JOURNAL OF

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Journal of Inorganic Biochemistry 98 (2004) 1987–1994

InorganicBiochemistry

Methoxy-phenyl substituted ansa-titanocenes as potentialanti-cancer drugs derived from fulvenes and titanium dichloride

Matthias Tacke a,*, Laurence P. Cuffe a, William M. Gallagher b, Ying Lou a,Oscar Mendoza a, Helge Muller-Bunz a, Franz-Josef K. Rehmann a, Nigel Sweeney a

a Chemistry Department, Centre for Synthesis and Chemical Biology (CSCB), University College Dublin, Belfield, Dublin 4, Irelandb Pharmacology Department, University College Dublin, Belfield, Dublin 4, Ireland

Received 30 June 2004; received in revised form 24 August 2004; accepted 2 September 2004

Available online 30 September 2004

Abstract

Starting from 6-(4 0-methoxyphenyl)fulvene (1a), 6-(2 0,4 0,6 0-trimethoxyphenyl)fulvene (1b), or 6-(3 0,5 0-dimethoxyphenyl)fulvene

(1c), [1,2-di(cyclopentadienyl)-1,2-di(4 0-methoxyphenyl)-ethanediyl] titanium dichloride (2a), [1,2-di(cyclopentadienyl)-1,2-

bis(2 0,4 0,6 0-trimethoxyphenyl)-ethanediyl] titanium dichloride (2b), and [1,2-di(cyclopentadienyl)-1,2-bis(3 0,5 0-dimethoxyphenyl)-

ethanediyl] titanium dichloride (2c) were synthesised. When titanocenes 2a–c were tested against pig kidney carcinoma cells

(LLC-PK) inhibitory concentrations (IC50) of 2.8 · 10�4, 3.6 · 10�4 and 2.1 · 10�4 M, respectively, were observed.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Anti-cancer drug; cis-Platinum; Titanocene; Fulvene; LLC-PK; DFT calculations

1. Introduction

Despite the resounding success of cis-platinum and

closely related platinum antitumor agents, the move-

ment of other transition-metal anti-cancer drugs to-

wards the clinic has been exceptionally slow [1–3].

Metallocene dichlorides (Cp2MCl2) with M = Ti, V,

Nb and Mo show remarkable antitumor activity [4,5].However, only titanocene dichloride has reached Phase

I clinical trials so far, with a maximum tolerable dose

of 315 mg/m2 per week. The dose limiting effects of

titanocene dichloride include nephrotoxicity and eleva-

tion of creatinine and bilirubin levels [6,7]. Unfortu-

nately, the efficacy of Cp2TiCl2 in Phase II clinical

trials in patients with metastatic renal-cell carcinoma

[8] or metastatic breast cancer [9] was too low to be pur-sued. Nevertheless, little synthetic effort has been em-

ployed to increase the cytotoxicity of any titanocene

0162-0134/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2004.09.001

* Corresponding author. Fax: +353 1 716 2127.

E-mail address: matthias.tacke@ucd.ie (M. Tacke).

dichloride derivative [10–12], despite the existence of a

novel method starting from titanium dichloride and ful-

venes [13–16], which allows direct access to highly sub-

stituted ansa-titanocenes [17–20]. Recently, using this

method we have synthesised [1,2-di(cyclopentadienyl)-

1,2-di(p-N,N-dimethylaminophenyl)ethanediyl] titanium

dichloride, which has an IC50 value of 2.7 · 10�4 M

when tested for cytotoxic effects on the LLC-PK cell line[21]. This paper reports the synthesis of novel methoxy

substituted [(1,2-diaryl-1,2-dicyclopentadienyl)-ethane-

diyl] titanium dichlorides, which combine the reactivity

of the titanium dichloride moiety with the ability of

hydrogen bonding towards DNA of the ammine ligand

of cis-platinum, if the aryl group is substituted

accordingly.

2. Experimental

Titanium tetrachloride (and as a 1 mol solution in

toluene for the synthesis of 2c) and n-butyl lithium (2

Table 1

Crystal data and structure refinement for 1b

Empirical formula C15H16O3

Formula weight 244.28

Temperature (K) 293(2)

Wavelength 0.71073

Crystal system Monoclinic

Space group P21/c (#14)

Unit cell dimensions

a (A) 7.3402(6)

b (A) 18.5478(16)

c (A) 9.5450(8)

b (�) 94.0230(10)

Volume (A3) 1296.30(19)

Z 4

Density (calculated) (mg/m3) 1.252

Absorption coefficient (mm�1) 0.086

F(000) 520

Crystal size (mm3) 0.50 · 0.30 · 0.30

H range for data collection 2.20–28.25.

Index ranges �9 6 h 6 9,

�24 6 k 6 24,

�12 6 l 6 12

Reflections collected 21,083

Independent reflections 3100 [Rint = 0.0280]

Completeness to theta = 28.25 96.3%

Absorption correction Numerical

Max. and min. transmission 0.9745 and 0.9581

Refinement method Full-matrix least-squares on F2

Data/restraints/parameters 3100/0/227

Goodness-of-fit on F2 1.044

Final R indices [I>2r(I)] R1 = 0.0500, wR2 = 0.1295

R indices (all data) R1 = 0.0622, wR2 = 0.1377

Largest diff. peak and hole (e A�3) 0.260 and �0.188

1988 M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994

mol solution in hexane for 2a and 2b and in cyclohex-

ane for 2c) were obtained commercially from Aldrich

Chemical Co. THF and toluene were dried over and

distilled from Na/benzophenone prior to use. 4-meth-

oxybenzaldehyde, 2,4,6-trimethoxybenzaldehyde and

3,5-dimethoxybenzaldehyde were obtained commer-cially from Aldrich Chemical Co. Cyclopentadiene

was collected under an atmosphere of nitrogen from

freshly cracked dicyclopentadiene and pyrrolidine was

distilled under argon prior to use. Manipulation of

air and moisture sensitive compounds was carried out

using standard Schlenk techniques under an argon

atmosphere. Nuclear magnetic resonance (NMR) spec-

tra were measured on a Varian 300 MHz spectrometer.Chemical shifts are reported in ppm and are referenced

to tetramethylsilane (TMS). Infrared (IR) spectra were

recorded on a Perkin–Elmer Paragon 1000 FT-IR Spec-

trometer employing a KBr disk. UV/Vis spectra were

recorded on an UNICAM UV/Vis Spectrometer in

either methanol (fulvenes) or acetonitrile (titanocenes).

The gaschromatography–mass spectra (GS–MS) were

measured on a FINNIGAN TRACE GCMS 2000Se-ries (70 eV) and 1 · 10�5 M solutions in ethyl acetate

were used.

A single crystal of fulvene 1b suitable for X-ray dif-

fraction experiments was grown by slow evaporation

of petroleum spirit (40–60) from an open sample bot-

tle. X–ray diffraction data for 1b were collected on a

BRUKER Smart Apex diffractometer at room temper-

ature for 1b. A semi-empirical absorption correctionon the raw data was performed using the program SA-

DABS [22]. The crystal structure was then solved by

direct methods (SHELXS-NT 97) [23] and refined by

full-matrix least-squares methods against F2. Further

details about the data collection are listed in Table 1,

as well as reliability factors. Further details are availa-

ble free of charge from the Cambridge structural data-

base under the CCDC No. 241169. With a view toelucidate the structures, spectroscopic data, bonding

properties and energies of formation, the application

of theoretical methods is advantageous. For this pur-

pose, the GAUSSIAN 98 Revision A11 [24] running

under Red Hat Linux was used. Density functional

theory (DFT) calculations were performed at the

B3LYP level using the 6-31G** basis set for the species

of interest.

2.1. 6-(4 0-methoxyphenyl)fulvene (1a)

The syntheses of fulvenes 1a–c were carried out under

argon as outlined in reference [25]. Pyrrolidine (2.5 ml,

30.0 mmol) was added to asolution of 4-methoxybenzal-

dehyde (2.4 ml, 20.0 mmol) and cyclopentadiene (4.1 ml,

50.0 mmol) in 30 ml of methanol. After addition, thecolour of the solution immediately turned from colour-

less to red-orange. Large amounts of an orange solid

precipitated out of the solution. When thin layer chro-

matography (TLC) analysis showed only one product

band after 15 min, acetic acid (1.8 ml, 32.0 mmol) was

added. The reaction mixture was diluted with 20 ml of

a mixture of diethyl ether and water (1:1). The resultant

organic layer was separated and the aqueous layer was

washed with diethyl ether (3 · 20 ml). The combined or-

ganic extracts were washed with a saturated aqueousNaCl solution. The organic solution was dried over

magnesium sulfate. When the solvent was removed un-

der reduced pressure, 3.6 g of an orange product were

obtained (90% yield rel. to 4-methoxybenzaldehyde).

m.p. 48 �C.1H NMR(d ppm CDCl3): 6.70, 6.64, 6.45, 6.30 (C5H4,

4H m); 7.53, 7.50, 6.88, 6.85 (C6H4, 4H m); 3.75

((OCH3)2, 6H s); 7.10 (Ph–CH–Cp, 1H s). 13C NMR(d ppm CDCl3): 143.3, 134.9, 129.7, 127.5, 119.9

(C5H4); 160.6, 132.4, 129.5, 114.3 (C6H4); 55.2

(OCH3); 138.2 (Ph–CH–Cp). IR absorptions (cm�1

KBr): 3062 (w); 3013 (w); 2965 (w); 2840 (w); 1622

(C@C m); 1460 (s); 1348 (m); 832 (s); 818 (s). GC–MS:

184.1 (M, 100%); 169.1 ðMþ � CHþ3 25%Þ. UV/Vis

(methanol): kmax = 331 nm. Anal. Calc. for C13H12O:

C, 84.75; H, 6.57; Found: C, 84.42; H, 6.54.

M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994 1989

2.2. 6-(2 0,4 0,6 0-trimethoxyphenyl)fulvene (1b)

Pyrrolidine (1.3 ml, 15.0 mmol) was added to a solu-

tion of 2,4,6-trimethoxybenzaldehyde (2.0 g, 10.0 mmol)

and cyclopentadiene (2.1 ml, 26.0 mmol) in 30 ml of

methanol. After this addition the solution turned fromcolourless to clear orange-yellow. A yellow solid precip-

itated after 10 min. When TLC analysis showed only

one product band after 20 h, acetic acid (0.9 ml, 16.0

mmol) was added. The reaction mixture was diluted

with 20 ml of a mixture of dichloromethane and water

(1:1). The resultant organic layer was separated and

the aqueous layer was washed with dichloromethane

(3 · 20 ml). The combined organic extracts were washedwith a saturated aqueous NaCl solution. The organic

solution was dried over magnesium sulphate. The crude

product was redissolved in petroleum spirit (40–60) and

purified by column chromatography over silica gel 60

(0.063–0.200) and using a solution of petroleum spirit

(40–60) and dichloromethane (2:1) as the eluent. After

solvent removal under reduced pressure, 1.5 g of a yel-

low product was obtained. 1.5 g (60% yield rel. to2,4,6-trimethoxybenzaldehyde). m.p. 120 �C.

1H NMR (d ppm CDCl3): 6.45, 6.44, 6.38, 6.32

(C5H4, 4H m); 6.15 (C6H2, 2H s); 3.84 (p-OCH3, 3H

s); 3.80 (o-OCH3, 6H s); 7.17 (Ph-CH-Cp, 1H s). 13C

NMR (d ppm CDCl3): 132.2, 130.1, 129.5, 127.1,

123.0, 107.9, 90.6 (C5H4 and C6H2,); 55.62(o-OCH3);

55.39 (p-OCH3). IR absorptions (cm�1 KBr): 3060 (w);

2940(w); 1629 (C@C m); 1467 (s); 1328 (m); 1122 (s);949 (w); 813 (m); 753 (s). GC–MS: 244.2 (M+ 100%);

229.1 ðMþ � CHþ3 55%Þ. UV/Vis (methanol): kmax = 277

nm. Anal. Calc. for C15H18O3: C, 73.15; H, 7.36; Found:

C, 73.63; H, 7.04.

2.3. 6-(3 0,5 0-dimethoxyphenyl)fulvene (1c)

Pyrrolidine (2.5 ml, 30.0 mmol) was added to a solu-tion of 3,5-dimethoxybenzaldehyde (2.0 g, 10.0 mmol)

and cyclopentadiene (5.0 ml, 75 mmol) in 100 ml of

methanol. After this addition, the colour of the solution

turned from colourless to red. The mixture was stirred

under argon for 20 h. Acetic acid (1.8 ml, 30.1 mmol)

was added and the reaction mixture was diluted with a

mixture of dichloromethane and water (1:1). The result-

ant organic layer was separated and the aqueous layerwas washed with dichloromethane (3 · 50.0 ml). The

combined organic extracts were washed with a concen-

trated NaCl aqueous solution. The organic solution

was dried over sodium sulphate and the solvent was re-

moved under reduced pressure. The crude product was

purified by column chromatography over silica gel using

a mixture of pentane and dichloromethane (3:1) as the

eluent following the purification procedure for fulvene1c. 4.0 g of a red oil was obtained (62% yield rel. to

3,5-dimethoxybenzaldehyde).

1H NMR (d ppm CDCl3): 6.68, 6.64, 6.49, 6.29

(C5H4, 4H m); 6.71 (o-C6H3, 2H, d, 2.4 Hz); 6.47 (p-

C6H3, 1H, t, 2.4 Hz); 3.79 (OCH3, 6H s); 7.12 (Ph–

CH–Cp, 1H s). 13C NMR (d ppm CDCl3): 160.66,

145.39, 138.40, 138.24, 135.49, 131.05, 127.16, 120.32,

108.39, 101.34 (C5H4 and C6H2); 55.36 (o-OCH3). IRabsorption (cm�1 KBr): 3066 (w); 3000 (w); 2956 (m);

2934 (m); 2835 (m); 1621 (C@C s); 1591 (vs); 1454 (s);

1424 (s); 1377 (m); 1344 (m); 1319 (m); 1230 (s); 1122

(s); 1056 (m); 949 (w); 813 (s); 753 (s). GC–MS: 214.3

(M 100%); 199.3 ðMþ � CHþ3 83%Þ; 183.3

ðMþ �OCHþ3 35%Þ. UV/Vis (CHCl3): kmax = 260 nm.

Anal. Calc. for C14H14O2: C, 78.48; H, 6.59; Found:

C, 78.05; H, 6.58.

2.4. [1,2-Di(cyclopentadienyl)-1,2-di(4 0-methoxy-phe-

nyl)-ethanediyl] titanium dichloride [1,2-(p-OMe-

C6H4)2C2H2{g5-C5H4}2]TiCl2 (2a)

TiCl4 (0.90 ml, 8.1 mmol) was added to 40 ml of dry

toluene containing 5% dry THF. The solution turned

immediately from colourless to pale yellow. The solu-tion was stirred and cooled down to �78 �C, followedby drop wise addition of n-butyl lithium (8.15 ml, 16.2

mmol). The solution turned from yellow to brown dur-

ing addition. After this addition, the mixture was al-

lowed to warm up slowly to room temperature (r.t.).

The colour of the solution became finally black. After

20 h stirring at, a solution of 1a (3.20 g, 16.3 mmol)

in 35 ml of dry toluene was added to the TiCl2 Æ 2THFsolution at r.t. under argon. Then it was stirred under

reflux for another 20 h. The solvent was removed under

vacuum leaving a black residue. The residue was

washed with chloroform and the solution was filtered

through celite under reduced pressure. The colour of

the filtrate reddened slightly. It was filtered using grav-

ity filtration for at least four times until no further

black precipitate appeared on the filter paper and thefiltrate turned to dark red. Chloroform was removed

to leave dark-red solid 2a with 2.2 g (4.5 mmol, 44%

yield). The ratio of trans and cis isomers is 50% and

50%.1H NMR (d ppm CDCl3): 7.23–6.76 (C6H4, 8H m

and C5H4, 2H m); 6.53–6.12 (C5H4, 4H m); 5.40

(trans-PhCHCp, 2H s); 4.72 (cis-PhCHCp, 2H s);

3.76 (trans-O(CH3)2 6H s); 3.74 (cis-O(CH3)2 6H s).13C NMR (d ppm CDCl3): 158.8, 138.9, 137.8,

133.8, 132.6, 130.2, 130.0, 128.7, 128.2, 126.4, 117.1,

117.0, 114.1, 113.8, 109.8 (C6H4 and C5H4); 53.4

(cis-PhCHCp, 2H); 50.8 (trans-PhCHCp); 55.2 (cis

and trans-O(CH3)2). IR absorptions (cm�1 KBr):

3094 (w); 2997 (w); 2953 (w); 2833 (w); 1608 (s);

1510 (s); 1458 (m); 1302 (w); 1248 (s); 930 (m); 819

(s); 760 (m); 532 (m). UV/Vis (MeCN): kmax = 282nm. Anal. Calc. for C26H24Cl2O2Ti: C, 64.09; H,

4.96; Found: C, 64.96; H, 5.27.

1990 M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994

2.5. [1,2-Di(cyclopentadienyl)-1,2-bis(2 0-4 0-6 0-trimeth-

oxyphenyl)-ethanediyl] titanium dichloride [1,2-

(2 0,4 0,6 0-(MeO)3-C6H2)2C2H2{g5-C5H4}2]TiCl2 (2b)

TiCl4 (0.4 ml, 3.1 mmol) was added to 40 ml of dry

toluene containing 5% dry THF. The solution turnedimmediately from colourless to pale yellow. The solu-

tion was stirred and cooled down to �78 �C, and then

was treated dropwise with n-butyllithium (3.1 ml, 6.2

mmol). The solution turned from yellow to brown dur-

ing the addition. After this addition, the mixture was al-

lowed to warm up slowly to r.t. The colour of the

solution finally became black. After 20 h stirring, a solu-

tion of 1b (1.5 g, 6.2 mmol) in dry toluene was added tothe solution of TiCl2 Æ 2THF at r.t. under argon. Then it

was stirred under reflux for another 20 h. After perform-

ing the extraction procedure for 2a, the product was

washed with hexane yielding 1.1 g (1.8 mmol, 58.5%

yield) of a black product 2b. The ratio of trans and cis

isomers is 70% and 30%.1H NMR (d ppm CDCl3): 6.45(C6H2, 4H s); 7.02–

6.14 (C5H4, 8H m); 6.00 (trans-PhCHCp, 2H s); 5.92(cis-PhCHCp, 2H); 3.83 (o-CH3, 12H s); 3.72 (p-CH3,

6H s). 13C NMR (d ppm CDCl3): 158.9, 141.9, 139.3,

132.2, 125.1, 117.0, 111.2, 89.5 (C6H2 and C5H4); 62.3

(trans-PhCHCp); 54.1 (OCH3); 37.3 (cis-PhCHCp). IR

absorptions (cm�1 KBr): 3098 (w); 2963 (w); 2916 (w);

2864 (w); 1611 (m); 1478 (m); 1261 (s); 1100 (m); 1030

(m); 813 (m). UV/Vis (MeCN): kmax = 283 nm. Anal.

Calc. for C30H32Cl2O6Ti: C, 58.94; H, 5.31; Found: C,59.88; H, 5.58.

2.6. [1,2-Di(cyclopentadienyl)-1,2-bis (m-dimethoxyphe-

nyl)ethanediyl] titanium dichloride [1,2-(3,3 0-(MeO)2-

C6H3)2C2H2{g5-C5H4}2]TiCl2 (2c)

TiCl4 (3.0 ml, 6.0 mmol, 1 M solution in toluene) was

added to 100 ml of dry toluene containing 10% dryTHF. The solution turned immediately from colourless

to pale yellow. The solution was stirred and cooled

down to �78 �C, followed by drop wise addition of n-

butyl lithium (6.0 ml, 12.0 mmol). The solution turned

from yellow to brown during addition. After this addi-

tion, the mixture was allowed to warm up slowly to

room temperature. The colour of the solution became fi-

nally black. After 20 h stirring at r.t., a solution of 1c(2.5 g, 11.7 mmol) in 35 ml of dry toluene was added

to the TiCl2 Æ 2THF solution at r.t. under argon. Then

it was stirred under reflux for another 20 h. The solvent

was removed under vacuum leaving a black residue. The

residue was washed with chloroform and the solution

was filtered through celite under reduced pressure. The

colour of the filtrate reddened slightly. It was filtered

using gravity filtration for at least four times until nofurther black precipitate appeared on the filter paper

and the filtrate turned to dark red. Chloroform was re-

moved to leave red solid 2c with 1.6 g (2.9 mmol, 50%

yield). The ratio of trans and cis isomers is 86–14%.1H NMR (d ppm CDCl3): 7.23–6.02 (C6H3, 6H, m

and C5H4, 8H, m); 5.37 (trans-PhCHCp, 2H, s); 4.72

(cis-PhCHCp, 2H s); 3.70 (cis-OCH3, 12H, s); 3.66

(trans-OCH3, 12H, s). 13C NMR (d ppm CDCl3):159.73, 139.86, 136.86, 129.90, 126.77, 116.47, 112.84,

106.21, 105.12, 97.52 (trans-C6H4 and trans-C5H4);

54.29 (trans-OCH3); 50.16 (trans-PhCHCp). IR absorp-

tion (cm�1 KBr): 3098 (w); 2995 (w); 2956 (w); 2929(w);

2830 (w); 1593 (s); 1457 (m); 1424 (m); 1383 (w); 1342

(w); 1322 (w); 1289 (w); 1202 (m); 1155 (s); 1062 (m);

815 (m); 683 (w); 535 (w). UV/Vis (CHCl3): kmax = 271

nm. Anal. Calc. for C28H28Cl2O4Ti: C, 61.45; H, 5.16;Found: C, 62.17; H, 5.46.

3. MTT-based cytotoxicity tests

The pig kidney carcinoma cell line, LLC-PK, was ob-

tained from the American Tissue Culture Collection.

The cytotoxic activities of titanocenes 2a and 2bwere determined using an MTT-based assay. In more

detail, cells were seeded into a 96-well plate (5000

cells/well) and allowed to attach for 24 h. Subse-

quently, the cells were treated with various concentra-

tions of the cytotoxic agents. In order to prepare drug

solutions, drugs were first dissolved in dimethyl sulfox-

ide (DMSO), followed by dilution with medium to the

required maximum concentration, 5 · 10�4 M, with afinal concentration of DMSO not exceeding 0.7%.

From these stock solutions, solutions with lower con-

centrations were prepared by further dilution with

medium. Care was taken that the drug solutions were

applied within 1 h on the cells to avoid interference

with hydrolysed compounds. After 48 h, the relevant

drug was removed, the cells washed with PBS and

fresh medium was added for another 24 h for recovery.Viability of cells was determined by treatment with

MTT in medium (5 mg/11 ml) for 3 h. The purple

formazan crystals formed were dissolved in DMSO

and absorbance measured at 540 nm using a VIC-

TOR2 multilabel plate reader (Wallac). IC50 (inhibitory

concentration 50%) values were determined from the

drug concentrations that induced a 50% reduction in

light absorbance.

4. Results and discussion

4.1. Synthesis

Fulvenes 1a and 1b (Fig. 1) were synthesized, accord-

ing to reference [25], by reacting the corresponding benz-aldehydes with cyclopentadiene in the presence of

pyrrolidine as a base.

Fig. 2. (a) Gaussview plot of the optimised structure of titanocene 2a.

(b) Gaussview plot of the optimised structure of titanocene 2b. (c)

Gaussview plot of the optimised structure of titanocene 2c.

R2

R2

R3

R3

R1

1a: R1 = OMe, R2 = R3 = H

1b: R1 = R2 = OMe, R3 = H

1c: R1 = R2 = H, R3 = OMe

Fig. 1. Structure of fulvenes 1a–1c.

M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994 1991

Titanocenes 2a–c (Figs. 2(a)–(c)) were synthesizedby reductive dimerisation of fulvenes 1a–c with tita-

nium dichloride, respectively (Scheme 1). TiCl2 was ob-

tained by reduction of TiCl4 with nBuLi as described

in references [17,18] (Scheme 1). The determined cis–

trans ratio at the bridge is 50:50 for 2a, 70:30 for 2b,

and 84:16 for 2c. The higher trans-ratio in 2b and2c re-

flects the steric bulk of the methoxy groups in the ortho

and meta positions, making the trans geometry morefavoured.

4.2. Structural discussion

Density functional theory calculations were carried

out for compounds 1b, 2a, 2b and 2c at the B3LYP level

using the 6-31G** basis set. In addition, X-ray diffrac-

tion measurements were carried out for compound 1b(see Table 1 for further details about the data

collection).

Selected bond lengths of the optimised structure of

fulvene 1b (Fig. 3) can be found in Table 2. As expected,

the carbon–carbon bond lengths in the cyclopentadiene

system of fulvene 1b vary significantly, demonstrating

the absence of a significant resonance system within

the five-membered ring. This is confirmed by the lengthof the regular exocyclic double bond C(1)–C(6) found at

134.3 pm and the single bond carrying the phenyl sub-

stituent C(6)–C(7) is calculated as 146.8 pm (Table 2).

The corresponding values of the crystal structure deter-

mination tend to be around 1–3 pm shorter than the cal-

culated values (Table 2, Fig. 3). A distortion from

planarity is seen with a dihedral angle between the aryl

ring and the five-membered dienyl ring of 54.7�. Thecrystal structure confirms this twist with an even larger

angle of 82.2�. The significant difference between calcu-

lated and experimental angle may be due to packing ef-

fects in the crystal.

Optimised structures were also calculated for titan-

ocenes 2a–c (Scheme 2) at the B3LYP level using the

6-31G** basis set. Selected bond lengths of these struc-

tures are listed in Table 3.

The length of bonds between the metal centre and

the carbon atoms of the cyclopentadienyl rings bound

R1

R2

R1

H

TiCl4 + 2 nBuLi

TiCl2 · 2 THF + 2 TiCl

ClH

H

R1

R1

R2

R1

R1

R2

-78 ˚C,toluene,5 % THF

reflux

16 h

R3

R3

R3

R3

R3

R3

2a: R1 = OMe, R2 = R3 = H2b: R1 = R2 = OMe, R3 = H2c: R1 = R2 = H, R3 = OMe

Scheme 1. Synthesis of titanocenes 2a–c.

1992 M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994

to the metal ion are similar for the three titanocenes.

They vary between 236.9 and 245.2 pm for 2a,

237.6–244.3 pm for 2b and 236.4–245.2 pm for 2c, withvalues slightly different for the different cyclopentadie-

nyl rings. The same applies for the carbon–carbon

bonds of the cyclopentadienyl rings with bonds length

C15

O3C12

C11

C3 C2

C1C4C5

C6C7

O2

C10

C8 C9

C14

O1C13

Fig. 3. Molecular structure of 1b; thermal ellipsoids are drawn on the

50% probability level; from disordered methyl groups only one

orientation is shown.

Table 2

Selected bond lengths of the DFT-calculated structure and crystal

structure of fulvene 1b

Bond length (pm) (1b)

DFT structure Crystal structure

C(1)–C(2) 147.3 145.5

C(2)–C(3) 135.6 134.3

C(3)–C(4) 146.8 145.3

C(4)–C(5) 135.8 133.5

C(5)–C(1) 146.7 146.8

C(1)–C(6) 136.2 134.3

C(6)–C(7) 146.0 146.8

between 140.4 and 142.8 pm for 2a, 140.4–142.3 pm

for 2b and 140.5–142.9 for 2c. These values suggest

the titanocenes have no plane of symmetry bisectingthe Cl–Ti–Cl plane and that the calculated structures

exhibit C2 symmetry. 2a–c have similar ansa bridge

lengths (C(6)–C 0(6)) of 156.2, 156.1 and 156.2 pm,

respectively. These values are in agreement with the

corresponding value calculated previously for [(1,2-

diphenyl-1,2-dicyclopentadienyl)ethanediyl] titanium

dichloride [21]. The steric bulk of the phenyl rings at-

tached to the bridging carbons causes a lengthening ofthe bond, in order to relieve the resultant steric strain.

The titanium–chlorine bond length are almost identical

for 2a–c, with values of 234.7 and 234.4 pm for 2a,

235.7 and 235.8 pm for 2b, and 234.7 and 234.5 pm

for 2c. For 2a the TiCl2 angle was calculated to be

97.7� and the dihedral angle between the aryl rings

to be 61.3�. The corresponding values for 2b are

97.3� and 57.7�, respectively. For 2c these values are97.6� and 60.3�. For 2a, the angle formed by the bonds

between C(1), C(6) and C(6 0) is 107.9�, between C(7),

Ti

Cl'

ClH

H

R

R

1

23

4

5

1'

2'

3'

4'5'

6

6'

Scheme 2. Numbering scheme for structural discussion of titanocenes

2a–2c.

Table 3

Selected bond lengths from the DFT-calculated structures of com-

plexes 2a–c

Bond length (pm) DFT structure

(2a) (2b) (2c)

Ti–C(1) 241.5 241.2 241.2

Ti–C(2) 236.9 237.0 236.4

Ti–C(3) 243.9 244.0 243.8

Ti–C(4) 244.9 245.2 245.2

Ti–C(5) 241.4 240.1 241.9

Ti–C(1 0) 242.2 242.2 242.2

Ti–C(2 0) 237.1 237.0 237.2

Ti–C(3 0) 243.6 244.0 243.4

Ti–C(4 0) 245.2 245.2 245.1

Ti–C(5 0) 241.3 240.1 241.0

C(1)–C(2) 142.8 142.9 142.9

C(2)–C(3) 142.1 142.1 142.0

C(3)–C(4) 140.4 140.2 140.5

C(4)–C(5) 142.4 142.3 142.3

C(5)–C(1) 141.5 142.0 141.4

C(10)–C(20) 142.5 142.9 142.4

C(20)–C(30) 142.3 142.1 142.3

C(30)–C(40) 140.3 140.2 140.3

C(40)–C(50) 142.4 142.3 142.3

C(50)–C(10) 141.7 141.6 141.7

Ti–Cl 234.7 235.7 234.7

Ti–Cl 0 234.4 235.8 234.5

C(6)–C(60) 156.2 156.1 156.2

C(1)–C(6) 151.4 151.7 151.3

1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-30,0

0,2

0,4

0,6

0,8

1,0

1,2

cis-Pt, IC50

: (3.3+/-0.5)E-6 Cp

2TiCl

2, IC

50: (2.0+/-1.0)E-3

2a, IC50

: (2.8+/-0.2)E-4

Nor

mal

ized

cel

l via

bilit

y

log10

of drug concentrations

1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-30,0

0,2

0,4

0,6

0,8

1,0

1,2

cis-Pt, IC50

: (3.3+/-0.5)E-6 Cp

2TiCl

2, IC

50: (2.0+/-1.0)E-3

2b, IC50

: (3.6+/-0.4)E-4

Nor

mal

ized

cel

l via

bilit

y

log10

of drug concentrations

1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-30,0

0,2

0,4

0,6

0,8

1,0

1,2

cis-Pt, IC50

: (3.3+/-0.5)E-6 Cp

2TiCl

2, IC

50: (2.0+/-1.0)E-3

2c, IC50

: (2.1+/-0.3)E-4

Nor

mal

ized

cel

l via

bilit

y

log10

of drug concentrations

Fig. 4. (a) Cytotoxicity curves from typical MTT assays showing the

effect of cis-platin, Cp2TiCl2, and compound 2a on the viability of pig

kidney carcinoma (LLC-PK) cells. (b) Cytotoxicity curves from typical

MTT assays showing the effect of cis-platin, Cp2TiCl2, and compound

2b on the viability of pig kidney carcinoma (LLC-PK) cells. (c)

Cytotoxicity curves from typical MTT assays showing the effect of cis-

platin, Cp2TiCl2, and compound 2c on the viability of pig kidney

carcinoma (LLC-PK) cells.

M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994 1993

C(6), and C(6 0) is 113.8, and between C(7), C(6) and

C(1) is 113.4�. The corresponding calculated values

for 2b are 107.8�, 116.7� and 113.0� and for 2c:

107.6�, 113.6 and 114.0�. The C(7)–C(6)–C 0(6) angle

for 2b is slightly larger than that of 2a because of

the ortho-substituted para-methoxy groups increasing

the steric bulk of the phenyl rings. The angles formedbetween the centroids of the cyclopentadienyl rings are

almost identical, with values of 128.0� for 2a and

128.4� in 2b, indicating bent ansa-metallocene type

geometry.

4.3. Cytotoxicity studies

The in vitro cytotoxicity of compounds 2a–2c weredetermined by an MTT-based assays [26] involving a

48 h drug exposure period, followed by 24 h of recovery

time. Compounds were tested for their activity on pig

kidney carcinoma (LLC-PK) cells. The IC50 values

found vary between 2.1 · 10�4 and 3.6 · 10�4 M (Figs.

4(a)–(c)) and are similar to the inhibition value found

previously for [1,2-di(cyclopentadienyl)-1,2-di(p-N,N-

dimethylaminophenyl)ethanediyl] titanium dichloride,which has an IC50 value of 2.7 · 10�4 M [21]. Under

identical conditions, cis-platin showed an IC50 value of

3.3 · 10�6 M, whereas the activity of Cp2 TiCl2 was at

least one order of magnitude lower than 2a–c [21].

1994 M. Tacke et al. / Journal of Inorganic Biochemistry 98 (2004) 1987–1994

5. Conclusion and outlook

Compounds 2a–2c have IC50 values in the lower 10�4

M range, which are significantly more cytotoxic than

unsubstituted titanocene dichloride against LLC-PK,

for which Phase I/II clinical trials have been performed.The use of methoxy-substituted ligands overcomes the

problem of low water solubility. It is intended to per-

form further in vitro cellular tests with these compounds

to evaluate its potential for testing in animal models and

additionally to search for differently substituted titan-

ocenes also derived from fulvenes.

Acknowledgements

The authors want to thank the Higher Education

Authority (HEA) and the Centre for Synthesis and

Chemical Biology (CSCB) for funding through the

HEA PRTLI cycle 3 and Science Foundation Ireland

(SFI) for funding through 04/BR/C0682.

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