Titanium(IV) carboxylate complexes: Synthesis, structural characterization and cytotoxic activity

Article abstract of DOI:10.1016/j.poly.2009.05.068

Four titanium(IV) carboxylate complexes [Ti(η⁵-C₅H₅)₂ (O₂CCH₂SMes)₂] (1), [Ti(η⁵-C₅H₄Me)₂ (O₂CCH₂SMes)₂] (2), [Ti(η⁵-C₅H₅)(η⁵ -C₅H₄SiMe₃)(O₂CCH₂SMes )₂] (3) and [Ti(η⁵-C₅Me₅)(O₂CCH ₂SMes)₃] (4; Mes = 2,4,6-Me₃C₆H₂) have been synthesised by the reaction of the corresponding titanium derivatives [Ti(η⁵-C₅H₅)₂Cl ₂], [Ti(η⁵-C₅H₄Me) ₂Cl₂], [Ti(η⁵-C₅H₅)(η⁵ -C₅H₄SiMe₃)Cl₂] and [Ti(η⁵-C₅Me₅)Cl₃] and two (for 1-3) or three (for 4) equivalents of mesitylthioacetic acid. Complexes 1-4 have been characterized by spectroscopic methods and the molecular structure of the complexes 1, 2 and 4 have been determined by X-ray diffraction studies. The cytotoxic activity of 1-4 was tested against tumor cell lines human adenocarcinoma HeLa, human myelogenous leukemia K562, human malignant melanoma Fem-x, and normal immunocompetent cells, that is peripheral blood mononuclear cells PBMC and compared with those of the reference complexes [Ti(η⁵-C₅H₅)₂Cl ₂] (R1), [Ti(η⁵-C₅H₄Me)₂Cl ₂] (R2), [Ti(η⁵-C₅H₅)(η⁵ -C₅H₄SiMe₃)Cl₂] (R3) and cisplatin. In all cases, the cytotoxic activity of the carboxylate derivatives was higher than that of their corresponding dichloride analogues, indicating a positive effect of the carboxylato ligand on the final anticancer activity. Complexes 1-4 are more active against K562 (IC₅₀ values from 72.2 to 87.9 μM) than against HeLa (IC₅₀ values from 107.2 to 142.2 μM) and Fem-x cells (IC₅₀ values from 90.2 to 191.4 μM).

Full text of DOI:10.1016/j.poly.2009.05.068

Polyhedron 29 (2010) 354–360
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Titanium(IV) carboxylate complexes: Synthesis, structural characterization and
cytotoxic activity
b
c
b
c
Santiago Gómez-Ruiz a,b, , Beatriz Gallego , Zeljko Zizak , Evamarie Hey-Hawkins , Zorica D. Juranic ,
*
ˇ
ˇ
ˇ
´
d,e,
*
´
Goran N. Kaluderovic
a Departamento de Química Inorgánica y Analítica, E.S.C.E.T., Universidad Rey Juan Carlos, 28933 Móstoles, Madrid, Spain
b Institut für Anorganische Chemie der Universität Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
c Institute of Oncology and Radiology of Serbia, 11000 Belgrade, Serbia
d Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle, Germany
e Department of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 14, 11000 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 June 2009
Fourtitanium(IV)carboxylate complexes[Ti(
g
5-C5H5)2(O2CCH2SMes)2] (1), [Ti(
g
5-C5H4Me)2(O2CCH2SMes)2]
(2), [Ti(
2,4,6-Me3C6H2) have been synthesised by the reaction of the corresponding titanium derivatives
[Ti(
5-C5H5)2Cl2], [Ti( 5-C5H4Me)2Cl2], [Ti( 5-C5H5)( 5-C5H4SiMe3)Cl2] and [Ti( 5-C5Me5)Cl3] and two
g g g
5-C5H5)( 5-C5H4SiMe3)(O2CCH2SMes)2] (3) and [Ti( 5-C5Me5)(O2CCH2SMes)3] (4; Mes =
Keywords:
g
g
g
g
g
Anticancer drugs
Titanocene complexes
Cytotoxic activity
Carboxylato ligands
(for 1–3) or three (for 4) equivalents of mesitylthioacetic acid. Complexes 1–4 have been characterized
by spectroscopic methods and the molecular structure of the complexes 1, 2 and 4 have been determined
by X-ray diffraction studies. The cytotoxic activity of 1–4 was tested against tumor cell lines human
adenocarcinoma HeLa, human myelogenous leukemia K562, human malignant melanoma Fem-x, and
normal immunocompetent cells, that is peripheral blood mononuclear cells PBMC and compared with
those of the reference complexes [Ti(
g g g
5-C5H5)2Cl2] (R1), [Ti( 5-C5H4Me)2Cl2] (R2), [Ti( 5-C5H5)
(
g
5-C5H4SiMe3)Cl2] (R3) and cisplatin. In all cases, the cytotoxic activity of the carboxylate derivatives
was higher than that of their corresponding dichloride analogues, indicating a positive effect of the
carboxylato ligand on the final anticancer activity. Complexes 1–4 are more active against K562 (IC50
values from 72.2 to 87.9
(IC50 values from 90.2 to 191.4
l
M) than against HeLa (IC50 values from 107.2 to 142.2
M).
lM) and Fem-x cells
l
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
addition, there are other interesting studies that report potential
binding of
[25,26].
a ligand-bound titanium(IV) complex to proteins
Since the pioneering work of Köpf and Köpf-Maier [1–3] and the
phase I clinical trials carried out for titanocene dichloride in 1993
[4–8], metallocene and non-metallocene titanium(IV) complexes
have been studied intensively due to their anticancer properties
[9–15], even though clinical trials in patients did not have a suc-
cessful outcome [16,17].
Mechanistic studies have indicated that one of the possible
pathways is that titanium may reach the cells assisted by the major
iron transport protein ‘‘transferrin” [18–21], binding to DNA which
may be the biological target of these complexes [22–24]. In
Thus, one of the most active fields in titanium(IV) anticancer
chemistry is focused in the design of new compounds with differ-
ent substituents which may increase their cytotoxicity in compar-
ison with that of titanocene dichloride [9–15,27–29]. It has been
observed that titanocene complexes which present polar substitu-
ents or electron withdrawing groups in their structure, present
higher in vitro anticancer activity [11]. In addition, we have re-
cently reported an increase of the cytotoxicity in titanocene and
ansa-titanocene complexes that have pendant alkenyl substituents
on the cyclopentadienyl rings [27–29].
However, almost all the studied titanium(IV) complexes are
dichloride derivatives and the number of studies on the anticancer
activity of titanium(IV) alkoxo or carboxylate derivatives is still rel-
atively small [10,30–37].
In view of our good results on the synthesis, characterization
and evaluation of the cytotoxic activity of metal carboxylate
complexes [38–40], here we present the synthesis, characterization
* Corresponding authors. Addresses: Departamento de Química Inorgánica
y
Analítica, E.S.C.E.T., Universidad Rey Juan Carlos, Móstoles (Madrid), Spain (S.
Gómez-Ruiz); Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg,
Kurt-Mothes-Straße 2, D-06120 Halle, Germany (G.N. Kaluderovic´). Fax: +34
914888143 (S. Gómez-Ruiz), tel.: +49 345 5525678; fax: +49 345 5527028 (G.N.
´
Kaluderovic).
E-mail addresses: santiago.gomez@urjc.es (S. Gómez-Ruiz), goran.kaluderovic@
chemie.uni-halle.de, goran@chem.bg.ac.rs (G.N. Kaluderovic´).
0277-5387/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2009.05.068

S. Gómez-Ruiz et al. / Polyhedron 29 (2010) 354–360
355
and study of the influence of the substituents on the
cyclopentadienyl ring and the carboxylato ligand in the stability
and cytotoxicity of four new titanium(IV) carboxylate complexes.
tyl), 129.3 (C-3 and C-5 of mesityl), 138.3 (C-2 and C-6 of mesi-
tyl), 143.0 (C-1 of mesityl), 186.2 (COO); FAB MS: m/z (%) 625 (1)
[M+], 545 (13) [M+ÀC5H4Me], 415 (100) [M+ÀOOCCH2SMes]. Anal.
Calc. for C34H40O4S2Ti: C, 65.37; H, 6.45. Found: C, 64.88; H,
6.31%.
2. Experimental
2.4. Preparation of [Ti(g g
5-C5H5)( 5-C5H4SiMe3)(O2CCH2SMes)2] (3)
2.1. General manipulations
The synthesis of 3 was carried out in an identical manner to 1
All reactions were performed using standard Schlenk tube
techniques in an atmosphere of dry nitrogen. Solvents were dis-
tilled from the appropriate drying agents and degassed before
use. Na(C5H5) and Na(C5H4Me) were prepared according to liter-
starting from mesitylthioacetic acid [42] (0.84 g, 4.02 mmol),
[Ti(g g
5-C5H5)( 5-C5H4SiMe3)Cl2] (0.64 g, 2.01 mmol) and NEt3
(0.58 mL, 4.02 mmol). Yield: 0.94 g, 69%. FT-IR (KBr): 1645 (s) (ta
COOÀ), 1335 (s) (ts COOÀ); 1H NMR (400 MHz, CDCl3, 25 °C): d
0.12 (s, 9H, SiMe3), 2.16 (s, 6H, p-Me of mesityl), 2.47 (s, 12H, o-
Me of mesityl), 3.34 (s, 4H, CH2), 6.24 (s, 5H, C5H5), 6.51, 6.55 (m,
2H each, C5H4SiMe3), 6.84 (s, 4H, m-H of mesityl); 13C{1H} NMR
(100.6 MHz, CDCl3, 25 °C): d 5.2 (SiMe3), 21.0 (p-Me of mesityl),
21.8 (o-Me of mesityl), 38.8 (CH2), 102.9, 113.7, 114.3 (C5H4SiMe3),
118.3 (C5H5), 128.9 (C-4 of mesityl), 129.2 (C-3 and C-5 of mesityl),
138.6 (C-2 and C-6 of mesityl), 142.9 (C-1 of mesityl), 186.0 (COO);
FAB MS: m/z (%) 669 (1) [M+], 603 (5) [M+ÀC5H5], 531 (6)
[M+ÀC5H4SiMe3], 459 (100) [M+ÀOOCCH2SMes]. Anal. Calc. for
C35H44O4S2SiTi: C, 62.85; H, 6.63. Found: C, 62.69; H, 6.59%.
ature procedures [41]. Li(C5H4SiMe3) [42], [Ti(
and [Ti(
5-C5Me5)Cl3] [44] were prepared as previously reported.
[Ti(
5-C5H5)2Cl2] (R1) and [Ti( 5-C5H4Me)2Cl2] (R2) were syn-
thesised by the reaction of two equivalents of Na(C5H5) or
g
5-C5H5)Cl3] [43]
g
g
g
Na(C5H4Me) with [TiCl4(THF)2], respectively. [Ti(
g
5-C5H5)(g5
-
-
C5H4SiMe3)Cl2] (R3) was prepared by the reaction of [Ti(g5
C5H5)Cl3] and Li(C5H4SiMe3). Mesitylthioacetic acid was prepared
with slight modification of the literature procedure [45]. IR spec-
tra (KBr pellets prepared in a nitrogen-filled glove box) were re-
corded on a Perkin–Elmer System 2000 FT-IR spectrometer in the
range 350–4000 cmÀ1 1H and 13C{1H} NMR spectra were re-
.
2.5. Preparation of [Ti(g
5-C5Me5)(O2CCH2SMes)3] (4)
corded on a Varian Mercury FT-400 spectrometer or on a Bruker
AVANCE-400 and referenced to the residual deuterated solvent.
Microanalyses were carried out with a Perkin–Elmer 2400 or
LECO CHNS-932 microanalyzer. FAB MS spectra were recorded
A solution of mesitylthioacetic acid [42] (0.63 g, 3.00 mmol) in
toluene (70 mL) was added dropwise over 15 min to a solution of
[Ti(g
5-C5Me5)3Cl] (0.29 g, 1.00 mmol) in toluene (30 mL) at room
on
a MASPEC II spectrometer with 3-nitrobenzylalcohol as
matrix.
temperature. The reaction mixture was stirred for 20 min, subse-
quently NEt3 (0.43 mL, 3.00 mmol) was added dropwise. The reac-
tion was then stirred at room temperature overnight. The mixture
was decanted and filtered and the filtrate evaporated to dryness.
The resulting yellow oily solid was dissolved in diethyl ether
(30 mL), filtered, concentrated to 10 mL and cooled to À30 °C to
give crystals of the title complex which were isolated by filtration.
Yield: 0.61 g, 76%. FT-IR (KBr): 1556 (s) 1531 (s) (ta COOÀ), 1451
(vs) (ts COOÀ); 1H NMR (400 MHz, CDCl3, 25 °C): d 1.92 (s, 15H,
C5Me5), 2.13 (s, 9H, p-Me of mesityl), 2.51 (s, 18H, o-Me of mesityl),
3.30 (s, 6H, CH2), 6.90 (s, 6H, m-H of mesityl); 13C{1H} NMR
(100.6 MHz, CDCl3, 25 °C): d 11.3 (C5Me5), 20.9 (p-Me of mesityl),
21.9 (o-Me of mesityl), 38.6 (CH2), 129.1 (C-4 of mesityl), 132.6
(C-3 and C-5 of mesityl), 135.7 (C5Me5), 138.3 (C-2 and C-6 of mesi-
tyl), 143.0 (C-1 of mesityl), 186.5 (COO); FAB MS: m/z (%) 811 (1)
[M+], 601 (100) [M+ÀOOCCH2SMes], 544 (35) [M+ÀOOCCH2SMes
– 4 Â Me]. Anal. Calc. for C43H54O6S3Ti: C, 63.69; H, 6.71. Found:
C, 63.45; H, 6.56%.
2.2. Preparation of [Ti(g
5-C5H5)2(O2CCH2SMes)2] (1)
A solution of mesitylthioacetic acid [42] (0.84 g, 4.02 mmol) in
toluene (50 mL) was added dropwise over 15 min to a solution of
[Ti(g
5-C5H5)2Cl2] (0.50 g, 2.01 mmol) in toluene (50 mL) at room
temperature. The reaction mixture was stirred for 20 min, subse-
quently NEt3 (0.58 mL, 4.02 mmol) was added dropwise. The
reaction was then stirred at 80 °C overnight. The mixture was
decanted and filtered and the filtrate concentrated (10 mL) and
cooled to À30 °C to give crystals of the title complex which were
isolated by filtration. Yield: 0.51 g, 43%. FT-IR (KBr): 1635 (s) (ta
COOÀ), 1334 (s) (ts COOÀ); 1H NMR (400 MHz, CDCl3, 25 °C): d
2.23 (s, 6H, p-Me of mesityl), 2.55 (s, 12H, o-Me of mesityl),
3.38 (s, 4H, CH2), 6.33 (s, 10H, C5H5), 6.92 (s, 4H, m-H of mesi-
tyl); 13C{1H} NMR (100.6 MHz, CDCl3, 25 °C): d 21.4 (p-Me of
mesityl), 22.0 (o-Me of mesityl), 41.6 (CH2), 118.5 (C5H5), 129.1
(C-4 of mesityl), 129.3 (C-3 and C-5 of mesityl), 138.4 (C-2 and
C-6 of mesityl), 142.9 (C-1 of mesityl), 186.4 (COO); FAB MS:
m/z (%) 596 (1) [M+], 531 (5) [M+ÀC5H5], 403 (8)
[M+ÀCOCH2SMes], 387 (100) [M+ÀOOCCH2SMes]. Anal. Calc. for
C32H36O4S2Ti: C, 64.42; H, 6.08. Found: C, 64.00; H, 6.01%.
2.6. Data collection and structural refinement of 1, 2 and 4
The data of 1, 2 and 4 were collected with a CCD Oxford Xcalibur
S (k(Mo Ka) = 0.71073 Å) using x and u scans mode. Semi-empir-
ical from equivalents absorption corrections were carried out with
SCALE3 ABSPACK [46]. All the structures were solved by direct methods
[47]. Structure refinement was carried out with SHELXL-97 [48]. All
non-hydrogen atoms were refined anisotropically, and hydrogen
atoms were calculated with the riding model and refined isotropi-
cally. Table 1 lists crystallographic details.
2.3. Preparation of [Ti(g
5-C5H4Me)2(O2CCH2SMes)2] (2)
The synthesis of 2 was carried out in an identical manner to 1
starting from mesitylthioacetic acid [42] (0.84 g, 4.02 mmol),
[Ti(g
5-C5H4Me)2Cl2] (0.56 g, 2.01 mmol) and NEt3 (0.58 mL,
4.02 mmol). Yield: 0.36 g, 55%. FT-IR (KBr): 1648 (s) (ta COOÀ),
1332 (s) (ts COOÀ); 1H NMR (400 MHz, CDCl3, 25 °C): d 1.96 (s,
6H, Me of Cp), 2.17 (s, 6H, p-Me of mesityl), 2.47 (s, 12H, o-Me
of mesityl), 3.34 (s, 4H, CH2), 6.05, 6.28 (m, 4H each, C5H4Me),
6.84 (s, 4H, m-H of mesityl); 13C{1H} NMR (100.6 MHz, CDCl3,
25 °C): d 15.7 (Me of Cp), 20.9 (p-Me of mesityl), 21.9 (o-Me of
mesityl), 39.9 (CH2), 113.4, 115.7, 121.7 (Cp), 129.0 (C-4 of mesi-
2.7. In vitro studies
2.7.1. Preparation of complex solutions
Stock solutions of the studied titanium complexes were made in
dimethyl sulfoxide (DMSO) at a concentration of 20 mM, filtered
through Millipore filter, 0.22
ent medium to various working concentrations. DMSO was used
lm, before use, and diluted by nutri-

356
S. Gómez-Ruiz et al. / Polyhedron 29 (2010) 354–360
Table 1
dient centrifugation. Interface cells, washed three times with
Haemaccel (aqueous solution supplemented with 145 mM Na+,
5.1 mM K+, 6.2 mM Ca2+, 145 mM ClÀ and 35 g/L gelatine polymers,
pH 7.4) were counted and resuspended in nutrient medium.
Crystallographic data for 1, 2 and 4.
1
2
4
Formula
Fw
T (K)
Crystal system
Space group
a (Å)
C32H36O4S2Ti
596.63
130(2)
monoclinic
P2(1)/c
13.833(5)
7.979(5)
26.555(5)
90
97.499(5)
90
C34H40O4S2Ti
624.68
130(2)
monoclinic
P2(1)/c
1.3197(5)
7.934(5)
28.594(5)
90
95.286(5)
90
2.98(1)
4
C43H54O6S3Ti
810.94
130(2)
monoclinic
P2(1)
8.20190(10)
17.8397(3)
14.2823(2)
90
95.758(2)
90
2.07923(5)
2
1.295
0.402
860
2.7.3. Cell sensitivity analysis
HeLa and Fem-x (2000 cells per well) were seeded into 96-well
microtitre plates and 20 h later, after the cell adherence, five differ-
ent concentrations of the studied compounds were added to the
wells. Final concentrations were in the range from 12.5 to
b (Å)
c (Å)
a
(°)
200 lM. The studied compounds were added to a suspension of
b (°)
leukemia K562 cells (3000 cells per well) 2 h after cell seeding, in
the same final concentrations applied to HeLa and Fem-x cells.
All experiments were carried out in triple triplicate. PBMC were
seeded (150000 cells per well) in nutrient medium enriched with
c
(°)
V (nm3)
Z
2.91(1)
4
1.364
0.474
Dc (Mg mÀ3
l
)
1.392
0.466
1320
(mmÀ1
)
(5 lg/mL) phytohaemaglutinin (PHA – Welcome Diagnostics, Eng-
F (0 0 0)
Crystal dimension (mm)
h range (°)
hkl ranges
1256
land) in 96-well microtitre plates and 2 h later, the studied com-
pounds were added to the wells, in triplicate, to five final
concentrations. Only nutrient medium was added to the cells in
the control wells. Nutrient medium with corresponding concentra-
tions of compounds, but void of cells was used as blank.
0.4 Â 0.3 Â 0.2
2.67–28.28
À18 6 h 6 18
À10 6 k 6 10
À35 6 l 6 35
7207/358/0
0.35 Â 0.03 Â 0.02 0.3 Â 0.1 Â 0.04
2.67–25.35
À15 6 h 6 15
À9 6 k 6 9
2.70–26.02
À10 6 h 6 10
À22 6 k 6 21
À17 6 l 6 17
8177/492/1
À34 6 l 6 34
5453/378/0
Data/parameters/
restraints
Goodness-of-fit on F2
0.823
0.833
0.821
2.7.4. Determination of target cell survival
Final R indices [I > 2
r
(I)]
R1 = 0.0322,
wR2 = 0.0564
R1 = 0.0801,
wR2 = 0.0615
R1 = 0.0486,
wR2 = 0.0657
R1 = 0.1313,
wR2 = 0.0788
R1 = 0.0353,
wR2 = 0.0542
R1 = 0.0566,
wR2 = 0.0570
Cell survival was determined by MTT test according to the
method of Mosmann [49] and modified by Ohno and Abe [50],
72 h after complex addition. Immediately afterwards, 20 lL of
MTT solution (5 mg/mL in phosphate buffered saline) was added
to each well. Samples were incubated for a further 4 h at 37 °C in
R indices (all data)
Largest difference in peak 0.233 and
0.448 and À0.396 0.366 and
and hole (e ÅÀ3
)
À0.327
À0.265
Absolute structure
parameter
À0.010(17)
a humidified atmosphere with 5% CO2. Then, 100 lL of 10% SDS
was added to the wells. Absorbance was measured at 570 nm the
next day. To achieve cell survival percentages, absorbance at
570 nm of a sample with cells grown in the presence of various
concentrations of agent was divided with absorbance of control
sample (the absorbance of cells grown only in nutrient medium),
having subtracted from absorbance of a corresponding sample
with target cells the absorbance of the blank.
due to solubility problems. Nutrient medium was RPMI 1640
medium, without phenol red, supplemented with L-glutamine
(3 mM), streptomycin (100 lg/mL), penicillin (100 IU/mL), 10% fe-
tal bovine serum (FBS) and 25 mM Hepes, and was adjusted to pH
7.2 by bicarbonate solution. MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) was dissolved (5 mg/mL) in
phosphate buffer saline pH 7.2, and filtered through Millipore fil-
3. Results and discussion
3.1. Synthesis and characterization of the titanium(IV) complexes 1–4
ter, 0.22
lm, before use. All reagents were purchased from Sigma
Chemicals.
Titanium(IV) carboxylate complexes [Ti(
(1), [Ti(
5-C5H4Me)2(O2CCH2SMes)2] (2) and [Ti(
C5H4SiMe3)(O2CCH2SMes)2] (3) were synthesised by the reaction
of the corresponding titanium derivatives [Ti(
5-C5H5)2Cl2],
[Ti(
5-C5H4Me)2Cl2] and [Ti( 5-C5H5)( 5-C5H4SiMe3)Cl2], respec-
tively, and two equivalents of mesitylthioacetic acid in toluene at
80 °C (Scheme 1). Analogously, [Ti(
5-C5Me5)(O2CCH2SMes)3] (4)
was prepared by the reaction of one equivalent of [Ti(g5
C5Me5)Cl3] and three equivalents of mesitylthioacetic acid in
g
5-C5H5)2(O2CCH2SMes)2]
5-C5H5)(g5
g
g
-
2.7.2. Cell culture
Human cervix adenocarcinoma HeLa and malignant melanoma
Fem-x cells were cultured as monolayers in the nutrient medium,
while human myelogenous leukemia K562 cells were maintained
as suspension culture. The cells were grown at 37 °C in 5% CO2
and humidified air atmosphere. Peripheral blood mononuclear
cells (PBMC) were separated from whole heparinized blood from
healthy volunteers by Lymphoprep (Nycomed, Oslo, Norway) gra-
g
g
g
g
g
-
Scheme 1. Synthesis of 1–3.

S. Gómez-Ruiz et al. / Polyhedron 29 (2010) 354–360
357
Scheme 2. Synthesis of 4.
toluene at room temperature (Scheme 2). Complexes 1–4 were
isolated as orange (1–3) or yellow (4) crystalline solids of high
purity.
These structural parameters are similar to those reported for
other titanocene carboxylate complexes with monodentate coordi-
nation [52–57].
In the 1H NMR spectra of 1–4, the resonances of the carboxylato
ligand were observed as four signals at different chemical shifts.
Two singlets at ca. 2.2 and 2.5 corresponding to the protons of
the methyl groups of the mesityl moiety, one slightly broad singlet
at ca. 3.3 assigned to the methylene protons and one singlet at
6.9 ppm for the aromatic protons. In addition to these signals,
the protons of the cyclopentadienyl ligands show the expected dif-
ferent patterns for the four different complexes.
Table 2
Selected bond lengths (Å) and angles (°) for 1, 2 and 4.
1
2
4
Ti(1)–Cent(1)
Ti(1)–Cent(2)
Ti(1)–O(1)
Ti(1)–O(2)
Ti(1)–O(3)
Ti(1)–O(4)
Ti(1)–O(5)
Ti(1)–O(6)
O(1)–C(11)
O(2)–C(11)
O(1)–C(13)
O(2)–C(13)
O(3)–C(22)
O(4)–C(22)
O(3)–C(24)
O(4)–C(24)
O(5)–C(33)
O(6)–C(33)
2.042
2.053
1.956(1)
3.556
1.943(1)
3.480
2.035
2.025
1.907(2)
3.637
1.904(2)
3.722
2.063
2.173(2)
2.143(2)
2.161(2)
2.099(2)
2.178(2)
2.145(2)
126.6(3)
125.9(3)
The 13C{1H} NMR spectra of 1–4 show eight signals correspond-
ing to the carboxylato ligand. A signal at ca. 40 ppm corresponding
to the carbon atom of the methylene group and six signals, two be-
tween ca. 20 and 23 ppm and four between 128 and 144 ppm, cor-
responding to the carbon atoms of the mesityl moiety, finally the
signal assigned to the carbon atom of the COO moiety is observed
at ca. 186 ppm. In addition, the 13C{1H} NMR spectra of 1–4 show
the expected signals for the Cp ligands.
The IR spectra of the complexes 1–3 show strong bands in two
different regions between 1648–1635 and 1335–1332 cmÀ1, which
correspond to the asymmetric and symmetric vibrations, respec-
tively, of the COO moiety. The differences, in all cases of more than
200 cmÀ1, between the asymmetric and symmetric vibrations,
indicate monodentate coordination of the carboxylato ligand
[51]. This phenomenon was also confirmed by single crystal
X-ray diffraction studies (see Section 3.2). In contrast, IR spectrum
of complex 4 show two strong bands at 1556 and 1451 assigned to
the asymmetric and symmetric vibrations of the COO moiety, in
this case the difference of less than 200 cmÀ1, indicate bidentate
coordination of the carboxylato ligand [51].
1.287(2)
1.219(2)
1.271(4)
1.200(4)
1.299(2)
1.211(2)
125.9(3)
126.4(3)
1.283(4)
1.190(4)
125.9(3)
126.1(3)
Cent(1)–Ti(1)–Cent(2)
Cent(1)–Ti(1)–O(1)
Cent(1)–Ti(1)–O(2)
Cent(1)–Ti(1)–O(3)
Cent(1)–Ti(1)–O(4)
Cent(1)–Ti(1)–O(5)
Cent(1)–Ti(1)–O(6)
Cent(2)–Ti(1)–O(1)
Cent(2)–Ti(1)–O(2)
Cent(2)–Ti(1)–O(3)
Cent(2)–Ti(1)–O(4)
O(1)–Ti(1)–O(2)
O(1)–Ti(1)–O(3)
O(1)–Ti(1)–O(4)
O(1)–Ti(1)–O(5)
O(1)–Ti(1)–O(6)
O(2)–Ti(1)–O(3)
O(2)–Ti(1)–O(4)
O(2)–Ti(1)–O(5)
O(2)–Ti(1)–O(6)
O(3)–Ti(1)–O(4)
O(3)–Ti(1)–O(5)
O(3)–Ti(1)–O(6)
O(4)–Ti(1)–O(5)
O(4)–Ti(1)–O(6)
O(5)–Ti(1)–O(6)
O(1)–C(11)–O(2)
O(1)–C(13)–O(2)
O(3)–C(22)–O(4)
O(3)–C(24)–O(4)
O(5)–C(33)–O(6)
131.1
106.6
92.2
108.5
91.9
133.0
104.3
104.3
107.6
84.2
101.9
106.8
167.0
106.6
102.7
105.3
107.8
97.4
104.7
94.8
33.9
90.74(5)
126.3
106.7
86.5
105.3
115.5
30.2
Complexes 1–4 were also characterized by FAB MS. The mass
spectra showed the molecular ion peaks. Fragments indicative of
the loss of different number of carboxylato or cyclopentadienyl li-
gands were also observed (see Section 2).
60.28(6)
75.99(6)
73.46(6)
143.53(6)
132.71(6)
82.71(6)
129.45(7)
131.19(7)
75.10(6)
61.35(6)
75.20(6)
84.07(6)
75.04(6)
129.12(6)
59.92(6)
118.3(2)
92.7(1)
111.0
3.2. Structural studies
124.6
159.8
120.2
141.1
Molecular structure of complexes 1, 2 and 4 have been deter-
mined by X-ray diffraction studies. Selected bond lengths and an-
gles of 1, 2 and 4 are given in Table 2.
35.8
26.5
Molecular structure of 1 (Fig. 1) and 2 (Fig. 2) confirm the bent
metallocene conformation of these complexes. Titanium atoms are
in a distorted tetrahedral environment and present two O-mono-
dentate carboxylate groups [Ti–O 1.956(1) and 1.943(1) Å for 1
and 1.907(2) and 1.904(2) Å for 2]. These distances are in agree-
ment with the monodentate coordination indicated by the large
difference of about 300 cmÀ1 between the asymmetric and
symmetric vibration of the COO moiety in the IR spectra (see
Section 3.1).
124.7(2)
124.2(3)
126.6(3)
124.6(4)
118.9(2)
117.9(2)

358
S. Gómez-Ruiz et al. / Polyhedron 29 (2010) 354–360
Fig. 1. Molecular structure and atom-labelling scheme for 1 with thermal ellipsoids
at 50% probability (hydrogen atoms are omitted for clarity).
Fig. 3. Molecular structure and atom-labelling scheme for 4 with thermal ellipsoids
at 50% probability (hydrogen atoms are omitted for clarity).
The pentagon formed by O(1)–O(2)–O(4)–O(5) and O(6) is a bit dis-
torted and not regular as a consequence of the different distances
and angles O–Ti–O. The axial positions of this pentagonal bipyra-
midal geometry are occupied by the centroid of the cyclopentadi-
enyl ring and O(3) with an Cent(1)–Ti–O(3) angle of 168.0°.
3.3. Cytotoxic studies
Titanocene complexes from this study 1–4 have been used in
order to understand the possible relationship between the differ-
ent substituents, the auxiliary ligands (carboxylato or chloride)
and the cytotoxic activity.
The in vitro cytotoxicities of complexes 1–4 against human ade-
nocarcinoma HeLa, human malignant melanoma Fem-x, human
myelogenous leukemia K562 and on normal immunocompetent
cells (human peripheral blood mononuclear cells, PBMC) were
determined by MTT-based assays (Table 3).
Fig. 2. Molecular structure and atom-labelling scheme for 2 with thermal ellipsoids
The investigated titanocene anti-tumor agents showed a dose-
dependent antiproliferative effect toward all cell lines and on hu-
man PBMC and stimulated PBMC. Estimations based on the IC50
values show that complexes 1–4 are more active against K562
at 50% probability (hydrogen atoms are omitted for clarity).
The distortion from regular geometry is evident from the Cent–
Ti–Cent angles of 131.1° for 1 and 133.0 for 2, and the O(1)–Ti–O(3)
angle of 90.74(5) for 1 and 92.7(1) for 2. The distances between
titanium atoms and the two non bound carboxylate oxygens,
O(2) and O(4), are longer than 3.5 Å, indicating no interaction.
Monodentate coordination of the carboxylato ligand is also con-
firmed by the significant differences in C–O bond lengths depend-
ing whether the oxygen is coordinated to titanium (between
1.271(4) and 1.299(2) Å) or not (between 1.190(4) and 1.219(2) Å).
Both carboxylate groups are bound to titanium atoms in slightly
different manner, one carboxylato ligand has a slightly longer Ti–O
bond and a smaller Ti–O–C angle. This may imply the presence of a
(IC50 values from 72.2 to 87.9
from 107.2 to 142.2 M) and Fem-x cells (IC50 values from 90.2
to 191.4 M).
lM) than against HeLa (IC50 values
l
l
From all the studied complexes, 3 and 4 present the highest
cytotoxic activity. The results corresponding to complex 4 were to-
tally unexpected, because monocyclopentadienyl titanium(IV)
complexes have been usually discarded as potential anticancer
drugs due to possible hydrolysis problems. Thus, with the observed
cytotoxic activities for complex 4, new ways in the anticancer
differing extent of Ti–O
p-interaction supporting the primary r-
Table 3
bonding for the two carboxylato ligands [55]. The average Ti–C
and C–C bond lengths of the cyclopentadienyl groups in 1 and 2
are in a good agreement with the values from structure determina-
tions of other titanocene derivatives [56–58].
IC50 (lM) for the 72 h of action of the studied compounds and cisplatin on HeLa, Fem-
x, K562 cells, on PBMC and PBMC stimulated with PHA determined by MTT test.
Compound IC50 SD
HeLa
Fem-x
K562
PBMC–PHA PBMC + PHA
Interestingly, the X-ray structure of the complex 4 (Fig. 3)
shows the bidentate coordination of the three carboxylato ligands.
The Ti–O bond lengths are all similar between 2.099(2) and
2.178(2) Å. In addition the distances C–O of the carboxylato ligands
are also very close with values of ca. 1.26 Å, indicating some multi-
ple bond character.
1
2
3
4
142.2 5.8
139.4 12.7 191.4 5.5 78.2 0.7
107.2 6.9
117.4 8.1
>200
>200
>200
4.4 0.3
164.9 9.4 86.8 0.3
146.2 3.8
162.0 3.7
104.6 5.3
132.9 0.6
>200
148.0 1.3
156.1 7.4
116.8 11.7
127.3 1.4
199.8 9.9
180.9 4.3
>200
90.2 6.8
87.9 3.6
123.0 5.2 72.2 1.7
177.7 4.9 >200
198.6 4.3 173.3 6.0 >200
R1
R2
R3
Cisplatin
>200
>200
>200
33.6
The geometry around titanium is
a distorted pentagonal
5.7 0.3
4.7 0.3
26
6
bipyramid and the metal attains the 18-electron configuration.

S. Gómez-Ruiz et al. / Polyhedron 29 (2010) 354–360
359
chemistry of monocyclopentadienyl titanium(IV) complexes can
be made out.
from the Ministerio de Educación y Ciencia, Spain (Grant No.
CTQ2008-05892/BQU) and Ministry of Science and Technological
Development of the Republic of Serbia (Grant Nos. 142010 and
145006).
In order to determine the selectivity in the in vitro cytotoxicity
of 1–4, some additional experiments were conducted on normal
and stimulated PBMC cells. Generally, complexes 1–4 showed mar-
ginal or no selectivity on HeLa or Fem-x cell lines, however, slight
selectivity is observed on K562 cells, especially with complex 2.
Titanocene complexes 1–4 are in all cases more active than
the reference complexes R1, R2 and R3, indicating that the
substitution of the chlorides by carboxylato ligands has a positive
effect on the anticancer activity of the studied complexes.
Carboxylate complexes 1–4 are about ca. 1.5–3 times more active
than the chloride derivatives R1, R2 and R3. In view of the
difference of the cytotoxic activity, it seems that the carboxylate
derivatives (or their corresponding hydrolysis product) present
either higher affinity to the binding to transferrin or albumin
[25,26], or higher stability to the decomposition in the biological
medium, which leads to a more effective action against the tu-
mor cell.
The cytotoxic activities of complexes 1–4 are not as high as the
activity reported by Tacke and coworkers in their oxali-titanocene
derivatives [35,36], however, they are comparable to those de-
scribed for other titanium(IV) carboxylate complexes [10,37].
On direct comparison with cisplatin, the cytotoxic activity of
complexes 1–4 is significantly lower, however, a higher tolerance
of relatively high titanium amounts in biological systems may be
possible, in comparison with the high number of side-effects asso-
ciated to very low concentrations of platinum. This may make
these results very promising for further experiments which will
be focused on the modification of the nature and number of car-
boxylato and cyclopentadienyl ligands, in order to find titanocene
complexes with higher cytotoxic activities.
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ˇ
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Four different titanium(IV) carboxylate complexes have been
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activity of monocyclopentadienyl titanium(IV) complex 4, opens
up the possibility on further investigations on this kind of
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