Naphthyl-substituted titanocene dichloride complexes: Synthesis, characterization and in vitro studies

Article abstract of DOI:10.1016/j.jorganchem.2011.12.015

The synthesis, characterization and the cytotoxic studies of several naphthyl-substituted titanocene complexes, [Ti(η⁵-C ₅H₅){η⁵-C₅H ₃R(CHR'(C₁₀H₇))}Cl₂] (R = H, R′ = Me (8), Ph (9); R = SiMe₃, R′ = Me (10), Ph (11)), are reported. In addition, the molecular structure of 9 has been determined by single-crystal X-ray diffraction studies. The cytotoxic activity of 9-11 has been examined against five human tumour cell lines: 8505C (anaplastic thyroid carcinoma), A549 (lung adenocarcinoma), A2780 (ovarian cancer), DLD-1 (colon carcinoma) and FaDu (head and neck carcinoma). Complex 11 showed the highest cytotoxic activity on all studied cell lines (IC₅₀ values from 35.65 ± 4.95 to 69.02 ± 1.67 μM). All compounds are more active than reference compound [Ti(η⁵-C₅H₅) ₂Cl₂] on all cell lines (except complex 8 against A549) leading to the conclusion that the presence of different substituents on the cyclopentadienyl ring may improve cytotoxicity of titanocene dichloride.

Full text of DOI:10.1016/j.jorganchem.2011.12.015

Journal of Organometallic Chemistry 700 (2012) 188e193
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Naphthyl-substituted titanocene dichloride complexes: Synthesis,
characterization and in vitro studies
,
,
,
c
,
a
ꢀb
Jesús Ceballos-Torres a b, Santiago Gómez-Ruiz a , Goran N. KaluCerovic
, Mariano Fajardo ,
*
*
Reinhard Paschke b, Sanjiv Prashar a
,
*
a Departamento de Química Inorgánica y Analítica, E.S.C.E.T., Universidad Rey Juan Carlos, 28933 Móstoles, Madrid, Spain
b Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany
c Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 22, 06120 Halle, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, characterization and the cytotoxic studies of several naphthyl-substituted titanocene
complexes, [Ti(h5eC5H5){h5eC5H3R(CHR’(C10H7))}Cl2] (R ¼ H, R0 ¼ Me (8), Ph (9); R ¼ SiMe3, R0 ¼ Me
(10), Ph (11)), are reported. In addition, the molecular structure of 9 has been determined by single-
crystal X-ray diffraction studies. The cytotoxic activity of 9e11 has been examined against five human
tumour cell lines: 8505C (anaplastic thyroid carcinoma), A549 (lung adenocarcinoma), A2780 (ovarian
cancer), DLD-1 (colon carcinoma) and FaDu (head and neck carcinoma). Complex 11 showed the highest
Received 10 August 2011
Received in revised form
8 December 2011
Accepted 13 December 2011
Keywords:
cytotoxic activity on all studied cell lines (IC50 values from 35.65 ꢀ 4.95 to 69.02 ꢀ 1.67
mM). All
Anticancer drugs
Cytotoxic activity
Metallocene
Titanium
compounds are more active than reference compound [Ti(h5eC5H5)2Cl2] on all cell lines (except complex
8 against A549) leading to the conclusion that the presence of different substituents on the cyclo-
pentadienyl ring may improve cytotoxicity of titanocene dichloride.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
DNA and leading to cell death [9]. Furthermore, binding studies of
biologically important molecules, e.g. albumin, with titanium(IV)
The majority of studies on metal complexes regarding anti-
tumour activity have been concentrated on cisplatin-like
complexes [1]. However, besides their positive effects they are
also associated with severe toxicity such as neurotoxicity, nephro-
toxicity and emesis. Therefore, recent studies have concentrated on
non-platinum metal compounds [2]. Amongst these, titanocene
derivatives were briefly studied in the 1980’s [3]. Due to the
limiting effects of titanocene dichloride which include nephrotox-
icity and a drastic elevation of creatinine and bilirubin levels [4],
clinical trials in patients with metastatic renal-cell carcinoma [5] or
metastatic breast cancer [6] did not produce the desired results.
Nevertheless, the interest in titanocene derivatives has recently
been rekindled with current efforts being focused on the design of
new compounds which may increase cytotoxicity by the modifi-
cation of the substituents of the cyclopentadienyl rings or the
auxiliary ligands directly bound to the metal centre [7].
complexes have been reported [10]. In addition, studies on hydro-
lysis of titanocene complexes in biological media [11], and the
excellent improvements in the cytotoxicity of titanocene deriva-
tives by the synthetic and biological work of many groups [7] have
led to a better understanding of their cytotoxic properties.
As a continuation of our work on titanocene complexes as
antitumoral agents [12], which was focused on the preparation of
alkenyl-substituted titanocene dichloride and titanocene carbox-
ylate derivatives, we present here the synthesis, characterization
and cytotoxic studies of titanocene dichloride complexes incorpo-
rating naphthyl-substituted cyclopentadienyl ligands. The incor-
poration of naphthyl substituents in the cyclopentadienyl moiety
may increase the DNA-intercalation properties of the correspond-
ing titanocene derivatives increasing their anticancer activity on
direct comparison with titanocene dichloride.
Mechanistic studies have indicated that titanium may reach the
cells assisted by the transport protein “transferrin” [8], binding to
2. Experimental
2.1. General manipulations
All reactions were performed using standard Schlenk tube
techniques in an atmosphere of dry nitrogen. Solvents were
distilled from the appropriate drying agents and degassed before
* Corresponding authors. Fax: þ34 914888143.
E-mail addresses: santiago.gomez@urjc.es (S. Gómez-Ruiz), goran.kaluderovic@
ꢀ
chemie.uni-halle.de (G.N. KaluCerovic), sanjiv.prashar@urjc.es (S. Prashar).
0022-328X/$ e see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2011.12.015

J. Ceballos-Torres et al. / Journal of Organometallic Chemistry 700 (2012) 188e193
189
use. Cyclopentadiene dimer, pyrrolidine, LiBun (1.6 M in hexane),
LiMe (1.6 M in Et2O), LiPh (1.8 M in dibutylether), SiMe3Cl and
C10H7CHO were purchased from Aldrich and used directly.
(C5H4)]CH(C10H7) (1) and Li{C5H4(CHMe(C10H7))} (2) were
synthesized as previously described by us [13]. [Ti(h5eC5H5)Cl3]
was prepared by the reaction of TiCl4 with C5H5SiMe3 [14]. IR
spectra were recorded on a Thermo Nicolet Avatar 330 FT-IR
spectrophotometer. 1H NMR and 13C{1H} NMR spectra were recor-
ded on a Varian Mercury FT-400 spectrometer. Microanalyses were
carried out with a PerkineElmer 2400 microanalyzer. EI-MS spec-
troscopic analyses were preformed on a MASPEC II system [II32/
A302] (m/z 50e1000).
2.2.5. Synthesis of Li{C5H3(CHPh(C10H7))(SiMe3)} (7)
The preparation of 7 was carried out in an identical manner to 6.
LiBun (2.60 mL, 4.16 mmol, 1.60 in hexane) and
C5H4(CHPh(C10H7))(SiMe3) (10) (1.34 g, 3.79 mmol). Yield 1.02 g,
75%. 1H NMR (400 MHz, d8-THF, 25 ꢁC):
d
¼ 0.09 (s, 9 H, SiMe3), 5.71,
5.83 and 5.91 (m, each 1 H, C5H3), 6.17 (s, 1 H, CHPh), 7.00e7.17 (m,
5 H, C6H5), 7.28e7.33 (m, 3 H, H in position 3, 6 and 7 of naphthyl
group), 7.28e7.33, 7.60, 7.77 and 8.42 (d, each 1 H, H in position 2, 4,
5 and 8 of naphthyl group) ppm. 13C{1H} NMR (100 MHz, d8-THF,
25 ꢁC):
d
¼ 0.7 (SiMe3), 48.9 (CpC), 108.2, 108.4, 110.6, 111.5, 124.1,
124.5, 124.6, 124.8, 125.1, 125.4, 125.8, 127.4, 127.6, 128.5, 129.0,
132.3, 134.3, 144.1 and 148.8 (C5H3, C6H5 and C10H7) ppm.
2.2. Synthesis of compounds
2.2.6. Synthesis of [Ti(h5eC5H5){h5eC5H4(CHMe(C10H7))}Cl2] (8)
A solution of Li{C5H4(CHMe(C10H7))} (2) (1.00 g, 4.43 mmol) inTHF
(50 mL) was added dropwise during 15 min to a solution of
[Ti(h5eC5H5)Cl3] (0.93 g, 4.24 mmol) in THF (50 mL) at 0 ꢁC. The
reaction mixture was allowed to warm to room temperature and
stirred for 2 h. Solvent was removed in vacuum and a toluene-hexane
9:1 mixture (50 mL) added to the resulting solid. The suspension was
filtered and the filtrate concentrated (20 mL) and cooled to ꢃ30 ꢁC to
give the title compound as a red solid. Yield 0.10 g, 73%. FT-IR (KBr):
2.2.1. Synthesis of Li{C5H4(CHPh(C10H7))} (3)
LiPh (9.44 mL, 16.99 mmol, 1.80 M in dibutylether) was added
dropwise to (C5H4)]CH(C10H7) (1) (3.15 g, 15.44 mmol) at 0 ꢁC. The
reaction mixture was allowed to warm to room temperature and
stirred for 4 h. The solvent was removed in vacuo and the resulting
white solid washed with hexane (2 ꢂ 50 mL) and dried under
vacuum to yield a free-flowing off white powder. Yield 4.05 g, 91%.
1H NMR (400 MHz, d-THF, 25 ꢁC):
C5H4), 6.16 (s, 1 H, CH), 7.00e8.40 (m, 12 H, C6H5 and C10H7) ppm.
13C{1H} NMR (100 MHz, d-THF, 25 ꢁC):
120.9, 124.5, 124.7, 125.1, 125.3, 125.6, 127.3, 127.5, 128.5, 129.1,
130.6, 132.4, 134.2, 144.4 and 149.0 (C5H4, C6H5 and C10H7) ppm.
d
¼ 5.55 and 5.67 (m, each 2 H,
n
¼ 3107 (nCH), 1629 (
n
C]C) cmꢃ1. 1H NMR (400 MHz, CDCl3, 25 ꢁC):
d
¼ 1.74 (d, 3 H, CHMe), 5.95(q,1 H, CHMe), 6.54(s, 5 H, C5H5), 6.40 and
d
¼ 49.1 (CpC), 102.1, 104.3,
6.88 (m, each 2 H, C5H4), 7.39, 7.52, 7.73 and 7.75 (m, 7 H, C10H7) ppm.
13C{1H} NMR (100 MHz, CDCl3, 25 ꢁC):
¼ 21.1 (CHMe), 36.9 (CpC),
d
119.9 (C5H5), 119.1, 120.4, 124.0, 125.5, 125.8, 125.9, 126.4, 127.4, 128.5,
129.1, 129.3, 131.2 and 134.3 (C5H4 and C10H7) ppm. EI-MS (m/e
(relative intensity)): 402 (1) [Mþ], 366 (24) [Mþ ꢃ Cl, e H], 337 (69)
[Mþ ꢃ Cp], 219 (33) [C5H4(CHMe(C10H7))þ], 203 (100) [Mþ ꢃ Ti, e
2 ꢂ Cl, e Cp, e Me, e H],183 (36) [Mþ ꢃ C5H4(CHMe(C10H7))],127 (7)
[C10Hþ7 ], 65 (22) [Cpþ]. C22H20Cl2Ti (402.9): calcd. C 65.54, H 5.00;
found C 65.21, H 4.77%.
2.2.2. Synthesis of C5H4(CHMe(C10H7))(SiMe3) (4)
SiMe3Cl (0.98 mL, 7.73 mmol) was added dropwise to a solution
of Li{C5H4(CHMe(C10H7))} (2) (1.46 g, 6.46 mmol) in THF at 0 ꢁC for
5 min. The reaction mixture was warmed to room temperature and
stirred for 6 h. The solvent was then removed in vacuo to give an
oily solid, which was extracted with hexane (2 ꢂ 50 mL). The
mixture was filtered and hexane removed from the filtrate to give
a yellow oily solid. Yield 1.91 g, 77%. 1H NMR (400 MHz, CDCl3,
2.2.7. Synthesis of [Ti(h5eC5H5){h5eC5H4(CHPh(C10H7))}Cl2] (9)
The preparation of 9 was carried out in an identical manner to 8.
Li{C5H4(CHPh(C10H7))} (1.08 g, 3.73 mmol) and [Ti(h5eC5H5)Cl3]
25 ꢁC, mixture of isomers):
d
¼ ꢃ0.05 and 0.00 (s, each 9 H, SiMe3),
1.67 and 1.70 (s, each 3 H, CHMe), 3.27 and 3.31 (s, each 1 H, HC5),
4.75 (q, 2 H, CHMe), 6.13, 6.18 and 6.44 (m, each 2 H, C5H3),
7.38e7.51, 7.72, 7.86 and 8.20 (m, 14 H, C10H7) ppm.
(0.78 g, 3.56 mmol). Yield 1.32 g, 80%. FT-IR (KBr):
n
¼ 3092 (nCH),
1694 (
n
C]C) cmꢃ1. 1H NMR (400 MHz, CDCl3, 25 ꢁC):
d
¼ 6.20 (s, 5 H,
C5H5), 6.49 (s, 1 H, CHPh), 6.92 (m, 2 H, C5H4), 7.03 (br s, 2 H, C5H4),
6.93e8.13 (m, 12 H, C6H5 and C10H7) ppm. 13C{1H} NMR (100 MHz,
2.2.3. Synthesis of C5H4(CHPh(C10H7))(SiMe3) (5)
CDCl3, 25 ꢁC):
d
¼ 48.6 (CHPh), 120.5 (C5H5), 124.3, 125.2, 125.6,
The preparation of 5 was carried out in an identical manner to 4.
SiMe3Cl (0.74 mL, 5.84 mmol) and Li{C5H4(CHPh(C10H7))} (3)
(1.40 g, 4.86 mmol). Yield 1.79 g, 78%. 1H NMR (400 MHz, CDCl3,
126.0, 126.4, 126.7, 127.5, 128.0, 128.5, 128.8, 129.0, 129.6, 131.5,
134.1, 140.0, 140.8 and 141.5 (C5H3, C6H5 y C10H7) ppm. EI-MS (m/e
(relative intensity)): 428 (100) [Mþ ꢃ Cl, e H], 399 (34) [Mþ ꢃ Cp],
25 ꢁC, for the predominant isomer):
d
¼ ꢃ0.03 (s, 9 H, SiMe3), 3.30
203 (38) [Mþ ꢃ 2 ꢂ Cl, e Cp, e C10H7, þ H], 183 (9) [Mþ
ꢃ
(d, 1 H, HC5), 5.84 (d, 1 H, CHPh), 6.00, 6.46 and 6.49 (m, each 1 H,
C5H3), 7.20e7.25 (m, 5 H, C6H5), 7.40e7.46, 7.75, 7.86 and 8.06 (m,
7 H, C10H7) ppm.
C5H4(CHPh(C10H7))], 91 (23) [C7Hþ7 ], 65 (11) [Cpþ]. C27H22Cl2Ti
(464.9): calcd C 69.70, H 4.77; found C 69.52, H 4.69%.
2.2.8. Synthesis of [Ti(h5eC5H5){h5eC5H3(CHMe(C10H7))(SiMe3)}]
(10)
2.2.4. Synthesis of Li{C5H3(CHMe(C10H7))(SiMe3)} (6)
LiBun (3.40 mL, 5.44 mmol, 1.60 M in hexane) was added
dropwise to a solution of C5H4(CHMe(C10H7))(SiMe3) (4) (1.45 g,
4.97 mmol) in hexane at ꢃ78 ꢁC. The mixture was warmed to 25 ꢁC
and stirred for 16 h. Solvent was then removed in vacuo giving
a white solid, which was washed with hexane (2 ꢂ 50 mL) and
dried under vacuum to give the title compound as a white powder.
The preparation of 10 was carried out in an identical manner to 8.
Li{C5H3(CHMe(C10H7))(SiMe3)}(0.89 g, 2.99mmol)and[Ti(h5eC5H5)
Cl3] (0.62 g, 2.83 mmol). Yield 0.41 g, 31%. FT-IR (KBr):
1594 (
C]C) cmꢃ1 1H NMR (400 MHz, CDCl3, 25 ꢁC, for the
predominant isomer):
n
¼ 3107 (nCH),
n
.
d
¼ 0.15 (s, 9 H, SiMe3),1.61 (d, 3 H, CHMe), 4.95
(m, 1 H, CHMe), 6.40 (s, 5 H, C5H5), 6.56, 6.92 and 7.00 (m, each 1 H,
Yield 0.90 g, 61%. 1H NMR (400 MHz, d8-THF, 25 ꢁC):
d
¼ 0.07 (s, 9 H,
C5H3), 7.46e7.91 (m, 7 H, C10H7) ppm. 13C{1H} NMR (100 MHz, CDCl3,
SiMe3), 1.66 (d, 3 H, CHMe), 4.93 (q, 1 H, CHMe), 5.83 (m, 3 H, C5H3),
7.34e7.40 (m, 3 H, H in position 3, 6 and 7 of naphthyl group), 7.50,
7.57, 7.77 and 8.36 (d, each 1 H, H in position 2, 4, 5 and 8 of
naphthyl group) ppm. 13C{1H} NMR (100 MHz, d8-THF, 25 ꢁC):
25 ꢁC, for the predominantisomer):
d
¼ 0.1 (SiMe3), 20.6 (CHMe), 34.2
(CpC),119.9 (C5H5),120.0,120.3,120.7,121.6,122.4,123.8,124.3,125.8,
126.4,127.3,127.8,129.1,131.2,138.1 and149.4 (C5H3 yC10H7)ppm. EI-
MS (m/e (relative intensity)): 474 (1) [Mþ], 438 (12) [Mþ ꢃ Cl, e H],
d
¼ 1.5 (SiMe3), 25.0 (CHMe), 36.2 (CHMe), 106.5, 108.2, 110.0, 110.9
409 (20), [Mþ ꢃ Cp], 402 (18) [Mþ ꢃ SiMe3, þ H], 183 (22) [Mþ
ꢃ
and 147.6 (C5H3), 124.5, 124.8, 125.4, 125.7, 126.0, 126.3, 128.4, 129.3,
132.5 and 135.1 (C10H7) ppm.
C5H3(CHMe(C10H7))(SiMe3)], 73 (100) [SiMeþ3 ], 65 (72) [Cpþ].
C25H28Cl2SiTi (474.9): calcd C 63.17, H 5.94; found C 63.00, H 5.99%.

190
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2.2.9. Synthesis of [Ti(h5eC5H5){h5eC5H3(CHPh(C10H7))(SiMe3)}]
(11)
with 10% foetal bovine serum (Biochrom AG) and penicillin/strep-
tomycin (PAA Laboratories).
The preparation of 11 was carried out in an identical manner to
8. Li{C5H3(CHPh(C10H7))(SiMe3)} (0.87 g, 2.42 mmol) and
[Ti(h5eC5H5)Cl3] (0.50 g, 2.28 mmol). Yield 0.51 g, 42%. FT-IR (KBr):
2.4.2. Cell lines and culture conditions
The cell lines 8505C, FaDu, A549, A2780 and DLD-1 are included
in this study. All these cell lines were kindly provided by Dr. Thomas
Mueller, Department of Hematology/Oncology, Martin-Luther
University of Halle-Wittenberg, Halle (Saale), Germany. Cultures
were maintained as monolayer in RPMI-1640 (PAA Laboratories,
Pasching, Germany) supplemented with 10% heat inactivated foetal
bovine serum (Biochrom AG, Berlin, Germany) and penicillin/
streptomycin (PAA Laboratories) at 37 ꢁC in a humidified atmo-
sphere of 5% (v/v) CO2.
n
¼ 3102 (nCH), 1597 (
n
C]C) cmꢃ1. 1H NMR (400 MHz, CDCl3, 25 ꢁC,
for the predominant isomer):
d
¼ 0.33 (s, 9 H, SiMe3), 6.14 (s, 1 H,
CHPh), 6.20 (s, 5 H, C5H5), 6.41, 6.70 and 6.81 (m, each 1 H, C5H3),
7.19e8.20 (m, 7 H, C10H7) ppm. 13C{1H} NMR (100 MHz, CDCl3,
25 ꢁC, for the predominant isomer):
d
¼ 0.3 (SiMe3), 49.0 (CHPh),
120.7 (C5H5), 124.2e141.1 (C5H3, C6H5 and C10H7) ppm. EI-MS (m/e
(relative intensity)): 500 (63) [Mþ ꢃ Cl, e H], 471 (14) [Mþ ꢃ Cp],
147 (78) [Mþ ꢃ Cl, e Cp, e SiMe3, e CHPh(C10H7), þ H], 91 (62)
[C7Hþ7 ], 73 (100) [SiMeþ3 ]. C30H30Cl2SiTi (536.9): calcd C 67.05, H
5.63; found C 66.29, H 5.20%.
2.4.3. Cytotoxicity assay
The cytotoxic activities of the titanium complexes 8e11, were
evaluated using the sulforhodamine-B (SRB, Sigma Aldrich) micro-
culture colorimetric assay [18]. Exponentially growing cells were
seeded into 96-well plates on day zero at the appropriate cell
densities to prevent confluence of the cells during the period of
experiment. After 24 h, the cells were treated with serial dilutions of
the studied compounds for 96 h. Final concentrations achieved in
2.3. Data collection and structural refinement of 9
Data were collected with a Bruker SMART APEX CCD system
(
l
(MoKa) ¼ 0.71073 Å, T ¼ 293(2) K) using
u and j scans. Empirical
absorption correction was carried out with SADABS [15]. The
structures were solved by direct methods [16]. Structure refine-
ments were carried out with SHELXL-97 [17]. All non-hydrogen
atoms were refined anisotropically and H atoms were located by
difference maps and refined isotropically. Table 1 lists crystallo-
graphic details.
treated wells were 1, 10, 20, 50, 100, 150 and 200
mmol/L. Each
concentration was tested in three triplicates on each cell line. The
final concentration of DMSO solvent never exceeded 0.5%, which
was non-toxic to the cells. The percentages of surviving cells relative
to untreated controls were determined 96 h after the beginning of
drug exposure. After 96 h treatment, the supernatant medium from
2.4. In vitro studies
2.4.1. Preparation of drug solutions
A solution of the studied titanium complexes was prepared in
dimethylsulfoxide (DMSO, Sigma Aldrich) at a concentration of
20 mM, filtered through Millipore filter (0.22 mm), before use, and
diluted by nutrient medium to various working concentrations.
Nutrient medium was RPMI-1640 (PAA Laboratories) supplemented
O
H
N
H
+
H
H2O
1
Table 1
Crystallographic data of 9.
LiR
Formula
Fw
C27H22Cl2Ti
465.22
T (K)
293(2)
Cryst syst
Space group
a (Å)
b (Å)
c (Å)
Monoclinic
P21/c
12.297(1)
13.035(1)
13.631(1)
90
93.139(1)
90
2184.9(3)
4
1.414
0.648
960
0.4 ꢂ 0.3 ꢂ 0.1
1.66 to 28.10
ꢃ15 ꢄ h ꢄ 15
ꢃ16 ꢄ k ꢄ 16
ꢃ17 ꢄ l ꢄ 17
24626
R
R
SiMe3Cl
LiCl
H
H
a
b
g
(deg)
(deg)
(deg)
V (Å 3
)
Z
SiMe3
Dc (Mg mꢃ3
)
LiBun
4 (R = Me)
5 (R = Ph)
(mmꢃ1
F(000)
Cryst dimens (mm)
range (deg)
)
Li
m
2 (R = Me)
3 (R = Ph)
BunH
q
hkl ranges
R
H
Collected reflections
Independent reflections
Completeness
5010
94.2%
Data/restraints/parameters
5010/0/271
1.028
Goodness-of-fit on F2
SiMe3
Final R indices [I > 2
s
(I)]
R1 ¼ 0.0448
wR2 ¼ 0.1018
R1 ¼ 0.0651
wR2 ¼ 0.1138
ꢃ0.160 and 0.336
Li
6 (R = Me)
7 (R = Ph)
Final R indices (all data)
Largest diff. peak and hole (e.Åꢃ3
)
Scheme 1. Synthesis of 1e7.

J. Ceballos-Torres et al. / Journal of Organometallic Chemistry 700 (2012) 188e193
191
the 96-well plates was thrown away and the cells were fixed with
a 10% TCA solution. For a thorough fixation, plates were then allowed
to stand at 4 ꢁC for approximately 2 h. After fixation the cells were
washed in a strip washer. The washing was carried out four times
with distilled water using alternate dispensing and aspiration
procedures. The plates were then dyed with 100 mL of a 0.4% SRB
solution for about 45 min. After dyeing, the plates were again
washed to remove the dye with a 1% acetic acid solution and allowed
to air dry overnight. Subsequently, 100 mL of 10 mM Tris base solu-
tions was added to each well of the plate and absorbance was
measured at 570 nm using a 96-well plate reader (Tecan Spectra,
Crailsheim, Germany). The IC50 values, defined as the concentrations
of the compound at which 50% cell inhibition was observed, were
estimated from the doseeresponse curves.
3. Results and discussion
3.1. Synthesis of metal complexes
Compounds 1e11 were prepared as indicated in Schemes 1
and 2.
In the 1H NMR spectra of all the prepared compounds, the
signals corresponding to the naphthyl fragment were observed in
the aromatic region between 7.0 and 8.5 ppm.
Fig. 1. Molecular structure and atom-labelling scheme for 9 (hydrogen atoms, except
at C(11), are omitted for clarity).
4 was isolated as a mixture of two isomers as confirmed by 1H
NMR spectroscopy where two singlets were observed at ꢃ0.05 and
0.00 ppm corresponding to trimethylsilyl group of each isomer.
However for 5, the singlet observed at ꢃ0.03 ppm, indicated the
predominance of one of the isomers (>90%). The lithium deriva-
tives of 3, 6 and 7 showed the expected chemical shifts in the 1H
NMR spectra (see Experimental Section).
The titanocene(IV) dichloride derivatives, [Ti(h5eC5H5)
{
h5eC5H3R(CHR’(C10H7))}Cl2] (R ¼ H, R0 ¼ Me (8), Ph (9); R ¼ SiMe3,
Cl
R0 ¼ Me (10), Ph (11)), were synthesized by the reaction of
[Ti(h5eC5H5)Cl3] and one molar equivalent of the corresponding
lithium derivative (2, 3, 6 and 7), respectively. 8e11 were isolated as
red solids.
Ti
Cl
9
Cl
Ti
In the 1H NMR spectra of the titanocene complexes one singlet,
corresponding to the unsubstituted cyclopentadienyl ring, was
observed between 6 and 6.5 ppm. The expected signals corre-
sponding to the methyl, phenyl and naphthyl fragments were also
present (see Experimental Section).
Cl
3
2
8
Table 2
Selected bond lengths (Å) and angles (ꢁ) for 9.a
Ti
Cl
Ti(1)eCl(1)
Ti(1)eCl(2)
2.3631(8)
2.3499(7)
2.079
Cl
Cl
Ti(1)eCent(1)
Ti(1)eCent(2)
C(1)eC(11)
C(11)eC(12)
C(11)eC(22)
2.064
7
1.516(3)
1.530(3)
1.529(3)
6
Cent(1)eTi(1)eCent(2)
Cent(1)eTi(1)eCl(1)
Cent(1)eTi(1)eCl(2)
Cent(2)eTi(1)eCl(1)
Cent(2)-Ti(1)eCl(2)
Cl(1)eTi(1)eCl(2)
C(1)eC(11)eC(12)
C(1)eC(11)eH(11)
C(1)eC(11)eC(22)
C(12)eC(11)eH(11)
C(12)eC(11)eC(22)
C(22)eC(11)eH(11)
Cent(1)eC(1)eC(11)
132.1
106.4
106.0
106.2
106.2
93.29(3)
110.4(2)
106.7(2)
111.5(2)
106(1)
114.4(2)
106.6(2)
176.5
Me3Si
Cl
Cl
Ti
11
Me3Si
Cl
Cl
Ti
10
Cent(1) and Cent(2) are the centroids of C(1)eC(5) and C(6)e
Scheme 2. Synthesis of 8e11.
C(10), respectively.

192
J. Ceballos-Torres et al. / Journal of Organometallic Chemistry 700 (2012) 188e193
Table 3
IC50
(mM) values for the 96 h of action of 8e11, titanocene dichloride and cisplatin on 8505C (anaplastic thyroid cancer), A549 (lung carcinoma), A2780 (ovarian cancer), DLD-1
(colon carcinoma) and FaDu (head and neck cancer) determined by sulforhodamine-B microculture colorimetric assay (SRB assay).
Compounds
IC50
[
mM]
8505C
A549
A2780
DLD-1
FaDu
8
9
10
11
cisplatin
[Ti(
194.51 ꢀ 4.80
105.22 ꢀ 1.51
124.57 ꢀ 3.24
45.19 ꢀ 1.26
5.02 ꢀ 0.23
>200
191.72 ꢀ 2.54
114.20 ꢀ 2.88
104.31 ꢀ 3.07
53.38 ꢀ 1.43
1.51 ꢀ 0.02
72.41 ꢀ 7.52
71.60 ꢀ 1.23
54.25 ꢀ 2.35
35.65 ꢀ 4.95
0.55 ꢀ 0.03
n.a.a
161.34 ꢀ 3.48
116.22 ꢀ 2.31
97.95 ꢀ 4.03
61.31 ꢀ 6.08
5.14 ꢀ 0.12
>200
>200
108.69 ꢀ 2.58
137.41 ꢀ 4.34
69.02 ꢀ 1.67
1.21 ꢀ 0.14
n.a.a
h
5-C5H5)2Cl2]
167.62 ꢀ 3.31
a
not analyzed.
The IR spectra of 8e11 showed bands corresponding to the
vibration frequency of CeH bond (nCH) (around 3100 cmꢃ1) and
3.3. Cytotoxicity studies
the stretching vibration of the C]C bonds (
n
C]C) (1600 cmꢃ1). In
The titanocene complexes were tested against the following
human tumour cell lines: 8505C (anaplastic thyroid carcinoma),
A549 (lung adenocarcinoma), A2780 (ovarian cancer), DLD-1 (colon
carcinoma) and FaDu (head and neck carcinoma). The IC50 param-
eters for 8e11 were obtained by SRB assay and are given in Table 3.
All complexes are active against all the studied cancer cell lines
in the examined range of concentrations, except 8 on cell line FaDu
(Fig. 2 and Table 3).
the UVevisible spectra of 8e11 two bands at ca. 300 and 400 cmꢃ1
corresponding to the chromophore groups and the MLCT band of
these molecules were observed. 8e11 were also characterized by
EI-MS and fragments indicative of the loss of cyclopentadienyl,
chloride, naphthyl and trimethylsilyl (for 10 and 11) were
observed.
As can be deduced from Table 3, 8e11 show a certain preference
against cell line A2780. Complex 11 is the most active of all the
tested compounds, with IC50 values between 35.65 ꢀ 4.95 and
3.2. Structural study
The molecular structure of 9 was established by single-crystal X-
ray diffraction studies. 9 crystallizes in the monoclinic P21/c space
group with four molecules located in the unit cell. The molecular
structure and atomic numbering scheme of 9 are shown in Fig. 1.
Selected bond lengths and angles for 9 are given in Table 2.
The molecular structure of 9 reveals that the titanium atom has
a distorted tetrahedral geometry with both C5 rings bound to the
metal atom in an h5 mode giving the typical bent metallocene
conformation observed in other titanocene dichloride complexes
[19]. TieCl bonds of 2.3631(8) and 2.3499(7) Å are in the range of
those observed for other similar titanocene derivatives [18]. The
bond angles about the chiral carbon atom C(11) are all proximate to
109ꢁ, confirming its sp3 hybridization. The phenyl and naphthyl
carbon atom bond angles are all close to 120ꢁ confirming sp2
hybridization and aromaticity of these fragments.
69.02 ꢀ 1.67
mM. The results obtained suggest that the presence of
phenyl and naphthyl groups in this type of complexes may lead to
an increase of the cytotoxic activity. These aromatic groups may be
able to intercalate between the DNA base pairs, hampering the
replication and propagation of cancer cells and thus increasing the
antitumoural effect of the titanocene complex [20]. An improve-
ment in cytotoxic activity of all complexes due to the presence of
trimethylsilyl group has been observed, confirming the previous
studies carried out by McGowan and coworkers [7h]. 8e11 are less
active than cisplatin but despite this, these results are encouraging
taking into account both the high tolerance of titanium in in vivo
biological systems, the notable number of negative side effects
associated with platinum-based drugs and the higher cytotoxic
activity of 8e11 compared to that of titanocene dichloride [21].
Fig. 2. Representative graphs showing survival of 8505C, A549, A2780, DLD-1 and FaDu cells grown for 96 h in the presence of increasing concentrations of the studied compounds
(8e11). Standard deviations (all of them less than 10%) are omitted for clarity.

J. Ceballos-Torres et al. / Journal of Organometallic Chemistry 700 (2012) 188e193
193
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4. Conclusions and Outlook
New naphthyl-substituted titanocene(IV) dichloride complexes
have been synthesized and characterized. The molecular structure
of 9 is described. The cytotoxic activity of these compounds has
been tested against human tumour cell lines observing a dose-
dependent cytotoxicity of all the studied compounds. Complex 11
showed the highest cytotoxic activity on all studied cell lines (IC50
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Acknowledgements
We gratefully acknowledge financial support from the Minis-
terio de Educación y Ciencia, Spain (Grant no. CTQ2008-05892/
BQU), the Ministerium für Wirtschaft und Arbeit des Landes
Sachsen-Anhalt, Germany (Grant No. 6003368706) and the Euro-
pean Union (Erasmus Program for J. C.-T.).
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(b) G.N. KaluCerovic, D. Pérez-Quintanilla, I. Sierra, S. Prashar, I. del Hierro,
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Z. Zizak, Z.D. Juranic, M. Fajardo, S. Gómez-Ruiz, J. Mater. Chem. 20 (2010) 806;
CCDC 837118 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
ꢁ ꢁ
ꢁ
(c) D. Pérez-Quintanilla, S. Gómez-Ruiz, Z. Zizak, I. Sierra, S. Prashar, I. del Hierro,
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