Synthesis and cytotoxicity studies of methoxy benzyl substituted titanocenes

Article abstract of DOI:10.1016/j.jorganchem.2007.11.037

From the reaction of 6(2-methoxy-phenyl)fulvene (1a), 6(3-methoxy-phenyl)fulvene (1b), 6(3,4-dimethoxy-phenyl)fulvene (1c) and 6(3,4,5-trimethoxy-phenyl)fulvene (1d) with LiBEt₃H, lithiated cyclopentadienide intermediates 2a-d were synthesised. These intermediates were then transmetallated to titanium with TiCl₄ to give benzyl substituted titanocenes bis-[(2-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3a), bis-[(3-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3b), bis-[(3,4-dimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3c) and bis-[(3,4,5-trimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3d). The three titanocenes 3a-c were characterised by single crystal X-ray diffraction, while the structure of the fourth titanocene 3d was elucidated through a DFT calculation. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in order to determine their IC₅₀ values. Titanocenes 3a-d were found to have IC₅₀ values of 97, 159, 88 and 253 μM, respectively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride.

Full text of DOI:10.1016/j.jorganchem.2007.11.037

Available online at www.sciencedirect.com
Journal of Organometallic Chemistry 693 (2008) 526–536
www.elsevier.com/locate/jorganchem
Synthesis and cytotoxicity studies of methoxy benzyl
substituted titanocenes
*
´
James Claffey, Megan Hogan, Helge Muller-Bunz, Clara Pampillon, Matthias Tacke
¨
Conway Institute of Biomolecular and Biomedical Research, The UCD School of Chemistry and Chemical Biology,
Centre for Synthesis and Chemical Biology (CSCB), University College Dublin, Belfield, Dublin 4, Ireland
Received 5 October 2007; received in revised form 13 November 2007; accepted 22 November 2007
Available online 3 January 2008
Abstract
From the reaction of 6(2-methoxy-phenyl)fulvene (1a), 6(3-methoxy-phenyl)fulvene (1b), 6(3,4-dimethoxy-phenyl)fulvene (1c) and
6(3,4,5-trimethoxy-phenyl)fulvene (1d) with LiBEt3H, lithiated cyclopentadienide intermediates 2a–d were synthesised. These intermediates
were then transmetallated to titanium with TiCl4 to give benzyl substituted titanocenes bis-[(2-methoxy-benzyl)cyclopentadienyl]tita-
nium(IV) dichloride (3a), bis-[(3-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3b), bis-[(3,4-dimethoxy-benzyl)cyclopenta-
dienyl]titanium(IV) dichloride (3c) and bis-[(3,4,5-trimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride (3d). The three
titanocenes 3a–c were characterised by single crystal X-ray diffraction, while the structure of the fourth titanocene 3d was elucidated through
a DFT calculation. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney
epithelial) cell line in order to determine their IC50 values. Titanocenes 3a–d were found to have IC50 values of 97, 159, 88 and 253 lM, respec-
tively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride.
Ó 2007 Elsevier B.V. All rights reserved.
Keywords: Anti-cancer drugs; Cisplatin; Titanocene; Hydridolithiation; Fulvene; RCC; LLC-PK
1. Introduction
patients with metastatic renal cell carcinoma [4] or meta-
static breast cancer [5] was too low to be pursued.
Beyond the field of platinum anti-cancer drugs there is
significant unexplored space for further metal-based drugs
targeting cancer. Titanium-based reagents have significant
potential against solid tumors. Budotitane ([cis-diethoxy-
bis(1-phenylbutane-1,3-dionato)titanium(IV)]) looked very
promising during its preclinical evaluation, but did not go
beyond Phase I clinical trials, although a Cremophor ELÒ
based formulation was found for this rapidly hydrolysing
molecule [1]. Much more robust in this aspect of hydrolysis
is titanocene dichloride (Cp2TiCl2), which shows medium
anti-proliferative activity in vitro but promising results
in vivo [2,3]. Titanocene dichloride reached clinical trials,
but the efficacy of Cp2TiCl2 in Phase II clinical trials in
The field got renewed interest with P. McGowan’s
elegant synthesis of ring-substituted cationic titanocene
dichloride derivatives, which are water-soluble and show
significant activity against ovarian cancer [6]. More recently,
novel methods starting from fulvenes and other precursors
[7,8] allow direct access to antiproliferative titanocenes via
reductive dimerisation with titanium dichloride [9–13],
hydridolithiation [14–17] or carbolithiation [18–26] of the
fulvene followed by transmetallation with titanium tetra-
chloride in the latter two cases.
Hydridolithiation of 6-anisyl fulvene and subsequent
reaction with TiCl4 led to bis-[(p-methoxybenzyl)cyclopen-
tadienyl]titanium(IV) dichloride (Titanocene Y) [14], which
has an IC50 value of 21 lM when tested on the LLC-PK
cell line, which has proven to be a good mimic of a kidney
carcinoma cell line and a reliable tool for the optimisation
of titanocenes against this type of cancer. The structures of
*
Corresponding author.
E-mail address: matthias.tacke@ucd.ie (M. Tacke).
0022-328X/$ - see front matter Ó 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2007.11.037

J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
527
Spectrometer. CHN analysis was done with an Exeter
Analytical CE-440 Elemental Analyser, while Cl was
determined in mercurimetric titrations. Density functional
theory (DFT) calculations were carried out for titanocene
3d at the B3LYP level using the 6-31G** basis set imple-
mented in the ab-initio programme package GAUSSIAN 03
[34]. X-ray diffraction data for the compounds 3a, 3b
and 3c were collected using a Bruker SMART APEX
CCD area detector diffractometer. A full sphere of reci-
procal space was scanned by phi-omega scans. Pseudo-
empirical absorption correction based on redundant
reflections was performed by the program SADABS [35].
The structures were solved by direct methods using SHEL-
XS-97 [36] and refined by full matrix least-squares on F2
for all data using SHELXL-97 [36]. In 3a all hydrogen atoms
were located in the difference fourier map and allowed to
refine freely. In 3b and 3c hydrogen atoms were added at
calculated positions and refined using a riding model.
Their isotropic temperature factors were fixed to 1.2 times
(1.5 times for methyl groups) the equivalent isotropic dis-
placement parameters of its parent atom. Anisotropic
thermal displacement parameters were used for all non-
hydrogen atoms. Further details about the data collection
are listed in Table 1, as well as reliability factors. Suitable
crystals of 3a were grown from a saturated dichlorometh-
ane solution, crystals of 3b were grown in saturated
dichloromethane solution with slow infusion of pentane
and 3c formed crystals from slow evaporation of a satu-
rated trichloromethane solution.
Ph
OMe
OMe
O
Me
Me
O
O
OEt
OEt
Cl
Cl
Cl
Cl
Ti
O
Ti
Ti
Ph
Budotitane
Titanocene Y
Titanocene Dichloride
Fig. 1. Structures of Budotitane, Titanocene Dichloride and Titanocene
Y.
Budotitane, Titanocene Dichloride and Titanocene Y are
shown in Fig. 1.
In addition, the anti-proliferative activity of Titanocene
Y and other titanocenes has been studied in 36 human
tumor cell lines [27] and against explanted human tumors
[28,29]. These in vitro and ex vivo experiments showed that
renal cell cancer is the prime target for this novel class of
titanocenes, but there is significant activity against ovary,
prostate, cervix, lung, colon, and breast cancer as well.
These results were underlined by first mechanistic studies
concerning the effect of these titanocenes on apoptosis
and the apoptotic pathway in prostate cancer cells [30].
Furthermore, it was shown, that titanocene derivatives give
a positive immune response by up-regulating the number of
natural killer (NK) cells in mice [31]. Recently, animal
studies reported the successful treatment of mice bearing
xenografted Caki-1 and MCF-7 tumors with Titanocene
Y [32,33].
2.2. Synthesis
Within this paper we present the synthesis and prelimin-
ary cytotoxicity studies of a series of four derivatives of
Titanocene Y.
Fulvene 1c was synthesised according to previously used
literature method [12].
2. Experimental
2.2.1. Synthesis of 6(2-methoxy-phenyl) fulvene,
C5H4–CH–C6H4–OCH3 (1a)
2.1. General conditions
3.02 g (22.5 mmol) of 2-methoxy-benzaldehyde was
dissolved in 100 ml of methanol to give a colourless solu-
tion. 4.00 ml (48.5 mmol) of freshly cracked cyclopentadi-
ene was added to the reaction solution, which remained
colourless. 3.10 ml (37.2 mmol) pyrrolidine was added to
the solution. The solution immediately changed colour
from colourless to yellow and finally reached a red colour.
The reaction was left to stir whilst being monitored by thin
layer chromatography (silica/dichloromethane), which
showed only one product spot after 3 h. 2.5 ml of acetic
acid was added to quench the reaction. One hundred mil-
liliter of water was added to the reaction mixture and the
organic product was extracted by 3 ꢀ 30 ml ether frac-
tions. The ether fractions were combined and the solution
was dried over magnesium sulphate and had its solvent
removed at reduced pressure to yield a red oil. The red
oil was purified by column chromatography with dichloro-
methane used as the eluent. The dichloromethane was
removed at reduced pressure to yield 3.61 g (87.0% yield,
19.6 mmol) of a red oil.
Titanium tetrachloride (1.0 M solution in toluene),
Super Hydride (LiBEt3H, 1.0 M solution in THF) and
the benzaldehyde derivatives were obtained from Aldrich
Chemical Company and used without further purification.
Diethyl ether and THF were dried over Na and benzo-
phenone and they were freshly distilled and collected
under an atmosphere of nitrogen prior to use. Pentane
was dried over sodium, benzophenone and di(ethlene-gly-
col)ethyl-ether and it was freshly distilled and collected
under an atmosphere of nitrogen prior to use. Manipula-
tions of air and moisture sensitive compounds were done
using standard Schlenk techniques, under a nitrogen
atmosphere. NMR spectra were measured on either a
Varian 300 or a 400 MHz spectrometer. Chemical shifts
are reported in ppm and are referenced to TMS. IR spec-
tra were recorded on a Perkin–Elmer Paragon 1000 FT-
IR Spectrometer employing a KBr disk or a liquid IR
cell. UV–Vis spectra were recorded on a Unicam UV4

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J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
Table 1
Crystallographic refinement data for titanocenes 3a–c
Identification code
3a
3b
3c
Empirical formula
Formula weight
Temperature (K)
C26H26Cl2O2Ti
489.27
100(2)
C26 H26O2Cl2Ti
489.27
100(2)
C28H30O4Cl2Ti
549.32
100(2)
˚
Wavelength (A)
0.71073
0.71073
0.71073
Crystal system
Space group
Unit cell dimensions
Monoclinic
C2/c (#15)
Triclinic
Monoclinic
C2 (#5)
ꢀ
P1 (#2)
˚
a (A)
24.011(3)
6.6699(10)
22.168(5)
a (°)
b (A)
90
6.8180(8)
113.259(2)
14.6413(18)
90
111.484(3)
13.4311(19)
100.355(4)
13.927(2)
95.618(4)
90
˚
6.4758(15)
101.074(4)
8.975(2)
90
b (°)
˚
c (A)
c (°)
Volume (A )
3
˚
2202.1(5)
1123.7(3)
1264.5(5)
Z
4
2
2
Dcalc (Mg/m3)
1.476
0.653
1016
0.30 ꢀ 0.20 ꢀ 0.05
1.85–29.00
ꢁ32 6 h 6 32,
ꢁ9 6 k 6 9,
ꢁ19 6 l 6 19
22773
1.446
0.640
508
1.443
0.583
572
0.80 ꢀ 0.15 ꢀ 0.01
1.87–28.25
ꢁ29 6 h 6 29,
ꢁ8 6 k 6 6,
ꢁ11 6 l 6 11
6065
Absorption coefficient (mmꢁ1
F(000)
Crystal size (mm3)
Theta range for data collection (°)
Index ranges
)
0.50 ꢀ 0.05 ꢀ 0.05
1.62–23.00
ꢁ7 6 h 6 7,
ꢁ14 6 k 6 14,
ꢁ15 6 l 6 15
7395
Reflections collected
Independent reflections [Rint
Completeness to theta = 29.00° (%)
Absorption correction
Maximum and minimum transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
]
2930 [0.0333]
99.9
3111 [0.0407]
99.4
2299 [0.0367]
98.3
Semi-empirical from equivalents
0.9681 and 0.6465
Full-matrix least-squares on F2
2930/0/193
1.075
Semi-empirical from equivalents
0.9687 and 0.8840
Full-matrix least-squares on F2
3111/0/282
1.118
Semi-empirical from equivalents
0.9942 and 0.6685
Full-matrix least-squares on F2
2299/1/161
1.059
R1 = 0.0336,
wR2 = 0.0849
R1 = 0.0365,
wR2 = 0.0866
0.493 and ꢁ0.226
R1 = 0.0539,
wR2 = 0.1167
R1 = 0.0682,
wR2 = 0.1234
0.691 and ꢁ0.318
R1 = 0.0412,
wR2 = 0.0960
R1 = 0.0480,
wR2 = 0.0997
0.699 and ꢁ0.417
R indices (all data)
ꢁ3
˚
Largest difference in peak and hole (e A
)
1H NMR (d ppm CDCl3, 300 MHz): 3.85 [s, 3H, C6H4–
OCH3], 6.43 [m, 2H, C5H4], 6.63 [s, 2H, C5H4], 6.87 [s,
1H, C5H4–CH], 6.89 [d, 1H, J = 8.4 Hz, C6H4], 6.99 [t,
1H, J = 7.5 Hz, C6H4], 7.33 [t, 1H, J = 7.2 Hz, C6H4],
7.58 [d, 1H, J = 7.5 Hz, C6H4].
10ꢁ2 mbar for 40 min and then to 90 °C for 20 min in a
Schlenk flask. The concentrated Super Hydride was dis-
solved in 30 ml of dry diethyl ether to give a cloudy white
suspension. 2.29 g (12.4 mmol) of the red oil 6(2-methoxy-
phenyl) fulvene was added to a Schlenk flask and was dis-
solved in 30 ml dry diethyl ether to give a red solution. The
red fulvene solution was transferred to the Super Hydride
solution via cannula. The solution was left to stir for 6 h
to give a white precipitate of the lithium cyclopentadienide
intermediate and the solution had changed colour from
orange/red to faint yellow. The precipitate was filtered on
to a frit and was washed with 15 ml of diethyl ether. The
white precipitate was dried briefly under reduced pressure
and was transferred to a Schlenk flask under nitrogen.
1.39 g (7.22 mmol, 58.1% yield) of the lithiated cyclopenta-
dienide intermediate was obtained. 3.60 ml (3.60 mmol) of
titanium tetrachloride was dissolved in 60 ml of dry THF
to give a yellow solution in a Schlenk flask. The lithium
cyclopentadienide intermediate was dissolved in 30 ml of
dry THF to give a colourless solution. The solution of tita-
nium tetrachloride solution was added to the lithium cyclo-
pentadienide intermediate solution via cannula to give a
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled):
55.8 [C6H4–OCH3], 110.9, 120.9, 120.9, 127.2, 130.8,
130.9, 132.7, 134.0, 135.1, 145.2, 148.3, 158.8.
IR absorptions (CH2Cl2, cmꢁ1): 3054, 1593, 1487, 1248,
760, 702.
UV–Vis (CH2Cl2, nm): 248 (e 1700), 269 (e 2600), 286
(e 2500), 307 (e 2400), 320 (e 2400), kmax 542 (weak).
Anal. Calc. for C13OH12: C, 84.8; H, 6.6. Found:
C, 84.8; H; 6.5%.
2.2.2. Synthesis of bis-[(2-methoxy-benzyl)
cyclopentadienyl]titanium(IV) dichloride,
[(g5-C5H4–CH2–C6H4–OCH3)]2TiCl2 (3a)
16.1 ml (16.1 mmol) of 1 M solution of Super Hydride
(LiBEt3H) in THF was concentrated by removal of the sol-
vent by heating it to 60 °C under reduced pressure of

J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
529
dark red solution. The dark red titanium solution was
refluxed for 16 h at 85 °C. The solution was then cooled
and the solvent was removed under reduced pressure.
The remaining brown residue was extracted with trichlo-
romethane (30 ml) and filtered through celite to remove
the remaining LiCl. The brown filtrate was filtered twice
more by gravity filtration. The solvent was removed under
reduced pressure to yield 1.35 g of a brown/black solid
(2.76 mmol, 76.4% yield).
UV–Vis (CH2Cl2, nm): 270 (e 2600), 287 (e 2700), 300 (e
2200), 318 (e 2100), kmax 336 (e 2200).
Micro Anal. Calc. for C13OH12: C, 84.7; H, 6.6. Found:
C, 84.3; H, 6.5%.
2.2.4. Bis-[(3-methoxy-benzyl)
cyclopentadienyl]titanium(IV) dichloride,
[(g5-C5H4-CH2-C6H4–OCH3)]2TiCl2 (3b)
16 ml (16.0 mmol) of 1 M solution of Super Hydride
(LiBEt3H) in THF was concentrated by removal of the
solvent by heating it to 60 °C under reduced pressure of
10ꢁ2 mbar for 40 min and then to 90 °C for 20 min in a
Schlenk flask. The concentrated Super Hydride was dis-
solved in 30 ml of dry diethyl ether to give a cloudy white
suspension. 2.03 g (11.0 mmol) of the red oil 6(2-methoxy-
phenyl) fulvene was added to a Schlenk flask and was dis-
solved in 30 ml dry diethyl ether to give a red solution.
The red fulvene solution was transferred to the Super
Hydride solution via cannula. The solution was left to stir
for 6 h in which time a white precipitate of the lithium
cyclopentadienide intermediate formed and the solution
had changed its colour from orange/red to yellow. The
precipitate was filtered on to a frit and was washed with
diethyl ether. The white precipitate was dried briefly
under reduced pressure and was transferred to a Schlenk
flask under nitrogen. 0.75 g (3.92 mmol, 35.5% yield) of
the lithiated cyclopentadienide intermediate was obtained.
2.0 ml (2.0 mmol) of titanium tetrachloride was dissolved
in 60 ml of dry THF to give a yellow solution in a
Schlenk flask. The lithium cyclopentadienide intermediate
was dissolved in 30 ml of dry THF to give a colourless
solution. The titanium tetrachloride solution was added
to the lithium cyclopentadienide intermediate solution
via cannula to give a dark red solution. The dark red tita-
nium solution was refluxed for 16 h. The solution was
then cooled and the solvent was removed under reduced
pressure. The remaining residue was extracted with tri-
chloromethane (30 ml) and filtered through celite to
remove the remaining LiCl. The brown filtrate was filtered
twice more by gravity filtration. The solvent was removed
under reduced pressure to yield 0.85 g of a brown/black
solid (1.74 mmol, 88.6% yield).
1H NMR (d ppm CDCl3, 400 MHz): 3.82 [s, 6H, C6H4
–OCH3], 4.01 [s, 4H, C5H4–CH2], 6.31 [s, 4H, C5H4],
6.38 [s, 4H, C5H4], 6.85 [d, 2H, J = 5.7 Hz, C6H4–
OCH3], 6.89 [t, 2H, J = 5.4 Hz, C6H4–OCH3], 7.14 [d,
2H, J = 5.4 Hz, C6H4–OCH3], 7.21 [t, 2H, J = 5.7 Hz,
C6H4–OCH3].
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled):
31.7 [C6H4–OCH3], 55.3 [C5H4–CH2], 110.4, 116.5,
120.6, 122.6, 127.9, 128.4, 130.5, 136.5, 157.3.
IR absorptions (KBr, cmꢁ1): 3076, 2360, 1585, 1489,
1437, 1250, 1105, 1026, 825, 752, 681.
UV–Vis (CH2Cl2, nm): 217 (e 9500), 229 (e 7100), 263
(e 6800), kmax 525 (weak).
Micro Anal. Calc. for TiC26O2H26Cl2: C, 63.8; H, 5.3;
Cl, 14.4. Found: C, 63.5; H, 5.2; Cl, 13.6%.
2.2.3. Synthesis of 6(3-methoxy-phenyl) fulvene,
C5H4–CH–C6H4–OCH3 (1b)
3.01 g (22.3 mmol) of 3-methoxy-benzaldehyde was dis-
solved in 80 ml of methanol to give a colourless solution.
3.90 ml (47.2 mmol) of fresh cracked cyclopentadiene was
added to the reaction solution, which remained colourless.
3.0 ml (36.0 mmol) of pyrrolidine was then added to the
solution. The solution slowly changed colour from colour-
less to yellow and finally reached a red/orange colour.
The reaction was left to stir for 28 h. 2.20 ml of acetic
acid was added to quench the reaction. 100 ml of water
was added to the reaction mixture and the organic prod-
uct was extracted by 4 ꢀ 30 ml ether. The ether solution
was dried with magnesium sulphate and had its solvent
removed at reduced pressure to yield a red oil. The red
oil was purified by column chromatography with dichlo-
romethane used as the eluent. The dichloromethane was
removed at reduced pressure to yield 3.92 g (95.5% yield,
21.3 mmol) of a red oil.
1H NMR (d ppm CDCl3, 300 MHz): 3.78 [s, 6H, C6H4–
OCH3], 4.07 [s, 4H, C5H4–CH2], 6.33 [s, 8H, C5H4], 6.76
[d, 2H, J = 4.5 Hz, C5H4], 6.77 [s, 2H, C5H4–CH], 6.80
[d, 2H, J = 5.7 Hz, C6H4], 7.21 [t, 2H, J = 5.7 Hz,
C6H4].
1H NMR (d ppm CDCl3, 300 MHz): 3.83 [s, 3H, C6H4
–OCH3], 6.41 [m, 2H, C5H5], 6.75 [d, 2H, J = 2.1 Hz,
C5H5], 6.79 [s, 1H, C5H5–CH], 6.89 [d, 1H, J = 1.5 Hz,
C6H4–OCH3], 7.11 [s, 1H, C6H4–OCH3], 7.18 [s, 1H,
C6H4–OCH3], 7.23 [t, 1H, J = 3.3 Hz, C6H4–OCH3].
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled):
55.9 [C6H4–OCH3], 110.8, 120.8, 120.9, 126.2, 127.1,
130.8, 130.8, 132.3, 132.7, 134.0, 135.0, 145.2.
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled):
36.0 [C6H4–OCH3], 54.2 [C5H4–CH2], 110.8, 113.9,
114.8, 120.3, 121.6, 128.6, 135.8, 140.0, 158.8.
IR absorptions (KBr, cmꢁ1): 3113, 2926, 2360, 1608,
1581, 1487, 1439, 1284, 1252, 1144, 1051, 825, 773.
UV–Vis (CH2Cl2, nm): kmax 263 (e 29000).
IR absorptions (CH2Cl2, cmꢁ1): 3050, 2838, 1625, 1597,
1465, 1207, 1158, 1049, 767, 697.
Micro Anal. Calc. for TiC26O2H26Cl2: C, 63.8; H, 5.4;
Cl, 14.5. Found C, 62.2; H, 5.5; Cl, 14.5%.

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J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
2.2.5. Synthesis of 6(3,4-dimethoxy-phenyl) fulvene,
[C5H4–CH–C6H3–(OCH3)2] (1c)
was dried briefly under reduced pressure and was trans-
ferred to a Schlenk flask under argon. 2.71 g (11.4 mmol,
95.5% yield) of the lithiated cyclopentadienide intermediate
was obtained. 5.6 ml (5.6 mmol) of titanium tetrachloride
was dissolved in 50 ml of dry THF to give a yellow solution
in a Schlenk flask. The lithium cyclopentadienide interme-
diate was dissolved in 50 ml of the dried THF to give a col-
ourless solution. The titanium tetrachloride solution was
added to the lithium cyclopentadienide intermediate solu-
tion via cannula to give a dark red solution. The dark red
titanium solution was refluxed for 16 h. The solution was
then cooled and the solvent was removed under reduced
pressure. The remaining residue was extracted with trichlo-
romethane (50 ml) and filtered through celite to remove the
remaining LiCl. The brown filtrate was filtered twice more
by gravity filtration. The solvent was removed under
reduced pressure to yield 2.71 g of an orange/brown solid
(4.6 mmol, 80.7% yield).
3.62 g (21.5 mmol) of 3,4-dimethoxy-benzaldehyde was
dissolved in 100 ml of methanol to give a colourless solu-
tion. 4.0 ml (48.4 mmol) of fresh cracked cyclopentadiene
was added to the reaction solution, which remained colour-
less. 3.0 ml (36.0 mmol) pyrrolidine was added to the solu-
tion. The solution slowly changed colour from colourless
to yellow and finally reached a red/orange colour. The
reaction was left to stir for 16 h. 3.0 ml of acetic acid was
added to quench the reaction. 100 ml of water was added
to the reaction mixture and the organic product was
extracted by 3 ꢀ 30 ml ether. The ether solution was dried
with magnesium sulphate and had its solvent removed at
reduced pressure to yield a red oil. The red oil was purified
by column chromatography with dichloromethane used as
the eluent. The dichloromethane was removed at reduced
pressure to yield 4.53 g (98.1% yield, 21.13 mmol) of an
orange solid.
1H NMR (d ppm CDCl3, 400 MHz): 3.85 [s, 12H,
C6H3–(OCH3)2], 4.05 [s, 4H, C5H4–CH2], 6.32 [s, 8H,
C5H4], 6.72 [s, 2H, C6H3–(OCH3)2], 6.77 [d, 2H,
J = 11.1 Hz, C6H3–(OCH3)2], 6.80 [d, 2H, J = 8.1 Hz,
C6H3-(OCH3)2].
1H NMR (d ppm CDCl3, 300 MHz): 3.93 [s, 6H, C6H3
–(OCH3)2], 6.41 [m, 2H, C5H4], 6.70 [m, 2H, C5H4],
6.90 [d, 1H, J = 6.3 Hz, C5H4–CH], 7.16 [d, 1H,
J = 6.9 Hz, C6H3–(OCH3)2], 7.18[s, 1H, C6H3
–(OCH3)2], 7.20 [d, J = 6.0 Hz, C6H3–(OCH3)2].
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled):
56.2 [C6H3–OCH3–OCH3], 56.2 [C6H3-OCH3–OCH3],
111.4, 113.4, 120.0, 125.0 [C5H4–CH], 127.7, 130.0,
130.1, 135.3, 138.7, 143.7, 149.3, 150.6.
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled):
36.7 [C6H3-(OCH3)2], 56.1 [C5H4–CH2], 111.5, 112.8,
116.0, 121.2, 122.7, 132.1, 137.8, 147.9, 149.2.
IR absorptions (KBr, cmꢁ1): 3107, 2956, 2933, 2835,
1589, 1514, 1464, 1414, 1333, 1263, 1234, 1140, 1024,
816, 752, 698.
UV–Vis (CH2Cl2, nm): 215 (e 18000), 234 (e 13000), 264
(e 14000), 278 (e 12000), 398 (e 1000), kmax 523 (e 180).
Micro Anal. Calc. for TiC28O4H30Cl2: C, 61.2; H, 5.5;
Cl, 12.9. Found: C, 60.0; H, 5.5; Cl, 12.3%.
IR absorptions (KBr, cmꢁ1): 2929, 2837, 2360, 1618,
1593, 1543, 1462, 1329, 1261, 1022, 891, 812, 769, 631,
584.
UV–Vis (CH2Cl2, nm): k 222 (e 4460), k 256 (e 4683), k
270 (e 4348), k 312 (e 4794), k 325 (e 5329), k 339 (e
5017), k 367 (e 5001), kmax 386 (e 3233).
Micro Anal. Calc. for C14O2H14: C, 78.5; H, 6.6. Found:
C, 77.5; H, 6.6%.
2.2.7. Synthesis of bis-[(3,4,5-trismethoxy-benzyl)
cyclopentadienyl]titanium(IV) dichloride,
[(g5-C5H4–CH2–C6H2–(OCH3)3)]2TiCl2 (3d)
2.2.6. Synthesis of bis-[(3,4-bismethoxy-benzyl)
cyclopentadienyl]titanium(IV) dichloride,
16.6 ml (16.6 mmol) of a 1 M solution of Super Hydride
(LiBEt3H) in THF was concentrated by removal of the sol-
vent by heating it to 60 °C under reduced pressure of
10ꢁ2 mbar for 40 min and then to 90 °C for 20 min. The
concentrated LiBEt3H solution was allowed to cool and
25 ml of dry diethyl ether was added to give a cloudy white
suspension in the Schlenk flask. 2.70 g (11.0 mmol) of the
red oil 6(3,4,5-trimethoxy-phenyl) fulvene was added to
another Schlenk flask and was dissolved in 60 ml dry
diethyl ether to give a red solution. The red solution was
transferred to the LiBEt3H solution via cannula. The solu-
tion was left to stir for 6 h to give a white precipitate of the
lithium cyclopentadienide intermediate and the solution
had changed colour from orange/red to faint yellow. The
lithium cyclopentadienide intermediate solution had its sol-
vent decanted off whilst remaining in an inert environment.
The white precipitate was washed with 2 ꢀ 20 ml dry pen-
tane, which was then also removed by decanting. The
[(g5-C5H4–CH2–C6H3–(OCH3)2)]2TiCl2 (3c)
15.8 ml (15.8 mmol) of 1 M solution of Super Hydride
(LiBEt3H) in THF was concentrated by removal of the sol-
vent by heating it to 60 °C under reduced pressure of
10ꢁ2 mbar for 40 min and then to 90 °C for 20 min. The
concentrated LiBEt3H was dissolved in 25 ml of dry diethyl
ether to give a colourless solution in a Schlenk flask. 2.55 g
(11.9 mmol) of the orange solid 6(3,4-dimethoxy-phenyl)
fulvene was added to a Schlenk flask and was dissolved
in 125 ml dry diethyl ether to give a red solution. The red
solution was transferred to the LiBEt3H solution via can-
nula. The solution was left to stir for 4 h to give a white pre-
cipitate of the lithium cyclopentadienide intermediate and
the solution changed colour from orange/red to faint yel-
low. The precipitate was filtered on to a frit and was
washed with 20 ml of diethyl ether. The white precipitate

J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
531
intermediate was dried briefly under reduced pressure.
5.6 ml (5.6 mmol) of titanium tetrachloride was dissolved
in 50 ml of dry THF to give a yellow solution in a Schlenk
flask. The lithium cyclopentadienide intermediate was dis-
solved in 80 ml of the dried THF to give a colourless solu-
tion. The solution of titanium tetrachloride solution was
added to the lithium cyclopentadienide intermediate solu-
tion via cannula to give a black solution. The black solution
was refluxed for 16 h. The solution was then cooled and the
solvent was removed under reduced pressure. The remain-
ing residue was extracted with trichloromethane (80 ml)
and filtered through celite to remove the remaining LiCl.
The black filtrate was filtered twice more by gravity filtra-
tion. The solvent was removed under reduced pressure to
yield 2.90 g of a black solid (4.76 mmol, 85.0% yield).
compare the cytotoxicity of the compounds presented in
this paper. This cell line was chosen based on their regular
and long-lasting growth behaviour, which is similar to the
one shown in kidney carcinoma cells. It was obtained from
the ATCC (American Tissue Cell Culture Collection) and
maintained in Dulbecco’s Modified Eagle Medium contain-
ing 10% (v/v) FCS (foetal calf serum), 1% (v/v) penicillin
streptomycin and 1% (v/v) L-glutamine. Cells were seeded
in 96-well plates containing 200 ll microtitre wells at a den-
sity of 5000-cells/200 ll of medium and were incubated at
37 °C for 24 h to allow for exponential growth. Then the
compounds used for the testing were dissolved in the mini-
mal amount of DMSO (dimethylsulfoxide) possible and
diluted with medium to obtain stock solutions of
5 ꢀ 10ꢁ4 M in concentration and less than 0.7% of DMSO.
The cells were then treated with varying concentrations of
the compounds and incubated for 48 h at 37 °C. Then, the
solutions were removed from the wells and the cells were
washed with PBS (phosphate buffer solution) and fresh
medium was added to the wells. Following a recovery
period of 24 h incubation at 37 °C, individual wells
were treated with a 200 ll of a solution of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in
medium. The solution consisted of 30 mg of MTT in
30 ml of medium. The cells were incubated for 3 h at
37 °C. The medium was then removed and the purple for-
mazan crystals were dissolved in 200 ll DMSO per well.
A Wallac Victor (Multilabel HTS Counter) Plate Reader
was used to measure absorbance at 540 nm. Cell viability
was expressed as a percentage of the absorbance recorded
for control wells. The values used for the dose–response
curves represent the values obtained from four consistent
MTT-based assays for each compound tested. Titanocenes
3a-d were found to have IC50 values of 97, 159, 88 and
253 lM, respectively (see Figs. 4 and 5).
1H NMR (d ppm CDCl3, 300 MHz): 3.82 [s, 6H,
(OCH3)2–C6H2–OCH3], 3.83 [s, 12H, OCH3–C6H2–
(OCH3)2], 4.06 [s, 4H, C5H4–CH2], 6.35 [s, 4H, C5H4],
6.46 [s, 4H, C6H2–(OCH3)3].
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled):
37.5 [C5H4–CH2], 56.4 [C6H2–(OCH3)2–(OCH3)], 61.1
[C6H2–(OCH3)2–OCH3], 106.4, 115.5, 123.1, 135.2,
136.9, 137.2, 153.5.
IR absorptions (cmꢁ1 KBr): 2960, 1591, 1506, 1460,
1419, 1331, 1261, 1124, 802.
Micro Anal. Calc. for TiC30O6H34Cl2: C, 59.1; H, 5.6;
Cl, 11.6. Found: C, 58.3; H, 5.7; Cl, 11.5% (see Figs. 2
and 3).
2.3. Cytotoxicity studies
Preliminary in vitro cell tests were performed on the cell
line LLC-PK (long-lasting cells-pig kidney) in order to
OCH3
OCH3
OCH3
OCH3
1b
1c
1a
OCH3 1d
H3CO
OCH3
Fig. 2. Structures of fulvenes 1a–1d.
H3CO
H3CO
OCH3
H3CO
OCH3
OCH3
H3CO
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Ti
Ti
Ti
Ti
OCH3
3d
3c
3a
3b
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
Fig. 3. Structures of titanocenes 3a–3d.

532
J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
tle [37] by the condensation of freshly cracked cyclopenta-
1.2
1.0
0.8
0.6
0.4
0.2
0.0
diene in the presence of a base in yields of 87–98%.
Pyrrolidine was used as the base to catalyse this reaction.
The four titanocenes were synthesised by the nucleo-
philic addition of hydride to the exocyclic double bond of
the fulvenes to form the required isolable lithium cyclopen-
tadienide intermediate in yields of 35–95%. These exocyclic
double bonds in the fulvenes have increased polarity, due
to the inductive effects of their respective phenyl groups.
This increased polarity allows for selective nucleophilic
attack at this double bond and not at the diene component
of the fulvenes.
Two equivalents of this lithium cyclopentadienide inter-
mediate can be transmetallated to 1 equiv. of TiCl4 result-
ing in the formation of 1 equiv. of the required benzyl
substituted titanocene in yields of 75–88% and 2 equiv. of
the by-product of lithium chloride following a 16 hour
reflux. The synthesis is shown in Scheme 1.
3a: IC50: (97+/-36)E-6
3b: IC50: (159+/-67)E-6
1E-10
1E-9
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
Log10 Drug Concentration (mol/L)
Fig. 4. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 3a and 3b on the viability of LLC-PK cells.
In the case of 6(3,4,5-trimethoxy-phenyl) fulvene 1d it
could not be isolated following hydridolithiation as the
lithiated cyclopentadienide intermediate 2d formed an
extremely compact solid upon precipitation which could
not be removed. In this case the TiCl4 solution was added
directly to the intermediate once dissolved in THF.
1.2
1.0
0.8
0.6
3.2. Structural discussion
The benzyl substituted titanocenes are a particular class
of titanocenes that are very promising in crystallisation
experiments due to the lack of stereoisomers. Suitable crys-
tals of 3a were grown from a saturated dichloromethane
solution, crystals of 3b were grown in saturated dichloro-
methane solution with slow infusion of pentane and 3c
formed crystals from slow evaporation of a saturated
trichloromethane solution. The length of the bond between
the titanium centre and the carbon atoms of the cyclopen-
tadienyl rings bound to the metal are very similar for 3a
3c: IC50: (88+/-7)E-6
3d: IC50: (253+/-51)E-6
0.4
0.2
0.0
1E-10
1E-9
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
Log10 Drug Concentration (mol/L)
˚
˚
and 3b. They vary from 2.344 A to 2.413 A for 3a and from
Fig. 5. Cytotoxicity curves from typical MTT assays showing the effect of
compounds 3c and 3d on the viability of LLC-PK cells.
˚
˚
2.342 A to 2.418 A for 3b. In 3c there is a slight elongation
to of these bonds to be seen to the metal ion with the bonds
˚
˚
3. Results and discussion
varying from 2.348 A to 2.487 A leading to a titanium cen-
˚
troid distance of 2.082 A. The carbon carbon bond lengths
3.1. Synthesis
of the cyclopentadienyl rings are more comparable to all
˚
three with 3a having lengths of 1.40–1.42 A, 3b having
˚
The synthesis of the four fulvenes were all done by well
established analogous synthetic processes of Stone and Lit-
lengths of 1.39–1.41 A and 3c having lengths of 1.40–
˚
1.42 A. The titanium to chlorine bond lengths are very
R
H
R
TiCl4
2LiBEt3H
Cl
2
2
Ti
Cl
THF
-2LiCl(s)
Et2O
-2BEt3
H
R
H
R
Li
Scheme 1. Synthesis of benzyl substituted titanocenes from fulvenes using the hydridolithiation reaction.

J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
533
0
˚
˚
similar varying from 2.355 A to 2.373 A. The Cl–Ti–Cl
there is no internal symmetry to be seen within the mole-
cule yet it was still possible to crystallise this compound
with no solvent present in the packing. The titanium ion
is featured on a C2 axis in 3c. For all three compounds
there are no p–p interactions to be seen between the substi-
tuted phenyl groups but some soft Van der Waals
bond angle was measured at 93.85° for 3a, 94.13° for 3b
and 96.03° for 3c (see Figs. 6–8).
The packing in 3a features a pronounced z shape of the
molecule. This z shape leads to a pseudo chain consisting of
two molecules of this titanocene per link of the chain. In 3b
Fig. 6. X-ray diffraction structure of bis-[(2-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride 3a (thermal ellipsoids are drawn on the 50%
probability level).
Fig. 7. X-ray diffraction structure of bis-[(3-methoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride 3b (thermal ellipsoids are drawn on the 50%
probability level).
Fig. 8. X-ray diffraction structure of bis-[(3,4-dimethoxy-benzyl)cyclopentadienyl]titanium(IV) dichloride 3c (thermal ellipsoids are drawn on the 50%
probability level).

534
J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
Table 2
Table 3
Selected bond lengths and angles from crystallographic structures of
titanocenes 3a–c
Selected bond lengths and angles from calculated structure of titanocene
3d
Identification code
3a
3b
3c
Identification code
3d
˚
Bond lengths (A)
˚
Bond lengths (A)
Ti–C(1)
Ti–C(2)
Ti–C(3)
Ti–C(4)
2.4133(13)
2.3921(15)
2.3848(15)
2.3440(15)
2.3858(15)
1.417(2)
1.402(2)
1.409(3)
1.423(2)
1.408(2)
1.508(2)
1.517(2)
2.3726(5)
2.3726(5)
2.059(1)
2.418(5)
2.391(5)
2.350(5)
2.342(5)
2.391(5)
1.410(7)
1.391(7)
1.400(7)
1.402(7)
1.412(7)
1.488(7)
1.516(7)
2.3635(15)
2.3652(15)
2.057(5)
2.487(3)
2.441(3)
2.376(3)
2.348(3)
2.361(3)
1.409(4
1.421(5)
1.400(5)
1.421(5)
1.412(5)
1.507(4)
1.525(5)
2.3552(9)
2.3551(9)
2.082(4)
Ti–C(1)
Ti–C(2)
Ti–C(3)
Ti–C(4)
2.4341
2.3753
2.3373
2.4288
2.4431
1.4395
1.4131
1.4279
1.4341
1.4199
1.5143
1.5211
2.3765
2.3812
2.4329
2.3834
2.3980
2.3757
2.4089
1.4408
1.4211
1.4219
1.4321
1.4201
1.5068
1.5313
Ti–C(5)
Ti–C(5)
C(1)–C(2)
C(2)–C(3)
C(3)–C(4)
C(4)–C(5)
C(5)–C(1)
C(1)–C(6)
C(6)–C(7)
Ti–Cl(1)
Ti–Cl(2)
Ti–Cent
C(1)–C(2)
C(2)–C(3)
C(3)–C(4)
C(4)–C(5)
C(5)–C(1)
C(1)–C(6)
C(6)–C(7)
Ti–Cl(1)
Ti–Cl(2)
*
Ti–C(1)
*
Ti–C(2)
Bond angles (°)
Cent(1)–Ti–Cl(1)
Cent(1)–Ti–Cl(2)
Cent(1)–Ti–Cent(2)
Cl(1)–Ti–Cl(2)
*
Ti–C(3)
105.67(1)
107.54(1)
130.54(1)
93.85(1)
105.50(5)
106.65(5)
130.85(5)
94.13(5)
105.87(2)
106.04(2)
131.48(1)
96.03(5)
*
Ti–C(4)
*
Ti–C(5)
*
*
*
*
*
*
*
*
*
*
*
*
*
*
C(1) –C(2)
C(2) –C(3)
C(3) –C(4)
C(4) –C(5)
C(5) –C(1)
C(1) –C(6)
C(6) –C(7)
interactions are present. The absence of solvent molecules
present in the three crystal unit cells is a particular advan-
tage when it comes to biological testing of these com-
pounds (see Table 2).
Bond angle (°)
Cl(1)–Ti–Cl(2)
Despite the efforts to crystallise titanocene 3d no crystal
structures were obtained. This could possibly be explained
by the solubility problems encountered with this isolated
titanocene. In order to acquire a structure density func-
tional theory (DFT) calculations were carried out for this
titanocene at the B3LYP level using the 6-31G** basis set.
Selected bond lengths of the optimised structure of this
titanocene are listed in Table 3 and the atom numbering
scheme is seen in Scheme 2. The calculated structure of
titanocene 3d is presented in Fig. 9.
93.3180
5
2
5'
4'
4
Cl
Ti
1
1'
6
6'
7'
7
3 Cl1
3'
2'
OCH3
OCH3
H3CO
H3CO
H3CO
OCH3
There are slight variations in the bond length between
the metal centre and the cyclopentadienyl carbons with
Scheme 2. Numbering scheme of 3d for the structural DFT discussion.
˚
˚
them varying from 2.3373 A to 2.4431 A. These slight bond
length differences are also seen between the carbon carbon
bonds of the cyclopentadienylrings with bond lengths vary-
MTT-based assays for each compound tested. As seen in
Figs. 1 and 2, titanocenes 3a–d showed IC50 values of 97,
159, 88 and 253 lM, respectively. When compared to
unsubstituted titanocene dichloride (IC50 value of
2000 lM), each of the newly synthesised titanocenes show
greater than an 8-fold decrease and up to 22-fold decrease
in magnitude in terms of the IC50 value. However, titanoc-
enes 3a–d do not reach the cytotoxicity with respect to the
class leader Titanocene Y with an IC50 value of 21 lM nor
to cisplatin with an IC50 value of 3.3 lM. This is likely to
be due to the loss of solubility in each of the four titanoc-
enes case showing the importance of the para methoxy phe-
nyl moiety in Titanocene Y itself. Also the change in shape
and size of the new titanocenes might also play a consider-
able part in the cause of the decrease in cytotoxicity
shown in the MTT based cell tests. In comparison to the
˚
˚
ing from 1.4131 A to 1.4408 A. All other bonds within the
calculated titanocene are of comparable lengths to those
whose X-ray structures were refined. The Cl–Ti–Cl angle
was calculated to be 93.32° which is highly comparable
to titanocenes 3a, 3b and 3c Cl–Ti–Cl bond angles.
In general, it is possible to calculate the molecular struc-
tures of titanocene dichloride derivatives within the error
margin of the X-ray crystal structure method, if the DFT
method, B3LYP, is used in combination with a large
enough basis set like 6-31G**.
3.3. Cytotoxicity studies
The values used for the dose–response curves of Figs. 1
and 2 represent the values obtained from four consistent

J. Claffey et al. / Journal of Organometallic Chemistry 693 (2008) 526–536
535
can be obtained free of charge from The Cambridge Crys-
tallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. Supplementary data associated with this
article can be found, in the online version, at
doi:10.1016/j.jorganchem.2007.11.037.
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The hydridolithiation of 6-aryl substituted fulvenes has
been found to be a very effective and reproducible way to
cytotoxic benzyl substituted titanocenes of high purity.
Following these investigative studies into the synthesis
and cytotoxicity of these methoxy phenyl substituted tita-
nocenes we have been able to establish that Titanocene Y
has the optimum positioning and number of methoxy
groups on the phenyl ring of this particular titanocene
class. Although all four of these titanocenes show an
increased cytotoxicity in comparison to unsubstituted
titanocene dichloride they do not compare as favourably
against Titanocene Y exhibiting an IC50 value of 21 lM.
´
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