Anti-tumour active gold(I), palladium(II) and ruthenium(II) complexes with thio- and selenoureato ligands: a comparative study†

We report the results of a comparative study of the biological activity of a series of gold(I), palladium(II) and ruthenium(II) complexes containing deprotonated thioand selenoureato ligands. A library of compounds was prepared and characterised by spectroscopic methods and the solid-state structures of several derivatives were determined by single crystal X-ray diffraction. The in vitro activity of these compounds was evaluated in mammary and ovarian carcinoma, acute lymphatic and acute and chronic myeloid leukemia cell lines. At lower concentrations Ruand Pd-containing compounds displayed stronger anticancer effects than the gold compounds. In all cases, the selenium derivatives proved to be more active than the corresponding sulfur compounds.


Synthesis and crystal structures
The new selenourea N,N-diethyl-N′-4-nitrobenzoylselenourea (denoted hereafter as HL Se ) was prepared from KSeCN, 4-nitrobenzoyl chloride and Et 2 NH in CH 2 Cl 2 in the presence of PEG-400 as phase-transfer catalyst following a literature procedure. 45Orange crystals of the pure material were obtained in about 35% yield after recrystallisation from EtOH.Alternatively, the compound can also be obtained in similar yield using the original method of Douglass. 1 The known thiourea N,N-diethyl-N′-4-nitrobenzoylthiourea (denoted hereafter as HL S ) was prepared analogously from KSCN, 4-nitrobenzoyl chloride and Et 2 NH in acetone.The identity and purity of HL Se was confirmed by 1 H, 13 C and 77 Se NMR spectroscopy as well as by single crystal X-ray diffraction.The molecular structure of the compound is shown in Fig. 1.Overall, the bond distances and angles of HL Se (Table S1 †) are similar to those reported for PhC(O)NHC(Se)NEt 2 46 with some minor differ- The compounds were characterised by 1 H, 13 C, 77 Se and 31 P NMR spectroscopy, electrospray mass spectrometry and several derivatives also by X-ray crystallography.In the gold(I) complexes the monoanionic thio-or selenoureato ligands coordinate only via the sulfur or selenium atom to the gold(I) centre.In case of the ruthenium and palladium species, bidentate coordination of both chalcogen atoms of the thio-and selenourea ligands is observed.In all the compounds reported here, the thio-or selenourea ligands are deprotonated, which is evident from the disappearance of the signal for the N-H proton in the 1 H NMR spectra of the compounds.For the gold derivatives, a shift of the position of the singlet due to the PTA ligand in the 31 P NMR spectra is observed upon coordination of the seleno-or thioureato ligands.This resonance is observed at −52.0 ppm in [AuCl(PTA)] and at −51.7 and −49.8 ppm in the thio-and selenoureato derivatives, respectively.In addition, the 13 C NMR signals of the quaternary carbon atom bound to the sulfur or selenium atom are shifted to 176.8 (S) or 167.1 (Se) ppm to higher field upon coordination.Whilst the 77 Se NMR signal for the unbound selenourea is found at 502 ppm, that of the gold selenoureato species is observed at 244 ppm and that of the ruthenium derivative is shifted upfield to −26 ppm.Unfortunately, the palladium species were not soluble enough to obtain any 77 Se NMR spectra.The metal compounds were stable in solution for at least 48 h, as observed by NMR spectroscopy.Furthermore, we did not observe any ligand exchange reactions in dmso solutions.X-ray quality crystals of two ruthenium and palladium complexes were obtained and studied by X-ray diffraction.In the organoruthenium complexes (Fig. 2), the typical piano stool conformation about the metal centre is observed.The metal is thus bound to the arene ring (in η 6 fashion), the chelating thio-or selenoureato ligand as well as to the phosphorus atom of the PPh 3 ligand.The presence of four different atoms around the metal centre results in metalcentred chirality.The O-Ru-E angles (E = S, Se) are comparable to those reported for homoleptic, octahedral ruthenium(III) tris(chelate) complexes. 34The distances between the ruthenium atom and the centre of the arene rings (S 1.745 Å, Se 1.746 Å) are typical for such η 6 -arene ruthenium compounds. 48e palladium complexes (Fig. 3) show square-planar coordination with one bidentate chalcogenoureato and a 1,10-phenanthroline ligand bound to the metal.In both the ruthenium and the palladium compounds, the chalcogenoureato ligands are nearly planar due to electron delocalisation in the ligand backbone.A comparison of the two C-N distances as well as the C-O and C-Se bond lengths of the free and coordinated ligand, shows that upon coordination to the metal atom the NH-CvSe unit is transformed into an NvC-Se − moiety (the selenolato tautomer).The C-Se bond distance increases from 1.82 Å to 1.88 Å (Pd) or 1.90 Å (Ru) which is in agreement with literature data for ruthenium-µ-phenylselenido compounds. 49Due to the slightly smaller radius of the sulfur atom, the analogous bond distances in the sulfur complexes are correspondingly shorter.The C-S bond distance increases from 1.66 Å (HL S ) to 1.73 Å in the complex.In contrast, the CvO and C(O)N bond distances remain virtually unchanged upon coordination when compared with those of the free seleno-or thiourea, respectively.A table listing important  Ellipsoids show 50% probability levels.Hydrogen atoms, the PF 6 − anions as well as the dmso of solvation have been omitted for clarity.
bond distances and angles of the metal complexes and the thio-and selenourea ligands is included in the ESI.†

Biological studies
The biological activity of HL S , HL Se as well as selected Au(I), Pd(II) and Ru(II) complexes including their chlorido-precursors (Chart 1) and cisplatin as further control was determined by their effect on proliferation, metabolic activity, programmed cell death and induction of oxidative stress of various tumor and leukemia cell lines.All Au(I), Pd(II) and Ru(II) compounds dose-dependently reduced the proliferation of mammary and ovarian carcinoma, acute lymphatic as well as acute and chronic myeloid leukemia cell lines (ESI Fig. S1A-S1F †), whereby, in general, the selenium derivatives induced stronger effects than the sulfur compounds.For example, a concentration of 5 µM Pd-S significantly reduced the proliferation of SUP-B15 cells from 100% (without compound; data not shown) to 44.5 ± 8.4% and a concentration of 10 µM to 18.7 ± 3.1% ( p < 0,05; Fig. 4) so that the IC50 was calculated as 4.9 ± 1.2 µM (Table 1).Interestingly, in the mammary carcinoma as well as the myeloid leukemia cell lines Pd-S was almost ineffective.In contrast, Pd-Se induced strong anti-proliferative effects and both Ru(II) compounds completely abolished the proliferation in all cell lines tested.Weaker anti-proliferative activity (again in all cell lines) was observed for the two Au(I) complexes.Cisplatin dose-dependently reduced the proliferation of the tumor cell lines, but did not show any activity on the leukemia cell lines.Whereas [RuCl 2 ( p-cymol)] 2 was completely ineffective in all cell lines, [AuCl(PTA)] and the thio-and selenoureas HL Se and HL S induced anti-proliferative effects at higher concentrations (    [PdCl 2 (1,10-Phen)] dose-dependently reduced the proliferation of all cell lines almost as effective as Pd-Se which may be explained by the ability of cis-[PdCl 2 (1,10-Phen)] to intercalate with DNA.The anti-proliferative activity of the substances was also examined on A-2780 cells resistant to cisplatin and revealed that the Au(I), Pd(II) and Ru(II) compounds have the potential to overcome cisplatin resistance (Table 2).
To evaluate if the reduced cell growth was also accompanied by cell death, the metabolic activity of the cell lines after compound incubation was analysed.Metabolic activity is determined by the reduction of tetrazolium salts into formazan derivatives.Since this process requires functional mitochondria, the assay discriminates living and dead cells whose mitochondria are deactivated within a few minutes after cell death.As shown in Fig. 5 and ESI Fig. S2A-S2F, † the metabolic activity of the cell lines was also dose-dependently diminished after 72 hours of incubation with the test compounds and cis-[PdCl 2 (1,10-Phen)].Again, the Ru(II) compounds and Pd-Se were the most active compounds with an IC50 of ∼4 µM (Table 1), whereas the chlorido precursors [AuCl(PTA)] and [RuCl 2 ( p-cymol)] 2 , HL S , HL Se as well as cisplatin were almost ineffective.
To confirm the cytotoxicity of the compounds, their capacity to induce programmed cell-death was determined by ssDNA apoptosis ELISA, which is based on the selective denaturation of DNA in apoptotic cells by formamide and further detection of denatured DNA with a monoclonal antibody to single-stranded DNA (ssDNA).Compounds which reduced proliferation and metabolic activity also induced apoptosis of the leukemia and tumor cells, whereby the sensitivity of the cell lines to programmed cell-death induction varied (ESI Fig. S3A-S3E †).For example, apoptosis of the cell line SUP-B15 was 3.7 ± 0.8 times higher after addition of 5 µM Pd-Se and 5.2 ± 0.6 times higher when 10 µM Pd-Se was added to the cultures in comparison to cells without compound incubation (Fig. 6).
A similar pro-apoptotic effect was observed for both Ru(II) compounds, whereas 10 µM Au-Se and 5 µM as well as 10 µM Pd-S caused a lower, but significant apoptosis rate.Consistent with the weak anti-proliferative and anti-metabolic activity, incubation with Au-S did not lead to programmed cell death in any of the cell lines.[AuCl(PTA)], [RuCl 2 ( p-cymol)] 2 and cisplatin were also ineffective in inducing apoptosis.Interestingly, despite no activity on proliferation and metabolism, HL S and HL Se significantly induced apoptosis in all leukemic cell lines, but not in the carcinoma cell lines (Fig. 6 and ESI Fig. S3A-S3E †).A similar effect was observed for cis-[PdCl 2 (1,10-Phen)], which decreased proliferation and metabolic activity of the cell lines, however, without inducing their cell death.These rather contradictory results are currently under investigation.
The pro-apoptotic effect of Ru-Se (Fig. 7A) and the lacking effect of [RuCl 2 ( p-cymol)] 2 (Fig. 7B) can be readily detected by morphological changes of the cisplatin-resistant A-2780 cells.
One mechanism involved in the induction of apoptosis is oxidative stress triggered by the formation of reactive oxygen   species (ROS) due to deregulated mitochondrial activity.It has been shown for a variety of gold, [50][51][52] ruthenium 31,32,[53][54][55] and palladium 56 complexes that their anti-tumour activity is mediated by an enhanced ROS production.Therefore, ROS levels were measured after a two-hour incubation of SUP-B15 cells with 10 µM of the respective compound.As shown in Fig. 8, both Ru(II) compounds and Pd-Se significantly induced oxidative stress in this cell line, in agreement with an increase of programmed cell death.Interestingly, the pro-apoptotic effect of HL S and HL Se cannot be explained by an increase in ROS formation.Neither the chlorido compounds nor cisplatin did induce oxidative stress of SUP-B15 cells.

Conclusions
In

Chemistry
Unless otherwise stated, all manipulations were carried out without taking precautions to exclude air and moisture.All chemicals and solvents (HPLC quality) were sourced commercially and used as received.[AuCl(PTA)] was prepared from the reaction of [AuCl(tht)] 57 with the appropriate amount of the phosphine.The other metal precursors cis-[PdCl 2 (1,10phen)], 58 [Ru( p-cymol)Cl 2 ] 2 59 as well as N,N-diethyl-N′-4-nitrobenzoylthiourea (HL S ) 60 were prepared by standard procedures described in the literature.The purity of all compounds was confirmed to be >95% by elemental analysis or high-resolution mass spectrometry.
IR spectroscopy.IR spectra were run as KBr pellets on a Bruker Tensor 27 instrument.Elemental analyses.Elemental analyses were performed by staff of the in-house microanalytical laboratory using an Elementar Vario EL analyser.
Mass spectrometry.High-resolution electrospray mass spectra were recorded on Bruker MicroTOF spectrometer in positive ion mode using solutions of the samples in MeCN.

Biological activity
Compounds.All compounds were dissolved in dmso, aliquoted and stored at −20 °C.Prior to use solutions of the respective compound were diluted at least 1 : 1000 with cell culture medium.Dmso in the appropriate concentration did not induce any effect on the cell lines.
Cell lines.The leukemic cell lines were purchased from DSMZ -German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.The mammary carcinoma cell lines were kindly provided by the Institute of Pharmacy, Free University of Berlin.The ovarian carcinoma cell lines were kindly provided by the Department of Gynecology, Medical University Innsbruck.The acute myeloid leukemia cell line HL-60 as well as the chronic myeloid leukemia cell line K-562, the mammary carcinoma cell lines MCF-7 and MDA-MB231 as well as the ovarian carcinoma cell lines A2780 (cisplatin-sensitive and cisplatin-resistant) were grown in RPMI 1640 without phenol red (PAA Laboratories, Pasching, Austria), supplemented with glutamine (2 mM), penicillin (100 U mL −1 ), streptomycin (100 µg mL −1 ) and fetal bovine serum (FBS; 10%; all from Invitrogen Corporation, Gibco, Paisley, Scotland) at 37 °C in a 5% CO 2 /95% air atmosphere and fed twice weekly.Cisplatin (1 µM) was added to the cisplatin-resistant A-2780 cell line every third passage.The acute lymphatic leukemia cell line SUP-B15 was grown in Mc Coy's 5A medium (Invitrogen) and supplemented with 20% FBS.
Analysis of metabolic activity and proliferation.Logarithmically growing cells were resuspended in culture medium at 1 × 10 6 cells per mL and plated in triplicate in U-bottomed or flat-bottomed microtiter plates (50 µL; Falcon, Becton Dickinson, Franklin Lakes, NJ, USA).Various concen-trations of compounds were added one hour thereafter.After a three-day culture at 37 °C in a 5% CO 2 /95% air atmosphere cultures were analyzed for metabolic activity using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (EZ4U kit; Biomedica, Vienna, Austria) according to the manufacturer's instructions and for cellular proliferation using [methyl-3 H]-thymidine uptake.Metabolic activity and proliferation in the absence of the compound was set to 100%.
Analysis of apoptosis.The plates for apoptosis were prepared analogously as described above and were evaluated with an enzyme-linked immunoadsorbent assay (ELISA) using the ssDNA apoptosis kit (Chemicon International, Hofheim, Germany) according to the manufacturer's instructions.Apoptosis rate was evaluated by dividing the optical density of the wells measured in the plates for ssDNA apoptosis by the optical density of the wells for metabolic activity.Apoptosis in the absence of the compounds was set to 1.
Detection of morphological changes.3 × 10 6 cells were adjusted to 1 × 10 6 cells per ml, placed in a 12-well plate and incubated at 37 °C in a humidified 5% CO 2 /95% air atmosphere for 24 hours to let the cells adhere.After 24 hours 10 µM compound was added and the cells were cultivated for another 24 hours.Every 30 minutes a picture was made by JuLI™ Live cell imaging system (NanoEnTek, Seoul, Korea).
Statistical analysis.The Wilcoxon Rank Sum Test was used to analyze the differences between proliferation, metabolic activity, apoptosis and ROS production in the absence and the presence of a variable concentration of the test compounds (NCSS software, Kaysville, UT, USA).
calculated as the concentration at which the compounds induce 50% inhibition of proliferation or metabolic activity of SUP-B15 cells in comparison to untreated cells.The IC50 values are presented as means ± standard error of four experiments.
summary, gold(I), palladium(II) and ruthenium(II) compounds containing anionic nitro-substituted acylseleno-or acylthioureas display strong anti-tumor and anti-leukemia effects.The ligands HL S , HL Se as well as the chlorido complexes [AuCl(PTA)] and [RuCl 2 ( p-cymol)] 2 induced only weak effects or only at the highest concentrations tested, whereas cis-[PdCl 2 (1,10-Phen)] reduced the proliferation and the metabolic activity almost as effective as the test compounds.The mechanisms of action include inhibition of proliferation and metabolic activity as well as induction of apoptosis and oxidative stress by ROS formation.The strongest effects were observed for the Ru(II) compounds followed by the Pd(II) complexes whereby the selenium-containing compounds displayed stronger biological activity than the sulfur derivatives.The Au(I) complexes were only effective at higher concentrations.

Table 1
IC50 values of the metal complexes in SUP-B15 cells