This journal is c The Royal Society of Chemistry 2013 Chem. Commun.

Cite this: DOI: 10.1039/c3cc38604j

A comparative chemical–biological evaluation oftitanium(IV) complexes with a salan orcyclopentadienyl ligand†

Julia Schur,a Cesar M. Manna,b Anthony Deally,c Reinhard W. Koster,d

Matthias Tacke,c Edit Y. Tshuvab and Ingo Ott*a

Titanium(IV) complexes with a salan or cyclopentadienyl ligand

showed different biological behaviour concerning binding to bio-

molecules, cellular accumulation and intracellular distribution.

Binding efficacy as well as trafficking on the cellular level are

crucial parameters for their biological effects.

Titanium complexes had been evaluated as potential anticancerdrugs soon after the launch of the platinum class of cancerchemotherapeutics and had reached the clinical trial stages.Unfortunately, the investigated complexes (e.g. titanocenedichloride TiCp2Cl2

1 or budotitane (bzac)2Ti(OEt)2,2 see Fig. 1)experienced solubility and stability problems and the clinical trialswere finally abandoned. Over the last few years new titanium(IV)complexes have been developed, with the aim of overcomingthe disadvantages of the initial lead compounds. Among those,titanium complexes with salan ligands3 and improved titano-cenes4 have shown promising potential and are undergoingfurther development.1–9

In this study we aimed to evaluate important parameters inmedicinal chemistry and preclinical research of two represen-tative examples, namely a titanium salan complex (Ti–Salan)and Titanocene Y (Ti–Y). Whereas Ti–Salan represents acomplex that is formed by N/O coordination of Ti(IV) to a salanbackbone, Ti–Y is an organometallic sandwich complex withadditional chlorido ligands. Both Ti–Salan10 and Ti–Y (unpublishedresults) have a high stability in water or water–solvent mixtures withhalf-life times of more than a week.

The experiments in this study include the investigation ofthe binding of the complexes to DNA and albumin, theircellular accumulation and intracellular distribution (uptakeinto mitochondria and nuclei) in comparison to their initiallydetermined toxicity. The archetypical titanium(IV) complexTiCp2Cl2 was used as a reference where appropriate.

First, the cytotoxicity of the complexes in two cancer celllines, namely MCF-7 breast cancer and HT-29 colon carcinomacells, was determined (see Table 1). Whereas TiCp2Cl2 was not

Fig. 1 Structures of titanium(IV) complexes.

Table 1 Cytotoxicity (as IC50 values) in MCF-7 and HT-29 cancer cells andbinding to macromolecules (DNA: 4 h, 50 mM of the compounds, albumin: 6 h,50 mM of the compounds)

CompoundsMCF-7(mM)

HT-29(mM)

DNA(pmol Ti/mg DNA)

Albumin(% bound)

Ti–Salan 1.1 � 0.4 0.7 � 0.2 18 � 4 27 � 10Ti–Y 4.1 � 2.4 5.9 � 1.1 290 � 80 76 � 21TiCp2Cl2 >500 >500 320 � 40 98 � 9

a Institute of Medicinal and Pharmaceutical Chemistry, Technische Universitat

Braunschweig, Braunschweig, Germany. E-mail: ingo.ott@tu-bs.de;

Tel: +49 5313912743b Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904,

Israel. E-mail: edit.tshuva@mail.huji.ac.il; Tel: +972 26586084c University College Dublin, UCD School of Chemistry and Chemical Biology,

Belfield, Dublin 4, Ireland. E-mail: matthias.tacke@ucd.ie; Tel: +353 17168428d Zoological Institute, Cell Physiology, Technische Universitat Braunschweig,

Braunschweig, Germany. E-mail: r.koester@tu-bs.de; Tel: +49 5313913230

† Electronic supplementary information (ESI) available: Experimental section,data of zebrafish embryo assays, albumin binding, comparative figure of cellularuptake. See DOI: 10.1039/c3cc38604j

Received 30th November 2012,Accepted 8th April 2013

DOI: 10.1039/c3cc38604j

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cytotoxic up to the highest concentration used (500 mM) Ti–Salanas well as Ti–Y triggered strong antiproliferative effects, whichwere more pronounced in the case of Ti–Salan. Next, zebrafishembryos were used to provide a preliminary study on thegeneral toxicity of the complexes during embryonic develop-ment. Interestingly, both Ti–Salan and Ti–Y did not lead tostrong lethality even after exposure to high concentrations andover extended exposure (>80% of the embryos survived thetreatment), only TiCp2Cl2 showed toxic effects at 100 mM (seeFig. 2). This screening thus provides an indication that Ti(IV)complexes could trigger antiproliferative effects in cancer cellsbut have low toxicity in whole organisms. In fact, for Ti–Y efficacyin xenograft models with moderate body weight loss no othersevere side effects has been reported.11–15 The activity of varioustitanium salan complexes was demonstrated in mice and thecomplexes were generally well tolerated.16,17

To establish the reactivity against biomolecules the bindingto DNA and albumin was studied by precipitation assays, whichprovide information on tight or irreversible binding to macro-molecules (see Table 1). Titanium was quantified by a method basedon high-resolution continuum source atomic absorption spectro-metry (HR-CS AAS). Interestingly, Ti–Salan showed a compar-ably low binding to DNA and albumin whereas both titanocenecomplexes demonstrated a much higher affinity. This wasparticularly the case for TiCp2Cl2, which was bound to bothbiomolecules in the highest amounts in good agreement withresults reported in the literature.1,2,18–20 Time dependentexperiments showed that the binding to albumin was alreadycomplete after the first hour of exposure for all three Ti(IV)complexes and values were stable over at least 6 h (see ESI†).Effective binding of Ti–Y to albumin had recently also beenobserved by means of competitive titration experiments.21

In order to evaluate the relevance of these binding propertiesand to shed more light on the biological fate of Ti–Salan andTi–Y, we performed experiments on the cellular titaniumaccumulation and its intracellular distribution. Fig. 3 showsthe time-dependent titanium levels of HT-29 cells exposed to10 mM of the complexes over a period of 27 h. The results are

expressed as nmol titanium per milligram cellular protein aswell as the estimated cellular titanium concentration thereof(see ESI† for experimental details). Since cell culture media areroutinely supplemented with fetal calf serum, which contains ahigh amount of albumin and other proteins, additional experi-ments were performed with serum free medium for bothcomplexes. The cellular titanium uptake from ‘‘normal’’ serumcontaining medium was highest for Ti–Salan (see Fig. 3, top),which showed a roughly 100-fold cellular accumulation comparedto the extracellular medium after 6 h that kept increasing furtherupon longer exposure to a more than 400-fold accumulation(24 h). Ti–Y led to an approximately 20-fold cellular titaniumaccumulation from serum containing media with stable valuesover the investigated period of 27 h (see Fig. 3, bottom). Incontrast to Ti–Salan and Ti–Y, with TiCp2Cl2 titanium was barelydetectable in the probes (see ESI† for a comparative presentation).It has to be noted that this uptake behaviour is in excellentagreement with the results of the cytotoxicity experiments incancer cells and with reported results on TiCp2Cl2.22

As could be expected from its efficient binding to albumin,the cellular titanium accumulation from serum free culturemedia was strongly enhanced for Ti–Y. Surprisingly, the cellulartitanium levels with Ti–Salan in serum free medium weresubstantially decreased over the whole incubation period.This suggests that albumin or also other serum proteins(e.g. transferrin)23–25 might act as transporters for Ti–Salanfacilitating its cellular accumulation and hence leading to itsstronger cytotoxicity.

Fig. 2 In vivo toxicity of 100 mM of the titanium(IV) complexes in 24–120 h postfertilization (hpf) zebrafish embryos. See ESI† for results over the whole investi-gated concentration range (0.2–100 mM).

Fig. 3 Time dependent cellular titanium accumulation in HT-29 cells exposed to10 mM of Ti–Salan (top) or Ti–Y (bottom) in serum containing and serum free cellculture media.

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To evaluate the intracellular distribution we determined thetitanium levels of the nuclei and the mitochondria isolated fromHT-29 cells exposed to Ti–Salan and Ti–Y. For Ti–Salan theseexperiments showed a slow but steady accumulation of titaniumin the mitochondria that resulted in high values after extendedexposure (24 h) and roughly matched the kinetics of the cellularuptake (see Fig. 4). Titanium levels in the nuclei also increased overtime (see Fig. 5). Ti–Y showed different biodistribution behaviour.Mitochondrial titanium levels remained low in comparison toTi–Salan even after extended exposure but the titanium amountin the nuclei was comparable during the first hours of exposurealthough followed by a strong decrease.

Titanium(IV) complexes with a salan (Ti–Salan) or cyclo-pentadienyl (Ti–Y) ligand show very different behaviour concerningtheir reactivity against biomolecules (DNA and albumin), theircellular uptake and their intracellular distribution. WhereasTi–Salan showed comparatively low binding to biomoleculesbut a high and serum dependent cellular uptake, Ti–Y affordedgenerally lower cellular accumulation with increased bindingefficacy to both albumin and DNA. Biodistribution studies(uptake into mitochondria and nuclei) indicated that for Ti–Ytransport into the nuclei and interaction with DNA are crucial

while for Ti–Salan mitochondrial targeting might be of higherrelevance. Overall these results clearly demonstrate thattitanium(IV) complexes do not generally follow a commonreactivity and biodistribution pattern and that their biologicalbehaviour with respect to proliferation inhibition can be sub-stantially influenced by the choice of the coordinated ligands.These findings should encourage future studies on additionaltitanium complexes of different ligand systems that may lead tovaluable reactivity by various mechanisms.

This joint research project was financially supported by theState of Lower-Saxony, Hannover, Germany.

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14 C. M. Dowling, J. Claffey, S. Cuffe, I. Fichtner, C. Pampillon,N. J. Sweeney, K. Strohfeldt, R. W. G. Watson and M. Tacke, Lett.Drug Des. Discovery, 2008, 5, 141–144.

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Fig. 4 Time dependent titanium accumulation in mitochondria of HT-29 cellsexposed to 25 mM of Ti–Salan or Ti–Y.

Fig. 5 Time dependent titanium accumulation in nuclei of HT-29 cells exposedto 25 mM of Ti–Salan or Ti–Y.

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