ANTICANCER RESEARCH 31: 825-830 (2011) Cytotoxicity of Ferrocenyl–Ethynyl Phosphine Metal Complexes of Gold and Platinum ELEANOR FOURIE1, ELIZABETH ERASMUS1, JANNIE C. SWARTS1, ALEXANDER JAKOB2, HEINRICH LANG2, GISELA K. JOONE3 and CONSTANCE E.J. VAN RENSBURG3 1Department of Chemistry, University of the Free State, Bloemfontein, 9300, Republic of South Africa; 2Institut für Chemie, Technishe Universität Chemnitz, 09111 Chemnitz, Germany; 3Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria, 0001, Republic of South Africa Abstract. Background: Ferrocene derivatives may possess Cl– ion has on the cytotoxicity of these neutral gold ≡ antineoplastic activity. Those with low ferrocenyl reduction complexes. The platinum complex [(Fc-C C-PPh2)2PtCl2], potentials often have the highest anticancer activity, as cell compound 5, resembling the structure of cisplatin, in components have to oxidise them to the active ferrocenium principle should exhibit good cytotoxicity, but was not tested species before cytotoxicity can be recorded. Some gold(I) due to its total insolubility in any biocompatible medium. complexes also possess anticancer activity. This study examined the cytotoxicity of ferrocenyl-ethynyl and Potentially good chemotherapeutic drugs frequently find ruthenocenyl-ethynyl complexes of gold and platinum. The limited clinical use due to the many negative medical and results were related to the ease of iron oxidation in the physical side-effects they exhibit. For cisplatin, [(H3N)2PtCl2], ferrocenyl fragment and compared with the cytotoxicity of such side-effects include inter alia poor aqueous solubility, a cisplatin, [(H3N)2PtCl2] and [Au(PPh2CH2CH2PPh2)2]Cl. high excretion rate from the body, loss of appetite (anorexia), Materials and Methods: Ferrocene-containing gold and development of drug resistance after continued drug dosage, ≡ 1 platinum complexes of the type Fc-C C-PPh2, , and high toxicity especially to the kidneys and bone marrow, and, ≡ ¡ II 5 Fc-C C-PPh2 M with Fc=ferrocenyl (Fe (η -C5H5) perhaps most important of all, the inability to distinguish 5 2 ≡ (η -C5H4)), Ph=phenyl (C6H5) and M=Au-Cl, , Au-C between healthy and carcinomatous cells (1). To combat these 3 ≡ 4 II 5 C-Fc, , or Au-C C-Rc, (Rc=ruthenocenyl, (Ru (η -C5H5) negative side-effects, new antineoplastic materials are 5 ≡ 5 (η -C5H4)) and the complex [(Fc-C C-PPh2)2PtCl2], , continuously being synthesised and evaluated (1), combination were investigated. Cytotoxicity tests were determined on the therapies are investigated in the hope of finding synergistic HeLa (human cervix epitheloid) cancer cell line, ATCC CCL- effects (2), new methods of delivering an active drug to a 2. Cell survival was measured by means of the colorometric malignant growth are developed (3, 4) and new techniques of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide cancer treatment, such as photodynamic cancer therapy (5), 1-4 assay. Results: The IC50 values of compounds from four are investigated. experiments causing 50% cell growth inhibition, ranged In terms of new antineoplastic material, it has been shown between 4.6 and 27 μmol dm–3. Drug activity was inversely that certain ferrocenium salts (6) have more favourable 50% proportional to the sum of all formal reduction potentials, lethal dosage values than cisplatin (7), while water-soluble o’ ≡ E , of the ferrocenyl groups of the Fc-C C-PPh2 and Fc- ferrocenium-containing carboxylates (8) induce good-to- C≡C- ligands coordinated to the gold centre. The Fc-C≡C- excellent cure rates against human adenocarcinoma, ¡ 2 PPh2 Au-Cl complex, compound , was most cytotoxic with squamous cell carcinoma and large-cell carcinoma of the –3 IC50=4.6 μmol dm , demonstrating the beneficial effect the lung in in vitro human tumour clonogenic assays. It has also been shown that by anchoring the antineoplastic ferrocene derivative 3-ferrocenylbutanoic acid on a water-soluble polymeric drug carrier, an increase in drug activity of almost Correspondence to: J.C. Swarts, Department of Chemistry, one order of magnitude is obtained (4). University of the Free State, Bloemfontein, 9300, Republic of South The cytotoxicity of ferrocene-containing complexes is Africa. Fax: +27 514446384, e-mail: [email protected] frequently dependent on the formal reduction potential of Key Words: Ferrocene, ethynyl phosphine, gold, platinum, the ferrocenyl group. Related to ferrocene-containing cytotoxicity, HeLa. alcohols, it was found that smaller Eo’ values lead to more 0250-7005/2011 $2.00+.40 825 ANTICANCER RESEARCH 31: 825-830 (2011) 1-7 favourable (higher) cytotoxicity (9). In contrast, the free β- Table I. Chemosensitivity of complexes expressed as IC50 (μmol dm- diketones FcCOCH COR followed exactly the opposite 3) valuesa after 7 days of incubation with the HeLa cancer cell line. 2 o’ trend (10). Two mechanisms by which the ferrocenyl group Formal reduction potentials, E , of the ferrocenyl group are vs. a Fc/Fc+ reference electrode. destroys antineoplastic growths were identified. The first o’ was shown to involve homolytic action, i.e. radical-induced Compound E IC50 electron transfer processes between a ferrocenium group and (V)b (μmol dm–3) water, inter alia to generate hydroxy radicals which cleave ≡ c DNA strands (11). This implies that, after being 1 (Fc-C C-PPh2) 0.300 13+1 2 (1¡AuCl) 0.350 4.6±0.7 administered to the body, a ferrocene-containing drug must 3 (1¡Au-C≡C-Fc) 0.000 (0.260)d 22±4 first be oxidised by redox-active enzymes in the body to the 4 (1¡Au-C≡C-Rc) 0.255 (0.270)e 27±4 ≡ ff ferrocenium species to show antineoplastic activity. Several 5 [(Fc-C C-PPh2)2PtCl2] f redox-active enzymes and/or proteins are able to oxidise the 6 Cisplatin [(H3N)2PtCl2] 0.19±0.01 f iron(II) centre of the ferrocenyl group to iron(III) to liberate 7 [Au(PPh2CH2CH2PPh3)2]Cl 0.14±0.01 the positively charged radical ferrocenium species (12-16). aData are presented as mean drug concentration causing 50% inhibition It is, therefore, irrelevant whether the bioactive agent of cell growth + standard error of the mean of four experiments. bData containing the ferrocenyl group is administered in the from ref 23. cUnpublished data by E Fourie, JC Swarts and H Lang. dThe reduced ferrocenyl or oxidised ferrocenium state, provided first formal reduction potential at 0.000 V of this diferrocene-containing ≡ that the formal reduction potential of the ferrocenyl group compound corresponds to the oxidation of the Fc group on the Au-C C- Fc molecular fragment. The second formal reduction potential is at 0.260 V is low enough to allow ferrocenyl oxidation inside a cell. ≡ e o’ and comes from the Fc-C C-PPh2 molecular fragment. The second E There are indications that the cut-off formal reduction value is for the ruthenocenyl group, Rc. fNot determined due to lack of potential of the ferrocenyl group where this cannot happen solubility, or the compound has no ferrocenyl group. any more is 0.52 V vs. Fc/Fc+ (9). Eo’ values of the ferrocenyl group of all four complexes 1-4 are less than 0.52 V (Table I), implying they may all have antineoplastic capability. In the second mechanism, the ferrocenyl group days, cell survival was measured by means of the colourometric 3- itself acts as a reducing agent when it reduces the tyrosyl (4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay radical of the R2 subunit of the enzyme ribonucleotide (24). Wells without cells and with cells but without drugs were reductase (17). The active site of dimeric R2 consists of a included as controls. Survival curves were plotted (Figure 2) as a tyrosil radical and two Fe(III) centers which are μ-oxo function of drug dose and the drug concentration that caused 50% bridged (18, 19). This enzyme catalyses the reduction of inhibition of cell growth (IC50) was estimated by extrapolation. ribonucleotides to deoxyribonucleotides, a key step in DNA syntheses (20) and its inactivation is, therefore, a goal in Results chemotherapy (21, 22). Since it is known that ferrocene-based drugs often need an Materials and Methods incubation period before they perform optimally in cell destruction, all the studies with complexes 1-4 were Compounds. Complexes 1-5 (Figure 1) were synthesised according performed utilising seven days of drug exposure to the cells to published procedures (23). in accordance with previous studies (4, 9). The cell growth inhibitory properties of ferrocenyl alkynyl complexes 1-4 Sample preparation. The samples were dissolved in dimethyl expressed as IC50 values are summarised in Table I and sulphoxide (DMSO) giving stock concentrations of 20 mmol dm–3 –3 ranged between 4.6 and 27 μmol dm . The lowest IC50 and diluted in the appropriate growth medium supplemented with values correspond to the more active compounds. The most foetal calf serum (FCS) to give final DMSO concentrations not exceeding 0.5% and drug concentrations of 20-2,000 μmol dm–3 active drug was found to be the mono ferrocenyl, chloride- ≡ ¡ prior to the cell experiments. containing gold complex 2. Complex 2, Fc-C C-PPh2 Au- –3 Cl with IC50=4.6 μmol dm , was three times more active ≡ Cell cultures. The human cervix epitheloid cancer cell line, HeLa than the free ligand Fc-C C-PPh2, 1, and clearly (ATCC CCL-2) (American Type Culture Collection, Manassas, VA, demonstrates the advantageous effect of introducing the USA) was grown as monolayer cultures from MEM. The growth gold(I) ion into anticancer drugs. Like the platinum(II) core media were maintained at 37˚C under 5% CO2 and fortified with of cisplatin [(H N) PtCl ], the gold(I) centre also possesses 10% FCS and 1% penicillin and streptomycin. 3 2 2 a d8 electronic configuration, although it exhibits a linear Cancer cells were seeded at 500 cells/well in 96-well microtitre plates in a final volume of 200 μl of growth medium in the presence or rather than square planar geometry.