Cytotoxicity of Ruthenium(II) Arene Complexes Containing Functionalized Ferrocenyl β-Diketonate Ligands

The synthesis and characterization of 24 ruthenium(II) arene complexes of the type [(p-cym)RuCl(Fc-acac)] (where p-cym = p-cymene and Fc-acac = functionalized ferrocenyl β-diketonate ligands) are reported, including single-crystal X-ray diffraction for 21 new complexes. Chemosensitivity studies have been conducted against human pancreatic carcinoma (MIA PaCa-2), human colorectal adenocarcinoma p53-wildtype (HCT116 p53+/+) and normal human retinal epithelial cell lines (APRE-19). The most active complex, which contains a 2-furan-substituted ligand (4), is 5x more cytotoxic than the analogs 3-furan complex (5) against MIA PaCa-2. Several complexes were screened under hypoxic conditions and at shorter-time incubations, and their ability to damage DNA was determined by the comet assay. Compounds were also screened for their potential to inhibit the growth of both bacterial and fungal strains.


Methods General
Synthetic procedures were conducted under aerobic conditions unless stated otherwise. All chemicals were supplied by Sigma-Aldrich Chemical Co., Acros Organics, Alfa Aesar, Fisher Chemicals and BOC gases used without further purification. Deuterated NMR solvents were purchased from Sigma-Aldrich Chemical Co. or Acros Organics. Chromatography columns were prepared using Fisher Chemicals 60A 35-70 micron silica gel.

Instrumentation
Nuclear magnetic resonance spectra were recorded using Bruker Advance 300, 400, 500, DPX300 and DPX500 MHz spectrometers. Chemical shifts are reported in parts per million (δ) downfield relative to the internal reference tetramethylsilane or referenced to the solvent signal. All NMR spectra were recorded in deuterated acetone at room temperature. Abbreviations used: br. = broad, d = doublet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, Q = quaternary, q = quartet, s = singlet, t = triplet. Mass spectra were recorded using a micromass ZMD 2000 spectrometer employing the electrospray (ES+/-) ionisation technique. Accurate molecular masses were obtained from Walters LCT, GCT or Bruker MicroTof spectrometers. Microanalyses were acquired by Mr. Stephen Boyer at the London Metropolitan University Elemental Analysis Service. UV-vis absorption spectra were acquired on a Cary Series UV-vis spectrophotometer using 1 cm path length quartz cuvettes.

X-ray Crystallography
A suitable single crystal was selected and immersed in inert oil. The crystal was then mounted to a goniometer head on an Agilent SuperNova X-ray diffractometer fitted with an Atlas area detector and a kappa-geometry 4circle goniometer, using mirror monochromated Mo-Kα radiation (λ = 0.71073 Å) or Cu-Kα (λ = 1.54184 Å) radiation. The crystal was cooled to 120 K by an Oxford cryostream low temperature device. 1 The full data set was recorded and the images processed using CrysAlis Pro. 2 Structure solution by direct methods was achieved through the use SHELXT and SHELXL programs, 3,4 and the structural model refined by full matrix least squares on F 2 using the program Olex2. 5 Molecular graphics were plotted, editing of CIFs and construction of tables of bond lengths and angles were achieved using Olex2. Unless otherwise stated, hydrogen atoms were placed using idealized geometric positions (with free rotation for methyl groups), allowed to move in a "riding model" along with the atoms to which they were attached, and refined isotropically. model" along with the atoms to which they were attached and refined isotropically. The SQUEEZE routine in Olex2 and was used to refine structures where diffuse electron density could not be adequately modelled as solvent of crystallization. 6 Cyclic Voltammetry A Metrohm Autolab PGSTAT30 potentiostat was used to conduct cyclic voltammetry measurements combined with the Nova software package (Metrohm, Version 2.1.5). A glassy carbon working electrode (diameter = 3 mm) and a glassy carbon rod counter electrode were used. As a reference electrode, a double junction Ag/AgCl electrode was used with a 2 M solution of LiCl in ethanol in the inner compartment and 0.1 M NBu4PF6 solution in acetonitrile in the outer compartment. All samples were measured in concentrations of 0.7-0.8 mg/mL for complexes and 0.5-0.6 mg/mL for ligands in 10 mL of a dried and degassed 0.1 M NBu4PF6 solution in acetonitrile. The voltammograms were corrected using ferrocene as internal standard with the Fc/Fc + couple at 0.40 V vs SCE (standard calomel electrode). 7 The reversible Fc*/Fc* + couple is measured between -0.5 -1.0 V with scanning speeds of 50, 100 and 250 mV/s. All other voltammograms are obtained using a scanning speed of 100 mV. The potential was scanned three times and the third run is presented using QtiPlot.

Hydrophobicity
Equal volumes of 1-octanol and sodium chloride saturated distilled water were stirred for 16 h and separated to give water-saturated octanol and octanol-saturated water. Standard solutions of each complex (5,10,20, 40 and 60 µM) were prepared in water-saturated octanol. The calibration curve of absorbance against concentration was determined from the maximum absorbance (λmax) of the standard solutions. Stock solutions of each complex (50 µM) in water-saturated octanol (25 mL) were prepared. Six independent samples were prepared by the addition of the stock solution (3 mL) to a 15 mL Falcon tube followed by layered addition of octanol-saturated water (3 mL). Samples were shaken at 1000 g/min for 2 h using an IKA Vibrax VXC basic shaker. The layers were separated, and the water-saturated octanol layer retained. The concentration of each sample in the watersaturated octanol layer was determined by UV-vis spectroscopy with reference to the individual calibration curves to give an average concentration for shaken samples ([C]final). The concentration of an unshaken sample of stock solution was determined to give [C]initial. The partition coefficient (LogP) was determined with by Eqn1.

Chemosensitivity under Hypoxic Conditions
The assay was conducted according to the protocol stated previously for normoxic conditions. However, all the incubations periods, the addition of the drug and the addition of the MTT solution were carried out inside a Don Whitley Scientific H35 Hypoxystation with the oxygen level set at 0.1 %. Cell culture media was conditioned for 24 hours at 0.1 % O2 prior to the start of the experiment.

Cell Uptake
Cell uptake assays were conducted using MIA PaCa-2 cells and these were maintained as described above. All assays were conducted in 6-well plates and cell concentrations of 1 x 10 5 cells/ well in 2 mL of complete cell media. These cell suspensions were added to the wells and incubated at 37°C and 5% CO2 for 24 h. The media was then removed, and 2 mL of media/copper compound was added to each well. For each compound, a concentration of 10 µM of ligands L1, L2, L4, L7 or complexes C1, C2, C4, C7 were used. The cells were incubated with the complexes for 48 h, before removing and discarding the media. The cell monolayers were then individually washed with PBS (3 x 1 mL) and all waste discarded. 0.25% Trypsin-EDTA (0.5 mL) was then added until the cells were removed from the surface of the wells and then diluted with media (1.5 mL). The contents of each well were transferred to separate Falcon tubes and centrifuged at 1500 rpm for 5 min. The supernatant was removed, the cell pellets were resuspended in PBS (1 mL) and centrifuged again at 1500 rpm for 5 min (this was completed 3 times). On the final time, the cells were counted before the last centrifuge and then pelleted and store in the freezer for ICP-MS analysis. All assays were conducted in triplicate.
Samples were then taken up in Optima high purity nitric acid, diluted to 10 mL with 1mL rhodium internal standard and 9 mL 18.2 Ultrapure water. The elemental contents of the samples were determined using ICP-MS-QQQ Thermo TQ spectrometer. Measurement of iron mass 56 and ruthenium isotope mass 100 was conducted by comparing solutions of known concentration and intensities. A series of standard calibration curves allows the concentration of each element to be determined. The standards were prepared by weight using 1000 mg/kg reference solutions. Operating conditions: cooling flow rate, 14.0 L/min, auxiliary gas flow rate, 0.8 L/min; sampling Depth 5 mm, additional gas flow 75 %, spray chamber 2.7 degrees, nebulizer flow rate, 1.144 L/min pump speed 15 rpm, RF power of 1,550 W.
Analysis of cellular DNA damage by the Comet assay Slides containing a layer of agarose were prepared in advance using 1% normal melting point agarose (NMPA, 500 mg/50 mL PBS). A concentration of 1 x 10 6 cells/ mL cell suspension was obtained, and 2 mL added to each well of a 6-well plate. The cells were incubated for 24 hours at 37C in a 5.0% CO2 atmosphere. Drug samples with concentrations ranging from 20-2.5 mM (+ untreated control) were prepared using high glucose DMEM. The medium was removed from the wells, and 2 mL of drug-containing media was added to each well and incubated for a further 48 hours at 37C in a 5.0% CO2 atmosphere. All the drug-containing media was removed and placed in centrifuge tubes, the wells were each washed with PBS (1 mL), which was also placed into the centrifuge tube. The media-free wells were then trypsinized (1 mL) for 3-5 minutes and then neutralized with high glucose DMEM (1 mL), these were all added to the centrifuge tube and centrifuged at 1500 rpm for 3 minutes. The supernatant was removed, and the pellet re-suspended in complete medium (1 mL). The cell suspensions can be stored at -80C until required (defrost and centrifuge at 16.1 rcf for 20 seconds). 0.5% low melting agarose (LMPA) was prepared (250 mg/50 mL PBS) and used to re-suspend the pellets (150-1000 mL, depending on size). Cell suspension (150 mL) was added to a previously coated glass slide (containing NMPA) and set using a coverslip and cool tray, the process was repeated with LMPA only. All slides were placed into a tray where freshly prepared neutral lysing solution and incubate for 1 h at 37C in dark conditions. The slides were then submerged in electrophoresis buffer for 30 minutes (x2). The slides were then placed in an electrophoresis chamber and freshly prepared electrophoresis buffer (pH 8.0) added and run at 24 V for 25 minutes. The slides were removed and rinsed with distilled water (x 3) and 100% ice cold ethanol (x1), then left to dry overnight. Staining solution (150 mL) was added to each slide and the Comets analysed using Comet assay III software. A minimum of 50 different comets were scored and the computer outputs an average on head and tail intensities and tail moments. Images were taken of the comets and the tail moments plotted against concentration of drug sample used. Reagents 1. Neutral Lysing Solution (2% sarkosyl, 0.5 M Na2EDTA, 0.5 mg/ mL proteinase K (pH 8.0)): Sarkosyl (2 g) and Na2EDTA (18.61 g) were added to distilled water (80 mL) and the pH adjusted to 8.0 with 10 M NaOH. Proteinase K (50 mg) was then added and made up to a final volume of 100 mL. 2. Electrophoresis Buffer (90 mM Tris buffer, 90 mM boric acid, 2 mM Na2EDTA (pH 8.0)): Tris base (32.707 g), boric acid (16.694 g) and Na2EDTA (2.233 g) were added to distilled water (2.5 L). The pH was adjusted to 8.0 and the volume made up to 3 L with distilled water. 3. Staining Solution: SYBR™ Gold solution (molecular probes inc, S-11494) (1 mL) was added to PBS (10 mL); this was made on the day of imaging. Statistical Analysis. Statistical analysis of the results was conducted using Student's t test, for p < 0.05 being considered as significant, and p < 0.01 as very significant.
Antibacterial Evaluation. Complexes were prepared in DMSO and water to a give a final concentration of 32 mg/mL in 384-well non-binding surface (NBS) plates. The final DMSO concentration was at a maximum of 1.0 %. All bacteria were cultured in Cation adjusted Mueller Hinton broth (CAMHB) at 37 °C overnight. A sample of each culture was diluted 40-fold in fresh broth and incubated at 37 °C for 1.5-3 h. The resultant mid-log phase cultures were diluted (CFU/mL measured by OD600), then added to each well of the compound containing plates, giving a cell density of 5 × 10 5 CFU mL -1 and a total volume of 50 μL. Colistin and vancomycin were used as positive bacterial inhibitor standards for Gram-negative and Gram-positive bacteria, respectively. Each standard was provided in four concentrations (above and below the MIC or CC50 value) and plated into the first 8 wells of column 23 of the 384-well NBS plates. All the plates were covered and incubated at 37 °C for 18 h without shaking and were carried out in duplicate. Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD600), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The significance of the inhibition values was determined by modified Z-scores, calculated using the median and MAD of the samples (no controls) on the same plate. Samples with inhibition S6 value above 80% and Z-Score above 2.5 for either replicate were classed as actives. Samples with inhibition values in the range 50-80% and Z-Score above 2.5 for either replicate were classed as partial actives.

Antibacterial HIT Confirmation
The preparation is as above, though with maximum DMSO concentrations of 0.5%. Results were analysed using the same methods and using the same controls. The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition ≥ 80%. In addition, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with partial activity. Hits were classified by MIC ≤ 16 mg/mL or MIC ≤ 10 mM in either replicate.

Antifungal Evaluation
Complexes were prepared in DMSO and water to a give a final concentration of 32 mg/mL in 384-well nonbinding surface (NBS) plate. The final DMSO concentration was at a maximum of 1.0%. Fungal strains were cultured for three days on yeast extract-peptone dextrose (YPD) agar at 30 °C. A yeast suspension of 1 x 10 6 to 5 x 10 6 CFU/mL (as determined by OD530) was prepared from five colonies. The suspension was diluted and added to each well of the compound-containing plates giving a final cell density of fungi suspension of 2.5 × 10 3 CFU/mL and a total volume of 50 mL. Fluconazole was used as a positive fungal inhibitor standard and provided in four concentrations (two above and two below the MIC or CC50 value) and plated into the first 8 wells of column 23 of the 384-well NBS plates. All the plates were covered and incubated at 35 °C for 36 h without shaking. All experiments were carried out in duplicate. Growth inhibition of C. albicans was determined measuring absorbance at 630 nm (OD630) and the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD600-570), after the addition of 0.001 % resazurin and incubation at 35 °C for an additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (fungi without inhibitors) on the same plate. The significance of the inhibition values was determined by modified Z-scores, calculated using the median and MAD of the samples (no controls) on the same plate. Samples with inhibition value above 80% and Z-Score above 2.5 for either replicate were classed as actives. Samples with inhibition values in the range 50-80% and Z-Score above 2.5 for either replicate were classed as partial actives.

Antifungal HIT Confirmation
The preparation is as above, though with maximum DMSO concentrations of 0.5%. Results were analysed using the same methods and using the same controls. The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition ≥ 80% for C. albicans and an inhibition ≥ 70% for C. neoformans. Due to a higher variance in growth and inhibition, a lower threshold was applied to the data for C. neoformans. In addition, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with marginal activity. Hits were classified by MIC ≤ 16 μg·mL -1 or MIC ≤ 10 mM in either replicate.

Cytotoxicity Assay against HEK-293
To assess the cytotoxicity against eukaryotic cells, HEK-293 cells were counted manually in a Neubauer haemocytometer and then plated in the 384-well plates containing the compounds to give a density of 5000 cells/well in a final volume of 50 mL. Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS was used as growth media and the cells were incubated together with the compounds for 20 hours at 37 °C in 5% CO2. Tamoxifen was used as a positive cytotoxicity standard in eight concentrations in 2-fold serial dilutions with 50 µg·mL -1 the highest concentration. All experiments were performed in duplicate. Cytotoxicity was measured by fluorescence with excitation at 560 nm and emission at 590 nm (F560/590), after addition of 5 μL of resazurin (25 mg/mL) and incubation for a further 3 h at 37 °C in 5% CO2. The fluorescence intensity was measured using a Tecan M1000 Pro monochromator plate reader, using automatic gain calculation. CC50 (concentration at 50%) were calculated by curve fitting the inhibition values against log(concentration) using sigmoidal dose-response function, with variable fitting values for bottom, top and slope. The maximal percentage of cytotoxicity is reported as DMax, indicating any compounds with partial cytotoxicity. The curve fitting was implemented using Pipeline Pilot's dose-response component. Any value with > indicates a sample with no activity (low DMax value) or samples with CC50 values above the maximum tested concentration (higher DMax value). Cytotoxic samples were classified by CC50 ≤ 32 μg·mL -1 or CC50 ≤ 10 μM in either replicate. In addition, samples were flagged as partial cytotoxic if DMax ≥ 50%, even with CC50 > the maximum tested concentration.

Haemolysis Assay
To assess blood toxicity, human whole blood was washed three times with three volumes of 0.9% NaCl and then resuspended to a concentration of 0.5 x 10 8 cells/mL, as determined by manual cell count in a Neubauer haemocytometer. The washed cells were then added to the 384-well compound-containing plates for a final volume of 50 µL. After a 10 min shake on a plate shaker the plates were then incubated for 1 h at 37 °C. Melittin was used as a positive haemolytic standard in eight concentrations in 2-fold serial dilutions with 50 µg·mL -1 the highest concentration. After incubation, the plates were centrifuged at 1000 g for 10 min to pellet cells and debris, 25 µL of the supernatant was then transferred to a polystyrene 384-well assay plate. Haemolysis was determined by measuring the supernatant absorbance at 405 mm (OD405). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC10 and HC50 (concentration at 10% and 50% haemolysis, respectively) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal doseresponse function with variable fitting values for top, bottom, and slope. In addition, the maximal percentage of haemolysis is reported as DMax, indicating any compounds with partial haemolysis. The curve fitting was implemented using Pipeline Pilot's dose-response component. Any value with > indicate sample with no activity (low DMax value) or samples with HC10 values above the maximum tested concentration (higher DMax value). Haemolysis samples were classified by HC10 ≤ 32 µg/mL or HC10 ≤ 10 µM in either replicate. In addition, samples were flagged as partial haemolytic if DMax ≥ 50%, even with HC10 > the maximum tested concentration.

Scheme S 1: Synthetic route for ferrocenyl -diketonate (Fc-acac) ligands L5, L6, L8, L11, L13, L13 and-L23,
where all other ligands have been previously reported. [6,7] Characterization for Ligands A functionalized ethyl ester (13.0 mmol) was added to a stirred solution of acetyl ferrocene (7.2 mmol) and sodium ethoxide (13.0 mmol) in ether (20 mL). The solution was stirred at reflux for 24-72 h after which time the product was isolated by one of two methods. 1) The solid precipitate was isolated by filtration, dissolved in distilled water (150 mL) and acidified with 10% hydrochloric acid until pH 5 which caused a red solid to precipitate out in solution. The solid was filtered and dried overnight under vacuum before purification. 2) The solution was acidified with 10% hydrochloric acid until pH 5 and added to water (50 ml). The product was extracted with ether (3 x 20 mL) and the organic layers were combined, dried over MgSO4 and filtered. Solvent was removed in vacuo to give a red solid product. 8,9 Ligand L5. Prepared using ethyl-3-furoate (13.0 mmol) and refluxing for 48 h, then worked up following method 1. The product was purified by column chromatography and eluting with 90:10 v/v hexane/ethyl acetate. The solvent was removed to yield the product as a red solid. X-ray Crystallography of Ligands Figure S 1: Molecular structures for ligands L5, L8, L11, L13, L14 and L23. Displacement ellipsoids are placed at the 50% probability level and shown only for the heteroatoms.    (7) 1.367 (7) 1.317(6)

Figure S 5: Molecular structures for compounds 16-18. Displacement ellipsoids are placed at the 50% probability level and shown only for the heteroatoms.
Supporting Information S15

S24
(IC50 values = 95 ± 9 µM (6) and 51 ± 4 µM (7)). It should be noted that when comparing to the recent work of Manikandan et al., complexes 1 (CH3) and 7 (CF3) follow the same trend, 10 whereby the activity of 7 > 1, however, their activities are significantly lower than what has been reported against HeLa (cervical), A2780 (ovarian) and A2780cisR (cisplatin resistant ovarian). This highlights potential selectivity towards these cell lines and highlights the need for further screening of our library against a wider range of cancerous cells.
In the case of the methyl complexes 8 (3'-Me) and 9 (3',5'-diMe), and chloro complexes 18 (3'-Cl) and 19 (3',5'-diCl), the di-meta substituted compounds (9 and 19) are more cytotoxic than the mono-meta substituted compounds (8 and 18) against MIA PaCa-2. Complexes 9 and 19 are ~4x more cytotoxic than the both monometa complexes 8 and 18 (Figure S10). Upon comparison of the compounds substituted in the meta and para positions, the cytotoxicity of the methyl, fluoro and chloro compounds increase when substituted in the para position ( Figure S11). The most significant increase in potency is observed for the fluoro complexes 15 (3'-F) cf. 17 (4'-F) and chloro complexes 18 (3'-Cl) cf. 20 (4'-Cl) when tested against MIA PaCa-2, whereby the cytotoxicity increases by >3-fold for the para substituted compounds (Figure S12). The same trend is not observed when tested against the HCT116 p53 +/+ cell line, and the cytotoxicity generally decreases when comparing substitution in the meta and para position.

Figure S 51: Complex 1 at initial (black) and 96 hours (orange) + 100 mM NaCl (90% DMSO-d6 + 10% D2O, 500 MHz).
DMSO + 10% D2O -comparison with and without 100 mM NaCl  1.65 V, -2.1 -1.65 V and -2.1 -1 V (from top to bottom). The wide scan CVs for compounds 1, 2, 4 and 7 exhibit up to three significant reduction peaks (PC) with a minor signal between -1.04 --1.13 V and one to two major signals between -1.52 --1.93 V (PC2 and PC3). All three reductions are tentatively assigned to the irreversible reduction of the respective acac ligands with an overall count of two electrons (determined by iPC). The reduction of the chloro complexes is observed in a single step (2,4) or two steps (1, 7), while the peak separation is very small for 7 and only observed as a shoulder around -1.40 V. PC1 is assigned to the respective solvolyzed complexes at much lower negative potentials between -1.04 --1.13 V. This assignment is based on a cyclic voltammetry experiment where the concentration of 1 is changed and a chloride source is added (Figure S 56). After double dilution of the compound the ratio iK(Fc*)/ik(PC1) increases from 0.2 to 0.3, pointing to larger amount of a potentially solvolyzed complex. Its identity is proven by addition of a Clsource (TBACl, 0.2 mmol). After addition, the peak disappears and the main reduction peak increases so that iPC2 ≈ iP(Fc*). Furthermore, the experiment shows that the anodic peak at -1.03 V is linked to the reduction(s) assigned as PC2 and PC3. Other minor cathodic signals in the area between -0.31 -0.25 V arise only after the irreversible 2-ereduction (see Figure S 55 and S 56). A similar signal is observed after the irreversible oxidation of Ru(II) → Ru(III) between 0.00 -0.14 V. Both irreversible redox reactions also lead to a broadening of the signal assigned to the Fc*/Fc* + redox couple so that the full reversibility is only visible in the narrow range scans (Figure S 54). All complexes were screened against S. aureus, Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii), and antifungal activity against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) (Figure 6B).

Figure S 55: Cyclic voltammograms of 1 (top left), 2 (top right), 4 (bottom left) and 7 (bottom right) in the electrochemical windows between -0.5 -
Although there are few distinct trends, generally, the most active complexes against S. aureus contain a Fc-acac ligand with neutral inductive aromatic ring systems (2, 3, 8-10). Also, the di-chloro complex 19 exhibits ca. 2-fold increase when compared to the mono-chloro β-diketonate complexes (18 and 20), which could suggest that increasing the number of chloride groups could affect the activity. A general trend of decreasing activity is observed when comparing the position and number of fluoride atoms around the ring of the ferrocenyl βdiketonate R substituent: 17 (para) > 15 (meta) > 14 (ortho) > 16 (di-meta). Interestingly, decreasing electronegativity of the halogen atoms F > Cl > Br > I cause a decrease in the activity of complexes when the halogen atom is located at the para position, yet an increase in activity of the complexes when the halogen atom is located at the meta position. The opposite observation is true for electron donating substituents (i.e., R = Me), and suggest that the inductive effects around the aromatic ring may be responsible to some degree in imparting the bacterial inhibition properties to the complexes. Complexes that were classified as active underwent HIT confirmation to determine their minimum inhibitory concentration (MIC, parentheses of Table S17), and complexes 2 and 9 are classified as active, with MIC values of 16 µg/mL. When addressing the fungal growth inhibition after incubation of complexes 1-24, complexes 2 and 9 were found to be active against the C. neoformans strain, with inhibition concentrations of 116% and 120% respectively. Generally, these complexes exhibit significantly lower growth inhibition when compared to bis(bipyridine)ruthenium ferrocenyl βdiketonato complexes, which exhibited inhibitions of 89-100%. Interestingly, complexes 2 and 9 were the only complexes to be classed as active during the antibacterial HIT confirmation studies. Complexes 2, 3, 8-10, 12 and 19 underwent additional hit confirmation to determine their MIC (parentheses of Table S17), yet they were all were inactive, with MIC values > 32 µg/mL.
To assess the complexes' potency towards normal cell types, screening was conducted against human embryonic kidney cell line, HEK293, after a 20 h incubation, and hemolysis assays conducted against human whole blood after a 1 h incubation period. The tested complexes showed varying degree of cytotoxicity towards HEK-293 cells, with complexes 9 and 12 exhibiting the highest (CC50 = 5.79 µg/mL) and lowest (CC50 = 28.37 µg/mL) cytotoxicity towards eukaryotic cells, respectively. These normal kidney results for compounds 1-24 are in contrast to the previous cytotoxicity studies performed on the ARPE-19 human retinal epithelial cells which showed no cytotoxicity at the maximum threshold of 100 µM. Hemolysis (Hm) results, on the other hand, were extremely positive and highlight that of the tested complexes, all exhibited no potency towards human blood at the maximum tested concentration of 32 µg/mL, which is important for the distribution of these complexes in the bloodstream.