Titanocene difluorides with improved cytotoxic activity

Article abstract of DOI:10.1021/ic9022163

Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me3SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.

Full text of DOI:10.1021/ic9022163

1292 Inorg. Chem. 2010, 49, 1292–1294
DOI: 10.1021/ic9022163
Titanocene Difluorides with Improved Cytotoxic Activity
‡
Silvia Eger,† Timo A. Immel,† James Claffey,‡ Helge Muller-Bunz, Matthias Tacke,‡ Ulrich Groth,† and
€
Thomas Huhn*,†
‡Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4,
†
€
Ireland and Fachbereich Chemie and Konstanz Research School Chemical Biology, Universitat Konstanz,
€
Universitatsstrasse 10, Fach 720, 78457 Konstanz, Germany
Received November 10, 2009
Titanocene difluorides can be obtained by halide metathesis of
the respective titanocene dichlorides with trimethyltin fluoride
(Me3SnF), giving access to a new class of cytotoxic active sub-
stances. Furthermore, an improved method for the synthesis of
diaryl-substituted titanocene dichlorides is presented.
for oxalititanocene Y (titanocene Y with both chloride ligands
substituted by the bidentate oxalate).6 We were interested in
the synthesis and cytotoxicity of fluorotitanocene derivatives
because of their anticipated higherstability againstsolvolysis;
the Ti-F bond is known to be more stable by 75 kcal/mol
than the Ti-Cl bond.7
Within this paper, we introduce several benzyl- and diaryl-
substituted titanocene difluorides that can be obtained by
fluorinating the respective TDCs with trimethyltin fluoride.
Preliminary cytotoxicity studies show that the fluorine ana-
logue of titanocene Y is 4-7 times more cytotoxic than
titanocene Y itself. Furthermore, we present an improved
method for the synthesis of diaryl-substituted TDCs.
For comparative cytotoxicity studies between TDCs and
titanocene difluorides, two different classes of titanocene
derivatives have been synthesized.
Three different benzyl-substituted TDCs [m- (3a), p- (3b),
and 3,5-dimethoxybenzyl (3c)] were synthesized according to
Sweeney et al.4 Selected fulvenes 1 are hydridolithiated by
LiB(Et)3H (SuperHydride) to give the corresponding lithium
cyclopentadienides 2, which were, in turn, transmetalated with
TiCl4 to yield bis(benzyl)titanocene dichlorides 3 (Scheme 1).
The second class of titanocene derivatives, diaryl-substi-
tuted titanocenes 7, was primarily synthesized according to a
one-pot procedure, consisting of halogen-lithium exchange,
fulvene addition, and transmetalation by TiCl4 published by
Pampillion et al.8
Neither thehalogenmetalexchangenor the carbolithiation
of the respective fulvene 1 might proceed to completeness;
i.e., the amount of lithium cyclopentadienide 6 is difficult to
estimate. However, an excess of titanium tetrachloride leads
to cyclopentadienyltitanium(IV) trichlorides, while an excess
of ligand facilitates the reduction of titanium(IV) to titanium-
(III) species. These byproducts hamper the isolation of the
target molecule because the respective TDCs 7 are difficult to
purify. Because the solubility of lithium cyclopentadienides 6
in diethyl ether is rather limited, the halogen-lithium ex-
change was set up with 2 equiv of tert-BuLi in Et2O instead of
Titanocene dichloride (TDC; Cp2TiCl2) attracted great
interest because it was the first non-platinum complex to
show promising results as an antitumor agent. It reached
clinical trials, but the efficacy of Cp2TiCl2 in phase II clinical
trials in patients with metastatic renal cell carcinoma1 or
metastatic breast cancer2 was too low to be pursued.
More recently, a large number of differently substituted
titanocene derivatives have been synthesized and tested for
their potential cytotoxicity.3 By substitution of the cyclopen-
tadienyl (cp) rings, the cytotoxicity in LLC-PK cells could be
increased by a factor of 1000. The p-methoxybenzyl-substi-
tuted titanocene Y (1b) shows an IC50 value of 21 μM,4 and
the dimethylamino-functionalized and heteroaryl-substi-
tuted titanocene C shows an IC50 value of 5.5 μM,5 compared
to 2 mM for Cp2TiCl2.
Furthermore, the cytotoxic activity can be influenced by
substitution of the two chloride ligands. The nature of these
two “not-cp-ligands” affects the hydrolytic stability and
thereby the bioavailability of the active substance. Ligands
that show a higher hydrolytic stability than chloride but can
still be hydrolyzed under physiological conditions seem to be
ideal. Recently, Claffey et al. reported an IC50 value of 1.6 μM
*To whom correspondence should be addressed. E-mail: thomas.huhn@
uni-konstanz.de. Fax: þ49-7531-884424.
(1) Lummen, G.; Sperling, H.; Luboldt, H.; Otto, T.; Rubben, H. Cancer
Chemother. Pharmacol. 1998, 42, 415–417.
€
(2) Kroger, N.; Kleeberg, U. R.; Mross, K. B.; Edler, L.; Sass, G.;
Hossfeld, D. K. Onkologie 2000, 23, 60–62.
(3) Strohfeld, K.; Tacke, M. Chem. Soc. Rev. 2008, 37, 1174–1187.
€
(4) Sweeney, N. J.; Mendoza, O.; Muller-Bunz, H.; Pampillon, C.;
Rehmann, F. K.; Strohfeldt, K.; Tacke, M. J. Organomet. Chem. 2005,
690, 4537–4544.
(5) Pampillon, C.; Sweeney, N.; Strohfeld, K.; Tacke, M. J. Organomet.
Chem. 2007, 692, 2153–2159.
(7) Hudlicky, M. Chemistry of Organic Fluorine Compounds;A Labora-
tory Manual; Ellis Horwood Ltd. and John Wiley & Sons: New York, 1976.
(8) Pampillon, C.; Mendoza, O.; Sweeney, N. J.; Strohfeldt, K.; Tacke, M.
Polyhedron 2006, 25, 2101–2108.
€
ꢀ
(6) Claffey, J.; Hogan, M.; Muller-Bunz, H.; Pampillon, C.; Tacke, M.
ChemMedChem 2008, 3, 729–731.
r
pubs.acs.org/IC
Published on Web 01/07/2010
2010 American Chemical Society

Communication
Inorganic Chemistry, Vol. 49, No. 4, 2010 1293
Scheme 1. Synthesis of Bis[(m-methoxybenzyl)cyclopentadienyl]-, Bis-
[(p-methoxybenzyl)cyclopentadienyl]-, and Bis[(3,5-dimethoxybenzyl)-
cyclopentadienyl]titanium(IV) Dichlorides 3a-3c
Scheme 2. Synthesis of Bis[bis(m-methoxyphenyl)methylcyclopenta-
dienyl] and Bis[bis(p-methoxyphenyl)methylcyclopentadienyl]titanium-
(IV) Dichlorides 7a and 7b
tetrahydrofuran (THF). After the addition of fulvene 1, the
precipitating lithium cyclopentadienide 6 was isolated by
gravity filtration over a sintered-glass funnel. After quantifi-
cation, the subsequent transmetalation was set up in a 2:1
stoichiometry with [TiCl4(THF)2] in THF under reflux.
A vigorously stirred suspension of 5 in Et2O reacts ex-
haustively with added fulvene 1 at room temperature, leading
to precipitation of the colorless lithium cyclopentadienide 6
in good yield. With this improved method, the synthesis of
m- and p-methoxydiaryl-substituted TDCs 7a and 7b pro-
ceeded in good yield and excellent purity (Scheme 2 and
Table 1).
Attempts to synthesize the 3,5-dimethoxydiaryl derivative
were met with failure. Apparently, the product from the
halogen metal exchange in the case of 3,5-dimethoxybromo-
benzene rearranges to the highly stabilized 2-lithio-1,3-di-
methoxybenzene.
Table 1. Yields of Isolated Chloro- and Fluorotitanocenes
3a
(X = Cl) (X = F)
8
7a
(X = Cl) (X = F)
9
a
b
c
m-MeO
p-MeO
3,5-MeO
53
74
58
82
83
60
a
b
m-MeO
p-MeO
77
76
66
79
a Yields are given with respect to the isolated lithium cyclopenta-
dienide.
Scheme 3. Use of Trimethyltin Fluoride for the Fluorination of
Benzyl- and Diaryl-Substituted TDCs (3 and 7) and Regeneration of
the Fluorinating Agent by Subsequent Recycling with an Aqueous KF
Solution
Subsequently, the TDCswere fluorinatedwith trimethyltin
fluoride utilizing a method reported first by Herzog et al.9
Trimethyltin fluoride is a nonodorous, colorless, crystalline
substance, and in contrast to other trimethyltin halogenides,
it is insoluble in most organic solvents because of its poly-
meric structure.10 It is known to be a powerful yet selective
fluorinating agent especially suitable for group IV metallo-
cene halogenides already effective at room temperature.11 It
reacts with Cpx MCl2 (M = Ti, Zr, Hf; Cpx = differently
the diminished yields (Table 1) result from repeated recrys-
tallizations to remove even trace amounts of impurities that
otherwise might obscure the biological assays. Following this
procedure, we were able to synthesize five pairs of TDCs (3a,
3b, 3c, 7a, and 7b) and titanocene difluorides (8a, 8b, 8c, 9a,
and 9b) for comparative cytotoxicity studies.
X-ray crystallographic studies established the molecular
structures of 8a and 8b (Figures 1 and 2).12 Neither the
different substitution of the benzyl rings nor the exchange of
the two chloride ligands with fluoride leads to a great
difference in the molecular structures. The distance between
2
substituted cp ligands) in toluene to Cpx MF2 in generally
2
good yields. The byproduct, the highly toxic trimethyltin
chloride, can be quantitatively recovered from the reaction
mixture by vacuum sublimation. Reaction with an excess
aqueous potassium fluoride solution in alcohol regenerates
the fluorinating compound; Me3SnF precipitates quantita-
tively from the solution as colorless crystals (Scheme 3).
Fluorination of benzyl-substituted TDCs 3 and 8 proved
to be suitable with a 10% molar excess of Me3SnF in a
toluene suspension. Because the fluorinating agent is inso-
luble in toluene, the excess was simply filtered off. The
1
completeness of the fluorination was traced by H and 19F
(12) Crystal data for 8a: C26H26O2F2Ti, Mr = 456.37 g/mol, monoclinic,
˚
˚
˚
space group C2, a = 20.841(2) A, b = 5.6814(6) A, c = 8.9748(9) A, β =
NMR spectroscopy. Yields are essentially quantitative, while
o
3
˚
91.267(2) , V = 1062.43(19) A , Z = 2, T = 100(2) K, Dcalc = 1.427 g/cm,
Rint=0.0167, 5682 reflections collected, R1 (wR2)=0.0343 (0.0857), and S=
1.044 for 2732 reflections with I>2σ(I). Crystal data for 8b: C26H26O2F2Ti,
(9) Herzog, A.; Liu, F.-Q.; Roesky, H. W.; Demsar, A.; Keller, K.;
Noltemeyer, M.; Pauer, F. Organometallics 1994, 13, 1251–1256.
(10) Krause, E. Ber. Dtsch. Chem. Ges. 1918, 51, 1447–1456.
(11) Roesky, H. W.; Herzog, A.; Liu, F.-O. J. Fluorine Chem. 1995, 72,
183–185.
˚
Mr = 456.37 g/mol, monoclinic, space group C2/c, a = 26.642(5) A, b =
o
3
˚
˚
˚
5.7514(10) A, c=14.681(3) A, β=109.766(3) , V=2117.0(6) A , Z=4, T=
100(2) K, Dcalc = 1.432 g/cm, Rint = 0.0346, 11 116 reflections collected, R1
(wR2)=0.0954 (0.2586), and S=1.198 for 3069 reflections with I>2σ(I).

1294 Inorganic Chemistry, Vol. 49, No. 4, 2010
Eger et al.
Figure 1. Molecular structure of 8b in the crystal (thermal ellipsoids are drawn on the 50% probability level).14 Hydrogen atoms are omitted for clarity.
˚
Selected bond lengths [A] and angles [deg]: Ti-F 1.866(3), Ti-centroid 2.060(1); F-Ti-F 96.61(19), centroid-Ti-F2 104.92(9), centroid-Ti-F1
105.71(9), centroid-Ti-centroid 133.20(1).
Table 2. Cytotoxicity Data of TDCs versus Titanocene Difluorides Estimated in
Two Different Cell Lines by an Alamar Blue Assay
IC50 Hela S3 [μM]
IC50 Hep G2 [μM]
compd
Cl
F
Cl
F
1
2
3
4
5
6
7
3a/8a
3b/8b
3c/8c
7a/ 9a
7b/9b
194 ( 36
59 ( 15
120 ( 4
35 ( 11
84 ( 25
1340 ( 501
7 ( 1
72 ( 10
13 ( 1
145 ( 34
34 ( 2
16 ( 1
191 ( 151
353 ( 63
211 ( 110
156 ( 39
42 ( 12
108 ( 30
1220 ( 744
6 ( 1
220 ( 68
30 ( 1
139 ( 51
56 ( 19
39 ( 11
432 ( 281
TD-X
cisplatina
Figure 2. Molecular structure of 8a in the crystal (thermal ellipsoids are
drawn on the 50% probability level).14 Hydrogen atoms are omitted for
˚
a Cisplatin was tested on all plates as an internal standard.
clarity. Selected bond lengths [A] and angles [deg]: Ti-F 1.8664(10),
Ti-centroid 2.068(1); F-Ti-F 96.97(7), centroid-Ti-F2 105.68(3),
centroid-Ti-F1 104.57(3), centroid-Ti-centroid 133.64(1).
Recently, it has been shown that the para-substitution
˚
the titanium center and the center of the cp rings is 2.068 A
pattern of titanocene Y is a prerequisite for coordination
onto the backbone of DNA.15 The strongly improved cyto-
toxicity of the 4-methoxyaryltitanocene difluorides (entries 2
and 5), therefore, seemed to be based on a favorable molec-
ular geometry as well as on enhanced hydrolytic stability.16
It should be noted that fluoride ions themselves are not
cytotoxic at concentrations below 10-3 M.17
In conclusion, the halide metathesis of functionalized
TDCs with Me3SnF proceeds smoothly and yields titanocene
difluorides in excellent yield. By suitable substitution of the
cp rings, selected difluorides showed a cytotoxicity 3-5-fold
higher than that of the respective dichlorides. Under the same
conditions, the “gold standard” cisplatin showed a cytotoxi-
city that was only 2-fold greater than our best hit
[IC50(cisplatin in HeLa S3) = 7 (( 1) μM]. Studies are now
underway in our laboratories to further investigate and
understand the influence of the substitution pattern and
hydrolytic stability on cytotoxicity.
˚
for 8a and 2.060 A for 8b, while the analogue bond mea-
13
˚
˚
sures 2.058 A in 3a and 2.060 A in 3b. As expected, the
average Ti-F bond in 8a and 8b is considerably shorter
˚
(1.866 A) than the average Ti-Cl bond in 3a and 3b (2.364
and 2.370 A). The F-Ti-F angle is slightly wider (96.97° for
˚
8a and 96.61° for 8b) than the corresponding Cl-Ti-Cl
angle in 3a (94.13°) and 3b (95.90°). The same tendency can
be observed for the centroid-Ti-centroid angle, which is
133.64° for 8a and 133.20° for 8b, while the corresponding
angle in the chloro analogues is 130.85° (3a) and 130.70° (3b).
All compounds were tested for their cytotoxicity on two
human cancer cell lines (HeLa S3 and Hep G2). IC50 values
were determined by Alamar Blue-based cytotoxicity assays.
The exchange of chloride with fluoride leads in most cases
to enhanced IC50 values. In general, the effect is more
pronounced for the sterically less demanding p-methoxybenzyl-
substituted titanocenes. Hence, difluorotitanocene Y 8b with
an IC50 value of 13 μM showed the highest cytotoxic activity,
closely followed by difluoride 9b with an IC50 value of 16 μM.
Both para-substituted fluorotitanocenes exhibit more than a
4-fold increased cytotoxicity compared to their chloro con-
geners (Table 2).
However, meta-substituted fluorotitanocene 8a showed
less improved activity (72 μM). The crystal structures of 8a
and 8b showed no influence of the methoxy substitution
pattern on the bonding length or angle. Therefore, the
observed differences in cytotoxicity are not based on the
electronic properties of the complexes. Fluoride 9a even has
the same cytotoxicity (35 μM) as its chloro counterpart 7a.
Here, as in the case of the 3,5-dimethoxybenzyltitanocene
3c/8c, sterical reasons seemed to limit the cytotoxicity.
Acknowledgment. S.E. thanks the EU program COST
D39 for funding of a “Short Term Scientific Mission” in
the laboratories of M.T. in Dublin. S.E. and T.A.I. thank
the Konstanz Research School Chemical Biology (KoRS-
CB) for financial support, scientific encouragement, and
academic training.
Supporting Information Available: Experimental details, a
table of bond lengths and bond angles, synthetic schemes of all
new compounds, NMR listings, and X-ray crystallographic
data (CIF). This material is available free of charge via the
Internet at http://pubs.acs.org.
(15) Tacke, M. Lett. Drug Des. Discovery 2008, 5, 332–335.
(16) Murray, J. H.; Harding, M. J. Med. Chem. 1994, 37, 1936–1941.
(17) (a) Khalil, A. M.; Da’dara, A. A. Arch. Environ. Contam. Toxicol.
1994, 26, 60–63. (b) Song, J.-S.; Lee, H.-Y.; Lee, E.; Hwang, H. J.; Kim, J. H.
Environ. Toxicol. Pharmacol. 2002, 11, 85–91.
€
(13) Claffey, J.; Hogan, M.; Muller-Bunz, H.; Pampillon, C.; Tacke, M.
J. Organomet. Chem. 2008, 693, 526–536.
(14) Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.