Metallocenes, a class of organometallic compounds in which metal atoms are bonded to aromatic ring structures in a "π-bond" (most commonly: pπ-dπ) fashion have for the past half-century come to play a pre-eminent part in theoretical and applied research. Ferrocene, di(η5- cyclopentadienyl)iron(II), has undisputedly been the most outstanding representative of the class. This organoiron compound owes its leading position in a good measure to the unique electron occupancies and energies of its highest occupied molecular orbitals, permitting the ferrocene-ferricenium couple to interact with its chemical and physical environment via unusual oxidation-reduction and electron exchange reactions. On account of these outstanding redox properties, the ferrocene-ferricenium system, both monomeric and polymeric, proved its value in years gone by as a pivotal role player in rocket technology, and more recently it has found its most rewarding function in the biosciences. Recognition in this laboratory of the significant antiproliferative and antineoplastic activities displayed by some water-soluble ferrocene derivatives, and, more particularly, by water-soluble ferricenium salts, set into operation a development program leading to the synthesis of macromolecular ferrocene conjugates in which the metallocene is bioreversibly connected to water-soluble, biocompatible and biodegradable carrier polymers. Carrier-drug conjugates of this type are specifically designed to act as prodrugs, providing enhanced therapeutic effectiveness through increased drug bioavailability at the target site, a feature of pre-eminent value in the chemotherapy of cancerous diseases. The synthesized conjugates are of two types. The first type comprises polymers characterized by tertiary amine side group terminals as hydrosolubilizing entities, while the second contains conjugates with hydroxyl-terminated side chains serving that same purpose. Cell culture tests, still ongoing, have been performed on conjugates of both types against the HeLa cell line commonly regarded as a drug-sensitive human cancer, and against the human Colo, LNCaP, and Caco lines representative of cancers distinguished by moderate to strong resistance to common anticancer drugs. Some preliminary results are now available. The general conclusion to be drawn from those findings is that all tested conjugates irrespective of structural particulars are remarkably active antiproliferative agents. Most outstanding are the results for the group of polymers possessing amine-type hydrosolubilizing units (as against those with hydroxyl functionality), which indicate activities equal or superior to those of cisplatin co-tested for comparison. The data, in addition, permit the conclusion that for each conjugate the activity findings hardly change as one goes from the sensitive HeLa to the remaining resistant lines. Comparing HeLa with Colo results specifically, one observes, in fact, a trend of ever so slightly increasing activity values in that direction, suggesting the tested conjugates in toto to show a propensity, however moderate, for overcoming resistance problems. Conjugates with amide-linked ferrocene as a group are superior performers in comparison with the ester-linked analogs. This trend could be associated in part with the preponderant fraction of hydroxyl-type solubilizing groups in the molecule, although the ester link must be implicated as well by some as yet unexplained mechanism. The findings here summarized show that the monomeric, neutral ferrocene compound, unable as such to achieve cell entry and exert cytotoxicity, will undergo efficient endocytosis in the carrier-bound state. Liberated from the carrier in the lysosomal-endosomal compartment, it will cause eventual cell death, most probably after lysosomal oxidation to the ferricenium state. Although the precise cytotoxic mechanism is not known, there is reasonable consensus to suggest that the affected cell's nuclear DNA is not, in fact, attacked by the ferricenium cation directly but rather by secondary, catabolically generated reactive oxygen species, such as the aggressive hydroxyl free radical. What next? Which specific research goals should be defined for the subsequent phase of development of these conjugates? How would one optimize their biological performance and prepare the way for eventual clinical trials? The immediate project goal must be the design, synthesis, and in vitro screening of a considerably enlarged number of carriers and conjugates. This will include variations of polymer backbone and side chain structures with a view to increasing basicity (for facilitated cell entry), protein repellence (for enhanced conjugate inertness and half-life during serum residence), and, most importantly, ease of drug release (e.g. by introduction of pH-sensitive cleavage sites in the drug-binding tether). Biological evaluation efforts, initially restricted to in vitro testing, will focus on the determination of cytotoxic activities against a number of additional human cancer cell lines, notably those derived from moderately to strongly drug-resistant intestinal and metastatic neoplasias. Also included should be cell cultures that are available both as sensitive parent lines and their drug-resistant sublines. In follow-on work further down the screening path, laying the foundation for combination therapies, leading ferrocene conjugate structures will be tested in combination with polymeric conjugates of other drug models, such as selected platinum compounds, methotrexate, 5-fluorouracil, or ARA-C. The results of such combination screens will form the basis for synthesis and bio-evaluation projects directed at macromolecular compounds in which ferrocene is co-conjugated to the same carrier together with other drug species. This will serve to create a unified pharmacokinetic pathway for the agents involved, leading to enhanced co-bioavailability. The ultimate goal of future programs, providing the preconditions for clinical trials, must logically be the determination of in vivo performance of selected conjugates and co-conjugates, including biodistribution studies, toxicological evaluation, and antineoplastic activity screening against explanted human cancers. The biological in vitro results available at this time augur exceptionally well for the future performance profile of ferrocene polymers in the fight against cancer. © 2005 Springer Science+Business Media, Inc.
