Ru, Rh and Ir metal complexes of pyridyl chalcone derivatives: Their potent antibacterial activity, comparable cytotoxicity potency and selectivity to cisplatin

1 Ru, Rh and Ir metal complexes of pyridyl chalcone derivatives: Their potent antibacterial 2 activity, comparable cytotoxicity potency and selectivity to cisplatin 3 4 Lincoln Dkhara, Venkanna Banothub, Emma Pinderc, Roger M. Phillipsc, Werner Kaminskyd, 5 Mohan Rao Kolliparaa* 6 7 aCentre for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong 793 022, 8 India. 9 bCentre for Biotechnology (CBT), Institute of Science & Technology (IST), Jawaharlal Nehru 10 Technological University Hyderabad (JNTUH), Kukatpally-500 085, Hyderabad, Telangana 11 State, India. 12 cDepartment of Pharmacy, School of Applied Sciences, University of Huddersfield, 13 Huddersfield, HD1 3DH UK. 14 dDepartment of Chemistry, University of Washington, Seattle, WA 98195, USA 15 E mail: mohanrao59@gmail.com 1 16 Abstract 17 Half sandwich ruthenium, rhodium and iridium complexes containing pyridyl chalcone 18 analogues (L1 and L2) are prepared by the reaction of [(arene)M(µ-Cl)Cl]2 (arene = benzene, p- 19 cymene, Cp*) and 20 mononuclear complexes (1-8) were obtained and characterized using FT-IR, 1H-NMR, 13C- 21 NMR, ESI mass and UV-Vis spectroscopic methods. The molecular structures of complexes 2, 22 4, 5 and 7 are established by single crystal X-ray diffraction studies. Antibacterial studies were 23 tested against three strains of bacterial microorganisms Staphylococcus aureus (gram +ve), 24 Klebsiella pneumoniae (gram -ve) and Escherichia coli (gram -ve). Further the cytotoxicity study 25 of the pyridyl chalcone derivatives and their complexes were evaluated against the human 26 colorectal cancer cell lines HT-29, HCT-116 p53+/+, HCT-116 p53-/- and ARPE-19 (non-cancer 27 retinal epithelium). 28 ___________________________________________________________________________ 29 Keywords: ruthenium, rhodium, iridium, pyridyl chalcone, antibacterial, anticancer (M =Ru, Rh/Ir)] with L1 and L2 in 1:2 (M:L) ratio. Eight neutral 2 30 1. Introduction 31 Cancer is one of the major causes of death in both developed and developing countries 32 [1]. Although the availability of drugs has increased over recent years, mortality rates remain 33 high and long-term responses are thwarted by problems of toxicity and the emergence of 34 resistance [2]. In recent years, there has been a resurgence interest in organometallic complexes 35 that have different modes of action compared to the platinates [3]. In our quest to find more 36 effective treatments for cancer, other metal-based compounds incorporating ruthenium, rhodium 37 and iridium have been developed and these have shown promising activity [4]. High selectivity 38 of ruthenium complexes towards cancer cells rather than healthy cells and their stable oxidation 39 states under bodily conditions showed that they are promising antitumor agents [5]. The 40 biological activity of the metal complexes of ruthenium, rhodium and iridium can be a 41 consequence of the coordination or intercalation mode of binding to the DNA [6]. Some of the 42 arene ruthenium complexes prepared by Sadler et al. showed promising anticancer activity both 43 in vitro and in vivo [7]. The anticancer activities of arene ruthenium complexes can be enhanced 44 based on the ligand scaffolds, which play an important role in controlling their activity such as 45 improving their water solubility. For example, bioactive compounds such as flavonoids, 46 isoflavonoids etc., can be incorporated to arene Ru(II) complexes to enhance their anticancer 47 activities [8]. Also, bioactive ligand scaffolds such as chalcone when combined with these arene 48 metal complexes exhibit better anticancer activity [9]. 49 The wide range of biological activities associated with chalcone based compounds, both 50 natural and synthetic, their ease of preparation, the potential of oral administration, safety and 51 profound natural abundance have led us to explore their therapeutic potential when combined 52 with metal complexes. Present-day studies have identified different chalcones and their hybrids 53 as the active component for anticancer and antibacterial activity. The increasing incidence of 54 infection caused by the rapid development of bacterial resistance to most of the known 55 antibiotics is a serious health problem [10]. Thus, research efforts for finding effective nature- 56 derived therapeutics against these multidrug-resistant microbes are required. Mai et al. have 57 reported a series of chalcone derivatives where -NH2 group on ring A (Chart 1) play an important 58 role in the anti-proliferative effect against HT-29 cancer cell [11]. However, in this work, ring A 59 contains either -NH2 group or -OH group and ring B is replaced with pyridyl substituent. 3 60 Keeping in mind the anti-proliferative effect of -NH2 substituent on ring A of chalcone 61 derivatives we are interested to explore this possibility by complexing pyridyl chalcone 62 derivatives (Chart 1) with arene ruthenium, rhodium and iridium complexes. The initial studies 63 designed to assess their cytotoxic potency against cancer and non-cancer cells are described. 64 Because of the reported activity of chalcones against microbes, the activity of these complexes 65 against bacterial strains is also reported. 66 Chart 1: Ligands used in this study 67 68 2. Experimental Section 69 2.1 Physical methods and materials 70 All the reagents were purchased from commercial sources and used as received. 3- 71 aminopyridine, 2-aminoacetophenone, 2-hydroxyacetophenone were obtained from Aldrich. The 72 solvents were purified and dried according to standard procedures. The starting precursor metal 73 complexes [Cp*MCl2]2 (M = Rh/Ir) were prepared by a new procedure using Anton Paar 74 Monowave 50 synthesis reactor. Infrared spectra were recorded on a Perkin-Elmer 983 75 spectrophotometer by using KBr pellets in the range of 400-4000 cm-1. 1H NMR spectra were 76 recorded on a Bruker Avance II 400 MHz spectrometer using DMSO-d6 and CDCl3 as solvents. 77 Absorption spectra were recorded on a Perkin-Elmer Lambda 25 UV/Visible spectrophotometer 78 in the range of 200-800 nm at room temperature in acetonitrile. Mass spectra were recorded 79 using Q-T of APCI-MS instrument (model HAB 273). Perkin-Elmer 2400 CHN/S analyzer was 80 used for elemental analyses of the complexes. All these mononuclear metal complexes were 81 synthesized and characterized by using FT-IR, 1H NMR, 13C-NMR, ESI mass, CHN, UV-Vis, 82 and Single-crystal X-ray diffraction techniques. 83 2.2 Single-crystal X-ray structures analyses 84 Single crystal X-ray diffraction data for the complexes (2), (4), (5) and (7) were collected 85 on an Oxford Diffraction Xcalibur Eos Gemini diffractometer at 293 K using graphite 86 monochromated Mo-Kα radiation (λ = 0.71073 Å). Suitable crystals were selected and each 4 87 mounted on a glass fiber. The strategy for the data collection was evaluated using the 88 CrysAlisPro CCD software [12]. Crystal data were collected by standard ‘‘phi–omega scan’’ 89 techniques and were scaled and reduced using CrysAlisPro RED software. The structures were 90 solved by direct methods using SHELXS-97 and refined by full-matrix least-squares with 91 SHELXL-97 refining on F2 [13, 14]. The positions of all the atoms were obtained by direct 92 methods. Metal atoms in the complex were located from the E-maps and non-hydrogen atoms 93 were refined anisotropically. The hydrogen atoms bound to the carbon were placed in 94 geometrically constrained positions and refined with isotropic temperature factors, generally 1.2 95 Ueq of their parent atoms. Figure 1 was drawn with the ORTEP3 program [15]. Figures 2-5 were 96 drawn with the MERCURY3.6 program [16]. 97 2.3 Cell lines testing, culture condition and cytotoxicity studies 98 The in vitro cytotoxicity of the pyridyl chalcone ligands (L1 and L2) and their 99 corresponding arene d6 metal complexes were performed at the University of Huddersfield 100 against the HCT-116 p53+/+, HCT-116 p53-/- and HT-29 human colorectal cancer lines. To 101 compare the activity of complexes against cancer cells compared to non-cancer cells, complexes 102 were also evaluated against the retinal epithelium cell line ARPE-19. HT-29 and ARPE-19 cells 103 were originally purchased from ATCC and HCT-116 cells (containing wild type p53 or deleted 104 p53) were obtained from Professor Bert Vogelstein's laboratory [17]. All reagents used were 105 purchased from Sigma Aldrich Co. Ltd (Dorset, UK) unless otherwise stated. Antiproliferative 106 activity of the compounds was evaluated using the standard MTT (3-(4,5-dimethylthiazol-2-yl)- 107 2,5-diphenyltetrazolium bromide) cellular viability assay as described elsewhere [18]. Briefly, 108 cells were seeded into 96 well plates of 1.5 x 103 cells per well and incubated for 24 hours at 109 37oC in an atmosphere of 5% CO2 before drug exposure. Generally, a stock solution was freshly 110 prepared by dissolving each of the compounds in DMSO at a concentration of 100 mM. The 111 highest concentration of drug tested was 100M and the final DMSO concentration applied to 112 cells was 0.1% (v/v), which is nontoxic to cells. Cisplatin was dissolved in phosphate-buffered 113 saline at a stock concentration of 25 mM. The cells were exposed to a range of drug 114 concentrations for 96 hours and cell survival was determined using the MTT assay [18, 19]. 115 Following drug exposure, 20 μL of MTT (0.5 mg/mL) in phosphate-buffered saline was added to 116 each well and it was further incubated at 37 °C for 4 hours in an atmosphere containing 5% CO2. 117 The solution was then removed and the formed formazan crystals were dissolved in 150 μM 5 118 DMSO. The absorbance of the resulting solution was recorded at 550 nm using an ELISA 119 spectrophotometer. The percentage of cell survival was calculated by dividing the true 120 absorbance of the treated cell by the true absorbance for controls (exposed to 0.1% DMSO). The 121 IC50 values presented in Table 1 were determined from plots of percentage survival against drug 122 concentration. Each experiment was performed in triplicate and a mean value obtained and stated 123 as IC50 (μM) ± SD. To compare the response of non-cancer cells to cancer cells, the selectivity 124 index (SI) presented in Table 2 was also calculated which is defined as the IC50 for ARPE 19 125 cells divided by the IC50 for each cancer cell line. Values >1 indicate that complexes have 126 selective activity against cancer compared to non-cancer cells in vitro. 127 2.4 In vitro antimicrobial evaluation 128 All the Gram-negative and Gram-positive bacterial strains used for the present study were 129 obtained from the Department of Microbiology, Osmania General Hospital, Hyderabad. All 130 strains were tested for purity by standard microbiological methods. The bacterial stock cultures 131 were maintained on Mueller-Hinton agar slants and stored at 4˚C. An agar-well diffusion method 132 [20] was employed for the evaluation of antibacterial activities of test compounds. DMSO was 133 used as a negative control. The bacterial strains were reactivated from stock cultures by 134 transferring into Mueller-Hinton broth and incubating at 37 ˚C for 18 h. A final inoculum 135 containing 106 colonies forming units (1 x 106 CFU/mL) was added aseptically to MHA medium 136 and poured into sterile petri dishes. Different test compounds at a concentration of 200 µg per 137 well were added to wells (8 mm in diameter) punched on an agar surface. Plates were incubated 138 overnight at 37 ºC and the diameter of inhibition zone (DIZ) around each well was measured in 139 mm. Experiments were performed in triplicates and these data were presented in Table 3. 140 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration 141 (MBC) was determined by the micro-broth dilution method done in 96 well plates according to 142 the standard protocol [21]. A 2-fold serial dilution of the compounds, with the appropriate 143 antibiotic, was prepared. Initially, 100 µl of MH broth was added to each well plate. Then 100 µl 144 of compound or antibiotic was taken from the stock solution and dissolved in the first well plate. 145 Serial dilution was done to obtain different concentrations. The stock concentrations of 2.0 146 mg/ml 24 hours culture turbidity was adjusted to match 0.5 McFarland standards which 147 correspond to 1×108 CFU/ml. The standardized suspension (100 µl) of bacteria was added to all 148 the wells except the antibiotic control well and the 96 well plates were incubated at 37 °C for 24 6 149 h. After 24 h of incubation 40 µl of MTT (3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltrazolium 150 bromide) reagent (0.1 mg/ml in 1x PBS) was added to all the wells. MIC was taken as the lowest 151 concentration which did not show any growth which was visually noted from the blue color 152 developed by MTT. Subcultures were made from clear wells and the lowest concentration that 153 yielded no growth after subculturing was taken as the MBC. The MIC and MBC values of tested 154 compounds were presented in Table 4. 155 2.5 Synthesis of rhodium and iridium dimer 156 In a sample test tube of size 10 ml 500 mg of RhCl3/IrCl3.nH2O, 0.4 ml of Cp* and 3 ml of 157 dry methanol were added and mix thoroughly. A small size Teflon coated magnetic stirrer was 158 inserted for stirring purpose. The mixture was sealed tightly and placed into an Anton Paar 159 Monowave 50 synthesis reactor. The reaction condition was adjusted by setting the temperature 160 to 110 oC and pressure will reach around 20 bars over 45 minutes. The instrument takes about 2- 161 3 minutes to heat up to the set temperature and the reaction proceeds smoothly for 45 minutes. 162 On completion, the reaction cools down to a temperature of 60 oC. A red-orange crystalline solid 163 was obtained. The solvent was decanted, washed three times with diethyl ether and air-dried. 164 Yield: 87% for Rhodium dimer and 90% for Iridium dimer 165 2.6 General procedure for the synthesis of metal complexes (1-8) 166 To a solution of metal precursor [(arene)MCl2]2 (arene = p-cymene / benzene) and 167 [Cp*MCl2]2 (M = Rh/Ir) complexes (0.1 mmol), pyridyl chalcone analogue (L1 and L2) (0.2 168 mmol) were added and stirred at room temperature in dry methanol (10 ml) for 5 hours (Scheme 169 1). The product starts to precipitate after 4 hours of stirring and was allowed to stir for another 170 one hour for the overall conversion of the reactant into the desired product. The solid precipitate 171 was centrifuged, washed thoroughly with diethyl ether and air-dried. 7 172 Scheme 1: Schematic representation for the synthesis of ligands and complexes 1-8 173 [(p-cymene)Ru(к1(N)-L1)Cl2] (1) 174 2.6.1 175 Color: Yellow; Yield: 81%; FT-IR (KBr, cm-1): 3406 (ʋN-H), 3291 (ʋN-H), 1651 (ʋC=O), 1616-1584 176 (ʋC=N); 1H NMR (400 MHz, DMSO-d6) δ 9.07 (d, J = 4 Hz, 1H), 8.68 (d, J = 4 Hz, 1H), 8.43 (d, 177 J = 8 Hz, 1H), 8.21 – 8.17 (m, 2H), 7.74 (d, J = 16 Hz, 1H), 7.57 (dd, J = 4, 4 Hz, 1H), 7.38 (t, J 178 = 8 Hz, 1H), 6.89 (d, J = 8 Hz, 1H), 6.68 (t, J = 8 Hz, 1H), 5.91 (d, J = 4 Hz, 2H), 5.87 (d, J = 4 179 Hz, 2H), 2.91 (sept, 1H), 2.17 (s, 3H), 1.27 (d, J = 8 Hz, 6H); ESI-MS (m/z): 459.21 [M-Cl]+- 180 HCl; UV-Vis {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 239 (4.896), 292 (5.588), 401 (2.013). 181 2.6.2 [(benzene)Ru(к1(N)-L1)Cl2] (2) 182 Color: Yellow; Yield: 78%; FT-IR (KBr, cm-1): 3421 (ʋN-H), 3312 (ʋN-H), 1650 (ʋC=O), 1615-1589 183 (ʋC=N); 1H NMR (400 MHz, DMSO-d6): δ 9.23 (s, 1H), 8.73 (d, J = 8 Hz, 2H), 8.23 (d, J = 16 184 Hz, 1H), 8.12 (d, J = 8 Hz, 1H), 7.83 (t, J = 8 Hz, 1H), 7.69 (d, J = 16 Hz, 1H), 7.30 (t, J = 8 Hz, 185 1H), 6.81 (d, J = 8 Hz, 1H), 6.59 (t, J = 8 Hz, 1H), 5.95 (s, 6H); 13C NMR (100 MHz, DMSO- 186 d6): δ = 189.6, 152.1, 144.7, 144.4, 140.7, 136.0, 134.7, 131.5, 127.7, 116.9, 114.5, 87.5; ESI- 187 MS (m/z): 403.03 [M-Cl]+-HCl; UV-Vis {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 240 (3.285), 188 291 (3.806), 402 (1.359). 189 2.6.3 [Cp*Rh(к1(N)-L1)Cl2] (3) 190 Color: Orange; Yield: 85%; FT-IR (KBr, cm-1): 3423 (ʋN-H), 3308 (ʋN-H), 1652 (ʋC=O), 1615-1584 191 (ʋC=N); 1H NMR (400 MHz, DMSO-d6) δ 8.92 (s, 1H), 8.49 (d, J = 4 Hz, 1H), 8.39 (d, J = 8 Hz, 192 1H), 7.97 (d, J = 16 Hz, 1H), 7.90 (d, J = 8 Hz, 1H), 7.50 (d, J = 8 Hz, 1H), 7.46 (d, J = 16 Hz, 193 1H), 7.09 (t, J = 8 Hz, 1H), 1.40 (s, 15H); ESI-MS (m/z): 461.24 [M-Cl]+-HCl; UV-Vis 194 {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 233 (4.831), 283 (3.675), 402 (1.540). 8 [Cp*Ir(к1(N)-L1)Cl2] (4) 195 2.6.4 196 Color: Yellow; Yield: 83%; FT-IR (KBr, cm-1): 3430 (ʋN-H), 3312 (ʋN-H), 1652 (ʋC=O), 1615-1583 197 (ʋC=N); 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.60 (d, J = 4 Hz, 1H), 8.37 (d, J = 8 Hz, 198 1H), 8.12-8.08 (m, 2H), 7.64 (d, J = 16 Hz, 1H), 7.50 (dd, J = 4, 4 Hz, 1H), 7.28 (t, J = 8 Hz, 199 1H), 6.80 (d, J = 12 Hz, 1H), 6.58 (t, J = 8 Hz, 1H), 1.62 (s, 15H); 13C NMR (100 MHz, DMSO- 200 d6 + CDCl3): δ = 198.75, 150.43, 134.00, 130.36, 124.51, 116.92, 114.89, 92.16, 85.18, 8.17; 201 ESI-MS (m/z): 551.30 [M-Cl]+-HCl; UV-Vis {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 235 202 (3.212), 290 (3.532), 398 (1.157). 203 2.6.5 [(p-cymene)Ru(к1(N)-L2)Cl2] (5) 204 Color: Yellow; Yield: 86%; FT-IR (KBr, cm-1): 3447 (ʋO-H), 1637 (ʋC=O), 1607-1581 (ʋC=N); 1H 205 NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 9.14 (s, 1H), 8.72 (d, J = 4 Hz, 1H), 8.47 (d, J = 8 206 Hz, 1H), 8.35 (d, J = 8 Hz, 1H), 8.26 (d, J = 16 Hz, 1H), 7.95 (d, J = 16 Hz, 1H), 7.68 (t, J = 8 207 Hz, 1H), 7.61 (t, J = 8 Hz, 1H), 7.13-7.10 (m, 2H), 5.91 (d, J = 4 Hz, 2H), 5.87 (d, J = 4 Hz, 2H), 208 2.91 (hept, J = 6.7 Hz, 1H), 2.17 (s, 3H), 1.27 (d, J = 8 Hz, 6H); ESI-MS (m/z): 460.23 [M-Cl]+- 209 HCl; UV-Vis {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 210 (6.895), 305 (6.406), 348 (2.558). 210 2.6.6 [(benzene)Ru(к1(N)-L2)Cl2] (6) 211 Color: Yellow; Yield: 78%; FT-IR (KBr, cm-1): 3445 (ʋO-H), 1647 (ʋC=O), 1585-1506 (ʋC=N); 1H 212 NMR (400 MHz, DMSO-d6): δ 12.37 (s, 1H, OH), 9.03 (s, 1H), 8.62 (d, J = 4 Hz, 1H), 8.25 (d, J 213 = 8 Hz, 1H), 8.12 (d, J = 12 Hz, 1H), 7.85 (d, J = 16 Hz, 1H), 7.57 (d, J = 8 Hz, 2H), 7.51 (dd, J 214 = 4, 4 Hz, 1H), 7.04-6.99 (m, 3H), 5.95 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ = 193.2, 215 161.7, 151.2, 150.5, 141.1, 136.4, 135.2, 130.9, 130.2, 128.2, 123.7, 120.7, 119.2, 117.7, 85.5; 216 ESI-MS (m/z): 404.05 [M-Cl]+-HCl; UV-Vis {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 201 217 (7.565), 305 (5.061), 348 (2.005). 218 2.6.7 [Cp*Rh(к1(N)-L2)Cl2] (7) 219 Color: Orange; Yield: 82%; FT-IR (KBr, cm-1): 3435 (ʋO-H), 1647 (ʋC=O), 1593-1489 (ʋC=N); 1H 220 NMR (400 MHz, DMSO-d6): δ 12.69 (s, 1H), 9.39 (s, 1H), 9.13 (s, 1H), 8.14 (d, J = 8 Hz, 1H), 221 8.03 (d, J = 8 Hz, 1H), 7.97 (d, J = 16 Hz, 2H), 7.87 (d, J = 16 Hz, 1H), 7.66 (t, J = 8 Hz, 1H), 222 7.57 (t, J = 8 Hz, 1H), 7.17 (d, J = 8 Hz, 1H), 7.10 (t, J = 8 Hz, 1H), 1.74 (s, 15H); UV-Vis 223 {Acetonitrile, λmax nm (ε/10-4 M-1 cm-1)}: 208 (6.325), 306 (5.466), 348 (2.361). 224 2.6.8 [Cp*Ir(к1(N)-L2)Cl2] (8) 9 225 Color: Yellow; Yield: 83%; FT-IR (KBr, cm-1): 3433 (ʋO-H), 1647 (ʋC=O), 1588-1491 (ʋC=N); 1H 226 NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 9.03 (s, 1H), 8.62 (d, J = 8 Hz, 1H), 8.37 (d, J = 8 227 Hz, 1H), 8.25 (d, J = 8 Hz, 1H), 8.16 (d, J = 16 Hz, 1H), 7.85 (d, J = 16 Hz, 1H), 7.58 (t, J = 8 228 Hz, 1H), 7.50 (dd, J = 4, 4 Hz, 1H), 7.03 – 7.00 (m, 2H), 1.62 (s, 15H); 13C NMR (100 MHz, 229 DMSO-d6 + CDCl3): δ = 193.19, 162.27, 150.96, 150.35, 141.19, 136.31, 135.15, 130.18, 230 123.70, 123.15, 118.90, 117.66, 91.96, 8.18; UV-Vis {Acetonitrile, λmax nm (ε/10-4M-1 cm-1)}: 231 210 (6.088), 305 (6.088), 348 (2.683). 232 3. Results and Discussion 233 3.1 Synthesis of complexes 234 In the present work, we have carried out the synthesis and biological activity evaluation of metal 235 complexes of ruthenium, rhodium and iridium bearing pyridyl chalcone derivatives. Pyridyl 236 chalcone derivatives (Scheme 1) were obtained following the procedure reported in the literature 237 [22]. Treatment of [(arene)Ru(µ-Cl)Cl]2, (arene = benzene, p-cymene), [Cp*M(µ-Cl)Cl]2 (M=Rh 238 and Ir) with ligand (L1 or L2) in 1:2 (M:L) ratio has yielded a series of neutral monodentate 239 mononuclear complexes 1-8 with the chemical formula [(arene)M{κ1(N)L1/L2}Cl2]. Despite 240 having extra binding sites of ligands towards metal, these ligands bind exclusively through 241 pyridyl nitrogen. Ruthenium and iridium complexes are yellow in color while rhodium 242 complexes are orange in color. These complexes are stable in the air as well as in solution and 243 they are soluble in MeOH and partially soluble in dichloromethane, chloroform and acetonitrile 244 and insoluble in solvents like hexane, diethyl ether and petroleum ether. The analytical data of 245 these compounds are consistent with the formulations. All complexes were fully characterized by 246 1H NMR, 13C NMR, IR, CHN and Mass spectroscopy. The molecular structure of the complexes 247 determined by single-crystal X-ray diffraction method, revealed the coordination of the metals to 248 the ligands (L1 and L2) only through the pyridyl nitrogen atom. 249 3.2 Spectral Studies of complexes (1-8) 250 3.2.1 Infrared Spectra (IR) of ligands and complexes 251 Information on the nature of the functional group attached to the metal atom was obtained 252 using IR spectroscopy. The ligand L1 IR spectrum displayed two bands at 3413 and 3284 cm-1 253 for the NH2 group while ligand L2 displayed one broadband at 3447 cm-1 for the OH group. Both 254 the carbonyl group of the ligands displayed a strong band at around 1648-1653 cm-1. All the 255 complexes exhibited characteristic bands corresponding to C=N, C=O, C=C, OH and NH2 10 256 stretching frequencies. The NH2 frequencies were observed in the range of 3150-3480 cm-1. The 257 C=N and C=C bond vibrated at a higher frequency region as compared to the free ligand around 258 1583-1616 cm-1 and 1450-1546 cm-1 indicating the coordination of the metals to the ligands 259 through the pyridine ring. 260 3.2.2 1H-NMR spectra of complexes 261 The 1H-NMR spectra of the complexes displayed signals associated with the ligand 262 protons and signals due to p-cymene and Cp* ring protons. The 1H NMR spectra of the ligand 263 L1 displayed one singlet at 9.40 ppm assigned to the CH proton of the pyridine ring, the NH2 264 proton signals were not observed due to solvent exchange with DMSO-d6, the trans-CH protons 265 with J = 16 Hz coupling constant were observed as a doublet at 7.68 and 8.26 ppm respectively 266 and the aromatic protons of the ligand L1 were observed in the downfield region around 6.55- 267 8.96 ppm. The 1H NMR spectra of the ligand L2 displayed one singlet at 12.68 ppm assigned to 268 the OH proton, a singlet at 8.89 ppm assigned to the CH proton of the pyridine ring, the trans-CH 269 protons with J = 16 Hz coupling constant were observed at 7.73 and 7.90 ppm respectively and 270 the aromatic protons of the ligand L2 were observed in the downfield region around 6.95-8.89 271 ppm. To further reveal the coordination behavior of these ligands to metals and the formation of 272 complexes, 1H NMR analyses of all these complexes were recorded in deuterated DMSO-d6 273 solvent at room temperature. The ligand aromatic protons of complexes 1-8 displayed the same 274 splitting pattern with a slight shift of proton signals towards the downfield or upfield region 275 which resulted from coordination of the metals to the ligand. The binding of the pyridyl chalcone 276 ligand to the ruthenium atom for complex 1 and 5 was confirmed by the distinct splitting of the 277 p-cymene ring protons upon coordination of the ligand to the p-cymene moiety. The signals 278 associated with the p-cymene ligand consisted of two doublets around 5.86-5.91 ppm for the ring 279 protons, one singlet at 2.17 ppm for the methyl protons and one doublet at 1.27 ppm for the 280 isopropyl group. Also, the methine proton of the p-cymene group exhibited septet at around 281 2.86-2.96 ppm. Ruthenium benzene complexes 2 and 6 displayed one singlet at 5.95 assigned to 282 the CH protons of the benzene ring. Rhodium and iridium complexes 3, 4, 7 and 8 bearing Cp* 283 analog displayed one singlet in the range of 1.40-1.74 ppm. The NH2 proton signals of the 284 complexes 1-4 were not observed due to solvent interaction with DMSO-d6 whereas the OH 285 proton signals of complexes 5-8 were observed in the range of 12.37-12.69 ppm. The 1H NMR 286 of all these complexes were given in supplementary data (Figure S1 to S10). 11 287 3.2.3 13 C-NMR spectra of complexes 288 The 13C-NMR spectra of the complexes further confirmed the formation and coordination 289 of the metals to the ligands. The 13C-NMR spectra of the complexes displayed signals associated 290 with the carbon of the ligands, the p-cymene moiety, and the Cp* group. The aromatic signals 291 for the ligand part of the complexes were observed in the range of 114.5-162.4 ppm. The 292 carbonyl group carbon of the complexes displayed a signal around 189.6-198.7 ppm. The ring 293 carbons of the p-cymene moiety displayed signals around 82.9-106.8 ppm whereas the methyl, 294 methine and isopropyl carbons signals were observed around 17.8-29.9 ppm. The benzene ring 295 carbons of the ruthenium benzene complexes displayed signals around 87.5-87.5 ppm. The Cp* 296 methyl carbons signal was observed around 8.1 ppm and the Cp* ring carbons signal was 297 observed at 91.9-92.1 ppm. These data are in good agreement with the previously reported 298 complexes done by our group [23] thus supporting the formation of the complexes. The 13C- 299 NMR spectra of some of the complexes were given in supplementary data (Figure S11 to S14). 300 3.2.4 Mass spectra of complexes 301 The mass spectra of the complexes further confirmed the formation of metal complexes. 302 The mass spectra of all the complexes except for complex 2 exhibited their predominant 303 molecular ion peaks at m/z value, which corresponds to [M-Cl]+-HCl ion peak. For instance, the 304 mass spectrum of complexes 1 displayed its predominant molecular ion peak at m/z: 459.21, 305 similarly for complexes 3, 4, 5 and 6 displayed their predominant molecular ion peak at m/z: 306 461.24, 551.30, 460.23 and 404.05 respectively which corresponds to [M-Cl]+-HCl ion peak. In 307 complex 2 the molecular ion peak was observed at m/z: 224.93, which is due to [M-Cl]+-HCl- 308 Ru-benzene ion peak. The mass ion peaks observed in these complexes are in accordance with 309 similar reported complexes [23]. The mass spectra of some of the complexes were given in 310 supplementary data (Figure S19 to S24). 311 3.2.5 UV-visible spectra of complexes 312 The electronic spectra of the monodentate mononuclear pyridyl chalcone complexes were 313 recorded in acetonitrile at 10-4 M concentration at room temperature and the respective plots are 314 shown in (Figure S25). The electronic spectra of the mononuclear complexes display two 315 absorption bands in the higher energy region around 230-240 nm which can be assigned as 316 ligand centered or intra ligand π-π* and low energy band at 290-305 nm corresponding to n-π* 317 transition [24, 25]. Medium intensity band at 340-410 nm were also observed corresponding to 12 318 metal to ligand charge transfer where from Ru (4d) orbitals to the low-lying empty orbitals π* 319 orbitals of the ligand. The low spin Rh(III) and Ir(III) complexes provide filled dπ (t2g) orbitals 320 which can interact with low lying π* orbitals of the pyridyl chalcone ligands and therefore we 321 can expect a band attributable to metal to ligand charge transfer MLCT band. 322 3.3. Description of the molecular structures of complexes 323 The solid-state structures of some of the mononuclear complexes were established by 324 single-crystal X-ray crystallography. The ORTEP view of the complexes along with the atom 325 numbering scheme is shown in Figure 1. The summary of the crystal data, data collection and 326 structure refinement parameters are summarized in Table S1. Selected bond lengths, bond angles 327 and metal atom involving ring centroid values are listed in Table S2. 328 Single crystal analyses for the complexes were carried out to have a deeper understanding 329 of the geometry of the complexes. By carrying out single-crystal analyses of the complexes we 330 were able to confirm the bonding modes associated with the ligand. Single crystals suitable for 331 X-ray diffraction analysis were obtained for complexes 2, 4, 5 and 7. These crystals are yellow, 332 orange and red in color and were obtained by the solvent diffusion method for all the complexes. 333 In all the complexes with both ligands, the preferable mode of coordination of the metal is 334 through pyridine nitrogen of the pyridyl chalcone forming monodentate mononuclear complexes. 335 All these half-sandwich complexes adopt a typical three-legged “piano-stool” geometry in which 336 the metal center is coordinated through one nitrogen donor atoms from the pyridine ring of the 337 ligand and two terminal chloride which represents the three-leg of a piano while the arene ring 338 (arene = p-cymene, benzene, Cp*) represent the seat of a piano. The molecular structure of these 339 mononuclear complexes strongly supports the formation of these complexes and the coordination 340 of ligands that occur through the pyridine nitrogen N(1) forming monodentate complexes (Figure 341 1). Complexes 2, 4 and 7 crystallizes in monoclinic system with P21/n and Cc space group while 342 complex 5 crystallizes in a triclinic system with P1̄ space group. The distance between the metal 343 to the centroid of the p-cymene/Cp* ring of complexes 2, 4, 5 and 7 is 1.657 Å, 1.779 Å, 1.667 Å 344 and 1.783 Å respectively. The M(1)-N(1) bond distances in these mononuclear complexes are in 345 the range of 2.116-2.146 Å. The M(1)-Cl(1) bond distances for complexes 2, 4 and 7 are in the 346 range of 2.410-2.4170 Å while that of complex 5 were found to be slightly longer at 2.4320(19) 347 Å. The M(1)-Cl(2) bond distances for complexes 2, 4 and 7 are in the range of 2.4167-2.439 Å 348 while that of complex 5 were found to be slightly shorter at 2.402(2) Å. These data are consistent 13 349 with the previously reported complexes of pyridyl-base ligands [26, 27]. The carbonyl (C=O) 350 bond distances for complexes 2, 4 and 7 are in the range of 1.228-1.247 Å, whereas complex 5 351 carbonyl bond distance is found to be slightly longer at 1.370(13) Å. The bond angle values 352 Cl(1)-M(1)-Cl(2) of these complexes are in the range of 86.99º-91.65º and the Cl(1)-M(1)-N(1) 353 and Cl(2)-M(1)-N(1) bond angle is also found to be in the range of 85.44º-88.22º which are 354 consistent with the piano stool arrangement of various groups about the metal center and is 355 comparable to previously reported values [26, 28]. 356 3.4. Non-covalent interaction 357 From the crystal packing of some of the complexes, it was observed that complex (2) 358 possessed intramolecular hydrogen bonding N-H∙∙∙∙∙O with 2.024 Å bond distance and 134.38º 359 bond angle. Non-covalent interaction C-H∙∙∙∙∙Cl with 2.774 Å bond distance and 143.76º was 360 observed between the CH group from the trans-CH=CH of ligand and chloride atom (Figure 2). 361 Interestingly the crystal packing in complex (2) formed a dimeric unit via π-π stacking (3.582 Å) 362 interaction known as supramolecular interaction. Complex 4, on the other hand, possessed 363 intramolecular hydrogen bonding N-H∙∙∙∙∙O with 2.010 Å bond distance and 128.93º bond angle 364 and intermolecular hydrogen bonding C-H∙∙∙∙∙Cl with 2.766 Å bond distance and 140.66º bond 365 angle. C-H∙∙∙∙∙ π interaction involving the methyl C-H of the cp* ring and the phenyl ring of the 366 ligand was also observed H3C∙∙∙∙∙H (4.861 Å) (Figure 3). Further, the crystal structure of complex 367 (5) crystallized with one CH3OH molecule which formed one intramolecular hydrogen bonding 368 O-H∙∙∙∙∙O with 1.804 Å bond distance and 146.61º bond angle and four different types of non- 369 covalent C-H∙∙∙∙∙Cl (2.852, 2.931, 2.613 and 2.884 Å) interactions between chloride of the metal 370 center and hydrogen atom from ligand and methanol molecule (Figure 4). Apart from these 371 interactions complex 5 displayed interaction between the solvent of crystallization (CH3OH) 372 oxygen and the hydrogen from the pyridine ring of the ligand forming inter hydrogen bonding 373 O∙∙∙∙∙H (2.613 Å). 374 3.5. 375 The response of cell lines to complexes 1-8, ligands 1-2 and cisplatin are presented in Figure 5. 376 Complexes 6-8 were inactive against all four cell lines at the highest dose tested (100 M) 377 whereas complex 5 and ligand 2 were moderately toxic to cells. In both cases, the activity of 378 complex 5 and ligand 2 was greater against ARPE-19 non-cancer cells (except for HCT-116 p53- 379 /- Cytotoxicity studies against cancer cell lines cells treated with ligand 2). This is reflected in the poor selectivity indices presented in Figure 14 380 6. In sharp contrast, ligand L1 and complexes 1-4 were potent cytotoxic agents with comparable 381 IC50 values to cisplatin. Except for complex 1, complexes 2-4 and ligand 1 showed comparable 382 or enhanced selectivity for cancer as opposed to non-cancer cells. Of particular interest is the 383 observation that HCT-116 p53-/- cells are less responsive to cisplatin than HCT-116 with wild 384 type p53 (Figure 5). In contrast, complexes 2-4 remain active against HCT-116 cells with 385 defective p53 and consequently retain their good selectivity index for these cancer cells 386 compared to ARPE-19 cells (Figure 6). As colorectal tumors with mutant or dysfunctional p53 387 are widely regarded as chemotherapy-resistant more aggressive cancers [29] this observation is 388 potentially significant and worthy of further investigation. In general the tested compounds for 389 anticancer activity against HCT-116 p53+/+, HCT-116 p53-/- and HT-29 human colorectal cancer 390 cell lines and were found that ligand L1 and its complexes 1-4 show excellent activity. Ligand 391 L2 and complex 5 displayed moderate activity whereas complexes 6-8 lacked activity even at a 392 higher dose (>100 µM). Furthermore, ligand L1 and complexes 2, 3 and 4 displayed a level 393 selectivity comparable to cisplatin. Mai et al. reported chalcone derivatives containing -NH2 394 group on ring A and phenyl group on ring B showed better cytotoxicity activity with IC50 = 395 4.39µM against HT-29 cancer cell [11]. However, in this work when ring A of chalcone 396 derivatives contain -NH2 group and ring B is replaced with pyridyl substituent an improved 397 cytotoxicity activity with IC50 = 2.10µM against HT-29 cancer cell was observed which is even 398 better than that of cisplatin. Hence, we can conclude that a slight change in the structure of the 399 compound can have a significant effect on the activities of the whole ligand and complexes. 400 Also, ligand L1 and ligand L2 differ only at ring A with R= NH2 for L1 and R= OH for L2 401 whereas the activity of L1 is 5 to 10 times more than that of L2. 402 3.6. In vitro antimicrobial activity of complexes and ligands 403 The synthesized ligands and complexes were evaluated for their in-vitro antibacterial 404 activity against gram-positive; Staphylococcus aureus and gram-negative; Escherichia coli, 405 Klebsiella pneumoniae strains by using standard techniques. The zones of inhibition (mm) in 406 comparison with ciprofloxacin were given in Table (3) and the chart representation was given in 407 Figure 7. All the compounds exhibited potent antibacterial activity against the tested organisms. 408 In-vitro assay results revealed that complex 5 (17 ± 0.58 mm) and complex 7 (16 ± 0.56 mm) 409 have better activity against gram-positive (Staphylococcus aureus). Complex 5 (17 ± 0.62 mm) 410 and complex 7 (16 ± 0.68 mm) also showed highest activity against gram-negative (Escherichia 15 411 coli) while complex 5 (16 ± 0.45 mm) and complex 7 (15 ± 0.35 mm) showed moderate activity 412 against gram-negative (Klebsiella pneumoniae). 413 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration 414 (MBC) results were listed in Table 4 and their chart representation was given in Figure 8. The 415 MIC & MBC values of the compounds ranged from 0.125 to 1.0 mg/mL against all three 416 organisms. Complex 3 and complex 7 values ranged from 0.125 to 0.25 mg/mL for S. aureus, E. 417 coli and Klebsiella pneumoniae. The MIC & MBC values of ciprofloxacin ranging from 0.031 to 418 0.062 mg/mL and 0.062 to 0.0125 mg/mL against the tested organisms were taken as standard. 419 4. Conclusion: 420 All together eight new complexes have been synthesized. All the complexes were isolated as 421 neutral compounds. The ligands and complexes were obtained in good yields and were 422 characterized by analytical and spectral methods. Single crystal X-ray diffraction study reveals 423 that the pyridyl chalcone ligands coordinated to the metal center only through the nitrogen atom 424 of the pyridine ring forming neutral monodentate complexes. Cytotoxicity studies against cancer 425 (HCT-116 p53+/+, HCT-116 p53-/- and HT-29 human colorectal cancer cells) and non-cancer 426 (ARPE-19 retinal epithelium) cells demonstrated that ligand L1 and its associated complexes 427 were more active compared to ligand L2 and its associated complexes. Furthermore, the 428 increased potency is accompanied by a significant increase in selectivity, particularly with 429 regards to HCT-116 cells with defective p53. Whilst potency and selectivity were comparable to 430 cisplatin, the fact that complexes 2-4 retained activity against HCT-116 p53-/- cells marks these 431 compounds as having different properties to cisplatin. Anti-bacterial studies for all the 432 compounds have also been carried out and they were found to be active against all the three 433 bacterial strains. 434 5. Acknowledgment 435 Lincoln Dkhar thanks SAIF-NEHU for spectral analyses and DST-PURSE SCXRD, India for 436 providing Single Crystal X-ray analysis. Lincoln Dkhar also thank the Department of Science 437 and Technology (DST), India for providing financial assistance under the Innovation in Science 438 Pursuit for Inspired Research (INSPIRE) fellowship. 439 6. Supplementary Material 16 440 CCDC 1908852 (2), 1908853 (3), 1908854 (5) and 1908855 (7) contains the supplementary 441 crystallographic data for this paper. These data can be obtained free of charge via 442 www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_request@ccdc.cam.ac.uk, or by 443 contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, 444 UK; Fax: +44 1223 336033. 445 7. References 446 [1] D.R. Williams, Chem. Rev., 72 (1972) 203–213. 447 [2] (a) A. Casini, C.G. Hartinger, A.A. Nazarov, P.J. Dyson, Top. Organomet. Chem., 32 448 (2010) 57. (b) P.J. Dyson, G. Sava, Dalton Trans., (2006) 1929. (c) A. Peacock, P.J. 449 Sadler, Chem. Asian J., 3 (2008) 1890. (d) L. Ronconi, P.J. Sadler, Coord. Chem. Rev., 450 251 (2007) 1633. (e) C.G. Hartinger, A.D. Phillips, A. Nazarov, Curr. Top. Med. Chem., 451 11 (2011) 2688. (f) C. Mu, S.W. Chang, K.E. Prosser, A.W.Y. Leung, S. Santacruz, T. 452 Jang, J.R. Thompson, D.T.T. Yapp, J.J. Warren, M.B. Bally, T.V. Beischlag, C.J. 453 Walsby, Inorg. Chem., 55 (2016) 177. (g) G.S. Smith, B. Therrien, Dalton Trans., 40 454 (2011) 10793. 455 [3] P. Zhang, P.J. Sadler, J Organomet Chem., 839 (2017) 5. 456 [4] (a) C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev., 38 (2009) 391. (b) G. Gasser, I. Ott, 457 N.J. Metzler-Nolte, Med. Chem., 54 (2011) 3. (c) A.F.A. Peacock, A. Habtemariam, R. 458 Fernandez, V. Wall, F.P.A. Fabbiani, S. Parsons, R.E. Aird, D.I. Jodrell, P.J. Sadler, J. 459 Am. Chem. Soc., 128 (2006) 1739. (d) R. Schuecker, R.O. John, M.A. Jakupec, V.B. 460 Arion, B.K. Keppler, Organometallics., 27 (2008) 6587. (e) I. Romero-Canelon, P.J. 461 Sadler, Inorg. Chem., 52 (2013) 12276. 462 [5] (a) G. Suss-Fink, Dalton Trans., 39 (2010) 1673. (b) C.S. Allardyce, P.J. Dyson, Platinum 463 Met. Rev., 45 (2001) 62. (c) M.J. Clarke, Coord. Chem. Rev., 236 (2003) 209. (d) B.T. 464 Loughrey, P.C. Healy, P.G. Parsons, M.L. Williams, Inorg. Chem., 47 (2008) 8589– 465 8591. 466 [6] H.K. Liu, P.J. Sadler, Acc. Chem. Res., 44 (2011) 349. 467 [7] (a) R.E. Morris, R.E. Aird, P.D. S. Murdoch, H. Chen, J. Cummings, N.D. Hughes, S. 468 Parsons, A. Parkin, G. Boyd, D.L. Jodrell, P.J. Sadler, J. Med. Chem., 44 (2001) 3616. 17 469 (b) O. Novakova, H. Chen, O. Vrana, A. Rodger, P.J. Sadler, V. Brabec, Biochemistry., 470 42 (2003) 11544. 471 [8] A. Kurzwernhart, W. Kandioller, S. Bachler, C. Bartel, S. Martic, M. Buczkowska, G. 472 Muhlgassner, M.A. Jakupec, H. B. Kraatz, P.J. Bednarski, V.B. Arion, D. Marko, B.K. 473 Keppler, C.G. J. Hartinger, J Med Chem., 55 (2012) 10512−10522. 474 [9] Madhu, RSC Adv., 6 (2016) 90982. 475 476 [10] M. Saleh, S. Abbott, V. Perron, C. Lauzon, C. Penney, B. Zacharie, Bioorg Med Chem Lett., 20 (2010) 945-949. 477 478 J. Pitchaimani, M.R.C. Raja, S. Sujatha, S.K. Mahapatra, D. Moon, S.P. Anthony, V. [11] C.W. Mai, M. Yaeghoobi, N.A. Rahman, Y.B. Kang, M.R. Pichika, Eur. J. Med. Chem., 77 (2014) 378. 479 480 [12] Crysalis P R O, release 2012 Version 1.171.36.20. Agilent Technologies, Yarnton. 481 [13] G.M. Sheldrick, (1997) SHELXL-97, Program for the Refinement of Crystal Structures. University of Göttingen, Germany. 482 483 [14] G.M. Sheldrick, Acta Crystallogr., (2015) C71 3. 484 [15] L.J. Farrugia, J. Appl. Crystallogr., 30 (1997) 565. 485 [16] L.J. Farrugia, J. Appl. Crystallogr., 32 (1999) 837. 486 [17] F. Bunz, A. Dutriaux, C. Lengauer, T. Waldman, S. Zhou, J. P. Brown, J. M. Sedivy, K. W. Kinzler, B. Vogelstein, Science., 282 (1998) 497–1501. 487 488 [18] Trans., 2012, 41, 13800. 489 490 [19] [20] [21] V. Banothu, C. Neelagiri, A. Adepally, J. Lingam, K. Bommareddy, Pharm Biol., 55 (2017) 1155-1161. 495 496 B. Venkanna, A. Uma, Ch. Suvarnalaxmi, N. Chandrasekharnath, R.S. Prakasham, L. Jayalaxmi, Curr. Trends Biotechnol. Pharm., 7 (2013) 782-792. 493 494 Z. Almodares, S.J. Lucas, B.D. Crossley, A.M. Basri, C.M. Pask, A.J. Hebden, R.M. Phillips, P.C. McGowan, Inorg. Chem., 53 (2014) 727. 491 492 S.J. Lucas, R.M. Lord, R.L. Wilson, R.M. Phillips, V. Sridharan, P.C. McGowan, Dalton [22] (a) A. Nie, J. Wang, Z.J. Huang, Comb. Chem., 8 (2006) 646-648. (b) N. Punna, K. 497 Harada, J. Zhou, N. Shibata, Org. Lett., 21 (2019) 1515-1520. (c) A.F.C. Ortiz, A.S. 498 Lopez, A.G. Rıos, F.C. Cabezas, C.E.R. Correa, J. Mol. Struct., 216 (2015) 1098. 18 499 [23] Chem. 32 (2018) 4362. 500 501 S. Adhikari, O. Hussain, R.M. Phillips, W. Kaminsky, M.R. Kollipara, Appl. Organomet. [24] P. Didier, I. Ortmans, A. Kirsch- De Mesmaeker, R. Watts, Inorg. Chem., 32 (1993) 5239-5245. 502 503 [25] B. Sullivan, D. Salmon, T. Meyer, Inorg. Chem., 17 (1978) 3334- 3341. 504 [26] C.M. Clavel, E. Paunescu, P. Nowak-Sliwinska, A.W. Griffioen, R. Scopelliti, P.J. Dyson, J. Med. Chem., 2015, 58, 3356− 3365. 505 506 [27] C.M. Clavel, E. Paunescu, P. Nowak-Sliwinska, A.W. Griffioen, R. Scopelliti, P.J. Dyson, J. Med. Chem., 2014, 57, 3546−3558. 507 508 [28] S. Adhikari, W. Kaminsky, M.R. Kollipara, J. Organomet. Chem., 848 (2017) 95-103. 509 [29] X.-L. Li, J. Zhou, Z.-R. Chen, W.-J. Chng, World J Gastroenterol., 21 (2015) 84–93. 510 19 Figure 1: ORTEP generated molecular structure of complexes 2, 4, 5 and 7 with 50% thermal ellipsoid probability. Hydrogen atoms (except on N2 and O2) are omitted for clarity purpose. 20 1 2 Figure 2: Crystal packing of complex 2 showing intramolecular and supramolecular interaction. 3 4 Figure 3: Crystal packing of complex 4 showing inter and intra molecular hydrogen bonding. 21 5 6 Figure 4: Crystal packing of complex 5 showing inter and intra molecular hydrogen bonding. 7 8 Figure 5: Response of a panel of cancer cells and ARPE-19 non-cancer cells following a 9 continuous exposure (96 hours) to complex 1 to 8, ligands 1 to 2 and cisplatin. Each value 10 represents the mean IC50 ± standard deviation for three independent experiments. Complex 6, 7 11 and 8 have IC50 values >100 µM which was the highest dose tested. 22 12 13 Figure 6: Selectivity ratios for complexes 1 to 5, ligands 1 and 2 and cisplatin. The selectivity 14 ratio is defined as the ratio of IC50 values for ARPE-19 divided by the IC50 for each cancer cell 15 line. Values greater than 1 (as indicated by the broken line) represent compounds that are 16 preferentially active against cancer cells compared to non-cancer ARPE-19 cells. As the 17 selectivity ratio is calculated using the mean IC50 values, no error bars are presented on this 18 figure. 19 20 Figure 7: Figure showing antimicrobial activity (Agar well) of the studied compounds 23 21 Figure 8: Figure showing MIB and MIC of tested compounds 22 23 24 Table 1: IC50 values of pyridyl chalcone (L1 and L2) and complexes 1-8 along with cisplatin 25 against HT-29, HCT-116 p53+/+, HCT-116 p53-/- cancer cell lines and non-cancer cell line 26 ARPE-19. Each value represents the mean ± standard deviation from three independent 27 experiments. Compounds 28 IC50 (µM) HT-29 HCT-116 p53+/+ HCT-116 p53-/ARPE-19 Ligand 1 2.10 (+/- 0.19) 4.44 (+/- 0.91) 4.56 (+/- 1.35) 11.21 (+/- 1.82) Ligand 2 54.18 (+/- 2.19) 43.83 (+/- 5.39) 28.06 (+/- 2.95) 39.39 (+/- 7.05) Complex 1 15.64 (+/- 0.88) 15.51 (+/- 1.64) 17.56 (+/- 4.04) 15.15 (+/- 4.49) Complex 2 3.35 (+/- 1.73) 3.44 (+/- 1.17) 2.17 (+/- 0.26) 6.18 (+/- 0.68) Complex 3 4.95 (+/- 0.86) 4.09 (+/- 1.38) 4.37 (+/- 1.45) 8.89 (+/- 4.18) Complex 4 4.82 (+/- 0.85) 4.23 (+/- 0.64) 5.17 (+/- 1.13) 7.35 (+/- 3.42) Complex 5 38.21 (+/- 3.42) 34.60 (+/- 1.73) 39.72 (+/- 3.28) 17.96 (+/- 1.55) Complex 6 >100 >100 >100 >100 Complex 7 >100 >100 >100 >100 Complex 8 >100 >100 >100 >100 Cisplatin 2.58 (+/- 0.72) 2.78 (+/- 1.4) 7.52 (+/- 0.65) 6.41 (+/- 0.95) NB - values at 100 = >100 indicates no IC50 at the highest dose tested on 100 µM 29 30 24 31 Table 2: Selectivity ratios of ligands and complexes along with cisplatin in HT-29, HCT- 32 116 p53+/+, HCT-116 p53-/- cancer cell lines. Compounds Selectivity Ratio (HT-29) Selectivity Ratio (HCT-116 p53+/+) Selectivity Ratio (HCT-116 p53+/+) Ligand 1 Ligand 2 Complex 1 Complex 2 Complex 3 Complex 4 Complex 5 Cisplatin 5.338 0.727 0.969 1.845 1.796 1.525 0.470 2.484 2.525 0.899 0.977 1.797 2.174 1.738 0.519 2.306 2.458 1.404 0.863 2.848 2.034 1.422 0.452 0.852 33 34 Table 3: Antibacterial activity (Agar well) of tested compounds S. No. Compound Names 1 Ligand-L1 Zone of inhibition (Diameter in mm) at conc. 200 μg S. aureus E. coli K. pneumoniae 14 ± 0.22 14 ± 0.18 13 ± 0.10 2 Ligand-L2 15 ± 0.35 15 ± 0.48 14 ± 0.15 3 Complex-1 15 ± 0.26 14 ± 0.16 14 ± 0.32 4 Complex-2 12 ± 0.12 13 ± 0.12 13 ± 0.23 5 Complex-3 14 ± 0.38 14 ± 0.24 14 ± 0.36 6 Complex-4 14 ± 0.26 13 ± 0.16 12 ± 0.08 7 Complex-5 17 ± 0.58 17 ± 0.62 16 ± 0.45 8 Complex-6 15 ± 0.35 16 ± 0.45 14 ± 0.22 9 Complex-7 16 ± 0.56 16 ± 0.68 15 ± 0.35 10 Complex-8 16 ± 0.29 15 ± 0.19 13 ± 0.15 11 Ciprofloxacin 32 ± 0.40 29 ± 0.10 31 ± 0.20 35 S. aureus = Staphylococcus aureus; E. coli = Escherichia coli; K. pneumoniae = Klebsiella 36 pneumoniae, NI: No Inhibition and Data are means (n = 3) ± Standard deviation of three 37 replicates. 38 25 39 Table 4: Antibacterial activity (MIC & MBC) of tested compounds Stock concentration in 2.0 mg/mL S. Compound S. aureus E. coli K. pneumoniae No. Names MIC MBC MIC MBC MIC MBC 1 Ligand-L1 0.25 0.5 0.25 0.5 0.25 0.5 2 Ligand-L2 0.5 1.0 0.25 0.5 0.5 1.0 3 Complex-1 0.25 0.5 0.25 0.5 0.25 0.5 4 Complex-2 0.5 1.0 0.25 0.5 0.25 0.5 5 Complex-3 0.125 0.25 0.125 0.25 0.125 0.25 6 Complex-4 0.25 0.5 0.125 0.25 0.125 0.25 7 Complex-5 0.25 0.5 0.25 0.5 0.25 0.5 8 Complex-6 0.5 1.0 0.25 0.5 0.25 0.5 9 Complex-7 0.125 0.25 0.125 0.25 0.125 0.25 10 Complex-8 0.25 0.5 0.25 0.5 0.125 0.25 11 Ciprofloxacin 0.062 0.125 0.031 0.062 0.062 0.125 40 S. aureus = Staphylococcus aureus; E. coli = Escherichia coli; K. pneumoniae = Klebsiella 41 pneumoniae. 42 26