Novel benzyl-substituted molybdocene anticancer drugs

Article abstract of DOI:10.1016/j.ica.2010.02.020

From the reaction of 6-(p-methoxyphenyl) fulvene (1a), 6-(3,4-dimethoxyphenyl) fulvene (1b) and 6-(3,4,5-trimethoxyphenyl) fulvene (1c) with LiBEt₃H, lithiated cyclopentadienide intermediates (2a-c) were synthesised. These intermediates were then transmetallated to molybdocene using MoCl₄ (synthesized in situ) to yield the benzyl-substituted molybdocenes bis-[(p-methoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3a), bis-[(3,4-dimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3b), and bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3c). The molybdocene 3a was characterised by single crystal X-ray diffraction. All three molybdocenes had their cytotoxicity investigated through MTT based preliminary in vitro testing on the human renal cell line Caki-1 in order to determine their IC50 values and compare them with the corresponding titanocene and vanadocene dichloride derivatives. Molybdocenes 3b-c were found to have the same IC50 values of 290 μM, while 3a yielded a value of 84 μM, respectively.

Full text of DOI:10.1016/j.ica.2010.02.020

Inorganica Chimica Acta 363 (2010) 1831–1836
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Novel benzyl-substituted molybdocene anticancer drugs
Brendan Gleeson, James Claffey, Anthony Deally, Megan Hogan, Luis Miguel Menéndez Méndez,
*
Helge Müller-Bunz, Siddappa Patil, 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 7 October 2009
Received in revised form 10 February 2010
Accepted 17 February 2010
Available online 24 February 2010
From the reaction of 6-(p-methoxyphenyl) fulvene (1a), 6-(3,4-dimethoxyphenyl) fulvene (1b) and 6-
(3,4,5-trimethoxyphenyl) fulvene (1c) with LiBEt3H, lithiated cyclopentadienide intermediates (2a–c)
were synthesised. These intermediates were then transmetallated to molybdocene using MoCl4 (synthe-
sized in situ) to yield the benzyl-substituted molybdocenes bis-[(p-methoxybenzyl)cyclopentadienyl]
molybdenum (IV) dichloride (3a), bis-[(3,4-dimethoxybenzyl)cyclopentadienyl] molybdenum (IV)
dichloride (3b), and bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3c).
The molybdocene 3a was characterised by single crystal X-ray diffraction. All three molybdocenes had
their cytotoxicity investigated through MTT based preliminary in vitro testing on the human renal cell
line Caki-1 in order to determine their IC50 values and compare them with the corresponding titanocene
and vanadocene dichloride derivatives. Molybdocenes 3b–c were found to have the same IC50 values of
Keywords:
Anticancer drug
Molybdocene
Hydridolithiation
Fulvene
290 lM, while 3a yielded a value of 84 lM, respectively
Caki-1
Cytotoxicity
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
in vitro but promising results in vivo [9,10]. Titanocene dichloride
reached clinical trials, but the efficacy of Cp2TiCl2 in Phase II clini-
cal trials in patients with metastatic renal cell carcinoma [11] or
metastatic breast cancer [12] was too low to be pursued. Novel
methods starting from fulvenes and other precursors allow direct
access to anti-proliferative titanocenes via reductive dimerisation
with titanium dichloride, carbolithiation or hydridolithiation of
the fulvene followed by transmetallation with titanium tetrachlo-
ride in the latter two cases [13]. Hydridolithiation of 6-anisyl
fulvene and subsequent reaction with TiCl4 led to bis-[(p-methoxy-
benzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene Y)
Renal Cell Carcinoma (RCC) is the third most common urologi-
cal cancer and is a particularly aggressive disease which has a poor
prognosis and resists conventional chemotherapy [1]. A 5-year sur-
vival rate for patients with advanced cases is estimated for 61% of
those patients, and the average overall survival rate for patients
diagnosed with the metastatic disease is less than a year [2]. In or-
der to find suitable treatments for such diseases, researchers have
explored the field of metal-based drugs, where Cisplatin is one of
the most successful and widely known compounds. Cisplatin is a
very effective anticancer drug and is widely used in the treatment
of many different types of cancers, particularly in testicular and
ovarian cancer [3–5]. However, problems are encountered when
treating RCC with Cisplatin due to its nephrotoxicity and its lack
of activity against tumours with natural or acquired resistance
[6,7]. As a result further examination into this area is needed.
Beyond the field of platinum anticancer drugs there is signifi-
cant unexplored space for further metal-based drugs targeting can-
cer. Titanium-based reagents have significant potential against
solid tumours. Budotitane ([cis-diethoxybis(1-phenylbutane-1,3-
dionato)titanium (IV)]) looked very promising during its preclinical
evaluation, but did not go beyond Phase I clinical trials [8]. Much
more robust in this aspect of hydrolysis is titanocene dichloride
(Cp2TiCl2), which shows medium anti-proliferative activity
[14], which has an IC50 value of 21 lM when tested on the
LLC-PK (long-lasting cells – pig kidney) 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.
Another aspect of these drug candidates is the difference in
anticancer activity that can be examined by changing the metal
centre. Vanadocene and vanadocene dichloride complexes have
proven to be effective antitumor agents [15–19]. Both titanocene
dichloride and vanadocene dichloride underwent extensive pre-
clinical testing against both animal and human cell lines [20–24].
In these studies Cp2VCl2 was found to be more active than Cp2TiCl2
in vitro.
Therefore, along with the titanocene dichloride derivatives,
similar vanadocene dichloride derivatives have been synthesised
using the same hydridolithiation route in order to examine the ef-
fect of changing the metal centre of these metallocenes. The most
* Corresponding author.
E-mail address: matthias.tacke@ucd.ie (M. Tacke).
0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.02.020

1832
B. Gleeson et al. / Inorganica Chimica Acta 363 (2010) 1831–1836
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Cl
Cl
Mo
Mo
Mo
Cl
Cl
Cl
OMe
OMe
OMe
3c
3b
Fig. 1. Structures of molybdocene dichlorides 3a–c.
3a
active compound in the series was found to be bis-[(p-methoxy-
benzyl)cyclopentadienyl] vanadium(IV) dichloride (Vanadocene
Y) [25]. This gave an IC50 value of 3.0 lM on the same LLC-PK cell
ray diffraction data for compound 3a were collected using a Bruker
SMART APEX CCD area detector diffractometer. A full sphere of re-
ciprocal space was scanned by phi–omega scans. Pseudo-empirical
absorption correction based on redundant reflections was per-
formed by the program SADABS [41]. The structures were solved by
direct methods using SHELXS-97 [42] and refined by full matrix
least-squares on F2 for all data using SHELXL-97 [42]. Hydrogen
atoms were added at calculated positions and refined using a rid-
ing 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 dis-
placement 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
the slow infusion of pentane into a saturated trichloromethane
solution.
line as Titanocene Y. Fluorinated derivatives of Vanadocene Y have
also been synthesised and tested on the human renal cell line Caki-
1, which displayed promising results [26].
Due to these significant increases in cytotoxicity, further exam-
ination of changes to the metal centre need to be carried out in or-
der to compare their anticancer activity with the titanocene and
vanadocene dichlorides.
While molybdocene compounds are well known for there cata-
lytic properties [27–29], there is also evidence that certain molyb-
docene dichlorides contain potential as anticancer agents, thanks
to pioneering work carried out by Harding and co-workers among
others [30–34]. Furthermore, the hydrolysis chemistry of Cp2MoCl2
has been well investigated [35–37]. The inherent stability of the Cp
ligands at physiological pH has allowed an extensive range of dif-
ferent biological experiments to be carried out and is an attractive
feature of these metallocenes. This is not the case for other metal-
locenes containing different metal centres, such as Cp2TiCl2. Stud-
ies have also previously been carried out in order to attempt to
derive the mechanism of antitumor action for Cp2MoCl2 [30,38–
40]. While these studies are not comprehensive, it is clear that
the different coordination chemistry of these metallocenes leads
to significantly different mechanisms, when the metal centre is
comprised of Ti, V or Mo.
Table 1
Crystal data and structure refinement for 3a.
Identification code
3a
Empirical formula
Formula weight
T (K)
C26H26O2Cl2Mo
537.31
100(2)
k (Å)
0.74450
Crystal system
Space group
Unit cell dimensions
a (Å)
monoclinic
C2/c (#15)
Within this paper we present the synthesis and preliminary
cytotoxicity studies of a series of three molybdocene dichloride
derivatives, modelled on Titanocene Y and Vanadocene Y, which
are shown in Fig. 1
25.583(5)
b (Å)
6.5331(11)
c (Å)
13.312(2)
a
(°)
90
b (°)
96.624(4)
c
(°)
90
2210.0(7)
V (Å3)
2. Experimental
Z
4
Dcalc (Mg/m3)
1.615
0.857
1096
2.1. General conditions
Absorption coefficient (mmÀ1
F(0 0 0)
)
Manipulations of air and moisture sensitive compounds were
performed under an inert atmosphere of nitrogen or argon using
standard Schlenk techniques. Molybdenum pentachloride (MoCl5)
and Super Hydride (LiBEt3H, 1.0 M solution in THF) were obtained
commercially from Sigma–Aldrich. All solvents were dried and dis-
tilled according to standard methods. 1H and 13C NMR spectra were
measured on a Varian 500 MHz spectrometer with CDCl3 as solvent
and TMS as internal standard. IR spectra were recorded on a Per-
kin–Elmer Paragon 1000 FT-IR Spectrometer employing NaCl discs.
UV–Vis spectra were recorded on a Unicam UV4 Spectrometer.
Electrospray ionization mass spectrometry (MS) was performed
on a quadrupole tandem mass spectrometer (Quattro Micro,
Micromass/Water’s Corp., USA), using solutions made up in 50%
dichloromethane and 50% methanol. MS spectra were obtained in
the ES+ (electron spray positive) mode for compounds 3a–c. X-
Crystal size (mm3)
0.50 Â 0.40 Â 0.3
Theta range for data collection
Index ranges
1.68–25.07°
À28 6 h 6 28, À7 6 k 6 7,
À15 6 l 6 15
Reflections collected
7021
Independent reflections (Rint
)
1701 (0.0306)
1423
99.6 %
Observed reflections (I > 2
Completeness to hmax
Absorption correction
r(I))
Semi-empirical from equivalents
Maximum and minimum transmission 0.7830 and 0.6512
Refinement method
Full-matrix least-squares on F2
1701/0/152
1.033
R1 = 0.0423, wR2 = 0.1049
R1 = 0.0516, wR2 = 0.1107
1.120 and À0.673
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2
r
(I)]
R indices (all data)
Largest difference in peak and hole
(e ÅÀ3
)

B. Gleeson et al. / Inorganica Chimica Acta 363 (2010) 1831–1836
1833
2.2. Synthesis
to a Schlenk flask and was dissolved in 90 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 16 h to give a white precipitate of the lithium cyclopentadie-
nide intermediate and the solution had changed colour from or-
ange/red to colourless with a white precipitate formed. The
precipitate was filtered on to a frit and was washed with 20 ml
of diethyl ether. The white precipitate was dried briefly under
reduced pressure and was transferred to a Schlenk flask under
nitrogen. 2.01 g (9.0 mmol, 80.7% yield) of the lithiated cyclopenta-
dienide intermediate 2b was obtained. The lithium cyclopentadie-
nide intermediate was dissolved in 60 ml of dry THF to give a
colourless solution. 1.07 g (4.5 mmol) of the black solid MoCl4
was dissolved in 25 ml of dry THF in a schlenk flask and the
lithiated cyclopentadienide intermediate solution was added via
cannula to yield a dark brown solution. This solution was then
refluxed for 20 h at 88 °C. After refluxing, the solution was allowed
to return to room temperature and the solvent was removed under
reduced pressure. The resulting brown solid was washed with
60 ml of dry pentane, redissolved in CH2Cl2 and the solution was
allowed to settle for 10 min. The solution was then filtered through
a frit to remove any remaining LiCl. The solvent was removed
under reduced pressure and the resulting brown/grey solid was
again washed with 60 ml of dry pentane and dried under reduced
pressure to yield a grey solid (0.87 g, 1.5 mmol, 32.3% yield) 3b.
Micro Analysis Calculated for MoCl2O4C28H30: C, 56.30; H, 5.06;
Cl, 11.87. Found: C, 55.91; H, 5.17; Cl, 11.53%.
The syntheses of 6-(p-methoxyphenyl) fulvene 1a, 6-(3,4-dime-
thoxyphenyl) fulvene 1b and 6-(3,4,5-trimethoxyphenyl) fulvene
1c were carried out according to already published procedures
[14,43].
2.2.1. Synthesis of bis-[(p-methoxybenzyl)cyclopentadienyl]
molybdenum(IV) dichloride, [g
5-C5H4–CH2–C6H4–O–CH3]2MoCl2 (3a)
Fifteen milliliters (15.0 mmol) of 1 molar solution of Super
Hydride (LiBEt3H) in THF was concentrated by removal of the
solvent by heating it to 60 °C under reduced pressure of
10À2 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. 2.40 g
(13.0 mmol) of the red solid 1a was added to a Schlenk flask
and was dissolved in 90 ml dry diethyl ether to give a red solu-
tion. The red fulvene solution was transferred to the Super Hy-
dride solution via cannula. The solution was left to stir for 8 h
to give a white precipitate of the lithium cyclopentadienide
intermediate and the solution had changed colour from orange/
red to colourless with a white precipitate formed. The precipitate
was filtered on to a frit and was washed with 20 ml of diethyl
ether. The white precipitate was dried briefly under reduced
pressure and was transferred to a Schlenk flask under nitrogen.
2.06 g (10.7 mmol, 82.2% yield) of the lithiated cyclopentadienide
intermediate 2a was obtained. The lithium cyclopentadienide
intermediate was dissolved in 60 ml of dry THF to give a colour-
less solution. 1.27 g (5.4 mmol) of the black solid MoCl4 was dis-
solved in 30 ml of dry THF in a Schlenk flask and the lithiated
cyclopentadienide intermediate solution was added via cannula
to yield a dark brown solution. This solution was then refluxed
for 20 h, allowed to return to room temperature and the solvent
was then removed under reduced pressure. The resulting brown
solid was washed with 60 ml of dry pentane, redissolved in
CH2Cl2 and the solution was allowed to settle for 10 min. The
solution was then filtered through a frit to remove any remain-
ing LiCl. The solvent was removed under reduced pressure and
the resulting brown/grey solid was again washed with 60 ml of
dry pentane and dried under reduced pressure to yield a grey
solid (1.19 g, 2.21 mmol, 41.5% yield) 3a.
1H NMR (d ppm CDCl3, 500 MHz): 3.67 [s, 4H, C5H4–CH2], 3.86
[d, J 5.4 Hz 12H, C6H3–(OCH3)2], 5.09 [d, 8H, J 3.9 Hz, C5H4–CH2],
6.80 [s, 4H, C6H3–(OCH3)2], 6.83 [s, 2H, C6H3–(OCH3)2].
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled): 35.3
[C5H4–CH2], 56.1 [C6H3–(OCH3)2], 95.8, 101.3 [C5H4–CH2], 111.3,
112.7, 121.2 [C6H3–(OCH3)2], 135.8, 144.2, 164.4.
MS (m/z, QMS-MS/MS): 563 [MÀCl]+
IR absorptions (KBr, cmÀ1): 3099 (w), 2996 (w), 2932 (m), 2833
(m), 1590 (m), 1515 (s), 1463 (s), 1418 (m), 1263 (s), 1236 (s), 1140
(s), 1026 (s), 810 (m).
UV–Vis (CH2Cl2, nm): k 230 (e 13,192), k 275 (e 6,714), k 353 (e
1,877).
2.2.3. Synthesis of bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl]
Micro Analysis Calculated for MoCl2O2C26H26: C, 58.12; H, 4.88;
Cl, 13.20. Found: C, 58.10; H, 4.89; Cl, 13.10%.
molybdenum (IV) dichloride, [g
5-C5H4–CH2–C6H2–(OCH3)3]2MoCl2
(3c)
1H NMR (d ppm CDCl3, 500 MHz): 3.67 [s, 4H, C5H4–CH2], 3.79
[s, 6H, C6H4–OCH3], 5.03 [s, 4H, C5H4–CH2], 5.09 [s, 4H, C5H4–
CH2], 6.84 [d, J 8.3 Hz, 4H, C6H4–OCH3], 7.17 [d, J 8.3 Hz, 4H,
C6H4–OCH3].
15.0 ml (15.0 mmol) of 1 molar solution of Super Hydride
(LiBEt3H) in THF was concentrated by removal of the solvent by
heating it to 60 °C under reduced pressure of 10À2 mbar for
40 min and then to 90 °C for 20 min in a Schlenk flask. The concen-
trated Super Hydride was dissolved in 30 ml of dry diethyl ether to
give a cloudy white suspension. 2.40 g (9.5 mmol) of the dark red
solid 1c was added to a Schlenk flask and was dissolved in 90 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 16 h to give a white precipitate of the
lithium cyclopentadienide intermediate and the solution had chan-
ged colour from orange/red to colourless with a white precipitate
formed. The precipitate was filtered on to a frit and was washed
with 20 ml of diethyl ether. The white precipitate was dried briefly
under reduced pressure and was transferred to a Schlenk flask
under nitrogen. 2.20 g (8.7 mmol, 82.1% yield) of the lithiated
cyclopentadienide intermediate 2c was obtained. The lithium
cyclopentadienide intermediate was dissolved in 60 ml of dry
THF to give a colourless solution. 1.04 g (4.4 mmol) of the black so-
lid MoCl4 was dissolved in 25 ml of dry THF in a schlenk flask and
the lithiated cyclopentadienide intermediate solution was added
via cannula to yield a dark brown solution. This solution was then
refluxed for 20 h at 88 °C. After refluxing, the solution was allowed
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled): 34.4
[C5H4–CH2], 55.3 [C6H4–OCH3], 96.4, 101.4 [C5H4–CH2], 114.1,
130.2, [C6H4–OCH3], 123.5, 140.7, 162.3.
MS (m/z, QMS-MS/MS): 503 [MÀCl]+
IR absorptions (KBr, cmÀ1): 3093 (m), 2960 (w), 2931 (w), 2834
(w), 1609 (m), 1511 (s), 1463 (w), 1439 (w), 1302 (m), 1247 (s),
1176 (s), 1106 (w), 1032 (s) 819 (m).
UV–Vis (CH2Cl2, nm): k 229 (
1,419).
e 8,473), k 270 (e 3,204), k 352 (e
2.2.2. Synthesis of bis-[(3,4-dimethoxybenzyl)cyclopentadienyl]
molybdenum(IV) dichloride, [g
5-C5H4–CH2–C6H3–(OCH3)2]2MoCl2
(3b)
15.0 ml (15.0 mmol) of 1 molar solution of Super Hydride (Li-
BEt3H) in THF was concentrated by removal of the solvent by heat-
ing it to 60 °C under reduced pressure of 10À2 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. 2.40 g (11.2 mmol) of the red solid 1b was added

1834
B. Gleeson et al. / Inorganica Chimica Acta 363 (2010) 1831–1836
to return to room temperature and the solvent was removed under
reduced pressure. The resulting brown solid was washed with
60 ml of dry pentane, redissolved in CH2Cl2 and the solution was
allowed to settle for 10 min. The solution was then filtered through
a frit to remove any remaining LiCl. The solvent was removed un-
der reduced pressure and the resulting brown solid was again
washed with 60 ml of dry pentane and dried under reduced pres-
sure to yield a light brown solid (1.86 g, 2.8 mmol, 64.8% yield) 3c.
Micro Analysis Calculated for MoCl2O6C30H34: C, 54.81; H, 5.21;
Cl, 10.79. Found: C, 54.63; H, 5.28; Cl, 10.42%.
Sn/Et2O
MoCl5
MoCl4(OEt2)2
MoCl4
Scheme 2. General Reaction scheme for the synthesis of MoCl4.
were incubated for 3 h at 37 °C. The medium was then removed
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 ex-
pressed 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.
1H NMR (d ppm CDCl3, 500 MHz): 3.70 [s, 4H, C5H4–CH2], 3.83
[s, 6H, C6H2–(OCH3)3], 3.85 [s, 12H, C6H2–(OCH3)3], 5.10 [d, 4H, J
2.5 Hz, C5H4–CH2], 5.14 [d, 4H, J 2.5 Hz, C5H4–CH2], 6.52 [s, 4H,
C6H2–(OCH3)3].
13C NMR (d ppm CDCl3, 125 MHz, proton decoupled): 35.8
[C5H4–CH2], 56.3, 60.9 [C6H2–(OCH3)3], 95.4, 101.3 [C5H4–CH2],
106.3 [C6H2–(OCH3)3], 124.6, 134.0, 153.4, 160.0.
3. Results and discussion
3.1. Synthesis
MS (m/z, QMS-MS/MS): 623 [M–Cl]+
IR absorptions (KBr, cmÀ1): 3102 (w), 2961 (m), 2936 (m), 2835
(m), 1591 (s), 1507 (s), 1457 (s), 1420 (s), 1331 (m), 1238 (s), 1130
(s), 1007 (s), 810 (w).
The lithium intermediates used in the synthesis of molybdo-
cene derivatives 3a–c were synthesised by the hydridolithiation
reaction of aryl-fulvenes with Super Hydride (LiBEt3H). This form
of nucleophilic addition to the exocyclic double bond of the fulvene
is highly selective due to the increased polarity as a result of the
inductive effect of the corresponding phenyl ring. There is no
nucleophilic attack seen at the diene element of the aryl-fulvenes.
The lithiated cyclopentadienide intermediate was isolated with
good yields of around 80%. Two equivalents of the lithiated cyclo-
pentadienide intermediate were transmetallated with 1 equiv. of
molybdenum tetrachloride to form the desired molybdocene
dichlorides in yields of 35–62% and lithium chloride as a by-prod-
uct. As shown in Scheme 1, derivatives 3a, 3b, and 3c were isolated
as air stable brown/grey solids. They are soluble in organic chlori-
nated solvents, however all three compounds displayed tendencies
to decompose slightly in CHCl3 solutions, when exposed to moist
air for 24 h.
UV–Vis (CH2Cl2, nm): k 230 (e 15,079), k 276 (e 7,184), k 353 (e
2,000).
2.3. Cytotoxicity studies
Preliminary in vitro cell tests were performed on the human re-
nal cell line Caki-1 in order to compare the cytotoxicity of the com-
pounds 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 streptomycin and 1% (v/v)
L-glutamine. Cells were seeded in 96-well plates containing
One of the problems encountered with this synthesis was that
the commercially available MoCl5 needed to be reduced to the
Mo(IV) tetrahalide. This was accomplished by using a novel syn-
thesis involving Sn powder and diethylether, which has been pub-
lish by Poli and co-workers [45], and is shown in Scheme 2. The
MoCl4(OEt2)2 intermediate was dried on a frit under reduced pres-
sure at 0 °C, transferred to a Schlenk flask and heated for 1 h at
80 °C to yield the desired MoCl4 product as a black powder.
200 l microtitre wells at a density of 5000-cells/200 l of medium
l
l
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 minimal amount of DMSO (dimethylsulfoxide) possible
and diluted with medium to obtain stock solutions of 5 Â 10À4 M
in concentration and less than 0.7% of DMSO. The cells were then
treated with varying concentrations of the compounds and incu-
bated 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
3.2. Structural discussion
treated with a 200
zol-2-yl)-2,5-diphenyltetrazolium bromide) [44] in medium. The
solution consisted of 20 mg of MTT in 30 ml of medium. The cells
l
l of a solution of MTT (3-(4,5-dimethylthia-
Despite the effort to crystallise molybdocenes 3b and 3c, no
single crystals could be isolated and hence no structures were ob-
tained. This could possibly be explained by the slow decomposition
R
H
R
MoCl4
Cl
2LiBEt3H
2
2
Mo
THF
Et2O
-2BEt3
Cl
H
-2LiCl(s)
R
H
R
Li
1a-c
2a-c
3a-c
a = 4-OMe
b = 3,4-(OMe)2
c = 3,4,5-(OMe)3
Scheme 1. General reaction scheme for the synthesis of benzyl-substituted molybdocene dichloride derivatives 3a–c.

B. Gleeson et al. / Inorganica Chimica Acta 363 (2010) 1831–1836
1835
Table 2
of the compound in solution as stated previously. However a suit-
Selected bond lengths and angles from the crystal structure of 3a.
able crystal structure of compound 3a has been isolated. Com-
pound 3a crystallises in the monoclinic space group C2/c (#15)
with four molecules in the unit cell and contains a twofold axis
(see Fig. 2). The absence of solvent molecules present in the unit
cell is a particular advantage when it comes to biological testing
of these compounds.
3a
Bond lengths (Å)
Mo–C(1)
Mo–C(2)
Mo–C(3)
Mo–C(4)
2.256(4)
2.242(4)
2.275(4)
2.364(6)
2.320(5)
1.956(1)
1.427(6)
1.419(6)
1.413(6)
1.363(7)
1.400(8)
1.493(6)
1.514(5)
2.478(3)
2.536(4)
The bond lengths between the molybdenum centre and the cen-
troids of the cyclopentadienyl rings were found to be 1.956 Å. This
is comparable to those found in the literature for Cp–Mo bonds
[46]. These bond lengths are also shorter than the corresponding
Ti–Cp(Centroid) bond, which has a bond length of 2.060 Å for
Titanocene Y and also the V–Cp(Centroid) bond length of 1.983 Å
in Vanadocene Y. This is due to the extra electron density at the
molybdenum d2 centre, which leads to back bonding at the Cp li-
gands, resulting in shorter bond lengths. The same characteristic
has the effect of slightly lengthening the Mo–Cl bonds to a range
of 2.478–2.536 Å (the chlorine atom is disordered over two posi-
tions with half occupancy each) for the molybdocene dichloride
structure, whereas the Ti–Cl bond lengths for Titanocene Y are in
the region of 2.370 Å, and the V–Cl bond lengths of Vanadocene
Y are in the range of 2.402 Å. This back bonding, along with the
bulkiness of the Cp ligands, also has an effect on the overall confor-
mation of the molecule, whereby the Cp(centroid)–Mo–Cp(cen-
troid) bond angle was widened to around 135.9. Compared to the
corresponding bond angle of 130.7° in Titanocene Y and 132.8°
in Vanadocene Y. Conversely the Cl–Mo–Cl bond angle is narrowed
in order to accommodate the broadened Cp(centroid)–Mo–Cp(cen-
troid) bond angle, and varies from 82.5° to 85.6° for 3a, while
Titanocene Y displayed a Cl–Ti–Cl bond angle of 95.9° and Vanado-
cene Y had a Cl–V–Cl bond angle of 88.5°. The carbon carbon bond
lengths of the cyclopentadienyl rings in 3a range from 1.363 to
1.427 Å. In summary it means, that the structure displays a dis-
torted tetrahedral conformation. The crystal data and refinement
details are found in Table 1, while selected bond lengths and angles
are displayed in Table 2.
Mo–C(5)
Mo–Cent
C(1)–C(5)
C(1)–C(2)
C(2)–C(3)
C(3)–C(4)
C(4)–C(5)
C(1)–C(6)
C(6)–C(7)
Mo–Cl(1)
Mo–Cl(2)
Bond angles (°)
Cent–Mo–Cent
Cent–Mo–Cl(1)
Cent–Mo–Cl(2)
Cl(1)–Mo–Cl(1)
Cl(2)–Mo–Cl(2)
Cl(1)–Mo–Cl(2)
135.87(1)
98.3(1)
115.2(1)
84.8(1)
85.6(1)
82.52(6)
the titanocenes and 9.1 and 8.3
tively. Compound 3a, which gave an IC50 value of 84
l
M for the vanadocenes, respec-
M, shows
l
a dramatic improvement in cytotoxicity when compared to the
other molybdocenes in the series. Upon comparison with its tita-
nium and vanadium equivalent however, which yielded IC50 val-
ues of 21 lM for Titanocene Y and 3.0 lM for Vanadocene Y,
molybdocene 3a (or Molybdocene Y) again does not reach the
same level of cytotoxic activity. This is also true for its tin equiva-
lent, which yielded an IC50 value of 15
conium derivative, Zirconocene Y, which displayed an IC50 value of
57 M [48]. From these comparisons, Vanadocene Y still displays
the best IC50 value of this class of metallocenes and is equivalent
lM [47], as well as its zir-
l
to that of Cisplatin, which gave an IC50 value of 3.3
the same LLC-PK cell line.
lM against
3.3. Cytotoxicity studies
These results do however emphasise the importance of the p-
methoxy phenyl moiety that is present in molybdocene 3a and
the other isostructural metallocenes. They also serve to highlight
the difference, in cytotoxic effects, of substituting the metal centre
of these metallocenes with different transition metals, in this case
molybdenum.
Fig. 3 displays the cytotoxicity curves for all three compounds,
in which one can observe that cell viability is reduced by more
than a factor of 3 relative to the drug concentration for compound
3a, compared to the other two molybdocenes, with the higher
concentrations resulting in almost total cell death. In general,
compounds 3a–c displayed good solubility and satisfactory mar-
gins of error.
Since the three molybdocenes 3a–c were found to be insoluble
in water, DMSO was the solvent used for the standard MTT based
assay tests. These compounds were tested on the human renal cell
line Caki-1, which is comparable with the previously used LLC-PK
cell line that was employed to evaluate similar metallocenes from
the group in the past. However since the cell lines are not identical,
this should be considered when examining the IC50 value compar-
isons between these molybdocene compounds and other isostruc-
tural metallocenes.
While displaying relatively low cytotoxic effects, molybdocenes
3b and 3c yielded the same IC50 values of 290
lM. Unfortunately,
this does not improve upon the corresponding titanocene and
vanadocene derivatives with IC50 values of 88 and 253
l
M for
C9
C13
C10
C8
C2
C3
O
C1
C5
C1
C11
C7
C6
Mo
C12
C4
Cl1
Cl1
Fig. 2. X-ray diffraction structure of 3a; thermal ellipsoids are drawn on the 50% probability level.

1836
B. Gleeson et al. / Inorganica Chimica Acta 363 (2010) 1831–1836
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The hydridolithiation of 6-aryl substituted fulvenes followed by
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Supplementary data
CCDC 736572 contains the supplementary crystallographic data
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