Novel zirconocene anticancer drugs?

Article abstract of DOI:10.1016/j.jorganchem.2008.08.020

From the reaction of Super Hydride (LiBEt₃H) with 6-(4-methoxyphenyl) fulvene (1a), 6-(2-fluoro-4-methoxyphenyl) fulvene (1b), and 6-(4-N,N-dimethylaminophenyl) fulvene (1c) lithiated cyclopentadienide intermediates (2a-c) were synthesised. The

Full text of DOI:10.1016/j.jorganchem.2008.08.020

Journal of Organometallic Chemistry 694 (2009) 828–833
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Novel zirconocene anticancer drugs?
*
Denise Wallis, James Claffey, Brendan Gleeson, Megan Hogan, Helge Müller-Bunz, Matthias Tacke
Conway Institute of Biomolecular and Biomedical Research, Centre for Synthesis and Chemical Biology (CSCB), UCD School of Chemistry and Chemical Biology,
University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 June 2008
Received in revised form 18 August 2008
Accepted 19 August 2008
Available online 23 August 2008
From the reaction of Super Hydride (LiBEt3H) with 6-(4-methoxyphenyl) fulvene (1a), 6-(2-fluoro-
4-methoxyphenyl) fulvene (1b), and 6-(4-N,N-dimethylaminophenyl) fulvene (1c) lithiated cyclopenta-
dienide intermediates (2a–c) were synthesised. These intermediates were then transmetallated to
zirconium with ZrCl4 to give benzyl-substituted zirconocenes bis-[(4-methoxybenzyl)cyclopentadienyl]
zirconium(IV) dichloride (3a), bis-[(2-fluoro-4-methoxybenzyl)cyclopentadienyl] zirconium(IV) dichlo-
ride (3b) and bis-[(4-N,N-dimethylaminobenzyl)cyclopentadienyl] zirconium(IV) dichloride (3c). All
three zirconocenes were characterised by single crystal X-ray diffraction and preliminary in vitro cell
tests were performed with the zirconocene derivatives on the LLC-PK cell line in order to determine their
This paper is dedicated to Prof. Dr. Gérard
Jaouen – the pioneer in the area of
Bioorganometallic Chemistry – on the
occasion of his 65th birthday.
cytotoxicity. Zirconocenes 3b and 3c did not show cytotoxicity up to a concentration of 170
exhibited an IC50 value of 57 M against LLC-PK.
lM, while 3a
l
Ó 2008 Elsevier B.V. All rights reserved.
Keywords:
Anticancer drugs
Zirconocene
Titanocene
Hydridolithiation
Cytotoxicity
LLC-PK
1. Introduction
with TiCl4 led to bis-[(p-methoxybenzyl)cyclopentadienyl] tita-
nium(IV) dichloride (Titanocene Y) [8], which has an IC50 value
Bioorganometallic compounds like Ferrocifen [1] are now being
explored as potential treatment against hormone dependant and
hormone independent breast cancer [2] following the pioneering
work in this field carried out by Gérard Jaouen.
of 21 lM when tested on the LLC-PK cell line. This particular cell
line was chosen as it has proven to be a good mimic of a kidney
carcinoma cell line and a reliable tool for the optimisation of tita-
nocenes against this type of cancer. Also, Titanocene Y is strongly
Another promising bioorganometallic anticancer drug candidate
is titanocene dichloride (Cp2TiCl2), which showed medium anti-
proliferative activity in vitro but promising results in vivo [3,4].
Titanocene dichloride reached clinical trials, but it’s efficacy in
Phase II clinical trials in patients with metastatic renal cell carci-
noma [4] or metastatic breast cancer [5] was too low to be pursued.
The field got renewed interest with P. McGowan’s elegant
synthesis of ring-substituted cationic titanocene dichloride deriva-
tives, which are water-soluble and show significant activity against
ovarian cancer [6]. More recently, novel methods starting from ful-
venes and other precursors allow direct access to antiproliferative
titanocenes via reductive dimerisation with titanium dichloride,
carbolithiation or hydridolithiation of the fulvene followed by
transmetallation with titanium tetrachloride in the latter two cases
[7]. Hydridolithiation of 6-anisyl fulvene and subsequent reaction
anti-angiogenic, exhibiting an IC50 value of 4.9 lM in the HUVEC
assay [9], which underlines that Titanocene Y is a possible drug
candidate against advanced renal cell cancer.
In addition, the anti-proliferative activity of Titanocene Y and
other titanocenes has been studied in 36 human tumor cell lines
[10] and against explanted human tumors [11,12]. 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 can-
cer 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 [13]. Further-
more, it was shown, that titanocene derivatives give a positive im-
mune response by up-regulating the number of natural killer (NK)
cells in mice [14]. Recently, animal studies reported the successful
treatment of mice bearing xenografted Caki-1 and MCF-7 tumors
with Titanocene Y [15,16] and the cytotoxicity could be improved
by an anion exchange leading to Oxali-Titanocene Y [17]. The
structures of Ferrocifen and Titanocene Y are shown in Fig. 1.
* Corresponding author.
E-mail address: matthias.tacke@ucd.ie (M. Tacke).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.08.020

D. Wallis et al. / Journal of Organometallic Chemistry 694 (2009) 828–833
829
Table 1
OH
Crystallographic refinement data for zirconocenes 3a–c
OCH3
OCH3
Identification code
3a
3b
3c
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C26H26O2Cl2Zr
532.59
100(2)
0.71073
Orthorhombic
Pbca (#61)
C26H24O2F2Cl2Zr
568.57
100(2)
0.71073
Monoclinic
C2/c (#15)
C28H32N2Cl2Zr
558.68
100(2)
0.71073
Orthorhombic
P212121 (#19)
Cl
Cl
Ti
Fe
O(CH2)3N(CH3)2
Ferrocifen
Titanocene Y
6.5846(5)
25.6492(19)
27.102(2)
90
90
90
4577.3(6)
8
1.546
0.735
17.9885(17)
6.7101(6)
19.3009(18)
90
92.508(2)
90
2327.5(4)
4
1.623
7.3449(6)
11.7907(10)
28.904(2)
90
90
90
2503.1(4)
4
1.482
0.672
b (Å)
c (Å)
Fig. 1. Structures of Ferrocifen and Titanocene Y.
a
(°)
b (°)
(°)
c
On the other hand, exchange of the titanium to the heavier me-
tal zirconium leads to zirconocene dichloride, which was tested on
Ehrlich’s ascites tumor (EAT) in vitro [18] and in vivo [19]. All
experiments showed no anticancer activity of the unsubstituted
zirconocene, which did not encourage further research into this
area. Within this paper, we present the synthesis and preliminary
cytotoxicity studies of a series of three substituted zirconocene
derivatives, which include the analogous derivative Zirconocene Y.
Volume (Å3)
Z
Dcalc (mg/m3)
Absorption
coefficient
0.740
(mmꢀ1
)
F(000)
2176
1152
1152
Crystal size (mm2)
h Range for mm
data collection
(°)
0.40 ꢁ 0.20 ꢁ 0.02 0.50 ꢁ 0.20 ꢁ 0.05 0.80 ꢁ 0.55 ꢁ 0.03
1.76–26.36
2.11–31.59
1.87–32.02
2. Experimental
Index ranges
–8 6 h 6 6,
–31 6 k 6 31,
–33 6 l 6 32
26197
–25 6 h 6 25,
–9 6 k 6 9,
–28 6 l 6 27
13076
–10 6 h 6 10,
–17 6 k 6 13,
–35 6 l 6 41
21387
2.1. General conditions
Reflections
collected
Zirconium tetrachloride, Super Hydride (LiBEt3H, 1.0 M solution
in THF) and the benzaldehyde derivatives were obtained from Al-
drich Chemical Company and used without further purification.
Diethyl ether and THF were dried over Na and benzophenone
and they were freshly distilled and collected under an atmosphere
of nitrogen prior to use. Pentane was dried over sodium, benzo-
phenone and di(ethylene–glycol)ethyl ether and it was freshly dis-
tilled and collected under an atmosphere of nitrogen prior to use.
Manipulations 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, 400 or
500 MHz spectrometer. Chemical shifts are reported in ppm and
are referenced to TMS. IR spectra 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 Spectrom-
eter. CHN analysis was done with an Exeter Analytical CE-440 Ele-
mental Analyser, while Cl was determined in mercurimetric
titrations. X-ray diffraction data for the compounds were collected
using a Bruker SMART APEX CCD area detector diffractometer. A
full sphere of reciprocal space was scanned by phi–omega scans.
Pseudo-empirical absorption correction based on redundant
reflections was performed by the program SADABS [20]. The struc-
tures were solved by direct methods using and refined by full ma-
trix least-squares on F2 for all data using SHELXL-97 [21]. In 3a and
3b all hydrogen atoms were located in the difference fourier
map and allowed to refine freely. In 3c the hydrogen atoms were
visible but not freely refined. Instead they were added at calcu-
lated positions and refined using a riding model. Further details
about the data collection are listed in Table 1, as well as reliability
factors. Further details are available free of charge from the Cam-
bridge structural database under the CCDC numbers 690596,
690597 and 690598 for 3a, 3b and 3c, respectively. Suitable crys-
tals of 3a–c were grown in saturated dichloromethane solution
with slow infusion of pentane.
Independent
reflections
Completeness to
hmax (%)
Absorption
correction
Maximum and
minimum
transmission
4657
[Rint = 0.0376]
99.7
3648
[Rint = 0.0237]
93.8
8074
[Rint = 0.0210]
94.8
Semi-empirical
from equivalents
0.9854 and
Semi-empirical
from equivalents
0.9639 and
Semi-empirical
from equivalents
0.9801 and
0.8211
0.8045
0.7205
Refinement method Full-matrix least-
Full-matrix least-
squares on F2
3648/0/198
Full-matrix least-
squares on F2
8074/0/302
squares on F2
4657/0/384
1.083
Data/restraints/
parameters
Goodness-of-fit on
F2
Final R indices
[I > 2r(I)]
1.050
1.053
R1 = 0.0316,
wR2 = 0.0686
R1 = 0.0404,
wR2 = 0.0718
R1 = 0.0271,
wR2 = 0.0673
R1 = 0.0297,
wR2 = 0.0686
R1 = 0.0296,
wR2 = 0.0702
R1 = 0.0311,
wR2 = 0.0710
R indices (all data)
Largest difference
peak and hole
0.458 and ꢀ0.307 0.762 and ꢀ0.305 0.806 and ꢀ0.327
(e Åꢀ3
)
2.2.1. Bis-[(4-methoxybenzyl)cyclopentadienyl] zirconium(IV)
dichloride, [(
5-C5H4–CH2–C6H4–OCH3)]2ZrCl2 (3a)
g
16.0 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 10ꢀ2 mbar for 40 min and then to
90 °C for 20 min in a Schlenk flask. The concentrated Super Hydride
was dissolved in 30 ml of dry diethyl ether to give a cloudy white
suspension. 2.25 g (12.2 mmol) of the orange solid 6-(4-methoxy-
phenyl) fulvene was added to a Schlenk flask and was dissolved
in 30 ml dry diethyl ether to give an orange solution. The fulvene
solution was transferred to the Super Hydride solution via cannula.
The solution was left to stir for 5 h to give a white precipitate of the
lithium cyclopentadienide intermediate and the solution had chan-
ged colour from orange/red to white. 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. 1.69 g (8.73 mmol,
71.5% yield) of the lithiated cyclopentadienide intermediate was
2.2. Synthesis
6-(4-Methoxyphenyl) fulvene (1a) was synthesised according
to the previously published procedure [22].

830
D. Wallis et al. / Journal of Organometallic Chemistry 694 (2009) 828–833
obtained. 1.02 g (4.36 mmol) of zirconium tetrachloride was dis-
solved in 60 ml of dry THF to give a colourless 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 zir-
conium tetrachloride solution was added to the lithium cyclopen-
tadienide intermediate solution via cannula to give a yellow
solution. The resulting yellow solution was heated under reflux
for 20 h. The solution was then cooled and the solvent was re-
moved under reduced pressure. The remaining orange residue
was extracted with 100 ml of chloroform to give a green solution
and filtered through celite to remove the remaining LiCl. The green
filtrate was filtered twice more by gravity filtration. The solvent
was removed under reduced pressure to yield 1.22 g of a cream/
green solid (2.76 mmol, 52.9% yield).
IR absorptions (CH2Cl2, cmꢀ1): 3099, 2901, 2805, 2282, 1616,
1524, 1436, 1361, 1233, 1066, 1045, 820, 816.
UV–Vis (CH2Cl2, nm): 234 (e 40641), 280 (e 32840), 428 (e
1114), kmax 555 ( 159).
e
Micro Anal. Calc. for ZrC26O2H24F2Cl2: C, 54.9; H, 4.3; Cl, 13.4.
Found C, 54.8; H, 4.2; Cl, 12.5%.
6-(4-N,N-dimethylaminophenyl) fulvene (1c) was synthesised
according to the already published procedure [24].
2.2.3. Bis-[4-dimethylaminobenzyl)cyclopentadienyl] zirconium(IV)
dichloride [(g
5-C5H4–CH2–C6H4–(N(CH3)2)]2ZrCl2 (3c)
8.4 ml (15.0 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 10ꢀ2 mbar for 40 min and then
to 90 °C for 20 min. The concentrated LiBEt3H was dissolved in
30 ml of dry diethyl ether to give a colourless solution in a Schlenk
flask. 1.50 g (7.6 mmol) of the orange solid 6-(4-dimethylamin-
ophenyl) fulvene was added to a 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 solution
was left to stir for 5 h to give a yellow precipitate of the lithium
cyclopentadienide intermediate and the solution changed colour
from orange/red to a clear solution with a yellow precipitate. The
precipitate was filtered on to a frit and was washed with 20 ml
of diethyl ether. The yellow precipitate was dried briefly under
reduced pressure and was transferred to a Schlenk flask under
nitrogen. 0.89 g (4.3 mmol, 56.6% yield) of the lithiated cyclopenta-
dienide intermediate was obtained. 0.51 g (2.2 mmol) of zirconium
tetrachloride was dissolved in 30 ml of dry THF to give a yellow
solution in a Schlenk flask. The lithium cyclopentadienide interme-
diate was dissolved in 30 ml of the dried THF to give a pale yellow
solution. The zirconium tetrachloride solution was added to the
lithium cyclopentadienide intermediate solution via cannula to
give a yellow solution. The yellow zirconium solution was refluxed
for 16 h. The solution was then cooled and the solvent was re-
moved under reduced pressure. The remaining residue was ex-
tracted with 100 ml of trichloromethane and filtered through
celite to remove the remaining LiCl. The yellow filtrate was filtered
twice more by gravity filtration. The solvent was removed under
reduced pressure to yield 0.40 g of a pale yellow/green solid
(0.71 mmol, 32.5% yield).
1H NMR (d ppm CDCl3, 300 MHz): 3.78 [s, 6H, C6H4–OCH3], 4.94
[s, 4H, C5H4–CH2], 6.17–6.21 [m, 4H, J 2.7 C5H4], 6.83 [d, 2H, J
8.2 Hz, C6H4–OCH3], 7.15 [d, 2H, J 8.2 Hz, C6H4–OCH3].
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled): 35.2
[C6H4–OCH3], 55.2 [C5H4–CH2], 112.8, 113.9, 116.7, 129.8, 158.2.
IR absorptions (KBr, cmꢀ1): 3099, 2967, 1690, 1511, 1462, 1300,
1246, 1176, 1029, 818.
UV–Vis (CH2Cl2, nm): 210 (
e
22631), 235 (e 26573), 281 (e
41176), 426 ( 1443), kmax 557 (
e
e 531).
Micro Anal. Calc. for ZrC26O2H26Cl2: C, 58.9; H, 4.9; Cl, 13.3.
Found: C, 59.8; H, 5.2; Cl, 12.1%.
6-(2-Fluoro-4-methoxyphenyl) fulvene (1b) has been synthes-
ised according to the established procedure [23].
2.2.2. Bis-[(2-fluoro-4-methoxybenzyl)cyclopentadienyl]
zirconium(IV) dichloride [(g
5-C5H4–CH2–C6H3F–OCH3)]2ZrCl2 (3b)
16.0 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 10ꢀ2 mbar for 40 min and then to
90 °C for 20 min in a Schlenk flask. The concentrated Super Hydride
was dissolved in 30 ml of dry diethyl ether to give a cloudy white
suspension. 2.25 g (12.0 mmol) of the orange solid 6-(2-fluoro-4-
methoxyphenyl) fulvene was added to a Schlenk flask and was dis-
solved in 60 ml dry diethyl ether to give an orange solution. The
fulvene solution was transferred to the Super Hydride solution
via cannula. The solution was left to stir for 16 h in which time a
white precipitate of the lithium cyclopentadienide intermediate
formed and the solution had changed its colour from orange to
clear. 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. 1.48 g (7.0 mmol, 71.2% yield) of the lithiated cyclopenta-
dienide intermediate was obtained. 0.82 g (3.5 mmol) of zirconium
tetrachloride was dissolved in 30 ml of dry THF to give a clear solu-
tion in a Schlenk flask. The lithium cyclopentadienide intermediate
was dissolved in 30 ml of dry THF to give a colourless solution. The
zirconium tetrachloride solution was added to the lithium cyclo-
pentadienide intermediate solution via cannula to give a pale yel-
low solution. After 24 h of reflux the solution became dark
yellow, was then cooled and the solvent was removed under re-
duced pressure. The remaining pale yellow residue was extracted
with 60 ml of trichloromethane and filtered through celite to re-
move the remaining LiCl. The yellow filtrate was filtered twice
more by gravity filtration. The solvent was removed under reduced
pressure to yield 1.26 g of a pale yellow solid (22.2 mmol, 63.4%
yield).
1H NMR (d ppm CDCl3, 500 MHz): 2.93 [s, 6H, C6H4–OCH3], 3.90
[s, 4H, C5H4–CH2], 6.17–6.20 [m, 8H, J 2.45 C5H4], 6.79 [d, 2H, J
7.3 Hz, C6H4], 7.09 [d, 2H, J 8.3 Hz, C6H4].
13C NMR (d ppm CDCl3, 100 MHz, proton decoupled): 41.4
[C6H4–(OCH3)], 35.2 [C5H4–CH2], 109.0, 112.7, 113.0, 116.6, 129.7,
129.6, 131.3, 134.4.
IR absorptions (KBr, cmꢀ1): 31071 2959, 2913, 2835, 2359,
2342, 1613, 1506, 1426, 1284, 1259, 1102, 1028, 820.
UV–Vis (CH2Cl2, nm): 202 ( 21354), 231 (
e
19974), 210 (
e
e
18972), 268 ( 41439), 424 ( 1692), kmax 554 (e 512).
e
e
Micro Anal. Calc. for ZrC28N2H32Cl2: C, 60.4; H, 5.8; Cl, 12.7; N,
5.0. Found: C, 59.3; H, 5.7; Cl 13.2; N, 4.8%.
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 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
containing 10% (v/v) FCS (foetal calf serum), 1% (v/v) penicillin
1H NMR (d ppm CDCl3, 500 MHz): 3.78 [s, 6H, C6H4–OCH3], 3.94
[s, 4H, C5H4–CH2], 6.22–6.24 [m, 8H, J 2.3 Hz C5H4], 6.58 [m, 2H,
C6H4], 7.09 [t, 1H, C6H4].
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled): 55.5
[C6H4–OCH3], 29.1 [C5H4–CH2], 101.8, 109.8, 112.8, 116.8, 131.3,
132.9, 159.7, 160.4, 162.2.
streptomycin and 1% (v/v)
well plates containing 200
L
-glutamine. Cells were seeded in 96-
l wells at a density of 5000-cells/
l

D. Wallis et al. / Journal of Organometallic Chemistry 694 (2009) 828–833
831
200
l
l of medium and were incubated at 37 °C for 24 h to allow for
The three zirconocenes were synthesised using the successfully
applied hydridolithiation route for the synthesis of titanocenes.
This route uses LiBEt3H to transfer a hydride to a fulvene through
nucleophilic addition to the exocyclic double bond of the fulvenes
1a–c to form lithium cyclopentadienyl intermediates. Two molar
equivalents of the lithiated intermediate underwent a transmetal-
lation reaction in each case when reacted with one molar equiva-
lent of ZrCl4 in THF under reflux, to give the appropriate non-
bridged substituted zirconocenes (Scheme 1). The zirconocenes
3a–c were synthesised using the unmodified route for the synthe-
sis of the analogous titanocenes. The NMR shifts for these com-
pounds are similar to that of their titanocene analogues [8] apart
from the cyclopentadienyl signals, which in the zirconocenes split
into a multiplet as opposed to the titanocenes, where they show as
one signal. This multiplet appears as a doublet of triplets in all
three compounds between 6.17 and 6.24 ppm. The zirconocenes
proved to be paler in colour than their titanocene analogues, rang-
ing from cream to pale green in colour and crystallised from satu-
rated dichloromethane solutions (see Fig. 3).
exponential growth. Then the compounds used for the testing were
dissolved in the minimal amount of DMSO (dimethylsulfoxide)
possible and diluted with medium to obtain stock solutions of
1.9 ꢁ 10ꢀ4 M in concentration and less than 0.7% of DMSO. The
cells were then treated with varying concentrations of the com-
pounds and incubated for 48 h at 37 °C. Then, the solutions were
removed from the wells and the cells were washed with PBS (phos-
phate 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 med-
ium. The solution consisted of 40 mg of MTT in 40 ml of medium.
The cells were incubated for 3 h at 37 °C. The medium was then re-
moved and the purple formazan 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 con-
trol wells. The values used for the dose response curves represent
the values obtained from four consistent MTT-based assays [25] for
each compound tested. While zirconocenes 3b and 3c showed no
cytotoxic effects at the concentrations given, zirconocene 3a was
3.2. Structural discussion
found to be quite cytotoxic and exhibited an IC50 value of 57
l
M.
For the purpose of X-ray diffraction, suitable single crystals of
3a–c were grown by vapour diffusion of pentane into a saturated
solution of each compound in dichloromethane. The collection
and refinement data for each crystal is shown in Table 1. The deter-
mined structures for all three zirconocenes did not contain solvent
molecules, which is advantageous for cell testing. Selected bond
lengths and angles are shown in Table 2.
3. Results and discussion
3.1. Synthesis
Fulvenes 1a–c were synthesised according to published proce-
The zirconocene complexes 3a–c have distorted tetrahedral
shapes with respect to their two halogenido ligands and two cyclo-
pentadienide groups. As their isostructural titanocene analogues
these derivatives are stable compounds despite they have only
16 valence electrons. The zirconium-cyclopentadienide centroid
distances were measured as 2.201 and 2.202 Å for zirconocene
3a, 2.197 Å for zirconocene 3b, and 2.195 and 2.206 Å for zircono-
cene 3c. The length of bonds between the zirconium and the car-
bon atoms of the cyclopentadienyl rings bound to the metal ion
are similar for all zirconocenes (Figs. 4–6); they vary only slightly
between 2.534 and 2.467 Å for 3a, between 2.544 and 2.447 Å for
3b and between 2.533 and 2.466 Å for 3c. The zirconium-to-chlo-
dures [22–24] and their structures are shown in Fig. 2.
F
N(CH )
OCH
OCH
3 2
3
3
1c
1a
1b
Fig. 2. Structures of fulvenes 1a–c.
R
H
R
ZrCl4
2LiBEt3H
Cl
2
2
Zr
Cl
THF
-2LiCl(s)
Et2O
-2BEt3
H
R
H
R
Li
Scheme 1. Synthesis of benzyl-substituted zirconocenes via the hydridolithiation of fulvenes.
F
OCH3
OCH3
OCH3
OCH3
N(CH3)2
N(CH3)2
Cl
Cl
Cl
Cl
Cl
Cl
Zr
Zr
Zr
3a
3b
3c
F
Fig. 3. Structures of zirconocenes 3a–c.

832
D. Wallis et al. / Journal of Organometallic Chemistry 694 (2009) 828–833
on the zirconium atom. The Cl–Zr–Cl0 bond angle on the other side
was measured at 98.16° for 3a, 95.31° for 3b and 96.60° for 3c; this
compression of between 10° and 15° can again be attributed to the
size of cyclopentadienide groups relative to the two chlorine li-
gands. Interestingly all the angles around the Zr atom are quite
similar to the ones around Ti in the corresponding titanium com-
pounds [8].
Table 2
Selected bond lengths and angles of zirconocenes 3a–c
3a
3b
3c
Bond length (Å)
Zr-Cent1
Zr–Cent2
Zr–Cl(1)
Zr–Cl(2)
2.201(1)
2.202(1)
2.459(6)
2.451(6)
2.197(2)
2.197(2)
2.449(4)
2.449(4)
2.195(1)
2.206(1)
2.449(5)
2.455(5)
The packing for zirconocenes 3a and 3b is similar and features a
z-shaped pattern. The zirconium ion is featured on a C2 axis in 3b,
however this is not the case for 3a or 3c. For zirconocene 3c, the
overall molecular shape is more rod-shaped than z-shaped due
to the alignment of the phenyl groups with the cyclopentadienyl
rings, although it is not an ideal rod shape because of this align-
ment. In zirconocene 3c the aniline groups are not planar, the
sum angles for these are 357.56° and 358.60°. In light of this, it is
interesting to note that the corresponding titanocene could only
Bond angle (°)
Cl(2)–Zr–Cl(1)
Cent(1)–Zr–Cent(2)
98.16(2)
129.21(1)
95.305(18)
129.59(1)
96.599(17)
128.88(1)
rine bond lengths are very similar for all three compounds varying
from 2.459 Å to 2.449 Å. All these bond lengths are significantly
larger than that of their titanocene analogues [8] due to the larger
size of the zirconium atom compared to the titanium atom. The
centroid-to-zirconium-to-centroid angle was measured to be
129.59° for 3a, 129.59° for 3b and 128.88° for 3c. These angles
are approximately 20° larger than that of a normal tetrahedral an-
gle (109.5°) due to the relatively bulky cyclopentadienide groups
be crystallized as its hydrochloride salt. There are no p–p interac-
tions to be seen between the substituted phenyl groups in any of
the compounds but some soft Van der Waals interactions are pres-
ent, this is also the case for the analogous titanocenes.
C26
C22
C13
O1
C9
C23
C10
C21
O2
C8
C18
C5
C1
C6
C4
C17
C14
C20
C24
C7
C12
C11
C19
C25
Zr
C16
C3
C15
C2
Cl2
Cl1
Fig. 4. X-ray diffraction structure of bis-[(p-methoxybenzyl)cyclopentadienyl] zirconium(IV) dichloride 3a; (thermal ellipsoids are drawn at the 50% probability level).
C4
C3
C2
Zr
C5
C11
C12
C7
C1
C1
C13
C10
C6
O
Cl
Cl
C8
C9
F
Fig. 5. X-ray diffraction structure of bis-[(2-fluoro-4-methoxybenzyl)cyclopentadienyl] zirconium(IV) dichloride 3b; (thermal ellipsoids are drawn at the 50% probability
level).
C27
C25
C26
C21
C24
C13
C14
Cl1
C20
C2
C3
C9
N2
C8
N1
C22
C6
C28
C16
C17
C1
C5
C23
C10
Zr
C15
C19
C7
C11
C12
C4
Cl2
C18
Fig. 6. X-ray diffraction structure of bis-[(4-N,N-dimethylaminobenzyl)cyclopentadienyl] zirconium(IV) dichloride 3c; (thermal ellipsoids are drawn at the 50% probability
level).

D. Wallis et al. / Journal of Organometallic Chemistry 694 (2009) 828–833
833
ferred to zirconocenes without change. Two of the three
zirconocenes 3a–b synthesised did not show cytotoxic activity in
the LLC-PK assay, as expected from the literature. But Zirconocene
Y showed a surprisingly high activity against this kidney cancer
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
cell line leading to an IC50 value of 57 lM. Future experiments
on a wider panel of cell lines are planned; these results will show,
whether Zirconocene Y has antitumor activity or will become an
anticancer drug candidate.
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3a: IC50: (57+/-17) E-6 M
3b
3c
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The solubility of 3a–c in DMSO is limited. For the cell testing
three times the amount of DMSO compared to their isostructural
titanocene analogues was required for each compound. In order
to keep the DMSO concentration below 0.7%, which protects the
cells from a cytotoxic effect through DMSO, three times the
amount of medium was added. As a result each solution of the zir-
conocene was three times less concentrated than that of its titano-
cene analogue reaching a maximum concentration of 170 lM. The
dose response curves shown in Fig. 7 represent the experimental
values obtained from two consistent MTT assays for each com-
pound tested. As seen in Fig. 7 compounds 3b and 3c did not show
a cytotoxic effect at the concentrations given, but 3a showed good
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a factor of three.
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4. Conclusions and outlook
The synthesis of titanocenes through the hydridolithiation reac-
tion of aryl-substituted fulvenes with Superhydride can be trans-