order to have a complex very similar to 4, but with a chelating
[
C
5
4 2 2 2
H -CH -Ph] TiCl , was prepared according to Scheme 2. The
synthesis of the phenylfulvene was carried out as outlined in the
2
2
ligand, which would prevent the intramolecular coordination of
phenyl groups to metal, and consequently produce a less stable
complex than 4 itself. In Figure 1b all the synthesized compounds
are reported.
literature and its lithium salt was obtained by reaction with
Super Hydride (LiBEt H) in dry diethyl ether. Then it was isolated
3
and subsequently reacted with half-equivalent of TiCl
dry THF.
4
Á2THF in
Complex 1 was synthesized to test the effect of phenoxy group,
Lastly, complex 5, [1,2-di(cyclopentadienyl)-1,2-bis-1,2-
2
instead of the methoxy one present in T , on the effectiveness of
di(phenyl)ethane-di-yl]titanium-dichloride), was prepared accord-
2
3
this titanocene, considering that this ligand may stabilize the tita-
nium cation either with oxygen atom or with phenyl ring which
can be easily verified.
ing to Scheme 3, following a literature method.
Hydrolytic stability of the titanocene complexes has been deter-
1
mined in aqueous solution, 90% DMSO by H NMR spectroscopy
Complexes 2 and 3 were synthesized so as to have different
leaving groups on titanocene, compared to chloride, and this could
improve the activity of the complexes, by means of favorable phar-
macokinetic properties.
(
Supplementary data Figs. S1 and S2), in order to possibly correlate
the chemical stability of these complexes with their observed cyto-
toxic activity. Since we can expect that rapid hydrolysis of the leav-
ing group (Cl) and cyclopentadienyl ligands could give way to
biologically inactive species, active species could be generated if
the Cp rings remain metal bound.
The hydrolysis of aromatic rings was evaluated by the integra-
tion of two signals of protons of cyclopentadienyl bound to metal,
compared to newly formed multiplet of substituted cyclopentadi-
ene (Table 1).
Complex 4 was synthesized to test if the ether group, which is
present on the titanocene Y, on T
, is necessary to have a significant higher cytotoxic activity or if
a more labile ligand, having greater ability of -donation, can pro-
1 2
and T , as well as on complex
1
p
duce a molecule with likewise interesting cytotoxic properties.
All the synthesized complexes were purified following common
procedures and isolated in good yield. Elemental analysis (C, H, N)
were in agreement with the proposed formulae. 1H COSY experi-
ments allowed the assignment of all the proton resonances of the
In order to investigate the effects on cancer cell growth of the
compounds synthesized, MCF7 and SkBr3 breast cancer cells were
used as model systems.
2
4,25
Cells were treated for 5 days with each
1
H NMR spectrum, whereas DEPT experiments were useful for
compound and were also exposed to cisplatin, in order to compare
the anticancer effects of the chemicals used to this well-known
chemotherapeutic. In dose-response and time course experiments,
most of the tested compounds showed the capability to inhibit, in a
dose-dependent manner, the proliferation of both cell lines, just
after a 24 h treatment (Supplementary Figs. S3 and S4). Among
all tested titanocene derivatives, complexes 1, 2 and 4 elicited
strong repressive effects on the proliferation of both cell lines
the attribution of 1 C NMR signals. The synthesized titanocenes
3
were also characterized by mass spectrometry. The set of these
21
data (see experimental part in references) allowed us to have
an unambiguous structural determination, as reported in Supple-
mentary Figure S1.
The synthesis of (bis-2-cyclopentadienyl-ethoxy-benzene)-
titanium-dichloride [C
in good yields by reaction of the lithium salt of the ligand
Li[C -CH CH OPh] with TiCl
The synthesis of (bis-2-cyclopentadienyl-ethoxy-benzene)-
titanium-bis-glycine [C -CH CH OPh] Ti(gly) (2) was carried
5 4 2 2 2
H -CH CH OPh]2-TiCl (1) was carried out
(Table 2 and supplementary data Figs. S3 and S4). In particular,
H
5 4
2
2
4
.
complex 4 showed quite similar patterns of response across the
H
5 4
2
2
2
2
Table 2
Cytotoxic activity of tested compounds on MCF7 and SkBr3 breast cancer cells after
one 24 h treatment, as determined by using the MTT assay. IC50 values were
2
calculated by probit analysis (P < 0.05,
v
test)
Compound
IC50
[lM]
MCF7
SkBr3
1
2
10 (±2)
9 (±1)
10 (±1)
6 (±2)
3
4
5
>100
38 (±5)
10 (±1)
25 (±5)
10 (±2)
10 (±3)
27 (±6)
29 (±8)
Cisplatin
Scheme 3. Synthesis of complex 5.

C. Saturnino et al. / Bioorg. Med. Chem. Lett. 24 (2014) 136–140
17. Huang, H.; Qian, Y. Synthesis 1987, 1987, 910.
139
two cancer cell lines (with IC50 values of 10 lM in both cell lines
1
1
2
2
8. Napoli, M.; Saturnino, C.; Sirignano, E.; Popolo, A.; Pinto, A.; Longo, P. Eu. J. Med.
Chem. 2011, 46, 122.
9. 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.
0. Schur, J.; Manna, C.; Deally, A.; Köster, R.; Tacke, M.; Tshuva, E.; Ott, I. Chem.
Comm. 2013, 49, 4785.
1. (a) Experimental section: The elemental analyses for C and H were recorded on a
ThermoFinnigan Flash EA 1112 series and performed according to standard
microanalytical procedures. H NMR, homodecoupled 1H NMR, 1H COSY and
after 24 h treatment) (Table 2), whereas complex 2 exhibited stronger
antiproliferative effects in SkBr3 cells with respect to those elicited
in MCF7 cells (IC50 values of 9
after a one-day-treatment (Table 2). Remarkably, a 24 h-treatment
with complex 2 (10 M) was able to fully prevent SkBr3 cell
lM in MCF7 and 6 lM in SkBr3 cells)
l
growth (Supplementary data Fig. S4). Finally, it should be pointed
out that complexes 1, 2 and 4 inhibited the proliferation of MCF7
cells more strongly compared to cisplatin (Supplementary Fig. S3).
The low activity of complex 5 can be justified considering that
the bridge between the two benzilic carbons prevents the coordi-
nation of the phenyl ring on the metal centre, making it less stable,
and therefore less active.
1
1
3
C NMR spectra were recorded at 298 K on a Bruker Avance 300 Spectrometer
1
13
operating at 300 MHz ( H) and 75 MHz
( C) 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 dissolved in acetonitrile and injected directly
into the electrospray source, using a syringe pump, which maintains constant
(
In conclusion, in this work, we synthesized and characterized
three new titanocene-dichloride complexes having a ethenyl-
phenoxide or a benzyl group as substituents of the cyclopentadi-
enyl rings. Both of these groups were able to stabilize the cationic
species that is supposed to be the pharmacologically active. More-
over, we also evaluated the influence of different leaving groups
flow at 5 ll/min. The temperature of the capillary was set at 220 °C. GC
analyses were carried out with a GC–MS 7890A/5975C spectrometer (Agilent
Technologies) equipped with an OPTIMA 17MS column (diphenylpolysiloxane/
dimethylpolysiloxane, 1:1, 30 m, 0.25 mm ID) and a mass-selective detector.
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 refluxing over
suitable drying agents, while NMR deuterated solvents (Euriso-Top products)
(
glycinate and oxalate) instead of chloride on the effectiveness of
4
were kept in the dark over molecular sieves. TiCl , Titanium(IV) chloride
bis-[2-cyclopentadienyl-ethoxy-benzene]titanium-complexes.
The synthesized compounds have been evaluated for their cyto-
toxic potential against two human breast cancer cell lines. Most of
these compounds showed significant anti-proliferative effects
compared to cisplatin when tested against both MCF7 and SkBr3
cells in MTT assays. In particular, 1, 2 and 4 represent the organo-
metallic complexes showing the highest inhibitory activity on the
growth of tested cancer cell lines. Notably, most of the compounds
used have demonstrated a stronger anti-proliferative effects on
both MCF7 and SkBr3 breast cancer cells than the titanocene deriv-
atives recently synthesized and evaluated on the same model sys-
tems.1 Therefore, as indicated by our results, the presence of a
coordinating group, such as phenyl or ether one, is necessary to
stabilize the active species and generate a cytotoxic complex. This
conclusion is supported by the lower activity of complex 5 (three
times less cytotoxic than the unbridged complex 4), in which the
bridge between the Cp ligands prevents the coordination of phenyl
group on the metal centre.
tetrahydrofuran complex and all chemicals were obtained from Aldrich
chemical Co. and used without further purification. Silver oxalate was
prepared by following the reported procedure.2
1b
Synthesis of (2-(cyclopenta-2,4-dienyl)ethoxy)benzene [C
5
H
5 2 2 6 5
-CH CH OC H ]
(I): 5.23 mL of 1-(2-chloroethoxy)benzene (38.45 mmol), previously distilled
2
on CaH , were dissolved in 50 mL of dry THF to give a colorless solution. 25 mL
of a solution 2 M of sodium cyclopentadienide in THF were added drop-wise at
À78 °C. The solution was warmed up to room temperature and left stirred
overnight to give a white precipitate and a dark pink solution. Afterwards the
mixture was quenched with methanol and cold water. The organic product
was extracted by 3x 50 mL ether fraction. The solution was dried over
magnesium sulphate and had its solvent removed at reduced pressure to yield
a brown oil (yield 89%). 1H NMR (300 MHz, CDCl
CH CH OC ),], 4.14 [t, 2H, C -(CH CH OC
) d 2.88 [t, 2H, C
)], 3.01-3.20 [m, 1H, C H
H
5 5
-
-
3
5
5
(
2
2
6
H
5
5
H
5
2
2
6
H
5
9
(CH CH OC H )], 6.17–6.52 [m, 4H, C H -(CH CH OC H )], 6.92–7.32 [m, 5H,
2
2
6
5
5
5
2
2
6
5
1
3
C
6
1
5
H
5
-(CH
2
CH
-(CH
2
OC
CH
34.4, 134.9, 159.1 [C
6
H
2
5
)]. C NMR (75 MHz, C
OPh)], 44.1 [C -(CH CH
-(CH CH
6
D
6 5 5 2 2
) d 30.1 [C H -(CH CH OPh)],
7.8 [C
5
H
5
2
5
H
5
2
2
OPh)], 114.8, 120.9, 128.1, 129.7,
+
5
H
5
2
2
OPh)]. Mass (GC–MS): 186.1 [M] .
Synthesis of (bis-2-cyclopentadienyl-ethoxy-benzene)-titanium-dichloride
[C -CH CH OC 2-TiCl (1): To solution of neutral ligand (0.45 g,
.41 mmol) in dry THF (40 mL), a stoichiometic amount of n-BuLi (2.5 M
H
5 4
2
2
6
5
H ]
2
a
2
solution in hexane, 1.5 mL) was slowly added at 0 °C. The solution was warmed
up to room temperature and left stirred overnight, obtaining a yellow lithium
intermediate. Afterward the solution was treated at À78 °C with 0.13 mL
(
4
1.21 mmol) of TiCl and stirred overnight. The solvent was then removed
Supplementary data
under reduced pressure. The remaining residue was extracted with
dichloromethane (30 mL) and filtered through celite to remove the LiCl. The
deep red filtrate was washed twice with hexane (20 mL) and then dried under
reduced pressure to give a deep red solid (yield 37%). 1H NMR (300 MHz, THF-
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
d
8
) d 3.58 [t, 4H, C
5
H
4
-(CH
-(CH
)]. 13C NMR (75 MHz, C
OC )], 114.0, 114.9, 128.9 [C
-(CH CH OC )]. Mass (E.I., 70 eV, m/z): 496
26Cl Ti (%): C, 63.83; H, 5.36. Found (%): C,
2
CH
2
OC
6
H
2
5
)], 4.68 [t, 4H, C
OC ),], 6.90–7.10 [m, 10H,
) d 25.1 [C -(CH CH OC
-(CH CH OC
5 4 2 2 6 5
H -(CH CH O C H )],
1
1.058.
6.75–6.85 [m, 8H,
C
5
H
4
2
CH
6
H
6
5
5 4
C H -
(
[
CH
C
2
CH OC
2 6
H
5
6
D
5
H
4
2
2
H
6 5
)], 67.5
H
5 4
2
-(CH CH
2
6
H
5
5
H
4
2
2
H )], 115.2,
6 5
References and notes
118.6, 120.2, 157.8, 159.4 [C
H
5 4
2
2
6 5
H
+
[
L
2
-TiCl
2
-Li] . Anal Calcd for C26
H
2 2
O
1
2
3
4
5
6
.
.
.
.
.
.
Gelasco, A.; Lippard, S. J. Top. Biol. Inorg. Chem. 1999, 1, 1.
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Canc. Agents Med. Chem. 2007, 7, 3.
64.02; H 5.32.
Synthesis of (bis-2-cyclopentadienyl-ethoxy-benzene)-titanium-bis-glycine
-CH CH OC Ti(gly) (2): In a round-bottom flask 50 mg of (bis-2-
cyclopentadienyl-ethoxy-benzene)-titanium-dichloride (0.16 mmol) was
[
C
H
5 4
2
2
6
H ]
5 2
2
dissolved in 30 mL of methanol (containing 1% of water). To this brown
solution 17 mg of glycine (0.23 mmol) was added at room temperature and the
mixture was stirred for 4 h. The solvent was removed in vacuum, obtaining a
7
8
9
.
.
.
Scilling, T.; Keppler, B. K.; Heim, M. E.; Niebch, G.; Dietzfelbinger, H.; Rastetter,
J.; Hanauske, A. R. Investig. New Drugs 1996, 13, 327.
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Pharmacol. 1998, 42, 415.
red/orange solid, the yield was quantitative. 1H NMR (300 MHz, DMSO-d
6
) d
)], 6.70–7.20
3
[
(
.16 [t, 4H, C
m, 18H,
75 MHz, THF-d
12.5, 114.3, 120.5, 129.1, 130.0, 159.5, 160.2 [C
OCO-CH -NH ], 47.1 [Ti-OCO-CH -NH ]. Anal. Calcd for C30
3.61; H 6.05. Found (%): C, 63.45; H 6.09.
Synthesis of (bis-2-cyclopentadienyl-ethoxy-benzene)-titanium-oxalate
CH CH OC Ti(C ) (3): Silver oxalate (0.04 g, 0.12 mmol) and (bis-
-cyclopentadienyl-ethoxy-benzene)-titanium-dichloride (0.04 g, 0.08 mmol)
5
H
4
-(CH
-(CH
) d 29.7 [C
2
CH
2
OC
6
H
5
)], 4.11 [t 4H, C
)]; 3.30 [s, 4H, Ti-OCO-CH
5
H
4
-(CH
2
CH
2
OC
-NH
-(CH
OC
6
H
5
13
C
5
H
4
2
CH
2
OC
6
H
5
2
2
]
C
NMR
)],
8
5
H
4
-(CH
2
2
CH OC
6
H
5
)], 68.3 [C
-(CH
5
H
4
2
CH
2
OC
6
H
5
Kröger, N.; Kleeberg, U. R.; Mross, K.; Edler, L.; Saß, G.; Hossfeld, D. Onkologie
1
5
H
4
2
CH
2
6
H
5
)], 185.6 [Ti-
Ti (%): C,
2
000, 23, 60.
2
2
2
2
H
34
N
2
O
6
1
1
1
0. Melendez, E. Crit. Rev. Oncol. Hematol. 2002, 42, 309.
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6
[
C
H
5 4
2
2
6
5
H ]
2
2 4
O
1
0023.
2
1
1
1
1
3. Top, S.; Kaloun, E. B.; Vessières, A.; Laios, I.; Leclercq, G.; Jaouen, G. J.
Organomet. Chem. 2002, 643, 350.
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were dissolved in THF (30 mL) in a round-bottom flask, shielded from the light.
The solution was left stirring for 24 h at room temperature. The suspension was
gravity filtered to give a red–orange colored filtrate. The solvent was removed in
2
008, 3, 729.
vacuum and a red–orange solid was obtained (yield 59.4%). 1H NMR (300 MHz,
5. Sweeney, N.; Gallagher, W. M.; Müller-Bunz, H.; Pampillón, C.; Strohfeldt, K.;
Tacke, M. J. Inorg. Biochem. 2006, 100, 1479.
6. Allen, O. R.; Knox, R. J.; McGowan, P. C. Dalton Trans. 2008, 39, 5293.
THF-d
.73–7.16 [m 18H, C
CH CH OC )], 68.3 [C
8
) d 3.16 [t, 4H, C
5
H
4
-(CH
-(CH CH
-(CH
2
CH
2
OC
6
H
5
)], 4.07 [t, 4H, C
5
H
4
-(CH
)]. C NMR (75 MHz, C ) d 30.8 [C
OC )], 115.4, 116.2, 119.7, 121.2, 130.0,
2 2 6 5
CH O C H )],
13
6
(
5
H
4
2
2
OC
6
H
5
6
D
6
5 4
H -
2
2
6
H
5
5
H
4
2
CH
2
6 5
H

1
40
C. Saturnino et al. / Bioorg. Med. Chem. Lett. 24 (2014) 136–140
1
30.2, 134.0, 159.1 [C
5
H
4
-(CH
2 2
CH OC
6
H
5
)], 160.2 [Ti-C
2
O
4
]. Anal. Calcd for
22. Stone, K. J.; Little, R. D. J. Org. Chem. 1984, 49, 11.
C
28
H
26
O
6
Ti (%): C, 66.41; H 5.18. Found (%): C, 66.21; H 5.21.
23. Tacke, M.; Cuffe, L. P.; Callagher, W. M.; Lou, Y.; Mendoza, O.; Muller-Bunz, H.;
Rehmann, F. K.; Sweeney, N. J. Inorg. Biochem. 1987, 2004, 98.
24. Caruso, A.; Chimento, A.; El-Kashef, H.; Lancelot, J. C.; Panno, A.; Pezzi, V.;
Saturnino, C.; Sinicropi, M. S.; Sirianni, R.; Rault, S. J. Enzym. Inhib. Med. Chem.
2012, 27, 60.
25. Cell culture: MCF7 breast cancer cells were maintained in DMEM/F-12
supplemented with 10% fetal bovine serum (FBS), 100 mg/mL penicillin/
streptomycin and 2 mM L-glutamine (Invitrogen, Gibco, Milan, Italy). SkBr3
breast cancer cells were cultured in RPMI-1640 medium supplemented with
10% FBS, 100 mg/mL penicillin/streptomycin and 2 mM L-glutamine
(Invitrogen, Gibco, Milan, Italy). Cells were switched to medium without
serum the day before experiments and thereafter treated in medium
supplemented with 1% FBS.
Synthesis of bis-[(benzyl)-cyclopentadienyl]-titanium-dichloride [C
TiCl (4): TiCl
Á2 THF (0.31 g, 0.93 mmol) was added to 20 mL of dry
THF. The solution turned immediately from colourless to pale yellow. 0.30 g
1.86 mmol) of the lithium cyclopenadienide intermediate were dissolved in
0 mL of dry THF and added via cannula to the solution containing the TiCl .The
5
H
4
-CH
2
-
C
6
H
5
]
2
2
4
(
3
4
solution turned from yellow to red during addition. After this addition ,the
mixturewas refluxedovernight and thencooled. Thesolvent was removedunder
reduced pression. The remaining residue was extracted with dichlorometane
(
30 mL) and filtered twice through celite to remove the LiCl. The filtrate was
washed twice with hexane (20 mL) and then dried under reduced pression to
1
give a dark red solid (yield 55%). H NMR (300 MHz, THF-d
CH -C ], 6.33–6.44 [m, 8H, C -CH -C ], 7.22–7.24 [m, 10H, C
): d 36.7 [C -CH -C
]. Mass (E.I., 70 eV, m/z): 426 [L
22Cl Ti (%): C, 67.16; H, 5.17. Found (%): C, 67.24;
8
) d 4.06 [s, 4H, C
5
H
4
-
-
2
6
H
5
5
H
4
2
6
H
5
5 4
H
-CH
2
1
3
C
1
3
6
H
5
]. C NMR (75 MHz, THF-d
28.0, 128.6, 140.6 [C -CH -C
-Ti] . Anal. Calcd for C24
8
5
H
4
2
H
6 5
], 110.9, 115.1, 122.6,
Inhibition of cell proliferation: The effects of each compound on cell viability
+
5
H
4
2
6
H
5
2
-TiCl
2
-Li] ;
were determined with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
+
26–29
57 [L
2
H
2
diphenyltetrazolium bromide] assay,
which is based on the conversion
H 5.13.
Synthesis
titanium-dichloride [1,2-(C
of MTT to MTT-formazan by mitochondrial enzyme. Cells were seeded in
quadruplicate in 96-well plates in regular growth medium and grown until 70–
80% confluence. Cells were washed once they had attached and then treated
of
[1,2-di(cyclopentadienyl)-1,2-bis-1,2-di(phenyl)ethanediyl]
5
6
H
)
5 2
C
2
2
H {
g
-C
5
4
H }
2
]TiCl
2
(5): TiCl
4
Á2 THF (1.08 g,
3
.24 mmol) was added to 20 mL of dry toluene containing 10% dry THF. The
with increasing concentrations (2–10 lM) of each compound for indicated
solution turned immediately from colourless to pale yellow. The mixture was
stirred and cooled down to À78 °C, followed by drop wise addition of n-
butyllithium (4 mL, 6.48 mmol). The solution turned from yellow to brown
during addition. After this addition the mixture was allowed to warm up slowly
to room temperature. The colour of the solution became finally black. After 20 h,
stirring at r.t, a solution of phenyl-fulvene (6.48 mmol, 1 g) in 10 mL of dry
time (for one day up to 5 days). Relative cell viability was determined by MTT
assay according to the manufacturer’s protocol (Sigma-Aldrich, Milan, Italy).
Mean absorbance for each drug dose was expressed as a percentage of the
control untreated well absorbance and plotted vs drug concentration. IC50
values represent the drug concentrations that reduced the mean absorbance at
570 nm to 50% of those in the untreated control wells.
toluene was added to the TiCl
stirred under reflux for another 16 hours. 10 mL of the solvent were removed and
then the remaining mixture was filtered through celite. The solvent was then
2
Á2 THF solution at r.t under nitrogen. Then it was
26. Carocci, A.; Catalano, A.; Bruno, C.; Lovece, A.; Roselli, M. G.; Cavalluzzi, M. M.;
De Santis, F.; De Palma, A.; Rusciano, M. R.; Illario, M.; Franchini, C.; Lentini, G.
Bioorg. Med. Chem. 2013, 21, 847.
removed under vacuum and washed with dichlorometane and finally twice with
27. Sinicropi, M. S.; Caruso, A.; Conforti, F.; Marrelli, M.; El-Kashef, H.; Lancelot, J.
C.; Rault, S.; Statti, G. A.; Menichini, F. J. Enzym. Inhib. Med. Chem. 2009, 24,
1148.
28. Catalano, A.; Desaphy, J.-F.; Lentini, G.; Carocci, A.; Di Mola, A.; Bruno, R.;
Carbonara, A.; De Palma, A.; Budriesi, R.; Ghelardini, C.; Perrone, M. G.;
Colabufo, N. A.; Conte Camerino, D.; Franchini, C. J. Med. Chem. 2012, 55,
1418.
1
hexane to give a dark brown solid (yield 49%). H NMR (300 MHz, CDCl
3
) d 5.54 [s,
], 6.20–6.37
]. C NMR (75 MHz,
-C(Hsyn)-H-C ], 110.0,
-CH -C ]. Mass (E.I.,
Ti (%):C, 67.48;H, 4.72.
1
H, anti-C
5
H
4
-C(Hanti)-H-C
-CH -C ], 6.89–7.29 [m, 10H, C
) d 54.2 [anti-C -C(Hanti)H-C ], 51.0 [syn-C
6 5 5 4 6 5
H ], 4.85[s, 1H, syn-C H -C(Hsyn)-H-C H
13
[
m, 8H, C
5
H
4
2
6
H
5
5
H
4
-CH -C
2 6
H
5
CDCl
3
5
H
4
6
H
5
5
H
4
6 5
H
1
7
13.4, 117.1, 127.5, 128.1, 128.3, 128.6, 133.8, 138.3 [C
5
H
2
4
2
6 5
H
+
0 eV, m/z):433.04 [L
2
-TiCl
2
-Li] . Anal.Calcd for C24
H
20Cl
Found (%): C, 67.39; H, 4.77.
b) Hogan, M.; Müller-Bunz, H.; Pampillon, C.; Tacke, M. ChemMedChem 2008, 3,
29.
29. 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.
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7