B. Gleeson et al. / Journal of Organometallic Chemistry 694 (2009) 1369–1374
1371
UV–Vis (CH2Cl2, nm): k 234 (
e
22,800), k 284 (
e
12,400), k 383 (
e
slowly at À78 °C to give a dark red solution. The dark red vanadium
solution was refluxed for 20 h at 88 °C. After refluxing, the solution
was allowed to return to room temperature and then cooled to
À78 °C where a light green precipitate formed. The precipitate
was filtered on to a frit and washed with 20 ml of THF and small
quantities of chloroform. The light green solid was then dissolved
in chloroform and filtered through a frit to remove any remaining
LiCl. The solvent was removed under reduced pressure to yield a
light green crystalline solid (0.89 g, 1.1 mmol, 35.2% yield) 3c.
Micro Anal. Calc. for VCl2O6C30H34: C, 58.83; H, 5.60; Cl, 11.58.
Found: C, 57.87; H, 5.65; Cl, 11.42%.
2,860), k 655 ( 460).
e
2.2.2. Synthesis of bis-[(3,4-dimethoxybenzyl)cyclopentadienyl]
vanadium(IV) dichloride [
5-C5H4–CH2–C6H3–(OCH3)2]2VCl2 (3b)
g
15.0 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 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 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 inter-
mediate and the solution had changed colour from orange/red to
colourless with a white precipitate formed. The precipitate was fil-
tered on to a frit and was washed with 20 ml of diethyl ether. The
white precipitate was dried briefly under reduced pressure and
ESR (CH2Cl2 solution, RT): 8-line hyperfine coupling,
giso = 2.015, Aiso = 7.47 mT.
MS (m/z, QMS-MS/MS): 576 [MÀCl]+.
IR absorptions (KBr, cmÀ1): 3099, 2947, 2832, 1637, 1589, 1506,
1457, 1419, 1330, 1238, 1140, 1116, 1054, 1000, 919, 803, 731.
UV–Vis (CH2Cl2, nm): k 236 (e 24,620), k 288 (e 12,380), k 386 (e
3,040), k 655 ( 520).
e
was transferred to
a
Schlenk flask under nitrogen. 1.92 g
2.3. Cytotoxicity studies
(8.6 mmol, 77.1% yield) of the lithiated cyclopentadienide interme-
diate 2b was obtained. The lithium cyclopentadienide intermediate
was dissolved in 60 ml of dry THF to give a colourless solution.
0.46 ml (4.3 mmol) of vanadium tetrachloride was added to the
lithium cyclopentadienide intermediate solution slowly at À78 °C
to give a dark red solution. The dark red vanadium solution was re-
fluxed for 20 h at 88 °C. After refluxing, the solution was allowed to
return to room temperature and then cooled to À78 °C where a
light green precipitate formed. The precipitate was filtered on to
a frit and washed with 20 ml of THF and small quantities of chlo-
roform. The light green solid was then dissolved in chloroform
and filtered through a frit to remove any remaining LiCl. The sol-
vent was removed under reduced pressure to yield a light green
crystalline solid (0.89 g, 1.6 mmol, 37.5% yield) 3b.
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 (fetal calf serum), 1% (v/v) penicillin
streptomycin and 1% (v/v)
well plates containing 200
200 l of medium and were incubated at 37 °C for 24 h to allow
L
-glutamine. Cells were seeded in 96-
l
l wells at a density of 5000-cells/
l
for exponential growth. Then the compounds used for the testing
were dissolved in the minimal amount of DMSO (dimethylsulfox-
ide) 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 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
Micro Anal. Calc. for VCl2O4C28H30: C, 60.88; H, 5.84; Cl, 12.84.
Found: C, 59.91; H, 5.37; Cl, 13.03%.
ESR (CH2Cl2 solution, RT): 8-line hyperfine coupling,
giso = 2.019, Aiso = 7.47 mT.
MS (m/z, QMS-MS/MS): 516 [MÀCl]+.
IR absorptions (KBr, cmÀ1): 3103, 3083, 2953, 2931, 2835, 1588,
1514, 1462, 1444, 1416, 1261, 1142, 1022, 874, 848, 767.
UV–Vis (CH2Cl2, nm): k 236 (
e
26,560), k 283 (
e
17,820), k 386 (
e
wells were treated with a 200 ll of a solution of MTT (3-(4,5-
3,580), k 655 ( 620).
e
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) [23] in
medium. The solution consisted of 30 mg of MTT in 30 ml of med-
ium. The cells were incubated for 3 h at 37 °C. The medium was
then removed and the purple formazan crystals were dissolved
2.2.3. Synthesis of bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl]
vanadium(IV) dichloride [
5-C5H4–CH2–C6H2–(OCH3)3]2VCl2 (3c)
g
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. 2.00 g (8.2 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 trans-
ferred 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 changed col-
our 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. 1.58 g (6.3 mmol, 76.3% yield) of the lithiated cyclopenta-
dienide intermediate 2c was obtained. The lithium cyclopentadie-
nide intermediate was dissolved in 60 ml of dry THF to give a
colourless solution. 0.34 ml (3.2 mmol) of vanadium tetrachloride
was added to the lithium cyclopentadienide intermediate solution
in 200 ll DMSO per well. A Wallac Victor (Multilabel HTS Counter)
Plate Reader was used to measure absorbance at 540 nm. Cell via-
bility was expressed as a percentage of the absorbance recorded for
control wells. The values used for the dose response curves repre-
sent the values obtained from four consistent MTT-based assays for
each compound tested.
3. Results and discussion
3.1. Synthesis
The lithium intermediates used in the synthesis of vanadocene
derivatives 3a–c were synthesised by the hydridolithiation reac-
tion 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