Synthesis, Molecular Structure and Cytotoxicity of Molecular Materials Based on Water Soluble Half-Sandwich Rh(III) and Ir(III) Tetranuclear Metalla-Cycles

The neutral dinuclear complexes [(η5-C5Me5)2Rh2(μ-dhnq)Cl2] (1) and [(η5-C5Me5)2Ir2(μ-dhnq)Cl2] (2) (dhnqH2 = 5,8-dihydroxy-1,4-naphthoquinone) were obtained from the reaction of [(η5-C5Me5)M(μ-Cl)Cl]2 (M = Rh, Ir) with dhnqH2 in the presence of CH3COONa. Treatment of 1 or 2 in methanol with linear ditopic ligands L (L = pyrazine, 4,4′-bipyridine or 1,2-bis(4-pyridyl)ethylene), in the presence of AgCF3SO3, affords the corresponding tetranuclear metalla-rectangles [(η5-C5Me5)4M4(μ-dhnq)2(μ-L)2]4+ (L = pyrazine, M = Rh, 3; M = Ir, 4; L = 4,4′-bipyridine, M = Rh, 5; M = Ir, 6; L = 1,2-bis(4-pyridyl)ethylene, M = Rh, 7; M = Ir, 8). All complexes were isolated as their triflate salts and were fully characterized by infrared, 1H and 13C NMR spectroscopy, and some representative complexes by single-crystal X-ray structure analysis. The X-ray structures of 3, 5 and 6 confirm the formation of the tetranuclear metalla-cycles, and suggest that complexes 5 and 6 possess a cavity of sufficient size to encapsulate small guest molecules. In addition, the antiproliferative activity of the metalla-cycles 3–8 was evaluated against the human ovarian A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) cancer cell lines and on non-tumorigenic human embryonic kidney HEK293 cells. All cationic tetranuclear metalla-rectangles were found to be highly cytotoxic, with IC50 values in the low micromolar range.


Introduction
The biological application of coordination-driven arene ruthenium metalla-materials is a flourishing area of research [1][2][3][4][5][6]. The tetranuclear assemblies have been found to possess good anticancer activity [7][8][9][10][11][12], to strongly interact with DNA [13,14], and to efficiently detect biologically relevant analytes [15][16][17]. Their DNA binding can occur through non-covalent interactions, novel modes of action also observed for di-and trinuclear organometallics [18][19][20], although fragmentation inside cells followed by coordination of the metal ion to DNA and/or other biomolecules cannot be excluded. Following the promising studies of arene ruthenium metalla-materials, a series of cationic arene osmium metalla-rectangles were recently reported [21]. These arene osmium derivatives display comparable cytotoxicity to the ruthenium-based analogues, suggesting that the biological applications of arene ruthenium metalla-rectangles can be extended to other transition metals.

Molecular structures of 3, 5 and 6
Dark green crystals of the metalla-rectangles [3](CF 3 SO 3 ) 4 , [5](CF 3 SO 3 ) 4 and [6](CF 3 SO 3 ) 4 , suitable for single-crystal X-ray structure analysis were obtained by addition of toluene to a dichloromethane solution of the respective complexes. All metalla-rectangles crystallize with solvent molecules and four triflate anions. The molecular structures of 3, 5 and 6 are presented in  respectively. Selected bond lengths and angles are listed in Table 1 and the crystallographic details are given in Table 2.

Antiproliferative Activity
The cytotoxicity of 3-8 and a Ru-analogue ( Figure 1) was evaluated against human A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) ovarian cancer cells, as well as against the non-tumorigenic HEK293 human embryonic kidney cells. The IC 50 values after 72 h are listed in Table  3. Regarding the neutral dinuclear complexes 1 and 2, their solubility in water was too low to allow a biological evaluation, and in DMSO the chlorido ligands were exchanged with solvent molecules. All the metalla-rectangles 3-8 are highly cytotoxic towards the three cell lines tested (0.06-0.31 μM), and are significantly more cytotoxic than the ruthenium analogue and cisplatin in all cases. One observable trend is that all the compounds were less active against the A2780cisR cell line compared to the A2780 cell line, indicative of a certain level of susceptibility of these compounds to the acquired cisplatin resistance mechanisms operating in the former, albeit with notably lower resistance factors (1.3-3.6) relative to cisplatin (15.6). The IC 50 values of the compounds in the HEK293 cell line are comparable to the corresponding IC 50 values for the A2780 cell line, with the exception of 3 which is almost three-fold more active in the A2780 cell line (0.06 μM) compared to the HEK293 cell line (0.17 μM), indicative of a moderate level of selectivity. For compounds 3-8 the cytotoxicity appears to be independent of the choice of metal (Rh or Ir) or the ditopic nitrogen ligand present, exemplified by the similarity in IC 50 values determined for 3-8 within the three cell lines. In contrast, the activity of the Ru-analogue is significantly lower than its Rh and Ir analogues, 5 and 6, in all three cell lines, indicating, in this case, the choice of metal (Rh and Ir versus Ru) is significant with respect to the level of cytotoxicity observed. Given these results it is tempting to speculate that compounds 3-8 exert their cytotoxic activity through a similar mechanism of action. Evidently on switching metal to yield the Ru-analogue the activity of the complex diminishes and is likely related to a fundamental difference in reactivity of this metalla-rectangle in vitro.

Cell Culture and Inhibition of Cell Growth
Human A2780 and A2780cisR ovarian carcinoma cells and HEK293 cells were obtained from the European Collection of Cell Cultures (ECACC) (Salisbury, UK). Cells were cultured in either RPMI-1640 with GlutaMAX (A2780, A2780cisR) or DMEM (Dulbecco's Modified Eagle Medium) high glucose with GlutaMAX (HEK 293) medium containing 10% fetal bovine serum (FBS) and penicillin at 37 °C and 5% CO 2 . Cytotoxicity was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay (see below). Cells were seeded in 96 well plates by the addition of cells as a suspension in their respective media containing 10% FBS (100 μL per well, approximately 4300 cells) and pre-incubated for 24 h.
Fresh stock solutions of the compounds were prepared in DMSO just before injections, then the stock solution were diluted by addition to the culture medium [RPMI (Roswell Park Memorial Institute medium) or DMEM for A2780 and A2780cisR or HEK 293, respectively]. The stock solutions were serially diluted to give compound solutions of the desired concentrations. Complex solutions (100 μL) were then added to plate wells (yielding final compound solutions in the range 0 to 5 μM) and the plates incubated for a further 72 h.
Subsequently, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (20 μL, 5 mg/mL in H 2 O) was added to each well and the plates incubated for a further 2 h. The culture medium was then aspirated and the violet formazan precipitate produced by mitochondrial dehydrogenases of living cells was dissolved by the addition of DMSO (100 μL) to each well. The absorbance of the resultant solutions at 590 nm, which is directly proportional to the number of surviving cells, was recorded using a multiwell plate reader. The percentage of surviving cells was determined by measurement of the absorbance of wells corresponding to untreated control cells. The reported IC 50 values are based on the mean values from two independent experiments; each concentration level per experiment was evaluated in triplicate, and those values are reported in Table 3.

Single-Crystal X-ray Structure Analysis
Crystals of compounds [3](CF 3 SO 3 ) 4 , [5](CF 3 SO 3 ) 4 and [6](CF 3 SO 3 ) 4 were mounted on a Stoe Image Plate Diffraction system equipped with a  circle goniometer, using Mo-Kα graphite monochromatic radiation (λ = 0.71073 Å) with  range 0-200°. The structures were solved by direct methods using the program SHELXS-97, while the refinement and all further calculations were carried out using SHELXL-97 [40]. The H-atoms were included in calculated positions and treated as riding atoms using the SHELXL default parameters. The non-H atoms were refined anisotropically, using weighted full-matrix least-square on F 2 . In 6, the solvent molecules were highly disordered and a data set corresponding to omission of the missing solvent was generated using the SQUEEZE algorithm [41] and the structure was refined to convergence. These missing solvent molecules are probably dichloromethane molecules, which fit perfectly with the size of the voids, the electron counts and the crystal packing of compound [5](CF 3 SO 3 ) 4 , which possesses two molecules of dichloromethane per asymmetric unit. Crystallographic details for [3]

Conclusions
The antiproliferative activities of a series of half-sandwich Rh(III) and Ir(III) tetranuclear metalla-cycles have been evaluated in vitro against the human ovarian A2780 (cisplatin sensitive) and A2780cisR (cisplatin resistant) cancer cell lines and on non-tumorigenic human embryonic kidney HEK293 cells. These metalla-rectangles have been found to be highly cytotoxic with IC 50 in the low micromolar range. The cationic charge, the size, the nature of the metal ion, and the presence of a hydrophobic cavity in these metalla-materials, are all plausible factors which can contribute to their high biological activity. These results further confirm the great potential of metalla-materials in the field of biology [1][2][3][4][5][6].