Ferrocene

Ferrocene

Ferrocene (or bis(η⁵-cyclopentadienyl)iron, (C₅H₅)₂Fe) is an orange powder and is probably one of the best studied metallocenes. As previously mentioned, its structure follows the 18-electron rule and it is a very stable complex. Its Cp⁻ ligands can be easily derivatised to introduce functional groups. Functionalised ferrocene derivatives are currently used as biosensors in blood glucose measuring equipment and they are also under intense research as potential anticancer agents.

Ferrocene was discovered by Paulson and Kealy in 1951. Cyclopentadienyl magnesium bromide and ferric chloride were reacted in a so-called Grignard reaction (reaction involving R-MgBr) in order to create a fulva-lene. Instead, they created ferrocene. At that time, it was difficult to identify the correct structure of ferrocene, but Wilkinson, Rosenblum, Whitting and Woodward managed to do this soon after its discovery .

2C₅H₅MgBr + FeCl₂ → Fe(C₅H₅)₂ + MgBr₂ + MgCl₂

Nowadays, ferrocene is synthesised via a so-called transmetallation reaction. Typically, commercially available sodium cyclopentadienide is deprotonated with KOH or NaOH, and the obtained anion is reacted with anhydrous ferrous chloride (FeCl₂). Instead of purchased sodium cyclopentadienide, freshly cracked cyclopentadiene is often used (Figure 8.7).

Ferrocene is a very stable complex and can be easily functionalised by derivatising its Cp⁻ ligands. The Cp⁻ ligands are aromatic, as previously mentioned, and therefore show a chemical behaviour similar to benzene. This means that reactions known for benzene chemistry can be used with ferrocene, such as the Friedel–Crafts acylation reaction. Ferrocene can be acylated by reacting it with the corresponding aluminium halide (AlX₃). Indeed, this chemical behaviour of ferrocene helped in identifying its real structure (Figure 8.8) .

Ferrocene and its derivatives are under intense screening for medicinal purposes. Research has shown that especially ferrocene derivatives exhibit very promising effects for a variety of clinical applications, such as antimalarial and anticancer agents as detailed below. Interestingly enough, ferrocene itself is not a particularly toxic compound, as it can be administered orally, injected or inhaled with no serious health concerns. It is believed to be degraded in the liver by cytochrome P₄₅₀, similar to benzene. Its degradation process involves the enzymatic hydroxylation of the cyclopentadienyl ligand. Animal studies on beagles have shown that treat-ment with up to 1 g/kg ferrocene did not result in acute toxicity or death, although it did lead to a severe iron overload, which was reversible .

Ferrocene can easily undergo oxidation to the ferrocenium cation in a one-electron oxidation process. The formed cation is fairly stable, and the whole process is reversible. This redox potential, together with a change in lipophilicity, is the main characteristic that makes ferrocene-based compounds interesting for a variety of potential clinical applications, especially the ones outlined in the following (Figure 8.9) .

Ferrocene and its derivatives as biosensors

Diabetes is a major health problem with hundreds of millions sufferers worldwide. As part of the illness, diabetic patients have increased glucose levels in their blood due to a lack of insulin or cells not reacting to insulin. Insulin promotes the uptake of glucose into the cells. There are several options to manage diabetes, but it is extremely crucial for the welfare of the patients that the blood glucose levels are closely monitored. In order to facilitate these regular measurements, a significant amount of research has gone into the development of portable and easy-to-use devices. Modern blood glucose monitors benefit from the technical advances of the so-called biosensor research, an area where the majority of the biosensors are used.

Biosensors are based on enzymes that contain redox-active groups. This means that the redox group can change its redox state as a result of a biochemical reaction. In nature, the enzymes glucose oxidase (GOx) or glucose dehydrogenase (GDH) are used as biosensors for blood glucose monitors. Typically, these enzymes accept electrons from the substrate, glucose in this case, and oxidise it. The enzyme changes to its reduced state, which normally deactivates the enzyme. In order to activate the enzyme again, electrons are transferred and the enzyme is oxidised. GOx and GDH in their reduced form transfer electrons to molecular oxygen, and hydrogen peroxide (H₂O₂) is produced. Oxygen or peroxide electrodes can then be used to measure any change of the substrate, which directly relates to the glucose levels present in the sample. Unfortunately, this method has problems, as, for example, molecular oxygen can be a limiting factor and a lack of oxygen can lead to wrong readings (Figure 8.10).

Enzymes such as GOx are very specific to the substrate they accept electrons from, that is, the substrates they oxidise, but they are more flexible to the substrate they donate electrons to. Therefore, a variety of inorganic redox-active compounds have been tested as so-called mediators. Mediators function by accepting electrons from the enzyme and thus oxidising the enzyme to its active form. They shuttle electrons from the enzyme to the electrode and are also called electron sinks. Electrodes can measure any changes in the redox potential of these mediators, and these changes can directly be related to the amount of glucose present in the sample. This technology excludes the need for molecular oxygen and problems connected to that (Figure 8.11) .

In 1984, the first ferrocene-based mediator in conjunction with GOx was used as a biosensor for glucose. Derivatives of ferrocene are still the most important examples for mediators in biosensors, mainly due to their wide range of redox potential, which is independent of any pH changes. Furthermore, the chemistry involved in synthesising these ferrocene derivatives is well explored and fairly straight forward. Additionally, the mediator must successfully compete with the natural mediator (molecular oxygen) in order to ensure accurate readings. From the point of its application as biosensor for blood glucose measurement, it is clear that ferrocene-based mediators can be used only once. This is due to the fact that, whilst ferrocene is relatively insoluble, the reduced form, the ferrocenium ion, is fairly soluble. Mediators should be insoluble in order to lead to reproducible results or, as in this case, can only be used once (Figure 8.12) [3, 4].

[Cp₂Fe] → [Cp₂Fe]⁺ + e⁻                           (8.1)

Equation 8.1: Oxidation of ferrocene to the paramagnetic ferrocenium ion.

Initially, ferrocene itself was used as mediator, but later on a variety of its derivatives were tested for their redox potential in biosensors. Some of these examples are shown below. This research has led to development of the modern blood glucose analysers, which are only the size of a pen and highly mobile devices. These devices use disposable strips and are simple to use (Figure 8.13) .

Ferrocene derivatives as potential antimalarial agent

A variety of compounds containing the ferrocene group have been synthesised and tested for their clinical properties, especially as antimalarial and anticancer agents. In this context, especially the ferrocene-based analogue of chloroquine, ferroquine, has shown significant promise. 

It successfully passed phase II clinical trials and is awaiting results from field testing. Chloroquine is a well-known drug used in the treatment of malaria caused by the parasite Plasmodium falciparum. Ferroquine is active against this parasite as well. Even more exciting is the fact that it is also active against the chloroquine-resistant strain of P. falciparum. The changed biological activity might be due to the changed lipophilicity and/or the redox action that is present after the introduction of the ferrocene group (Figure 8.14) .

Ferrocifen – a new promising agent against breast cancer?

Ferrocene and its derivatives were, and still are, under intense scrutiny as potential anticancer agents. Initially, a range of ferrocenium salts was tested for their cytotoxic activity. The mode of action is still unclear, but DNA, cell membrane and enzymes have been proposed as potential targets. Ferrocenium salts are believed to gen-erate hydroxyl radicals under physiological conditions. These may damage the DNA, possibly by oxidising the DNA. Furthermore, it is believed that the cell membrane might be a target. Research has shown that the counter-ion is crucial for the cytotoxic activity as well as their aqueous solubility. Ferrocenium salts such as the picrate and trichloroacetate derivates display good aqueous solubility and high cytotoxic activity. As part of this research, ferrocene was also successfully bound to polymers in order to improve their water solubility and therefore cytotoxic activity .

Jaouen and coworkers substituted phenyl rings in existing drugs and natural products by ferrocene groups in order to introduce a redox-active metal group into these molecules and to change their lipophilicity. A breakthrough was achieved when a phenyl group in tamoxifen, a selective oestrogen receptor modulator (SERM) used as front-line treatment of hormone-dependent breast cancer, was replaced by ferrocene. The active metabolite of tamoxifen is actually the hydroxylated form 4-hydroxytamoxifen, which is highly active in the fight against oestrogen-dependent breast cancer. Breast cancer can be divided into hormone-dependent (also called oestrogen-dependent, ER(+)), which is characterised by the presence of an oestrogen receptor, and hormone-independent (also called oestrogen-independent, ER(−)) [2, 5].

Selective oestrogen receptor modulators are defined as a class of compounds that interact with the oestro-gen receptor. This interaction can happen in various tissues leading to different actions.

The combination of tamoxifen derivatives with ferrocene was a very successful approach, and has led to the development of a class of compounds called ferrocifens. Whilst around two-thirds of patients are diagnosed with ER(+) breast cancer and can be treated with hormone therapy such as with SERMs, there is still an urgent need to develop drugs to be used against ER(−) breast cancer. Preclinical studies have shown that ferrocifen is active against the latter type of breast cancer which is not susceptible to the treatment with tamoxifen (Figure 8.15) [2, 5].

The cytotoxic effect of tamoxifen results from the competitive binding to the oestrogen receptor and repress-ing DNA transcription, which is mediated by oestradiol. It is believed that ferrocifen follows the same mode of action. Research has shown that the replacement of the phenyl group in tamoxifen by ferrocene results in a reduced binding affinity to the receptor (RBA, receptor binding affinity). Variation, and especially increase, of the chain length has a negative effect on the RBA and also on the bioavailability. The optimum chain length seems to be when n = 4. It is also important to note that the Z-isomer binds more strongly to the receptor. Very surprisingly, ferrocifen (with n = 4) showed also an antiproliferative effect when tested on the oestrogen-independent cell line MDA-MB231, which does not have oestrogen receptors and is not accessible for treatment with tamoxifen. This means that there must be an additional mode of action that is independent of the oestrogen receptor.

Replacing the ferrocenyl group by a ruthenium group resulted in a drop of cytotoxic activity, indicating that the iron group is important. It has been proposed that the additional mode of action of ferrocifen could rely on the redox activation of the ferrocenyl group and the presence of reactive oxygen species (ROS) .

These extremely promising results stimulated further research in this area. Tamoxifen was coupled to a variety of known metal-based compounds with potential anticancer activity, such as oxaliplatin, titanocene dichloride and others. Oxaliplatin contains the so-called DACH–Pt group (DACH, 1,2-diaminocyclohexane), which has been used as a basis for a new DACH–Pt–tamoxifen derivative.

Oxaliplatin showed a cytotoxic effect of 6.3 μM when tested on the oestrogen-dependent human breast cancer cell line MCF-7, whilst the tamoxifen-vectorised derivatives (see Figure 8.16; R = H, 14 μM and R = OH, 4 μM) also presented an antiproliferative effect at a similar magnitude. Looking in more detail, research shows that the derivative that contains the hydroxyl group displays a higher RBA and also a better IC₅₀ value. This shows that the hydroxyl group (also present in the active metabolite of tamoxifen) is important for the recognition by the oestrogen receptor. Nevertheless, the vectorisation of DACH–Pt does not really result in a significant improvement in comparison to oxaliplatin itself, and therefore this combination is not really beneficial as an SERM for the fight against breast cancer .

The titanocene-tamoxifen derivative has an RBA value of 8.5%, which means it should recognise the oestro-gen receptor well. The results of the cytotoxicity tests were very unexpected, where a proliferative effect was observed. The estrogenic effect was comparable to that of oestrogen itself. It is believed that this estrogenic effect is due to the titanium moiety and/or its hydrolysis products (Figure 8.17) .

In order to bring ferrocifen into clinical studies, it was important to find a suitable formulation. This is an area notoriously difficult for metal-based drugs, and OH-ferrocifen finally entered phase II clinical trials. A variety of pharmaceutical approaches have been researched, including the use of nanoparticles, cyclodextrins and lipid nanocapsules .