Fine-tuning the cytotoxicity of ruthenium(ii) arene compounds to enhance selectivity against breast cancers

Ruthenium-based complexes have been suggested as promising anticancer drugs exhibiting reduced general toxicity compared to platinum-based drugs. In particular, Ru(η6-arene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), or RAPTA, complexes have demonstrated efficacy against breast cancer by suppressing metastasis, tumorigenicity, and inhibiting the replication of the human tumor suppressor gene BRCA1. However, RAPTA compounds have limited cytotoxicity, and therefore comparatively high doses are required. This study explores the activity of a series of RAPTA-like ruthenium(ii) arene compounds against MCF-7 and MDA-MB-231 breast cancer cell lines and [Ru(η6-toluene)(PPh3)2Cl]+ was identified as a promising candidate. Notably, [Ru(η6-toluene)(PPh3)2Cl]Cl was found to be remarkably stable and highly cytotoxic, and selective to breast cancer cells. The minor groove of DNA was identified as a relevant target.


Synthesis and characterization of the screened compounds
All organometallic manipulations were carried out under a nitrogen atmosphere using standard Schlenk techniques. CH2Cl2 was dried catalytically under nitrogen using a solvent purification system, manufactured by Innovative Technology Inc. All other solvents were dried over molecular sieves (3 Å) and saturated with nitrogen prior to use.
[Ru(η 6 -toluene)Cl2]2 1 , [Ru(η 6 -toluene)(PPh3)Cl2] 2 and complexes 1 3 , 2 3 , 3 4 , 4 5 , 5 6 , 6 6 , 7 7 , 8 8 , and 9 2 were prepared according to reported procedures and the characterization data were in agreement with those reported. Compounds 11BF4 9 and 11PF6 2 have been previously reported, but in this work, they have been prepared following a different synthetic route. All chemicals were either of reagent or analytical grade and used as purchased from commercial sources without additional purification. RuCl3·3H2O was obtained from Precious Metals Online. NMR spectra were acquired on a Bruker Avance 400 MHz spectrometer at room temperature unless otherwise stated. Chemical shifts are reported in ppm relative to SiMe4 (δ = 0) and coupling constants (J) are reported in Hz.
The following abbreviations were used to designate multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets. High-resolution mass spectra (HRMS) were acquired by the MS service at EPFL, using either a Thermo Orbitrap Elite instrument with an LTQ-Orbitrap analyser or a Waters XEVO G2-S QTOF instrument with a QTOF analyser. Figure S1. Structures of the studied ruthenium arene compounds.
Light orange crystals suitable for X-ray diffraction were obtained by slow diffusion at 4°C of pentane into a solution of 10 in CH2Cl2.     The n-octanol-water partition ratio is the most common way of identifying the lipophilicity of a compound. This assay was performed following the Shake-flask method 10 . To a 0.1 mM of 11CA in a water-saturated n-octanol solution was added n-octanolsaturated MilliQ water in different organic: aqueous ratios (1:1; 1:2 and 2:1). The mixture was shaken for 2 minutes, and after that, the solution was centrifuged to separate the organic and aqueous phase. Aliquots of each phase were taken and analyzed separately by ICP-MS. Samples were submitted to acidic digestion with 2 mL of concentrated HNO3 and [ ] !#$ ! are the concentrations of ruthenium in the organic phase before and after mixing with the aqueous phase, respectively. All measurements were performed in triplicates.
The molar conductivity (ΛM, reported as S·cm 2 ·mol -1 ) of solutions of 11CA in acetonitrile (1 mM) were measured in a Radiometer Copenhagen CDM92 equipped with a conductivity cell CDC641T glass platinum electrode at room temperature (25 °C). The conductivity of the pure solvent was used as standard controls.              and either NAD + (0.88 mM, 2 eq.) and sodium formate (11.02 mM, 25 eq.), for transfer hydrogenation; or reduced glutathione (1.32 mM, 3 eq.), for oxidation, were added to a 10-mm NMR tube. The pD was adjusted to 7.2 ± 0.1. 1 H NMR spectra and 31 P NMR spectra were recorded at 310 K every 300 s for 8 h.

Molecular docking
The docking files were prepared using AutoDock Tools. After removing the solvent molecules, cocrystallized ligands, and cofactors, all the hydrogens atoms were added to DNA, Gasteiger charges were calculated, and then non-polar hydrogen atoms were merged. The free torsion tree was assigned, and the ruthenium atom parameters were added. A grid box was created with 96 × 96 × 96 points and a resolution of 0.375 Å (which is roughly a quarter of the length of a carbon-carbon single bond), in order to include the entire DNA fragment. Standard AutoDock force fields were used to calculate the energies and the best conformation was optimized by applying a Lamarckian Genetic Algorithm (LGA). With an initial population size of 300 and several evaluations of 2500000, the GA was allowed to run up to 27000 generations to find one best individual.
Each experiment was set to 100 GA runs. The gene mutation and crossover rate were fixed at 2% and 80%, respectively. Clustering was performed to analyze the convergence of the simulations. The resulting poses were visualized with Visual Molecular Dynamics (VMD) 19 Figure S42. 1 H NMR spectra of 10 in DMSO-d6. Figure S43. 31 P NMR spectra of 10 in DMSO-d6. Figure S44. 19 F NMR spectra of 10 in DMSO-d6. Figure S45. 13 C NMR spectra of 10 in DMSO-d6. Figure S46. 31 P NMR spectra of 10 in chloroform-d. Figure S47. 1 H NMR spectra of 11Cl in chloroform-d. Figure S48. 31 P NMR spectra of 11Cl in chloroform-d. Figure S49. 13 C NMR spectra of 11Cl in chloroform-d. Figure S50. 1 H NMR spectra of 11Cl in methanol-d4. Figure S51. 31 P NMR spectra of 11Cl in methanol-d4. Figure S52. 37 Cl NMR spectra of 11Cl in methanol-d4. Figure S53. 1 H NMR spectra of 11PF6 in chloroform-d. Figure S54. 31 P NMR spectra of 11PF6 in chloroform-d. Figure S55. 19 F NMR spectra of 11PF6 in chloroform-d. Figure S56. 13 C NMR spectra of 11PF6 in chloroform-d. Figure S57. 1 H NMR spectra of 11SbF6 in chloroform-d. Figure S58. 31 P NMR spectra of 11SbF6 in chloroform-d. Figure S59. 19 F NMR spectra of 11SbF6 in chloroform-d. Figure S60. 13 C NMR spectra of 11SbF6 in chloroform-d. Figure S61. 1 H NMR spectra of 11OTf in chloroform-d. Figure S62. 31 P NMR spectra of 11OTf in chloroform-d. Figure S63. 19 F NMR spectra of 11OTf in chloroform-d. Figure S64. 13 C NMR spectra of 11OTf in chloroform-d. Figure S65. 1 H NMR spectra of 11BF4 in chloroform-d. Figure S66. 31 P NMR spectra of 11BF4 in chloroform-d. Figure S67. 19 F NMR spectra of 11BF4 in chloroform-d. Figure S68. 11 B NMR spectra of 11BF4 in chloroform-d. Figure S69. 13 C NMR spectra of 11BF4 in chloroform-d. Figure S70. 1 H NMR spectra of 11BPh4 in chloroform-d. Figure S71. 31 P NMR spectra of 11BPh4 in chloroform-d. Figure S72. 11 B NMR spectra of 11BPh4 in chloroform-d. Figure S73. 13 C NMR spectra of 11BPh4 in chloroform-d. Figure S74. 1 H NMR spectra of 11NO3 in chloroform-d. Figure S75. 31 P NMR spectra of 11NO3 in chloroform-d. Figure S76. 13 C NMR spectra of 11NO3 in chloroform-d.