Regulating the anticancer properties of organometallic dendrimers using pyridylferrocene entities : synthesis , cytotoxicity and DNA binding studies †

A new series of eight firstand second-generation heterometallic ferrocenyl-derived metal–arene metallodendrimers, containing ruthenium(II)–p-cymene, ruthenium(II)–hexamethylbenzene, rhodium(III)– cyclopentadienyl or iridium(III)–cyclopentadienyl moieties have been prepared. The metallodendrimers were synthesized by first reacting DAB-(NH2)n (where n = 4 or 8, DAB = diaminobutane) with salicylaldehyde, and then the Schiff-base dendritic ligands were reacted in a one-pot reaction with the appropriate [(η-p-iPrC6H4Me)RuCl2]2, [(η-C6Me6)RuCl2]2, [(η-C5Me5)IrCl2]2 or [(η-C5Me5)RhCl2]2 dimers, in the presence of 4-pyridylferrocene. Heterometallic binuclear analogues were prepared as models of the larger metallodendrimers. All complexes have been characterized using analytical and spectroscopic methods. The cytotoxicity of the heterometallic metallodendrimers and their binuclear analogues were evaluated against A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant human ovarian cancer cell lines and against a non-tumorigenic HEK-293 human embryonic kidney cell line. The second generation Ru(II)-η-C6Me6 metallodendrimer is the most cytotoxic and selective compound. DNA binding experiments reveal that a possible mode-of-action of these compounds involves non-covalent interactions with DNA.


Introduction
Research on the design of heterometallic complexes as possible anticancer agents has flourished. [1][2][3][4][5][6] Due to its favourable electronic properties and ease of functionalization, 7-12 ferrocene has been incorporated in various biologically active systems, 9,13,14 in an effort to achieve a synergistic effect between the metal centers. [15][16][17][18][19][20] Furthermore, simple derivatives of ferrocene display good activity in vitro, with inhibition of tumors observed in vivo. 21,22 In the search for new tamoxifen-like drugs, Jaouen and co-workers 23,24 prepared ferrocifens from 1-[4-(2-dimethylaminoethoxy)]-1-( phenyl-2-ferrocenylbut-1-ene), which are highly active ferrocenyl-derivatives of the purely organic breast cancer drug tamoxifen. The increase in activity is attributed to the dual action of the organic drug and the Fenton chemistry (i.e. formation of singlet oxygen) of the Fe centre. 25,26 Moreover, the stability of ferrocene in aqueous and aerobic media, the facility with which a large variety of derivatives may be prepared, and its favorable electrochemical properties, has resulted in ferrocene becoming a promising molecule for incorporating in biological applications. 12 Furthermore, certain ferrocene-containing compounds exhibit cytotoxicities comparable to the benchmark anticancer drug, cisplatin.
The concept of combining organometallic complexes with compounds of known therapeutic value is a growing area of research, and coupled with the notion of multinuclearity that can result in enhanced therapeutic activity, 19,[59][60][61][62][63][64][65][66][67][68][69][70][71] the combination is an attractive drug-design strategy. Moreover, one way of introducing the notion of multinuclearity is to conjugate therapeutic agents onto dendritic scaffolds, 59,72-77 that could potentially exploit the enhanced permeability and retention (EPR) effect. [78][79][80] Recently, we reported tetranuclear and octanuclear N,Oand N,N-ruthenium-arene metallodendrimers, with ferrocene moieties functionalized on the periphery of the dendritic scaffold. The majority of the compounds efficiently inhibit the growth of both A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant human ovarian cancer cells (i.e. IC 50 < 5 μM). 81 In addition, these complexes displayed no cross-resistance to cisplatin, with the metallodendrimers having a better activity compared to their mononuclear complexes.
On the basis of the above observed results we hypothesized that preparing ferrocenyl-derived metal-arene metallodendrimers, where the ferrocenyl moiety is coordinated directly to the metal center rather than on the periphery, may further enhance the antiproliferative activity of the compounds. In this study, we describe the synthesis of a series of bidentate heterometallic ferrocenyl-derived dendrimers containing ruthenium(II)-p-cymene, ruthenium(II)-HMB (where HMB = hexamethylbenzene), rhodium(III)-Cp* or iridium(III)-Cp* (where Cp* = cyclopentadienyl) moieties [1][PF 6 ] 4 -[8]PF 6 ] 8 (see Scheme 1). The cytotoxicity of the complexes was evaluated against the A2780 and the A2780cisR human ovarian cancer cell lines, as well as against the non-cancerous human embryonic kidney (HEK-293) cell line. In order to investigate whether the antiproliferative activity is size-dependent, binuclear model analogues [9][PF 6 ]- [12][PF 6 ] were also prepared and evaluated.  8 . The aliphatic protons of the dendritic core and dendritic arms occur as broadened peaks, between 1.2 ppm and 3.0 ppm and at similar chemical shifts to those of the dendri-tic ligands L1 and L2.  8 show a loss of two-fold symmetry around the metal center upon coordination of the N,O-dendritic ligand. Evidence for this is provided by the presence of two sets of signals (one of the signals is masked by the singlet observed for the un-substituted Cp ring) for the -CH 2group adjacent to the imine nitrogen, due to the diastereotopic nature of these protons induced by the chiral metal center. As a result, the methyl protons of the isopropyl group, on the arene ring, of the Ru-p-cymene metallodendrimers [ 6 ] were prepared to evaluate whether there is a size dependency correlation between the complexes prepared and their biological activity (see below). Two equivalents of the monomeric ligand L3, 84 were reacted with one equivalent of the particular metaldimer, in the presence of triethylamine, in a one-pot reaction (Scheme 2). Subsequently, 4-ferrocenylpyridine was added to the reaction mixture, which generated a cationic complex, and the products were isolated as red or red-orange hexafluorophosphate salts.   8 was observed compared to their neutral Ru-HMB-Cl analogs (G1-Ru-HMB-Cl = 27 μM (A2780); 25 μM (A2780cisR); G2-Ru-HMB-Cl = 10 μM (A2780); 10 μM (A2780cisR)) reported previously, 83 with comparable activities to their cationic Ru-HMB-PTA analogs (G1-Ru-HMB-PTA = 9 μM (A2780); 25 μM (A2780cisR); G2-Ru-HMB-PTA = 6 μM (A2780); 12 μM (A2780cisR)). 73 There is a correlation between the size-dependency of the metallodendrimers and the cytotoxicity, with metallodendrimers [3][PF 6 ] 4 and [4][PF 6 ] 8 being the only compounds to exhibit a significant degree of cancer cell selectivity. A direct comparison between these systems (ferrocene at the metal center) and the previously prepared systems (ferrocene at the periphery) 81 cannot be made as different in vitro biological studies were performed. However, coordination of the pyridylferrocene moiety to the Ru center affords more cytotoxic compounds.

NMR stability study
Compound stability is important for biological applications and, in the case of metal-based drugs, many compounds are actually prodrugs and hence the identity of the compound that reaches the cell can be important. Following uptake into the cell, related ruthenium-arene-PTA complexes are activated via aquation, generating the aqua species, a process that can be monitored by NMR spectroscopy. 85 (Fig. 1) show that the compounds are stable in solution, with no side-products or the formation of the aqua-species observed over a period of 24 hours. In addition, these data show that the complexes are stable in deuterated dimethylsulfoxide, relevant during the time period between preparation of the compound stock solutions and dosing of the cancer cells in the assay.

UV-Vis study: interactions with a DNA model
DNA is a potential drug target for ruthenium-arene compounds and is an important target in cancer therapy. 87 Furthermore, some of the most cytotoxic ruthenium drugs act as DNA intercalators upon coordination to the suitable ancillary ligand. 40 In order to correlate the antiproliferative activity of the metallodendrimers to possible interactions with DNA, a UV-vis study was performed. The interactions of [4][PF 6 ] 8 (the most active compound) and its model analogue [10][PF 6 ] were selected for the study and their interaction with Red Salmon testes DNA monitored by UV-vis spectroscopy (Fig. 2).
The UV-vis spectra for both [4] 6 ]) decrease, with no substantial red-or blue-shift observed for λ max for both systems (Fig. 2). Similar hypochromic shifts were reported with Cu(II), Co(II) and Ni(II) complexes upon addition of increasing concentration of DNA, 88 whereas norepinephrine (DNA binding agent) displays a hyperchromic shift upon DNA addition. 89 A hyperchromic shift is usually associated with the partial uncoiling of the DNA double helix, which exposes more of the DNA base-pairs, which in turn results in the increase in absorbance at λ max . However, hypochromic effects are also associated with non-covalent interactions between the complex and DNA, such as intercalative binding, 90 indicating that DNA could be a relevant target for these compounds.
Heterometallic model complexes were also prepared and characterized (for comparison with the larger metallodendrimers). Antiproliferative studies performed on the metallodendrimers and the model binuclear complexes show that the model complexes display limited activity, whereas the metallodendrimers display higher cytotoxicities, particularly the second-generation ferrocenyl-derived ruthenium(II)-hexamethylbenzene metallodendrimer, which displayed the best activity of the series. The presence of ferrocene leads to more cytotoxic compounds compared to previously reported nonferrocenyl derivatives. In addition, these metallodendrimers displayed no cross-resistance to cisplatin and some are cancer cell selective, being less cytotoxic to the non-tumorigenic cells included in the study. Spectroscopic studies illustrate that these systems are stable in solution and additional DNA binding studies suggest that a possible mode-of-action of these metallodendrimers (at least for the most active derivative) involves possible non-covalent interactions (illustrated by UV-vis experiments) with the DNA.

General details
All solvents and reagents were purchased from Sigma-Aldrich; DAB-G 2 -PPI-(NH 2 ) 8 was purchased from SyMO-Chem and used without further purification. Ruthenium trichloride trihydrate, iridium trichloride trihydrate and rhodium trichloride trihydrate were obtained from Heraeus Limited. L1, 82 L2, 82 L3, 84 [(η 6 -p-iPrC 6 H 4 Me)RuCl 2 ] 2 , 91 [(η 6 -C 6 Me 6 )RuCl 2 ] 2 , 92 [(η 5 -C 5 Me 5 ) IrCl 2 ] 2 , 93 [(η 5 -C 5 Me 5 )RhCl 2 ] 2 , 93 and 4-ferrocenylpyridine (4-FcPyr) 16 were prepared according to literature procedures. Infrared (IR) spectra determined in the solid state on a Perkin-Elmer Spectrum 100 FT-IR spectrometer equipped with a SMART iTR ATR unit. The intensity of stretching vibrations are marked as strong (s), medium (m) and weak (w). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury XR300 spectrometer ( 1 H: 300.08 MHz; 13  Chemical shifts δ in ppm indicate a downfield shift relative to tetramethylsilane (TMS) and were referenced relative to the signal of the solvent. 94 Individual peaks are marked as singlet (s), doublet (d), doublet-of-doublet (dd), triplet (t), or multiplet (m). High-resolution electrospray ionization-mass spectrometry (HR-ESI-MS) was carried out on a Waters Synapt mass spectrometer. Data were recorded in positive ion mode. Elemental analysis (C, H, N) was carried out using a Thermo Flash 1112 Series CHNS-O Analyzer. For certain metallodendrimers, the analyses are outside acceptable limits, which is ascribed to the encapsulation of solvent molecules and/or other inorganic salts by the dendritic compounds. UV-vis absorption studies were carried out on a Cary UV-vis spectrophotometer using a 1 cm path length quartz cuvette to carry out the measurements.  NCH 2 CH 2 core , NCH 2 CH 2 core , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 1st branch , NCH 2 CH 2 CH 2 N 2nd branch , NCH 2 CH 2 CH 2 N 2nd branch ), 2.00 (br s, 144H, Ar HMB ), 3.57-3.75 (br m, 16H, NCH 2 CH 2 CH 2 N 2nd branch )     3 Cp* ); 24.6, 50.1, 51.8, seeded in 96-well plates as monolayers with 100 μL of cell solution (approximately 10 000 cells) per well and pre-incubated for 24 h in medium supplemented with 10% FBS. Compounds were prepared as DMSO solution, then dissolved in the culture medium and immediately serially diluted to the appropriate concentration, to give a final DMSO concentration of 0.5%. A 100 μL portion of drug solution was added to each well and the plates were incubated for another 72 h. Subsequently, MTT (5 mg mL −1 solution in PBS) was added to the cells and the plates were incubated for a further 4 h. The culture medium was aspirated, and the purple formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in DMSO. The optical density, directly proportional to the number of surviving cells, was quantified at 590 nm using a multiwell plate reader and the fraction of surviving cells was calculated from the absorbance of untreated control cells. The IC 50 values were determined by fitting the plot of the log of the ratio between the percentages of surviving cells, divided by the amount of dead cells, against the log of drug concentration using a linear threshold function. Evaluation is based on means from at least two independent experiments, each comprising four microcultures per concentration level.