Identification of an iridium(III) complex with anti-bacterial and anti-cancer activity.

Group 9 transition metal complexes have been widely explored as therapeutic agents due to their unique geometry, their propensity to undergo ligand exchanges with biomolecules and their diverse steric and electronic properties. These metal complexes can offer distinct modes of action in living organisms compared to carbon-based molecules. In this study, we investigated the antimicrobial and anti-proliferative abilities of a series of cyclometallated iridium(III) complexes. The iridium(III) complex 1 inhibited the growth of S. aureus with MIC and MBC values of 3.60 and 7.19 μM, respectively, indicating its potent bactericidal activity. Moreover, complex 1 also exhibited cytotoxicity against a number of cancer cell lines, with particular potency against ovarian, cervical and melanoma cells. This cyclometallated iridium(III) complex is the first example of a substitutionally-inert, Group 9 organometallic compound utilized as a direct and selective inhibitor of S. aureus . ## Results and Discussion Results and Discussion Synthesis The iridium(III) complexes 1–4 and rhodium(III) complex 5 are kinetically-inert, organometallic complexes with a general structure [M(C^N) 2 (N^N)] + (M = Ir or Rh). Complexes 1 and 2 contain a 5-amino-1, 10-phenathroline N^N ligand, while complexes 3–5 contain the bathophenanthroline N^N ligand. In regards to the C^N ligand, complexes 3 and 5 possess 2-phenylpyridine ligands, while complex 1 bears the related 2-( p -tolyl)pyridine ligand. Complexes 2 and 4 carry the bulkier 2-phenylquinoline and 1-isoquinoline C^N ligands, respectively. The syntheses and characterization of the iridium(III) or rhodium(III) complexes 1 – 5 are described in the Methods section. The spectroscopic data of the complexes are presented in Table 1 . Complexes 2 and 5 are novel whereas complexes 1 51 , 3 52 and 4 53 have been previously described. All complexes were synthesized and tested as racemic mixtures of enantiomers. Antibacterial activity The anti-bacterial activity of the complexes 1 – 5 was investigated using a disk diffusion assay. In this assay, four different bacterial strains ( Staphylococcus aureus , Escherichia coli , Enterococcus faecalis and Klebsiella pneumoniae ) were plated on an agar dish, and disks soaked with solutions of the metal complexes (11.5 μM) or ampicillin were placed onto the dish. The antimicrobial activity of the metal complexes would be manifested as the absence of bacterial growth around the disk, due to diffusion of the compounds into the surrounding agar. After 24 h, the growth inhibition zones of the test strains were determined. Encouragingly, the results showed that racemic complex 1 ( rac - 1 ) exhibited selective anti-bacterial activity towards S. aureus , with an average inhibition zone of 15 mm ( Fig. 2 ). On the other hand, the other metal complexes showed limited antibacterial activity. Determination of MIC and MBC values We next determined the minimum inhibitory concentration (MIC) value of complex 1 against S. aureus by the broth dilution method. Briefly, S. aureus inoculum in Luria Bertain broth was incubated with serial dilutions of complex 1 at 37 °C for 24  h. The MIC was determined as the lowest concentration of complex 1 inhibiting visible bacterial growth. The results showed that the MIC of S. aureus grown with complex 1 was 3.60 μM ( Fig. 3 ). To determine the MBC (minimum bactericidal concentration), the agar dilution method was used. Briefly, S. aureus inoculum treated with different concentrations of complex 1 were taken aseptically from tubes that had not presented visible turbidity and plated onto agar. The MBC was considered as the lowest concentration of complex 1 that allowed less than 0.1% of the original inoculum treated with complex 1 to grow on the surface of the agar. The results showed that the MBC of S. aureus grown with complex 1 was 7.19 μM. To determine the nature of antibacterial effect of a compound, the MBC/MIC ratio is typically used 54 . When this ratio is lower than 4, the compound is considered as a bactericidal. On the other hand, if this ratio is higher than 4, the compound is considered to be bacteriostatic. As the MBC/MIC ratio of complex 1 is 2, complex 1 is considered to be bactericidal. From these data, a brief structure-activity relationship analysis can be conducted. Complex 1 contains the 2-( p -tolyl)pyridine ligand C^N, which is relatively smaller compared to the quinoline or isoquinoline-based C^N ligands of complexes 2 and 4 , respectively. This suggests that smaller C^N ligands may be beneficial for antimicrobial activity. Additionally, complex 1 also contains an amino substituent on the phenanthroline N^N ligand. The amino group may confer the necessary hydrophilic/hydrophobic balance for the complex to penetrate the thick peptidoglycan cell wall of Gram-positive bacteria such as S. aureus . While complex 2 contains the same 5-amino-1,10-phenanthroline N^N substituent as complex 1 , its larger 2-phenylquinoline C^N ligand may have diminished its activity. Interestingly, a slightly increased zone area was observed when the iridium(III) center of 3 was replaced with rhodium(III) (as in congener 5 ), suggesting the importance of the metal center in contributing to antibacterial activity. However, complexes 3 – 5 , containing the relatively large bathophenanthroline N^N ligand, generally showed modest zone areas and were less active compared to complex 1 . In particular, complexes 2 – 5 showed only very limited activity against S. aureus . Additionally, complexes 2 – 5 at a concentration of 50–60 μM were not able to inhibit bacterial growth in LB broth. These complexes might exhibit antibacterial activity at higher concentrations, but this would not be practical for clinical use. Taken together, these findings suggest that the presence of the relatively bulky N^N ligand detracts from antibacterial activity. We propose that the lipophilicity and/or structure of complex 1 might allow it to effectively pass through the hydrophobic interior of the bacterial cell membrane. Partition Coefficients (log P ) The octanol-water partition coefficients (log P ) for complex 1 was determined. Complex 1 showed a log P value of 0.629, indicating that it is slightly lipophilic and also satisfies Lipinski’s lipophilicity criterion (log P  < 5) for drug likeness. Evaluation of cell proliferation Lipophilicity correlates with cytotoxic potency. Therefore, the cytotoxicity of complex 1 towards different cell lines was evaluated. By using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro cytotoxicity of complex 1 was evaluated in a panel of human cell lines, including ovarian cancer A2780 and SKOV3 cells, hepatocellular carcinoma HepG2 cells, cervical adenocarcinoma HeLa cells, and malignant melanoma A375 and A2058 cells. Cells were exposed to complex 1 in concentrations ranging from 0.01 to 10 μM and cellular proliferation was assessed after 72 h. The results showed that complex 1 inhibited cellular proliferation of A2780, HepG2, SKOV3, HeLa, A375, and A2058 cells in a dose-dependent manner with IC 50 values of 7.23 ± 1.30, 17.0 ± 1.39, 1.77 ± 1.70, 1.68 ± 1.29, 1.55 ± 0.15 and 1.24 ± 0.31 μM, respectively ( Fig. 4 ). The results indicate that the cytotoxic effect of complex 1 is cell type-dependent. Complex 1 selectively inhibits growth of ovarian, cervical and melanoma cells, but not hepatocarcinoma cells. Stability of complex 1 The most potent complex 1 was stable in H 2 O/acetonitrile (400:1) solution at 298 K for at least 24 h as revealed by UV-Vis spectroscopy. No significant change in absorption spectrum of complex 1 was observed over 24 h ( Fig. 5 ). The result indicates that complex 1 is stable at room temperature. ## Synthesis Synthesis The iridium(III) complexes 1–4 and rhodium(III) complex 5 are kinetically-inert, organometallic complexes with a general structure [M(C^N) 2 (N^N)] + (M = Ir or Rh). Complexes 1 and 2 contain a 5-amino-1, 10-phenathroline N^N ligand, while complexes 3–5 contain the bathophenanthroline N^N ligand. In regards to the C^N ligand, complexes 3 and 5 possess 2-phenylpyridine ligands, while complex 1 bears the related 2-( p -tolyl)pyridine ligand. Complexes 2 and 4 carry the bulkier 2-phenylquinoline and 1-isoquinoline C^N ligands, respectively. The syntheses and characterization of the iridium(III) or rhodium(III) complexes 1 – 5 are described in the Methods section. The spectroscopic data of the complexes are presented in Table 1 . Complexes 2 and 5 are novel whereas complexes 1 51 , 3 52 and 4 53 have been previously described. All complexes were synthesized and tested as racemic mixtures of enantiomers. ## Antibacterial activity Antibacterial activity The anti-bacterial activity of the complexes 1 – 5 was investigated using a disk diffusion assay. In this assay, four different bacterial strains ( Staphylococcus aureus , Escherichia coli , Enterococcus faecalis and Klebsiella pneumoniae ) were plated on an agar dish, and disks soaked with solutions of the metal complexes (11.5 μM) or ampicillin were placed onto the dish. The antimicrobial activity of the metal complexes would be manifested as the absence of bacterial growth around the disk, due to diffusion of the compounds into the surrounding agar. After 24 h, the growth inhibition zones of the test strains were determined. Encouragingly, the results showed that racemic complex 1 ( rac - 1 ) exhibited selective anti-bacterial activity towards S. aureus , with an average inhibition zone of 15 mm ( Fig. 2 ). On the other hand, the other metal complexes showed limited antibacterial activity. ## Determination of MIC and MBC values Determination of MIC and MBC values We next determined the minimum inhibitory concentration (MIC) value of complex 1 against S. aureus by the broth dilution method. Briefly, S. aureus inoculum in Luria Bertain broth was incubated with serial dilutions of complex 1 at 37 °C for 24  h. The MIC was determined as the lowest concentration of complex 1 inhibiting visible bacterial growth. The results showed that the MIC of S. aureus grown with complex 1 was 3.60 μM ( Fig. 3 ). To determine the MBC (minimum bactericidal concentration), the agar dilution method was used. Briefly, S. aureus inoculum treated with different concentrations of complex 1 were taken aseptically from tubes that had not presented visible turbidity and plated onto agar. The MBC was considered as the lowest concentration of complex 1 that allowed less than 0.1% of the original inoculum treated with complex 1 to grow on the surface of the agar. The results showed that the MBC of S. aureus grown with complex 1 was 7.19 μM. To determine the nature of antibacterial effect of a compound, the MBC/MIC ratio is typically used 54 . When this ratio is lower than 4, the compound is considered as a bactericidal. On the other hand, if this ratio is higher than 4, the compound is considered to be bacteriostatic. As the MBC/MIC ratio of complex 1 is 2, complex 1 is considered to be bactericidal. From these data, a brief structure-activity relationship analysis can be conducted. Complex 1 contains the 2-( p -tolyl)pyridine ligand C^N, which is relatively smaller compared to the quinoline or isoquinoline-based C^N ligands of complexes 2 and 4 , respectively. This suggests that smaller C^N ligands may be beneficial for antimicrobial activity. Additionally, complex 1 also contains an amino substituent on the phenanthroline N^N ligand. The amino group may confer the necessary hydrophilic/hydrophobic balance for the complex to penetrate the thick peptidoglycan cell wall of Gram-positive bacteria such as S. aureus . While complex 2 contains the same 5-amino-1,10-phenanthroline N^N substituent as complex 1 , its larger 2-phenylquinoline C^N ligand may have diminished its activity. Interestingly, a slightly increased zone area was observed when the iridium(III) center of 3 was replaced with rhodium(III) (as in congener 5 ), suggesting the importance of the metal center in contributing to antibacterial activity. However, complexes 3 – 5 , containing the relatively large bathophenanthroline N^N ligand, generally showed modest zone areas and were less active compared to complex 1 . In particular, complexes 2 – 5 showed only very limited activity against S. aureus . Additionally, complexes 2 – 5 at a concentration of 50–60 μM were not able to inhibit bacterial growth in LB broth. These complexes might exhibit antibacterial activity at higher concentrations, but this would not be practical for clinical use. Taken together, these findings suggest that the presence of the relatively bulky N^N ligand detracts from antibacterial activity. We propose that the lipophilicity and/or structure of complex 1 might allow it to effectively pass through the hydrophobic interior of the bacterial cell membrane. ## Partition Coefficients (log Partition Coefficients (log P ) The octanol-water partition coefficients (log P ) for complex 1 was determined. Complex 1 showed a log P value of 0.629, indicating that it is slightly lipophilic and also satisfies Lipinski’s lipophilicity criterion (log P  < 5) for drug likeness. ## Evaluation of cell proliferation Evaluation of cell proliferation Lipophilicity correlates with cytotoxic potency. Therefore, the cytotoxicity of complex 1 towards different cell lines was evaluated. By using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro cytotoxicity of complex 1 was evaluated in a panel of human cell lines, including ovarian cancer A2780 and SKOV3 cells, hepatocellular carcinoma HepG2 cells, cervical adenocarcinoma HeLa cells, and malignant melanoma A375 and A2058 cells. Cells were exposed to complex 1 in concentrations ranging from 0.01 to 10 μM and cellular proliferation was assessed after 72 h. The results showed that complex 1 inhibited cellular proliferation of A2780, HepG2, SKOV3, HeLa, A375, and A2058 cells in a dose-dependent manner with IC 50 values of 7.23 ± 1.30, 17.0 ± 1.39, 1.77 ± 1.70, 1.68 ± 1.29, 1.55 ± 0.15 and 1.24 ± 0.31 μM, respectively ( Fig. 4 ). The results indicate that the cytotoxic effect of complex 1 is cell type-dependent. Complex 1 selectively inhibits growth of ovarian, cervical and melanoma cells, but not hepatocarcinoma cells. ## Stability of complex 1 Stability of complex 1 The most potent complex 1 was stable in H 2 O/acetonitrile (400:1) solution at 298 K for at least 24 h as revealed by UV-Vis spectroscopy. No significant change in absorption spectrum of complex 1 was observed over 24 h ( Fig. 5 ). The result indicates that complex 1 is stable at room temperature. ## Methods Methods Materials and cell lines All chemicals, unless specified, were obtained from Sigma-Aldrich. Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , Klebsiella pneumonia , HepG2, SKOV3, HeLa, A375 and A2058 were purchased from American Type Culture Collection (ATCC). A2780, HepG2, SKOV3, HeLa, A375 and A2058 cells were routinely grown in suspension in 90% RPMI-1640 containing L-glutamine (2 nM) or DMEM, antibiotics (100 IU penicillin/mL, 100 μg streptomycin/mL) and supplemented with 10% (v/v) fetal bovine serum (FBS). The cells were maintained at a cell density of 0.2–1 × 10 6 cells/mL in a 5% CO 2 humidified atmosphere at 37 °C. General experimental Mass spectrometry was performed at the Mass Spectroscopy Unit at the Department of Chemistry, Hong Kong Baptist University, Hong Kong (China). Melting points were determined using a Gallenkamp melting apparatus and are uncorrected. Deuterated solvents for NMR purposes were obtained from Armar and used as received. 1 H and 13 C NMR were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz ( 1 H) and 100 MHz ( 13 C). 1 H and 13 C chemical shifts were referenced internally to solvent shift (acetone- d 6 : 1 H δ 2.05, 13 C δ 29.8, 206.1). Chemical shifts (δ) are quoted in ppm, the downfield direction being defined as positive. Uncertainties in chemical shifts are typically ±0.01 ppm for 1 H and ±0.05 for 13 C. Coupling constants are typically ±0.1 Hz for 1 H- 1 H and ±0.5 Hz for 1 H- 13 C couplings. The following abbreviations are used for convenience in reporting the multiplicity of NMR resonances: s, singlet; d, doublet; t, triplet; m, multiplet. All NMR data was acquired and processed using standard Bruker software (Topspin). Photophysical measurement Emission spectra and lifetime measurements for complex 1 were performed according to the previously described method 53 . Synthesis These complexes were synthesized using a modified literature method 55 . MCl 3· 3H 2 O was heated to 150 °C with 2.2 equivalents of C^N ligand in 3:1 methoxymethanol and deionized water under a nitrogen atmosphere for 12 h. The product was filtered off and washed with ether (3 × 50 mL) and then with deionized water (3 × 50 mL). A suspension of [M 2 (C^N) 4 Cl 2 ] (0.2 mmol) and the N^N ligand (0.44 mmol) in a mixture of dichloromethane:methanol (1:1, 20 mL) was refluxed overnight under a nitrogen atmosphere. The resulting solution was then allowed to cool to room temperature, and filtered to remove unreacted cyclometallated dimer. To the filtrate, a solution of ammonium hexafluorophosphate (0.5 g in 5 mL methanol) was added and the filtrate was reduced in volume by rotary evaporation until precipitation of the crude product occurred. The precipitate was then filtered and washed with several portions of water (2 × 50 mL) followed by diethyl ether (2 × 50 mL). The product was recrystallized by acetonitrile:diethyl ether vapor diffusion to yield the titled compound. All complexes were characterized by 1 H-NMR, 13 C-NMR, high resolution mass spectrometry (HRMS) and elemental analysis. Complex 1 Reported 51 . Complex 2 Yield: 61%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 9.22 (d, J  = 8.4 Hz, 1H), 9.12 (s, 1H), 9.06 (d, J  = 8.0 Hz, 1H), 8.98 (d, J  = 4.0 Hz, 1H), 8.94 (d, J  = 4.0 Hz, 1H), 8.59 (d, J  = 8.8 Hz, 2H), 8.52 (d, J  = 8.8 Hz, 2H), 8.34 (d, J  = 8.0 Hz, 2H), 8.31-8.25 (m, 2H), 7.84 (d, J  = 8.0 Hz, 2H), 7.34-7.25 (m, 6H), 6.93 (dd, J  = 6.0 Hz, 1.2 Hz, 2H), 6.70 (d, J  = 8.0 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 171.4, 152.6, 152.1, 149.4, 148.6, 148.4, 147.2, 147.1, 145.3, 144.5, 141.9, 141.1, 141.0, 136.1, 135.6, 135.5, 134.1, 133.8, 131.5, 131.4, 131.3, 130.1, 130.0, 128.6, 128.3, 128.2, 127.6, 127.5, 127.3, 126.4, 125.3, 125.2, 124.7, 123.8, 123.7, 119.0, 118.9, 103.7. HRMS: Calcd. for C 42 H 29 IrN 5 [M–PF 6 ] + : 796.2052 Found: 796.2091. Anal. (C 42 H 29 N 5 IrPF 6 +2H 2 O): C, H, N: calcd. 51.64, 3.40, 7.17, found: 51.65, 3.15, 7.41. The elemental analysis result ( Figure S1 ) was shown in the supporting information . Complex 3 Reported 52 . Complex 4 Reported 53 . Complex 5 Yield: 58%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 8.58 (d, J  = 5.2 Hz, 2H), 8.33-8.30 (m, 4H), 8.06-8.02 (m, 6H), 7.80 (d, J  = 4.8 Hz, 2H), 7.70-7.66 (m, 10H), 7.19 (t, J  = 3.6 Hz, 2H), 7.09 (dd, J  = 6.0 Hz, 1.2 Hz, 4H), 6.52 (d, J  = 7.6 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 164.6, 164.3, 153.7, 150.0, 148.9, 145.8, 139.3, 137.4, 135.5, 133.4, 129.8, 129.6, 129.4, 129.0, 128.1, 126.9, 126.8, 125.7, 124.1, 123.1, 121.4; HRMS: Calcd. for C 46 H 32 RhN 4 [M–PF 6 ] + : 743.1682 Found: 743.2193. Anal. (C 46 H 32 N 4 RhPF 6 +0.5H 2 O): C, H, N: calcd. 61.55, 3.71, 6.24, found: 61.51, 3.66, 6.28. The elemental analysis result ( Figure S2 ) was shown in the supporting information . Disk diffusion susceptibility test The disk diffusion method was used to determine sensitivity of bacteria to complexes 1 – 5 . S. aureus , E. faecalis , E. coli and K. pneumoniae direct colony suspensions were prepared in Luria Bertain broth for less than 18 h. The turbidity of four bacterial suspensions were standardized to match 1.0 × 10 8   UFC/mL (or 0.5 McFarland turbidity units). A sterile swab dipped in fresh bacterial suspensions was inoculated to the entire surface of Mueller-Hinton agar and the inoculum was distributed evenly. The plates were incubated with standardizing inoculum suspension for 15 min at room temperature. 10 μL complexes 1 – 5 (11.5 μM) or ampicillin (28 μM) were impregnated on sterile 6 mm paper disks for 15 min. Subsequently, the paper disks were aseptically placed on and pressed down firmly to the inoculated plates. Plates were incubated for 24 h in a 37 °C incubator. After the incubation, the inhibition zones were measured. Ampicillin was used as a positive control for bacterial inhibition. The presence or absence of growth around the disks was a measure of the ability of complexes 1 – 5 to inhibit growth of bacterial. All experiments were done in triplicate. MIC and MBC testing Complex 1 was examined for its antimicrobial activity against S. aureus . The MIC was estimated by the broth dilution method in Luria Bertain broth using the standardized method 56 57 58 59 60 . Briefly, serial dilutions of complex 1 were prepared in distilled water with concentrations ranging from 1.8 to 57.6 μM. 1 mL of an S. aureus inoculum (1 × 10 8  UFC/mL) and 0.1 mL of serially diluted complex 1 were added to 2.9 mL of Luria Bertain broth. Controls without complex 1 were also prepared. After 24 h of incubation at 37 °C under agitation in hermetic tubes, the MIC was determined as the lowest concentration of complex 1 that inhibited visible bacterial growth. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the MBC, 10 μL of bacterial inoculum treated with different concentrations of complex 1 was taken aseptically from tubes that had not presented visible turbidity and was plated onto Mueller-Hinton agar. The MBC was considered as the lowest concentration of complex 1 that allowed less than 0.1% of the original inoculum treated with complex 1 to grow on the surface of the Mueller-Hinton agar. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the nature of antibacterial effect of complex 1 , the MBC/MIC ratio was used; when the ratio was lower than 4, complex 1 was considered as a bactericidal complex 1 and when the ratio was higher than 4, it was considered as a bacteriostatic complex 1 . Log P Determination This experiment was carried out according to a reported method and the partitions were detected by HPLC 61 . The partition coefficients of the Ir(III) complexes were calculated using the equation log P  = log([Ir] WSO /[Ir] OSW ), where [Ir] WSO means the concentration of Ir(III) complex dissolved in octanol-saturated water (WSO), and [Ir]OSW means the concentration of Ir(III) complex dissolved in water-saturated octanol (OSW). Cell cytotoxicity Complex 1 was dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 10 mM and serially diluted with low FBS medium to the working concentrations. The evaluation of cells proliferation was conducted using MTT assay. In brief, the cells were seeded in 96-well plates at a density of 8 × 10 4 cells/mL in complete medium for overnight. Serial dilution of complex 1 in low FBS medium was then added to the wells. After 72 h, 20 μL MTT (5 mg/mL) was added to each well. The plates were then incubated for an additional 4 h at 37 °C while avoiding direct light. Subsequently, the medium in each well was removed and 100 μL DMSO was added to each well. The plates were shaken gently for 10 min at room temperature to completely dissolve the purple formazan in the mitochondria of living cells. The color intensity was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices). ## Materials and cell lines Materials and cell lines All chemicals, unless specified, were obtained from Sigma-Aldrich. Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , Klebsiella pneumonia , HepG2, SKOV3, HeLa, A375 and A2058 were purchased from American Type Culture Collection (ATCC). A2780, HepG2, SKOV3, HeLa, A375 and A2058 cells were routinely grown in suspension in 90% RPMI-1640 containing L-glutamine (2 nM) or DMEM, antibiotics (100 IU penicillin/mL, 100 μg streptomycin/mL) and supplemented with 10% (v/v) fetal bovine serum (FBS). The cells were maintained at a cell density of 0.2–1 × 10 6 cells/mL in a 5% CO 2 humidified atmosphere at 37 °C. ## General experimental General experimental Mass spectrometry was performed at the Mass Spectroscopy Unit at the Department of Chemistry, Hong Kong Baptist University, Hong Kong (China). Melting points were determined using a Gallenkamp melting apparatus and are uncorrected. Deuterated solvents for NMR purposes were obtained from Armar and used as received. 1 H and 13 C NMR were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz ( 1 H) and 100 MHz ( 13 C). 1 H and 13 C chemical shifts were referenced internally to solvent shift (acetone- d 6 : 1 H δ 2.05, 13 C δ 29.8, 206.1). Chemical shifts (δ) are quoted in ppm, the downfield direction being defined as positive. Uncertainties in chemical shifts are typically ±0.01 ppm for 1 H and ±0.05 for 13 C. Coupling constants are typically ±0.1 Hz for 1 H- 1 H and ±0.5 Hz for 1 H- 13 C couplings. The following abbreviations are used for convenience in reporting the multiplicity of NMR resonances: s, singlet; d, doublet; t, triplet; m, multiplet. All NMR data was acquired and processed using standard Bruker software (Topspin). ## Photophysical measurement Photophysical measurement Emission spectra and lifetime measurements for complex 1 were performed according to the previously described method 53 . ## Synthesis Synthesis These complexes were synthesized using a modified literature method 55 . MCl 3· 3H 2 O was heated to 150 °C with 2.2 equivalents of C^N ligand in 3:1 methoxymethanol and deionized water under a nitrogen atmosphere for 12 h. The product was filtered off and washed with ether (3 × 50 mL) and then with deionized water (3 × 50 mL). A suspension of [M 2 (C^N) 4 Cl 2 ] (0.2 mmol) and the N^N ligand (0.44 mmol) in a mixture of dichloromethane:methanol (1:1, 20 mL) was refluxed overnight under a nitrogen atmosphere. The resulting solution was then allowed to cool to room temperature, and filtered to remove unreacted cyclometallated dimer. To the filtrate, a solution of ammonium hexafluorophosphate (0.5 g in 5 mL methanol) was added and the filtrate was reduced in volume by rotary evaporation until precipitation of the crude product occurred. The precipitate was then filtered and washed with several portions of water (2 × 50 mL) followed by diethyl ether (2 × 50 mL). The product was recrystallized by acetonitrile:diethyl ether vapor diffusion to yield the titled compound. All complexes were characterized by 1 H-NMR, 13 C-NMR, high resolution mass spectrometry (HRMS) and elemental analysis. Complex 1 Reported 51 . Complex 2 Yield: 61%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 9.22 (d, J  = 8.4 Hz, 1H), 9.12 (s, 1H), 9.06 (d, J  = 8.0 Hz, 1H), 8.98 (d, J  = 4.0 Hz, 1H), 8.94 (d, J  = 4.0 Hz, 1H), 8.59 (d, J  = 8.8 Hz, 2H), 8.52 (d, J  = 8.8 Hz, 2H), 8.34 (d, J  = 8.0 Hz, 2H), 8.31-8.25 (m, 2H), 7.84 (d, J  = 8.0 Hz, 2H), 7.34-7.25 (m, 6H), 6.93 (dd, J  = 6.0 Hz, 1.2 Hz, 2H), 6.70 (d, J  = 8.0 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 171.4, 152.6, 152.1, 149.4, 148.6, 148.4, 147.2, 147.1, 145.3, 144.5, 141.9, 141.1, 141.0, 136.1, 135.6, 135.5, 134.1, 133.8, 131.5, 131.4, 131.3, 130.1, 130.0, 128.6, 128.3, 128.2, 127.6, 127.5, 127.3, 126.4, 125.3, 125.2, 124.7, 123.8, 123.7, 119.0, 118.9, 103.7. HRMS: Calcd. for C 42 H 29 IrN 5 [M–PF 6 ] + : 796.2052 Found: 796.2091. Anal. (C 42 H 29 N 5 IrPF 6 +2H 2 O): C, H, N: calcd. 51.64, 3.40, 7.17, found: 51.65, 3.15, 7.41. The elemental analysis result ( Figure S1 ) was shown in the supporting information . Complex 3 Reported 52 . Complex 4 Reported 53 . Complex 5 Yield: 58%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 8.58 (d, J  = 5.2 Hz, 2H), 8.33-8.30 (m, 4H), 8.06-8.02 (m, 6H), 7.80 (d, J  = 4.8 Hz, 2H), 7.70-7.66 (m, 10H), 7.19 (t, J  = 3.6 Hz, 2H), 7.09 (dd, J  = 6.0 Hz, 1.2 Hz, 4H), 6.52 (d, J  = 7.6 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 164.6, 164.3, 153.7, 150.0, 148.9, 145.8, 139.3, 137.4, 135.5, 133.4, 129.8, 129.6, 129.4, 129.0, 128.1, 126.9, 126.8, 125.7, 124.1, 123.1, 121.4; HRMS: Calcd. for C 46 H 32 RhN 4 [M–PF 6 ] + : 743.1682 Found: 743.2193. Anal. (C 46 H 32 N 4 RhPF 6 +0.5H 2 O): C, H, N: calcd. 61.55, 3.71, 6.24, found: 61.51, 3.66, 6.28. The elemental analysis result ( Figure S2 ) was shown in the supporting information . ## Complex 1 Complex 1 Reported 51 . ## Complex 2 Complex 2 Yield: 61%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 9.22 (d, J  = 8.4 Hz, 1H), 9.12 (s, 1H), 9.06 (d, J  = 8.0 Hz, 1H), 8.98 (d, J  = 4.0 Hz, 1H), 8.94 (d, J  = 4.0 Hz, 1H), 8.59 (d, J  = 8.8 Hz, 2H), 8.52 (d, J  = 8.8 Hz, 2H), 8.34 (d, J  = 8.0 Hz, 2H), 8.31-8.25 (m, 2H), 7.84 (d, J  = 8.0 Hz, 2H), 7.34-7.25 (m, 6H), 6.93 (dd, J  = 6.0 Hz, 1.2 Hz, 2H), 6.70 (d, J  = 8.0 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 171.4, 152.6, 152.1, 149.4, 148.6, 148.4, 147.2, 147.1, 145.3, 144.5, 141.9, 141.1, 141.0, 136.1, 135.6, 135.5, 134.1, 133.8, 131.5, 131.4, 131.3, 130.1, 130.0, 128.6, 128.3, 128.2, 127.6, 127.5, 127.3, 126.4, 125.3, 125.2, 124.7, 123.8, 123.7, 119.0, 118.9, 103.7. HRMS: Calcd. for C 42 H 29 IrN 5 [M–PF 6 ] + : 796.2052 Found: 796.2091. Anal. (C 42 H 29 N 5 IrPF 6 +2H 2 O): C, H, N: calcd. 51.64, 3.40, 7.17, found: 51.65, 3.15, 7.41. The elemental analysis result ( Figure S1 ) was shown in the supporting information . ## Complex 3 Complex 3 Reported 52 . ## Complex 4 Complex 4 Reported 53 . ## Complex 5 Complex 5 Yield: 58%. 1 H NMR (400 MHz, Acetone- d 6 ) δ 8.58 (d, J  = 5.2 Hz, 2H), 8.33-8.30 (m, 4H), 8.06-8.02 (m, 6H), 7.80 (d, J  = 4.8 Hz, 2H), 7.70-7.66 (m, 10H), 7.19 (t, J  = 3.6 Hz, 2H), 7.09 (dd, J  = 6.0 Hz, 1.2 Hz, 4H), 6.52 (d, J  = 7.6 Hz, 2H); 13 C NMR (100 MHz, Acetone- d 6 ) δ 164.6, 164.3, 153.7, 150.0, 148.9, 145.8, 139.3, 137.4, 135.5, 133.4, 129.8, 129.6, 129.4, 129.0, 128.1, 126.9, 126.8, 125.7, 124.1, 123.1, 121.4; HRMS: Calcd. for C 46 H 32 RhN 4 [M–PF 6 ] + : 743.1682 Found: 743.2193. Anal. (C 46 H 32 N 4 RhPF 6 +0.5H 2 O): C, H, N: calcd. 61.55, 3.71, 6.24, found: 61.51, 3.66, 6.28. The elemental analysis result ( Figure S2 ) was shown in the supporting information . ## Disk diffusion susceptibility test Disk diffusion susceptibility test The disk diffusion method was used to determine sensitivity of bacteria to complexes 1 – 5 . S. aureus , E. faecalis , E. coli and K. pneumoniae direct colony suspensions were prepared in Luria Bertain broth for less than 18 h. The turbidity of four bacterial suspensions were standardized to match 1.0 × 10 8   UFC/mL (or 0.5 McFarland turbidity units). A sterile swab dipped in fresh bacterial suspensions was inoculated to the entire surface of Mueller-Hinton agar and the inoculum was distributed evenly. The plates were incubated with standardizing inoculum suspension for 15 min at room temperature. 10 μL complexes 1 – 5 (11.5 μM) or ampicillin (28 μM) were impregnated on sterile 6 mm paper disks for 15 min. Subsequently, the paper disks were aseptically placed on and pressed down firmly to the inoculated plates. Plates were incubated for 24 h in a 37 °C incubator. After the incubation, the inhibition zones were measured. Ampicillin was used as a positive control for bacterial inhibition. The presence or absence of growth around the disks was a measure of the ability of complexes 1 – 5 to inhibit growth of bacterial. All experiments were done in triplicate. MIC and MBC testing Complex 1 was examined for its antimicrobial activity against S. aureus . The MIC was estimated by the broth dilution method in Luria Bertain broth using the standardized method 56 57 58 59 60 . Briefly, serial dilutions of complex 1 were prepared in distilled water with concentrations ranging from 1.8 to 57.6 μM. 1 mL of an S. aureus inoculum (1 × 10 8  UFC/mL) and 0.1 mL of serially diluted complex 1 were added to 2.9 mL of Luria Bertain broth. Controls without complex 1 were also prepared. After 24 h of incubation at 37 °C under agitation in hermetic tubes, the MIC was determined as the lowest concentration of complex 1 that inhibited visible bacterial growth. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the MBC, 10 μL of bacterial inoculum treated with different concentrations of complex 1 was taken aseptically from tubes that had not presented visible turbidity and was plated onto Mueller-Hinton agar. The MBC was considered as the lowest concentration of complex 1 that allowed less than 0.1% of the original inoculum treated with complex 1 to grow on the surface of the Mueller-Hinton agar. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the nature of antibacterial effect of complex 1 , the MBC/MIC ratio was used; when the ratio was lower than 4, complex 1 was considered as a bactericidal complex 1 and when the ratio was higher than 4, it was considered as a bacteriostatic complex 1 . ## MIC and MBC testing MIC and MBC testing Complex 1 was examined for its antimicrobial activity against S. aureus . The MIC was estimated by the broth dilution method in Luria Bertain broth using the standardized method 56 57 58 59 60 . Briefly, serial dilutions of complex 1 were prepared in distilled water with concentrations ranging from 1.8 to 57.6 μM. 1 mL of an S. aureus inoculum (1 × 10 8  UFC/mL) and 0.1 mL of serially diluted complex 1 were added to 2.9 mL of Luria Bertain broth. Controls without complex 1 were also prepared. After 24 h of incubation at 37 °C under agitation in hermetic tubes, the MIC was determined as the lowest concentration of complex 1 that inhibited visible bacterial growth. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the MBC, 10 μL of bacterial inoculum treated with different concentrations of complex 1 was taken aseptically from tubes that had not presented visible turbidity and was plated onto Mueller-Hinton agar. The MBC was considered as the lowest concentration of complex 1 that allowed less than 0.1% of the original inoculum treated with complex 1 to grow on the surface of the Mueller-Hinton agar. The experiments were performed in triplicate. The complete data are shown in Table S2 in the Supplementary Information . To determine the nature of antibacterial effect of complex 1 , the MBC/MIC ratio was used; when the ratio was lower than 4, complex 1 was considered as a bactericidal complex 1 and when the ratio was higher than 4, it was considered as a bacteriostatic complex 1 . ## Log Log P Determination This experiment was carried out according to a reported method and the partitions were detected by HPLC 61 . The partition coefficients of the Ir(III) complexes were calculated using the equation log P  = log([Ir] WSO /[Ir] OSW ), where [Ir] WSO means the concentration of Ir(III) complex dissolved in octanol-saturated water (WSO), and [Ir]OSW means the concentration of Ir(III) complex dissolved in water-saturated octanol (OSW). ## Cell cytotoxicity Cell cytotoxicity Complex 1 was dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 10 mM and serially diluted with low FBS medium to the working concentrations. The evaluation of cells proliferation was conducted using MTT assay. In brief, the cells were seeded in 96-well plates at a density of 8 × 10 4 cells/mL in complete medium for overnight. Serial dilution of complex 1 in low FBS medium was then added to the wells. After 72 h, 20 μL MTT (5 mg/mL) was added to each well. The plates were then incubated for an additional 4 h at 37 °C while avoiding direct light. Subsequently, the medium in each well was removed and 100 μL DMSO was added to each well. The plates were shaken gently for 10 min at room temperature to completely dissolve the purple formazan in the mitochondria of living cells. The color intensity was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices). ## Conclusion Conclusion In summary, we have identified a metal-based complex that selectively inhibited the growth of S. aureus . The iridium(III) or rhodium(III) complexes 1 – 5 were synthesized by the reaction of MCl 3· n H 2 O with the C^N ligand to yield the dimeric compound [M 2 (C^N) 4 Cl 2 ], which was treated with the corresponding N^N ligand to give the targeted complexes. Complexes 2 and 5 are novel and have been characterized for the first time. Complex rac - 1 inhibited the growth of S. aureus with MIC and MBC values of 3.60 and 7.19 μM. As the MBC/MIC ratio of complex 1 is approximately 2, complex 1 is considered to be bactericidal. Moreover, complex 1 also exhibited cytotoxicity against a number of cancer cells, with particular potency against ovarian, cervical and melanoma cells but not hepatocarcinoma cells, suggesting that its cytotoxic activity was cell type-dependent. To our knowledge, this cyclometallated iridium(III) complex is the first example of a substitutionally-inert, Group 9 organometallic compound utilized as a direct inhibitor of S. aureus . We anticipate that cyclometallated iridium(III) complex 1 may serve as a useful scaffold for the further development of highly potent inhibitors of S. aureus as potential anti-bacterial agents and anti-cancer agents. ## Additional Information Additional Information How to cite this article : Lu, L. et al. Identification of an iridium(III) complex with anti-bacterial and anti-cancer activity. Sci. Rep. 5 , 14544; doi: 10.1038/srep14544 (2015). ## Supplementary Material Supplementary Material Supplementary Information