Differential cytotoxicity induced by the Titanium(IV)Salan complex Tc52 in G2-phase independent of DNA damage.

BACKGROUND: Chemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown. METHODS: Four different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38. RESULTS: Here we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton. CONCLUSIONS: In summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.

Heptacoordinate Heteroleptic Salan (ONNO) and Thiosalan (OSSO) Titanium(IV) Complexes: Investigation of Stability and Cytotoxicity.

Seven heptacoordinate titanium(IV) complexes were synthesized based on the concept of hetero-bis-chelate stabilization of salan (ONNO) and thiosalan (OSSO) titanium(IV)alkoxides with 2,6-pyridinedicarboxylic acid (dipic) and derivatives thereof. The resulting compounds were investigated in a solid by X-ray diffraction and in solution by NMR spectroscopy. A thiosalan (OSSO) titanium(IV) complex could be isolated and its conformational stabilization by dipic was shown by (1)H NMR spectroscopy to lead to nonfluxional behavior even at room temperature. The stability of selected complexes was assessed at pH 1.9, 6.8, and 12.1 by an UV-vis monitored hydrolysis study with >5 Mio. equivalents of water. Even at pH 12.1 [L(1)Ti(dipic)(1)] showed t1/2 of more than 2 days. The cytotoxicity of all compounds was investigated in two human carcinoma cell lines. IC50-values in the range of cisplatin were achieved by all tested compounds except for [L(4)Ti(dipic)(1)], which was proven to be nontoxic. The functionalization of dipic was thus well tolerated and did neither interfere with the stability nor the cytotoxicity of the heteroleptic complexes.

Synthesis and X-ray structure analysis of a heptacoordinate titanium(IV)-bis-chelate with enhanced in vivo antitumor efficacy.

Chelate stabilization of a titanium(IV)-salan alkoxide by ligand exchange with 2,6-pyridinedicarboxylic acid (dipic) resulted in heptacoordinate complex 3 which is not redox-active, stable on silica gel and has increased aqueous stability. 3 is highly toxic in HeLa S3 and Hep G2 and has enhanced antitumor efficacy in a mouse cervical-cancer model.

Cytotoxic dinuclear titanium-salan complexes: structural and biological characterization.

Controlled hydrolysis of donor-substituted titanium-salan complexes led to the formation of well-defined dinuclear complexes. Structure determination by means of X-ray and NMR-studies revealed the presence of a single µ-oxo bridge and one labile alkoxide ligand per titanium center. Concomitant cytotoxicity assays of the isolated dinuclear complexes showed cytotoxicities in the low micro-molar region, surpassing in this respect even their monomeric ancestors, thus making them possible highly active metabolites of titanium-salan anti-cancer drugs.

Titanium salan complexes displays strong antitumor properties in vitro and in vivo in mice.

The anticancer activity of titanium complexes has been known since the groundbreaking studies of Köpf and Köpf-Maier on titanocen dichloride. Unfortunately, possibly due to their fast hydrolysis, derivatives of titanocen dichloride failed in clinical studies. Recently, the new family of titanium salan complexes containing tetradentate ONNO ligands with anti-cancer properties has been discovered. These salan complexes are much more stabile in aqueous media. In this study we describe the biological activity of two titanium salan complexes in a mouse model of cervical cancer. High efficiency of this promising complex family was demonstrated for the first time in vivo. From these data we conclude that titanium salan complexes display very strong antitumor properties exhibiting only minor side effects. Our results may influence the chemotherapy with metallo therapeutics in the future.

Small Molecule Inhibitors of Human DNA Polymerase λ.

To discover chemical probes to further under-stand the function of individual DNA polymerases, we established a generally applicable high-throughput screening. By applying this technique we discovered three novel inhibitor classes of human DNA polymerase λ (DNA Pol λ), a key enzyme to maintain the genetic integrity of the genome. The rhodanines, classified as an excellent drug scaffold, were found to be the most potent inhibitors for DNA Pol λ. Importantly, they are up to 10 times less active against the highly similar DNA polymerase ß. We investigated basic structure activity relationships. Furthermore, the rhodanines showed pharmacological activity in two human cancer cell lines. So the here reported small molecules could serve as useful DNA Pol λ probes and might serve as starting point to develop novel therapeutic agents.

Dimethyl titanocene Y: a valuable precursor for libraries of cytotoxic titanocene derivatives.

Reaction of the known titanocene Y 2 with methyl lithium at -15 degrees C yields bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) dimethyl (dimethyl titanocene Y, 3), a hitherto unknown, surprisingly robust titanium (IV) dimethyl species. Dimethyl titanocene Y was utilized in the preparation of several bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) dicarboxylates by the reaction with the free carboxylic acids in fair to good yields. Cytotoxicity of all new compounds has been estimated in Hela S3 cells.

Cytotoxic titanium salan complexes: surprising interaction of salan and alkoxy ligands.

The synthesis, biochemical evaluation, and hydrolysis studies of a wide selection of alkyl- and halogen-substituted titanium salan alkoxides are presented herein. A systematic change in the employed alkoxides revealed that both the bulk of the salan ligands and the steric demand of the labile ligands are of great importance for the obtained biological activity. Surprisingly, these two factors are not independent from each other; lowering the steric demand of the alkoxide of a hitherto nontoxic complex renders it cytotoxic. Therefore, our data suggest that the overall size of the complex exerts a strong influence on its biological activity. To decide whether the correlation between the cytotoxicity and the steric demand of the whole complex is merely based on an altered hydrolysis or on the interaction with biomolecules, the behavior of selected complexes under hydrolytic conditions and the influence of transferrin were investigated. Complexes differing only in their labile alkoxy ligands gave the same hydrolysis products with similar hydrolysis rates but displayed cytotoxicities that differed in the range of one order of magnitude. Thus, it seems that the hydrolysis product is not the active species but rather that the unhydrolysed complex is important for the first interaction with a biomolecule. This promoted the idea of hydrolysis being a detoxification pathway. In accordance with the above conclusion, chloro-substituted complex [Ti(Ph(Cl)N(Me))(2)(O(iPr))(2)] displayed a high cytotoxicity (IC(50) approximately 5 microM) and surprisingly high hydrolytic stability (t(1/2)=108 h). These findings, together with the observed cytotoxicity in a cisplatin-resistant cell line, make halo-substituted salan complexes an interesting target for further studies.

Titanocene difluorides with improved cytotoxic activity.

Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me(3)SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.