Titanocenes

Titanocenes

Titanium, with chemical symbol Ti and atomic number 22, is a member of the d-block metals and belongs to group 4 of the periodic table of elements (Figure 8.18).

Titanium is a metal, which puzzles the bioinorganic scientists, as it has no known native role in the human body (or any organism) despite it is the ninth most abundant element in the earth’s crust . The metal titanium is extremely corrosion-resistant and has a remarkable biocompatibility, which has led to its clinical applications. These include endoprostheses of knees and hips, dental implants, heart pacemakers and many more. Titanium complexes are used for their catalytic properties, and there are complexes that interact with biomolecules (this will be shown below). Nevertheless, there is no known function of titanium as a native essential metal [6, 7].

The most stable oxidation state is Ti(IV), and most titanium compounds with lower oxidation states are easily oxidised to Ti(IV). It is very characteristic that these Ti(IV) species easily undergo hydrolysis and form Ti—O bonds and fairly often polymeric structures . Titanium oxides and many Ti(IV) compounds do not show high solubility in water and therefore have only limited bioavailability. This might explain why titanium has no known biological role despite its high natural abundance, and is not an essential metal for any life form .

Titanium dioxide (TiO₂) is a white solid with a low aqueous solubility and is the natural occurring oxide of titanium present in minerals such as rutile (composed mainly of TiO₂). TiO₂ is used as a white pigment in paints, food colouring, sunscreens and toothpaste. For the purification of crude TiO₂, the raw material is reduced with carbon and then oxidised in the presence of chlorine. The resulting titanium tetrachloride (TiCl₄) is purified by distillation and reacted with oxygen at around 2000 K to form TiO₂ in its pure form.

Current research into the medicinal applications of titanium complexes mainly focusses on their use as anticancer agents. It is important to note that these complexes are different from the above-described titanium alloys/metals or TiO₂, which are highly insoluble in aqueous media. The titanium complexes described below contain functional groups that can be easily exchanged and/or interact with biomolecules in order to fulfil their medicinal role.

History of titanium-based anticancer agents: titanocene dichloride and budotitane

The success of cisplatin stimulated the search for further metal-based anticancer drugs. Two titanium-based complexes, namely titanocene dichloride (Cp₂TiCl₂) and budotitane (Ti(IV)(bzac)₂(OEt)₂, bzac, 1-phenylbutane-1,3-dionate), were identified as promising drug candidates and entered clinical trials . Both compounds contain labile groups in the cis position analogous to cisplatin. Unfortunately, hydrolysis in titanium compounds is faster than in cisplatin, and in these cases can lead to the formation of hydroxo-bridge species and potentially to TiO₂, which is insoluble in water. This represents the main challenge for the clinical application of this class of compounds (Figure 8.19) .

Cp₂TiCl₂ was first synthesised in 1954 and is a red crystalline solid in its pure form. The Ti(IV) centre is coordinated by two ⁵-Cp ligands and two chlorine ligands. Cp₂TiCl₂ can be synthesised by reacting titanium tetrachloride (TiCl₄) with NaCp . Compared to those of ferrocene, the planar cyclopentadienyl ligands are coordinated to the titanium centre in a bent sandwich configuration . Furthermore, Cp₂TiCl₂ is an exception to the 18 VEs rule, as it has only 16 VEs, but it is still a stable complex. Each Cp⁻ ligand contributes six elec-trons, whilst each Cl⁻ ligand contributes two electrons. Ti(IV) has no VEs left, which makes a total of 16 VEs.

2NaC₅H₅ + TiCl₄ → (C₅H₅)₂TiCl₂ + 2NaCl

Compared to cisplatin, much less is known on the mode of action of titanium-based drug candidates. In fact, the active species responsible for the anticancer activity of Cp₂TiCl₂ in vivo has yet not been identified, nor has the coordination mechanism to DNA or eventual repair processes. Studies have shown that titanium will accumulate near phosphorus-rich areas, indicating a titanium–DNA interaction takes place. Crystallo-graphic studies suggest that, within Cp₂TiCl₂, the Cp groups sterically prevent the cross-linking of DNA. A direct comparison between Cp₂TiCl₂ and cisplatin shows that their spectrum of activity differs, leading to the conclusion that their mode of action must also differ .

Current studies have confirmed the uptake of Ti(IV) by transferrin and transport to the cancer cells. Within the cell, it is believed that Ti(IV) may bind, in contrast to cisplatin, to the DNA backbone, which means coordinating to the negatively charged phosphates. Generally, it is believed that this coordination is not very strong .

Cp₂TiCl₂ showed very promising results in preclinical studies. In vitro studies have been carried out by Köpf-Maier on a variety of human cancers including human lung and renal cell cancer carcinomas xenografted into mice. The results were very encouraging, as a significant response of the tumour to the chemotherapeutic agent was observed and Cp₂TiCl₂ seemed to be an interesting candidate to bring into the clinic . The major challenge for a clinical application of Cp₂TiCl₂ as an anticancer agent is presented by its hydrolytic instability at pH below 5.5, where both chlorides are lost. Even at neutral pH, research has shown that the Cp rings are lost quickly, resulting in the formation of hydrolysed products which are water insoluble . Therefore, it was necessary to develop a formulation before this compound could be brought into clinical trials. For its use in clinical trials, Cp₂TiCl₂ was formulated as a lyophilised powder in malate buffer (pH = 3.2) or malic acid (pH = 3.5). Cp₂TiCl₂, formulated as a water-soluble powder, has entered phase I and phase II clinical trials [7]. Results from phase I clinical trials were used to establish the maximum tolerable dose and to define the pharmacokinetic properties. Dose-limiting toxicities have been identified, as reversible damage to the liver and kidneys. Phase II clinical trials concentrated on the effect of Cp₂TiCl₂ treatment of renal cell cancer and metastatic breast cancer. Unfortunately, the encouraging effect seen in preclinical studies could not be repeated in the clinical studies. The tumours did not show a significant response to Cp₂TiCl₂, and therefore clinical trials were discontinued .

Budotitane is an octahedral Ti(IV) complex that was developed as a potential anticancer agent around the same time as the anticancer properties of Cp₂TiCl₂ were discovered. Budotitane can be synthesised by reacting TiCl₄ with the appropriate diketonate in an anhydrous solvent. Important structural features are the unsubstituted aromatic rings, which positively contribute to the cytotoxic activity, and the hydrolysable group. The latter group seems to be less important in regard to the cytotoxicity of budotitane but crucial for formu-lation purposes as it seems to determine the aqueous stability. Research has shown that the aqueous stability increases with the hydrolysable group in the order: I⁻ < Br⁻ < Cl⁻ < F⁻ < OR⁻ (Figure 8.20) .

Figure 8.20 Synthesis of budotitane

Another major drawback for the clinical use of budotitane is the presence of isomers. There is the possi-bility of a cis or trans arrangement of the —OC₂H₅ group, with the cis arrangement believed to be the more stable one. Furthermore, the β-diketonato ligand is not symmetrical. This means that there are eight possible arrangements in this octahedral scheme. In solution, there is a mixture of isomers and it is difficult to isolate the isomers. So far, it is not known which isomer exhibits the anticancer activity (Figure 8.21) .

Preclinical studies using human xenografts in nude mice highlighted the potential of budotitane as novel chemotherapeutic agent in 1984. Budotitane showed cytotoxic activity comparable to cisplatin. Keppler et al. intensively studied its antitumour activity in a variety of animal models and found specific activity against Ehrlich ascetic tumour and colon cancer. Budotitane, similar to Cp₂TiCl₂, is prone to hydrolysis in conjunction with a low aqueous solubility. Therefore, significant research went into the development of an appropriate for-mulation . Several clinical studies were performed using cremophor EL as a nonionic surfactant, which has been used as a vehicle for the solubilisation of a wide variety of hydrophobic drugs (cremophor). Furthermore, 1,2-polyethyleneglycol has been added to encourage the coprecipitation of the drug. All three ingredients (budotitane, cremophor EL and 1,2-polyethyleneglycol) are dissolved in anhydrous ethanol and mixed, and the solvent is then evaporated off. Using this procedure, micelles containing 100–200 mg/100 ml of active pharmaceutical ingredient are formed, which are stable for a few hours. Budotitane went through several phase I and phase II clinical trials. Budotitane was administered as intravenous (IV) infusion twice a week to patients with solid tumours refractory to previous treatments. Cardiac arrhythmia was identified as the dose-limiting

2Hbzac + TiCl₄ → [Ti(bzac)₂(OEt)₂] + 4HCl

side effect. Unfortunately, problems with the formulation, such as the existence of isomers and the difficulty in analysing and characterising the loaded micelles, led to a discontinuation of the clinical trials .

Poor water solubility and fast hydrolysis are the main problems for the use of titanocene or titanium-based compounds as anticancer agents. Nevertheless, this intensive research in the 1980s and 1990s has encouraged further research as described in the following.

Further developments of titanocenes as potential anticancer agents

As mentioned, poor aqueous solubility and instability in water are the main problems that restrict titanocenes in clinical applications. McGowan and coworkers renewed the interest in research in this area with their elegant synthesis of ring-substituted cationic titanocene dichloride derivatives. The idea was based on the introduction of charges in order to improve the aqueous solubility. Indeed, the resulting cationic titanocenes are more water-soluble and show a significant cytotoxic activity especially against cisplatin-resistant ovarian cancer (Figure 8.22) .

Following on from these results, Tacke and coworkers based their research on novel synthetic methods starting from substituted fulvenes. A fulvenes is an organic molecule with the chemical formula C₆H₆, which consists of a five-membered ring system and can be used to easily introduce functional groups into titanocenes (Figure 8.23) .

Reaction of substituted fulvenes with titanium dihalides via a reductive dimerisation process leads to the so-called ansa-titanocenes, which are normally used as catalysts. These ansa-titanocenes are characterised by a carbon–carbon bridge between the cyclopentadienyl (Cp) rings, which restricts the geometry of the Cp rings. 

In vitro testing showed that they exhibit a moderate cytotoxic activity, which is similar or better than Cp₂TiCl₂ when tested against a model of renal cell cancer (Figure 8.24) .

The reaction of substituted fulvenes with superhydride (LiBEt₃H) in a so-called hydridolithiation reaction or the reaction with an organolithium compound in a carbolithiation reaction followed by transmetallation with titanium tetrachloride allows access to a variety of substituted titanocenes. Hydridolithiation allows access to benzyl-bridged titanocenes, whilst titanocenes obtained via carbolithiation typically contain more functional groups. This can be of advantage, for example, for the introduction of groups that can be eas-ily ionised in order to improve the water solubility of these titanocenes, but can also be a disadvantage as additional stereocentres are potentially introduced (Figures 8.25 and 8.26).

These titanocenes have been intensively studied with regard to their potential anticancer activity. It has been shown that some of these compounds are active against a variety of human cancer types. The so-called titanocene Y has been the most intensively studied titanocene of this series. It has shown good activity against a model of renal cell cancer as well as other human cancer cell lines in a variety of in vitro experiments (Figure 8.27).

In general, titanocenes obtained via these methods reached IC₅₀ values in the low micromolar range when tested against a renal cell cancer model. This represents an up to 2000-fold improvement compared to Cp₂TiCl₂. Nevertheless, the main problems are the potential presence of stereocentres and still a poor aqueous solubility. Further research has been carried out with the aim to replace the chloride ligands by other groups. This includes the incorporation of chelating ligands similar to the research undertaken for cisplatin. Nevertheless, this research did not result in any major improvement with regard to the cytotoxic activity. Also, some initial formulation studies have been carried out with only limited success. The mode of action of these titanocenes is so far not clear. It is believed that they coordinate to DNA via its phosphate backbone. It has also been shown that they can use transferrin as a transporter molecule into the cancer cell .