Structural Determinants of p53-Independence in Anticancer

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Structural Determinants of p53-Independence in Anticancer Ruthenium-Arene Schiff-base Complexes Mun Juinn Chow, Maria V. Babak, Daniel Yuan Qiang Wong, Giorgia Pastorin, Christian Gaiddon, and Wee Han Ang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00348 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 16, 2016

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Molecular Pharmaceutics

Structural Determinants of p53-Independence in Anticancer RutheniumArene Schiff-base Complexes Mun Juinn Chow1, 2, Maria V. Babak1, Daniel Yuan Qiang Wong1, Giorgia Pastorin2, 3, Christian Gaiddon*4, 5 and Wee Han Ang*,1, 2 1

Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore

2

NUS Graduate School for Integrative Sciences and Engineering

3

Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 Singapore

4

U1113 INSERM, 3 Avenue Molière, Strasbourg 67200, France

5

Oncology section, FMTS, Université de Strasbourg, Strasbourg, France

KEYWORDS Ruthenium Arene Schiff-Base Complexes, Anticancer, Structure-Activity Relationship Studies, p53-independent activity.

1 Environment ACS Paragon Plus


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ABSTRACT p53 is a key tumor supressor gene involved in key cellular processes and implicated in cancer therapy. However, it is inactivated in more than 50% of all cancers due to mutation or overexpression of its negative regulators. This leads to drug resistance and poor chemotherapeutic outcome as most clinical drugs act via a p53-dependent mechanism of action. An attractive strategy to circumvent this resistance would be to identify new anticancer drugs that act via p53-independent mode-of-action. In the present study, we identified 9 Ru (II)-Arene Schiff-base (RAS) complexes able to induce p53-independent cytotoxicity and discuss structural features that are required for their p53-independent activity. Increasing hydrophobicity led to an increase in cellular accumulation in cells with a corresponding increase in efficacy. We further showed that all 9 complexes demonstrated p53-independent activity. This was despite significant differences in their physicochemical properties suggesting that the iminoquinoline ligand, a common structural feature for all the complexes, is required for the p53-independent activity.


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INTRODUCTION p53 is a key tumor supressor gene involved in the mediation of cellular DNA repair, cell cycle arrest, apoptosis-induction and neurotoxicity of anticancer drugs.1-2 It is inactivated in more than 50% of all cancers due to mutation or overexpression of p53 negative regulators.3 As a result, these cancers are more resistant to drugs that act via p53-dependent pathways. For instance, p53dependent drugs such as oxaliplatin (OXP) or doxorubicin (DOX) are less effective in inducing apoptosis in these cancer types even after causing significant DNA damage.4-5 Various strategies have been explored for more effective treatment of cancers with defective p53 function. One such strategy involves the resensitization of these cancers to chemotherapy by restoring p53 activity. This could be done through (i) the inhibition of the interaction between p53 and its negative regulators or (ii) restoring misfolded mutant p53, using targeted small molecules or selective peptide sequences.6 However, such treatment is often limited by the off-target effects, low efficacy or development of resistance towards these ‘p53-activators’.6-7 A more attractive strategy would be to identify new anticancer candidates that are able to bypass this resistance mechanism entirely by inducing p53-independent antiproliferation. The success of Ru (III)-containing compounds such as KP1019, which received favorable evaluations in preclinical studies,8-9 has lead to recent interest in the development of more active Ru (II) complexes as anticancer agents.10-12 Some prominent examples of cytotoxic Ru (II) complexes that have undergone preclinical studies are RAPTA-C, RM175 and RDC11.13-16 However, many of these Ru (II) complexes have p53-dependent activity and are subjected to the same resistance mechanism of p53-mutated cancers. Previous studies indicated that RAPTA-C, RM175 and RDC11 induced p53 and its target genes to various degrees.17-19 Many other reported Ru (II) complexes also exert their antiproliferation effect via p53-dependent pathways.20-26 In



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contrast, only a limited number of Ru (II) complexes have been reported to demonstrate p53independent activity (Fig 1). Several Ru (II)-arene complexes bearing azopyridine, iminopyridine or chloroquine ligand, and a imidazole-bearing phenanthroline Ru (II) complex demonstrated similar toxicity in both wild type colorectal carcinoma HCT116 and p53-null lineage.27-29 Another Ru (II)-arene complex bearing iminophosphorane ligand induced apoptotic cell death without p53-induction.30 Other metal complexes of Pt and Os have also been shown to act via p53-independent mechanism.27, 31-33 In all cases, the metal centre has little or no influence on the p53-dependence of these complexes; the p53-independence of these complexes could often be ‘switched on’ or ‘switched off’ by structural modification to the ligands.27, 33 In light of this, a greater understanding of the structural determinents of p53-independence could lead to the identification of new organoruthenium compounds with p53-independent activity, adding to the limited pool of drugs for the effective treatment of resistant cancers with mutated p53 status. Previously, a Ru (II)-Arene Schiff-base (RAS) complex bearing 1,3,5-triisopropylbenzene (TPB) and iminoquinoline ligands, RAS-1T, was identified as a promising lead cytotoxic compound with an unique mode-of-action.34 Preliminary investigations suggested that RAS-1T acted via a mechanism distinct from classical alkylating agents including cisplatin (CDDP) and other reported anticancer Ru (II) complexes. RAS-1T did not induce p53 overexpression and was equipotent in epithelial cells BJwt (p53+/+) and BJshp53 (p53-/-). Further investigation of RAS-1T and its hexamethylbenzene (HMB) analogue, RAS-1H, revealed that both compounds induced non-apoptotic cell death via ER-stress pathways but the pathways were different for each compound.35 In this work we report a structure-activity relationship study on an expanded set of RAS complexes that are structurally related to RAS-1H (4) and RAS-1T (5), aimed to (i) investigate


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if p53-independent activity is a general feature of this class of iminoquinoline-containing Ru (II) complexes and (ii) identify the key structural features that lead to their p53-independence. We further discuss their activity against a panel of colorectal (HCT116 and SW480) and gastric (AGS and KATOIII) cancer cells, the influence of the arene and chelating ligands on RAS complexes’ stability to aquation and biological nucleophiles, as well as intracellular accumulation.

RESULTS & DISCUSSION Synthesis and Characterization of RAS Complexes RAS complexes 1 – 9 (Fig 2) were synthesized using the general reaction route as shown in Scheme 1. Recently, we reported the synthesis and characterization of 4, 5 and 9.34-35 2Quinolinecarboxaldehyde and different aniline derivatives were reacted in dry EtOH or MeOH to give the bidentate imine ligand with varying degree of purity. Most of the imine ligands could not be isolated as pure compounds due to the facile hydrolysis of the imine bond and the crude product was used directly for synthesis of the RAS complexes by addition of stoichiometric amount of the corresponding [Ru(η6-arene)Cl2]2 in MeOH. Complexes 1 – 9 were further purified and isolated via flash column chromatography. In addition, we report an improved synthetic protocol of 8 and 9 that did not require purification by column chromatography. Briefly, the imine ligand was prepared from 2-quinolinecarboxaldehyde and excess 3chloroaniline in toluene using a Dean-Stark apparatus and the crude ligand treated directly with [Ru(η6-arene)Cl2]2 in MeOH. The product was filtered through a short plug of neutral alumina



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and recrystallized using vapour diffusion of diethyl ether into a saturated CH2Cl2 solution at 4 ºC. The 1H NMR spectra of 1 – 9 showed resonances typical of RAS complexes.34 The disappearance of the singlet peak corresponding to quinolinecarboxaldehyde at ca. 10.2 ppm and the appearance of the peak corresponding to the imine proton at ca. 8.5-9.5 ppm indicated the formation of the chelate ligand. The desymmetrization of the Ru complex due to chelation resulted in additional splitting pattern of the arene proton signals (arene = toluene, cymene, TPB). ESI-MS spectra of these compounds also showed the characteristic [M]+ molecular ion peaks with Ru and Cl isotopic pattern. The purity of the complexes was confirmed by either elemental analysis (EA) or RP-HPLC to be >95%. Solid-state structural information was obtained for 4 and 9 via single crystal X-ray diffraction studies (Fig 3) and selected structural data are given in Table 1 and Table 2. Single crystals were obtained via slow diffusion of diethyl ether into saturated CH2Cl2 solution at 4 ºC. RAS complexes 4 and 9 adopted the classical ‘piano-stool’ structure similar to most reported Ru(II)arene complexes with some notable deviations.26, 30, 36 The Ru-Carene bond ranges for 4 and 9 of 2.196-2.283 Å and 2.175-2.280 Å, respectively, are longer than RM175 (biphenyl) and RAPTAC (cymene) of 2.161-2.231 Å and 2.180-2.262 Å, respectively, presumably due to the sterically encumbered HMB ligand. All corresponding bond length and angles between 4 and 9 were similar, suggesting that changing the 4-OMe group to a 3-Cl group did not alter the core RAS structure. The conformation adopted by the iminoquinoline ligands could be described by the torsion angle θC22-N2-C23-C24 of 37.6(9)˚ and 34.6(6)˚ for 4 and 9, respectively, disrupting the extended conjugation within the chelate ligand. This twisted conformation was presumably due


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to the steric bulk of the arene ligand. The same structural feature was observed in the recently reported RAS complexes.34

Effect of structural variation on stability and Log POW Aqueous stability and Log POW are important factors that affect a potential drug’s bioavailability and cell permeability,37 which can potentially influence the molecular target and mode-of-action. To determine the stability of the 9 RAS complexes under investigation towards aquation and biological nucleophiles, we monitored their UV-Vis profile in double distilled H2O (ddH2O) and Hanks Balanced Salt Solution (HBSS) containing 10% fetal bovine serum (FBS) over 24 h. Any reaction would manifest as a shifts in the UV-Vis profile, producing isosbestic point(s) in the overlapping spectrums at different time points. The investigated RAS complexes were generally stable in water (Fig S1). Slight shifts in the spectrums and the formation of several isosbestic points were observed for 6, 7 and 8 after 24 h in water, which suggests that the aquation of the Ru-Cl bond in 6, 7 and 8 occurred very slowly in the absence of other competing nucleophiles at r. t.. Although all RAS complexes could be considered to be resistant to aquation in water, only some could be considered stable towards interfering nucleophiles as seen in their stability in serum-containing HBSS (Fig S2). This is particularly well illustrated by focusing on the slight variations implemented when comparing 4, 5, 8 and 9 (Fig 4). In general, compounds with more π-donating arene (HMB, TPB) and less π-acidic iminoquinoline (-OMe) ligands stabilized the Ru-Cl bonds making it less susceptible towards substitution. For instance, 5 was stable in HBSS with 10% FBS. However, changing the 4-OMe to the more π-acidic 3-CF3 or 3-Cl group (7 and



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8) labilised the Ru-Cl bond. Replacing the TPB arene with the more π-donating HMB in 9 resulted in a more stable complex resistant to Ru-Cl aquation. Furthermore, it cannot be ruled out that compounds bearing labile benzene or toluene arene ligands (1 and 2) decomposed in serumcontaining buffer due to the displacement of arene ligand by biological nucleophiles (Fig S2). Log POW of 1 – 5 and 9 were determined using the shake-flask method.38 Log POW of 6, 7 and 8 could not be determined as their spectra were significantly shifted after the partitioning and phase separation (data not shown), presumably due to non-specific interactions with the noctanol phase. The trend in the Log POW studies were as expected, where RAS complexes with more hydrophobic ligands displayed higher Log POW values (Table 3).

Effects of ligand structural variations on cytotoxicity profile To determine the effects of structural variation to cytotoxicity, we obtained the IC50 values of the nine RAS complexes in colorectal cancer cell lines HCT116 and SW480, and gastric cancer cell lines AGS and KATOIII (Table 3). In general, RAS complexes bearing the more hydrophobic HMB and TPB arene ligands demonstrated the highest cytotoxicity in the cell lines, with IC50 values in the low µM range. Complex 5 displayed the greatest efficacies with IC50 ca. 1.0 µM in HCT116, AGS and KATOIII and 4.1 µM in SW480, which was 34- and 7-fold more cytotoxic than CDDP in AGS and KATOIII, respectively. For RAS complexes with 4-OMe (1 – 5), IC50 values decreased with the increasing hydrophobicity of the arene ligand, from the least hydrophobic benzene in 1 to the most hydrophobic TPB in 5. Interestingly, starting from complex 3 in the reverse order, the IC50 became higher in HCT116 and AGS versus SW480 and


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KATOIII, suggesting that hydrophobicity also affected selectivity between different cancer types. Both 4 and 9 demonstrated biphasic dose-response curve in AGS and KATOIII cells, respectively. AGS cell viability decreased with increasing concentration of 4 to reach a plateau of 40% between 5 µM and 20 µM before dropping drastically to 0% above 20 µM (Fig 5a).34 A similar biphasic profile was seen in KATOIII cells treated with 9 (Fig 5b). In contrast, the TPB analogues (5 and 8) did not display such biphasic dose-response profiles. The basis for this unique concentration-dependent mechanism of antiproliferative activity induced by HMBbearing RAS complexes (4 and 9) remains to be elucidated. This biphasic dose-response was not observed in benzene, toluene, cymene or TPB RAS analogues. Few reported anticancer compounds exhibit such biphasic dose-response profiles. One such compound is anti-tubule taxane Docetaxal, which induced mitotic catastrophe at lower doses and p53-independent apoptosis at higher doses in prostate cancer (PC3, DU145) and breast cancer cell lines (MCF7, MDA-MB-231).39-42 A similar concentration-dependent dual mode-of-action for 4 and 9 would be highly conceivable.

RAS complexes accumulate in cells mainly by passive diffusion One possibility for the higher cytotoxicity of compounds bearing hydrophobic arene ligands could be attributed to better cellular uptake via passive diffusion.43 More efficient cellular accumulation of the RAS compounds may result in a higher efficacy. The RAS complexes evaluated were considered hydrophilic compounds by virtue of their Log Pow values. We investigated whether varying the hydrophobicity of the arene ligands (1 – 5) affected cellular



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uptake in HCT116 cells, with 6 and OXP for comparison. Cell samples were centrifuged, washed, and digested separately in concentrated HNO3 before their Ru/Pt contents were analyzed by ICP-MS (Table S1). There was a positive correlation between hydrophobicity, cellular accumulation and cytotoxicity for 1 – 5 (Fig 6a-b). When treated at the same concentration of 1.5 µM, the most hydrophobic complex 5 accumulated 7.5-times more than 1, the least hydrophobic complex. Interestingly, complex 6 accumulated 3 times less than complex 4 but had a higher cytotoxicity. Hence, with the exception of complex 6, the higher uptake also corresponded with lower IC50 suggesting that uptake was partially responsible for the higher cytotoxicity of the more hydrophobic RAS complexes. In addition, there was an interesting abrupt improvement of drug intake at a Log Pow just above -1.5, suggesting that this value might be considered as a goal for designing cytotoxic Ru (II) arene compound. Since the RAS complexes are cationic and bear hydrophobic ligands, we investigated if organic cation transporters (OCT) could be responsible for their activity in colorectal cancer in a similar manner as OXP. OXP was effective in the treatment of colorectal cancer compared to CDDP and attributed to the fact that OXP was a substrate for OCT1/2 and would be more efficiently accumulated in OCT-overexpressing colorectal cancer cells.44 As the RAS complexes are positively charged and contained hydrophobic ligands, they could possibly be substrates of OCTs in a similar fashion to OXP. Hence HCT116 cells were treated with 5 and 6 for 7 h in the presence and absence of OCT2-inhibitor cimetidine and compared to OXP as a positive control. Cimetidine did not affect the viability of cells treated with 5 and 6, suggesting that both are not substrates of OCT2 (Fig 7a-b). In contrast, the viability of cells treated with OXP increased in the presence of cimetidine (Fig 7c), indicating reduced uptake of the cytotoxic OXP. Taken


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together, the data suggested that RAS complexes accumulated in the cell mainly via passive diffusion. A general trend indicated that increasing hydrophobicity resulted in increased cellular uptake and higher toxicity, which was characteristic of entry by passive diffusion. However, exception such as in complex 6 indicated that additional mechanisms and physicochemical determinants might exist and could participate in the uptake process. Although, OCT transporters were not involved in the uptake of RAS complexes, the possibility that other energy-dependent uptake pathways are involved in the cellular accumulation of RAS complexes cannot be excluded.

Structural determinant of p53-Independent mode-of-action We determined previously that 4 and 5 induced cell death in AGS and HCT116 cells without concomitant induction of p53.34-35 To further verify the p53-independence of the RAS complexes, we evaluated the expression of p53 as well as downstream, cell cycle-arresting and apoptosis-associated biomarkers in treated HCT116 cells. Cells were exposed to 4, 5, 8 and 9 for 2 h, 6 h and 24 h at their IC50 and IC75 values. In addition, their activities were probed in the absence and presence of p53-inhibitor pifithrin-α. OXP was used as a positive control. Western blot analysis was performed against p53 and its targets, cell cycle-regulating cyclin D1 and p21 (Fig 8a).45-47 In general, there was a lack of induction of these biomarkers by 4, 5, 8 and 9 although p53-independent induction of p21 was observed with 4, 5 and 9 after 24 h exposure.48 In contrast, OXP exhibited a concentration and time-dependent upregulation of p53, cyclin D1 and p21. RT-qPCR analyses were performed on p53-regulated anti-apoptotic gene BCL2 and pro-apoptotic genes NOXA and BAX (Fig 8b).49-51 Complexes 4 and 9 induced a slight



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increase in NOXA expression at the later time points (6 h and 24 h) while 5 and 8 did not cause significant change in NOXA expression. Cells treated with 4, 5, 8 and 9 also induced negligible change in expression of BAX and BCL2. In contrast, OXP treatment at IC75 led to 3-fold induction of NOXA, 5.5-fold increase in BAX and a significant reduction in BCL2 after 24 h exposure. To demonstrate functionally that the cytotoxic activities of 4, 5, 8 and 9 were independent of p53, cells were co-incubated with pifithrin-α. Co-treatment of 4, 5, 8 and 9 with pifithrin-α improved the efficacies of RAS complexes while in the case of OXP, co-treatment incurred a 3-fold increase in IC50 suggesting that OXP exerted its cytotoxicity via a p53dependent pathway. The lack of significant induction of p53 and its downstream targets implied that these RAS complexes exerted their antiproliferative effects via p53-independent pathways. On the other hand, OXP significantly increased the expression of p53, cyclin D1, p21, NOXA and BAX and decreased the expression of BCL2. This was in agreement with previous studies, which showed that oxaliplatin induced p53-mediated cycle arrest and (to a smaller degree) p53-mediated apoptosis in HCT116 cells.52-53 Furthermore, p53 inhibition with pifithrin-α did not adversely affect RAS cytotoxicities compared to OXP. This further confirmed that the RAS complexes acted via a p53-independent mode-of-action. To ascertain that the p53-independent activity is a general characteristic of this class of iminoquinoline-containing RAS complexes, we measured p53 expression following 24 h treatment at IC50 concentrations for the remaining complexes 1, 2, 3, 6 and 7 as well as measured viabilities of treated cells after 48 h in the absence and presence of pifithrin-α. Likewise, OXP was used as a positive control. There was a lack of p53 induction by these RAS complexes and their activities not negatively impacted by the presence of pifithrin-α, as seen by the similar or


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lower cell viabilities in cells co-treated with pifithrin-α (Fig S3), in keeping with the other RAS complexes. In general, all RAS complexes tested in this studies demonstrated p53-independent activity, even though their physicochemical properties such as stability to nucleophiles and hydrophobicity differed significantly. A similar class of Ru (II) arene complexes bearing iminoand azo-pyridine ligands (Fig 1; top-right) has been shown to demonstrate p53-independent activity only when the chloride ligand was substituted with iodide.27 It was reported that the choice of halide ligand strongly influenced the p53-dependence on their class of compound. The key difference with RAS complexes was that they demonstrated p53-independent activities without the need for iodide substitution. The iminoquinoline and Ru (II)-arene functionalities common to all 9 RAS complexes were crucial for the observed p53-independence cytotoxicity and structural tuning at the non-halide sites could be used to fine-tune their antiproliferative modes-of action. CONCLUSION The current study gives insights to the structural determinants of cytotoxicity and p53independence in Ru (II)-arene complexes through a detailed chemical and in vitro SAR study of 9 RAS complexes. In general, the more hydrophobic complexes displayed higher cellular accumulation and cytotoxicity across a panel of cancer cell lines. In addition, the lack of OCT involvement in the uptake of RAS complexes suggested that entry occurred mainly via passive diffusion, despite the fact that these complexes were hydrophilic in nature. RAS complexes also displayed tolerance towards aquation, although analogues with benzene or toluene ligands were significantly less stable towards biological nucleophiles as these arenes were much more prone



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to displacement. These complexes also exerted their anticancer activities via p53-independent pathways as seen in the lack of activation of p53 and downstream targets, and their unchanged activity in the presence of small molecule p53-inhibitors. Compounds such as this may have important implication in the treatment of resistant cancers with mutated p53 or p53-null status.

EXPERIMENTAL Materials. All experimental procedures were carried out without additional precautions to exclude air or moisture unless otherwise specified. All chemicals and solvents were used as received unless otherwise specified. Dry ethanol and methanol were obtained by drying them over molecular sieves 3-4 Å 24 h before use. RuCl3.xH2O was purchased from both Precious Metals

Online.

[(η6-benzene)RuCl2]2,

hexamethylbenzene)RuCl2]2

and

[(η6-toluene)RuCl2]2,

[(η6-cymene)RuCl2]2,

[(η6-1,3,5-triisopropylbenzene)RuCl2]2 were

[(η6-

synthesized

according to previously reported protocols.43, 54-56 Thiazolyl blue tetrazolium bromide (MTT), Trizma® Base BioUltra, Nonidet P-40, DL-Dithiothreitol, Non-fat Dried Milk Bovine, TWEEN® 20, Ponceau S and Cimetidine, were purchased from Sigma-Aldrich. All other chemicals used were purchased from Sigma-Aldrich (Singapore). Ultrapure water used was purified by a Milli-Q UV purification system (Sartorius Stedim Biotech SA). Gibco® Versene solution, Gibco® Trypsin/EDTA solution, 10% SDS solution, Penicillin-Streptomycin (10 000 U/mL), Dulbecco's Phosphate-Buffered Saline (10x), TRIzol® Reagent and Applied Biosystem® High Capacity cDNA Reverse Transcription Kit were purchased from Life Technologies. HycloneTM RPMI 1640, DMEM medium and Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific Inc. Bio-rad Protein Assay Dye Reagent Concentrate,


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40% Acrylamide/Bis solution, 10x Tris/glycine buffer, TEMED, 4x Laemmli Sample Buffer, Nitrocellulose Membrane, 0.2 µm and 0.45 µm were purchased from Bio-rad Laboratories. cOmplete, mini Protease Inhibitor Cocktail Tablets, RNase A, FastStart Universal Probe Master (Rox) were purchased from Roche Diagnostics. LuminataTM Classico Crescendo Western HRP Substrate were purchased from Merck Millipore Corporation. Instrumentation. 1H NMR spectrums were obtained using either a Bruker AMX 300, Avance 400 or AMX 500 spectrometer and the chemical shifts (δ) were reported in parts per million with reference to residual solvent peaks. Electrospray-ionization Mass Spectrometry (ESI-MS) spectra were obtained using Thermo Finnigan MAT ESI-MS System. UV-vis spectra were obtained using the Shimadzu UV-1800 UV Spectrophotometer with a TCC-240A Temperate Controlled Cell Holder. Ru concentrations were determined using the Optima ICP-OES (Perkin-Elmer) operated by CMMAC, NUS. Elemental analyses of selected Ru complexes were carried out using a Perkin-Elmer PE 2400 elemental analyzer by CMMAS, NUS. HPLC analysis of compound purity. Determination of the purity of the RAS Complexes were done using analytical HPLC on a Shimadzu Prominence System equipped with a DGU-20A3 Degasser, two LC-20AD Liquid Chromatography Pump, a SPD-20A UV/Vis Detector and a Shim Pack GVP-ODS 2.0 mm 18 column (5 µM, 120Å, 250 mm x 4.60 mm i.d.) at r.t. at a flow rate of 1.0 mL/min with detection at both 214 nm and 254 nm. The gradient elution conditions were as follows: 20-80% solvent B over 30 min, where solvent A is 10 mM NH4OAc pH 7.0 and solvent B is CH3CN. Synthesis of 1, [(η6-benzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 2Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and p-anisidine (61.5 mg, 0.5 mmol) was added



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to dry EtOH (10 mL) and and stirred at r.t. over 24 h. The solvent was removed in vacuo and dried. The resultant crude 4-methoxy-N-(2-quinolinylmethylene)-aniline (94 mg, approx. 0.36 mmol) was treated with [(η6-benzene)RuCl2]2 (89.6 mg, 0.18 mmol) in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo and the resulting solid purified by gradient elution column chromatography (1:1 v/v EtOH/CHCl3, Rf = 0.2; EtOH, Rf = 0.5). The purified product was redissolved in CHCl3 and filtered through a syringe column to remove trace amount of dissolved silica, before the solvent was removed in vacuo. The final product was then dried in vacuo for 1 h to give a dark red solid. Yield: 40 mg (22%). 1H NMR (400 MHz, D2O): δ 8.68 (d, 3J= 9 Hz, 1H, quinolinyl), 8.57 (s, 1H, PhN=CH), 8.35 (d, 3JHH = 8 Hz, 1H, quinolinyl), 8.07 (t, 3JHH = 8 Hz, 1H, quinolinyl), 8.01 (d, 3JHH = 8 Hz, 1H, quinolinyl), 7.87 (t, 3JHH = 8 Hz, 1H, quinolinyl), 7.81 (d, 3JHH = 8 Hz, 1H, quinolinyl), 7.74 (d, 3JHH = 9 Hz, 2H, C6H4OMe), 7.05 (d, 3JHH = 9 Hz, 2H, C6H4OMe), 5.83 (s, 6H, C6H6), 3.88 (s, 3H, OCH3) ppm. ESI-MS (+ve mode): m/z = 477 [M]+. ). Purity of the complex was determined to be >95% pure by RP-HPLC (% Purity): 95.8% at 214 nm and 96.4% at 254 nm; tr = 12.5 min. Synthesis of 2, [(η6-toluene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 2 was obtained with [(η6-toluene)RuCl2]2 (94.6 mg, 0.18 mmol) and crude 4-methoxy-N-(2quinolinylmethylene)-aniline (94 mg, approx. 0.36 mmol) using the same protocol as used for 1 (Gradient elution: 1:4 v/v EtOH/CHCl3, Rf = 0.2; EtOH, Rf = 0.7). The final product was then dried in vacuo for 1 h to give a dark red solid. Yield: 65 mg (35%). 1H NMR (400 MHz, DMSOd6): δ 9.16 (s, 1H, PhN=CH), 8.89 (d, 3JHH = 8 Hz, 1H, quinolinyl), 8.76 (d, 3JHH = 9 Hz, 1H, quinolinyl), 8.31 (t, 3JHH = 8 Hz, 2H, quinolinyl), 8.15 (t, 3JHH = 7 Hz, 1H, quinolinyl), 7.99 (m, 3H, quinolinyl /C6H4OMe), 7.22 (d, 3JHH = 9 Hz, 2H, C6H4OMe), 6.07 (t, 3JHH = 6 Hz, 1H, C6H5), 6.01 (d, 3JHH = 6 Hz, 1H, C6H5), 5.81 (t, 3JHH = 6 Hz, 1H, C6H5), 5.71 (t, 3JHH = 6 Hz, 1H,


Page 17 of 40

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C6H5), 5.37 (d, 3JHH = 6 Hz, 1H, C6H5), 3.90 (s, 3H, ArOCH3), 2.17 (s, 3H, CH3) ppm. ESI-MS (+ve mode): m/z = 491 [M]+. Purity of the complex was determined to be >95% pure by RPHPLC (% Purity): 98.1% at 214 nm and 95.8% at 254 nm; tr = 14.4 min. Synthesis of 3, [(η6-cymene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 3 was obtained with [(η6-cymene)RuCl2]2 (109.72 mg, 0.179 mmol) and crude 4-methoxy-N-(2quinolinylmethylene)-aniline (94 mg, approx. 0.358 mmol) using the same protocol as used for 1 (Gradient elution: 1:4 v/v EtOH/CHCl3, Rf = 0.3; 1:1 v/v EtOH/CHCl3, Rf = 0.5). The final product was then dried in vacuo for 1 h to give a dark orange solid. Yield: 125 mg (61%). 1H NMR (400 MHz, DMSO-d6): δ 9.15 (s, 1H, PhN=CH), 8.91 (d, 3JHH = 8 Hz, 1H, quinolinyl), 8.73 (d, 3JHH = 9 Hz, 1H, quinolinyl), 8.32 (t, 3JHH = 7 Hz, 2H, quinolinyl), 8.17 (t, 3JHH = 7 Hz, 1H, quinolinyl), 8.01 (t, 3JHH = 7 Hz, 1H, quinolinyl), 7.96 (d, 3JHH = 9 Hz, 2H, C6H4OMe), 7.23 (d, 3JHH = 9 Hz, 2H, C6H4OMe), 6.11 (d, 3JHH = 6 Hz, 1H, i-PrC6H4Me), 5.89 (d, 3JHH = 6 Hz, 1H, i-PrC6H4Me), 5.75 (d, 3JHH = 6 Hz, 1H, C6H4), 5.39 (d, 3JHH = 6 Hz, 1H, C6H4), 3.91 (s, 3H, ArOCH3), 2.32 (sept, 1H, 3JHH = 7 Hz, CH(Me)2), 2.24 (s, 3H, i-PrArCH3), 0.90 (d, 3JHH = 7 Hz, 3H, CH(CH3)2), 0.74 (d, 3JHH = 7 Hz, 3H, CH(CH3)2) ppm. ESI-MS (+ve mode): m/z = 533 [M]+. Purity of the complex was determined to be >95% pure by RP-HPLC (% Purity): 99.4% at 214 nm and 96.0% at 254 nm; tr = 19.0 min. Synthesis

of

4,

[(η6-hexamethylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-

aniline)]Cl. Complex 4 was synthesized according to published procedure.35 Synthesis of 5, [(η6-1,3,5-triisopropybenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)aniline)]Cl. Complex 5 was synthesized according to published procedure.34



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Page 18 of 40

Synthesis of 6, [(η6-1,3,5-triisopropybenzene)RuCl(N-(2-quinolinylmethylene)-1-naphthylamine)]Cl. 2-Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and 1-napthylamine (53.2 mg, 0.5 mmol) was added to dry EtOH (10 mL) and and stirred at r.t. over 24 h. The solvent was removed in vacuo and dried. The resultant crude N-(2-quinolinylmethylene)-1-naphthylamine (141 mg, approx. 0.5 mmol) was treated with [(η6-1,3,5-triisopropylbenzene)RuCl2]2 (188 mg, 0.25 mmol) in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo and the resulting solid purified by column chromatography (1:4 v/v EtOH/CHCl3, Rf = 0.6). The purified product was redissolved in CHCl3 and filtered through a syringe column to remove trace amount of dissolved silica, before the solvent was removed in vacuo. The final product was then dried in vacuo for 1 h to give a dark red solid. Yield: 198 mg (62%). 1H NMR (400 MHz, CD3OD): δ 9.12 (s, 1H, PhN=CH), 8.91 (d, 3JHH = 12 Hz, 1H, quinolinyl), 8.86 (d, 3JHH = 11 Hz, 1H, quinolinyl), 8.18 (m, 8H, quinolinyl/napthyl), 7.72 (m, 3H, quinolinyl/napthyl), 5.86 (s, 3H, C6H3), 2.18 (sept, 3JHH = 9 Hz, 3H, CHMe2), 1.07 (d, 3JHH = 9 Hz, 9H, CH(CH3)2), 0.98 (d, 3JHH = 9 Hz, 9H, CH(CH3)2) ppm. ESI-MS (+ve mode): m/z = 623 [M]+. Purity of the complex was determined to be >95% pure by elemental analysis. Anal. Calcd for C35H38Cl2N2Ru·H2O (%): C 62.12, H 5.96, N 4.14; Found: C 62.29, H 5.59, N 4.18. Synthesis

of

7,

[(η6-1,3,5-triisopropybenzene)RuCl(3-trifluoromethyl-N-(2-quinolinyl-

methylene)aniline)]Cl.

2-Quinolinecarboxaldehyde

(78.6

mg,

0.5

mmol)

and

3-

(trifluoromethyl)aniline (62.5 µl, 0.5 mmol) was added to dry EtOH (10 mL) and and stirred at r.t. over 24 h. The solvent was removed in vacuo and dried. The resultant crude 3trifluoromethyl-N-(2-quinolinylmethylene)aniline (150 mg, approx. 0.5 mmol) was treated with [(η6-1,3,5-triisopropylbenzene)RuCl2]2 (188 mg, 0.25 mmol) in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo and the resulting solid purified by gradient elution


Page 19 of 40

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column chromatography (1:4 v/v EtOH/CHCl3, Rf = 0.4; 1:1 v/v EtOH/CHCl3, Rf = 0.7). The purified product was redissolved in CHCl3 and filtered through a syringe column to remove trace amount of dissolved silica, before the solvent was removed in vacuo. The final product was then dried in vacuo for 1 h to give a light brown solid. Yield: 156 mg (46%). 1H NMR (400 MHz, DMSO-d6): δ 9.23 (s, 1H, PhN=CH), 8.96 (d, 3JHH = 9 Hz, 1H, quinolinyl), 8.91 (d, 3JHH = 8 Hz, 1H, quinolinyl), 8.53 (s(broad), 1H, C6H4), 8.43 (d, 3JHH = 8 Hz, 1H, C6H4), 8.31 (t, 3JHH = 8 Hz, 2H, quinolinyl), 8.16 (t, 3JHH = 9 Hz, 1H, quinolinyl/C6H4), 8.01 (t, 3JHH = 8 Hz, 2H, quinolinyl/C6H4), 7.91 (t, 3JHH = 8 Hz, 1H, quinolinyl/C6H4), 5.60 (s, 3H, C6H3), 2.37 (sept, 3JHH = 7 Hz, 3H, CHMe2), 1.16 (d, 3JHH = 7 Hz, 9H, CH(CH3)2), 0.84 (d, 3JHH = 7 Hz, 9H, CH(CH3)2) ppm. ESI-MS (+ve mode): m/z = 641 [M]+. Purity of the complex was determined to be >95% pure by elemental analysis. Anal. Calcd for C32H35Cl2F3N2Ru·3H2O (%): C 52.60, H 5.66, N 3.83; Found: C 52.43, H 5.46, N 3.84. Synthesis of 8, [(η6-1,3,5-triisopropybenzene)RuCl(3-chloro-N-(2-quinolinylmethylene)aniline)]Cl. 2-Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and 3-chloroaniline (52.9 mg, 0.5 mmol) was added to dry EtOH (10 mL) and stirred at r.t. over 24 h. The solvent was removed in vacuo and dried. The resultant crude 3-chloro-N-(2-quinolinylmethylene)aniline (133 mg, approx. 0.5 mmol) was treated with [(η6-1,3,5-triisopropylbenzene)RuCl2]2 (188 mg, 0.25 mmol) in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo and the resulting solid purified by column chromatography (1:4 v/v EtOH/CHCl3, Rf = 0.6). The purified product was redissolved in CHCl3 and filtered through a syringe column to remove trace amount of dissolved silica, before the solvent was removed in vacuo. The final product was then dried in vacuo for 1 h to give a dark red solid. Yield: 89 mg (28%). 1H NMR (400 MHz, MeOD-d4): δ 9.02 (d, 3JHH = 9 Hz, 1H, quinolinyl), 9.00 (s, 1H, PhN=CH), 8.79 (d, 3JHH = 8 Hz, 1H,



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Page 20 of 40

quinolinyl), 8.25 (m, 3H, quinolinyl/C6H4), 8.15 (t, 3JHH = 8 Hz, 1H, quinolinyl/C6H4), 8.06 (dt, 3

JHH = 5 Hz, 1H, quinolinyl/C6H4), 7.99 (t, 3JHH = 8 Hz, 1H, quinolinyl/C6H4), 7.65 (d, 3JHH = 5

Hz, 2H, quinolinyl/C6H4), 5.60 (s, 3H, C6H3), 2.49 (sept, 3JHH = 7 Hz, 3H, CHMe2), 1.23 (d, 3JHH = 7 Hz, 9H, CH(CH3)2), 0.97 (d, 3JHH = 7 Hz, 9H, CH(CH3)2) ppm. ESI-MS (+ve mode): m/z = 607 [M]+. Purity of the complex was determined to be >95% pure by elemental analysis. Anal. Calcd for C31H35Cl3N2Ru·3H2O (%): C 53.41, H 5.93, N 4.02; Found: C 53.02, H 5.66, N 3.87. Synthesis

of

9,

[(η6-hexamethylbenzene)RuCl(3-chloro-N-(2-quinolinylmethylene)-

aniline)]Cl. Complex 9 was synthesized according to published procedure.34 Alternative synthetic route for 8 and 9. 2-Quinolinecarboxaldehyde (314 mg, 2 mmol) and 3chloroaniline (268 mg, 4 mmol) were placed to a 50 mL round-bottom flask equipped with a Dean-Stark apparatus. 25 mL of toluene was added to dissolve the reactants and the resulting brown solution was refluxed overnight. The mixture was allowed to cool and concentrated under reduced pressure to give the dark brown oil which was dissolved in a minimal amount of diethyl ether. The black insoluble residue was filtered and the solution was again concentrated under reduced pressure and dried in vacuo to give a brown semi-solid. The crude product containing small amount of unreacted 3-chloroaniline was used without additional purification. 1H NMR (300 MHz, d6-DMSO): δ 8.79 (s, 1H, Ph-N=CH), 8.53 (d, J = 8.6 Hz, 1H, quinolinyl), 8.29 (d, J = 8.6 Hz, 1H, quinolinyl), 8.12 (dd, J = 15.9, 7.9 Hz, 2H, quinolinyl), 7.90 – 7.83 (m, 1H, quinolinyl), 7.76 – 7.69 (m, 1H, quinolinyl), 7.54 – 7.47 (m, 2H, quinolinyl), 7.43 – 7.35 (m, 2H, quinolinyl).

The

corresponding

[(η6-arene)RuCl2]2

(0.15

mmol)

and

3-chloro-N-(2-

quinolinylmethylene)-aniline (180 mg, 0.675 mmol) were dissolved in 10 mL of dry MeOH under N2 atmosphere and stirred for 48 h. A resulting brown solution was evaporated under reduced pressure and the brown residue was washed with copious amounts of diethyl ether. The


Page 21 of 40

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solid was subsequently dissolved in minimal amount of CH2Cl2 and eluted through a short layer of

neutral

Al2O3 (5 cm)

by

CH2Cl2

to

remove

unreacted

3-chloro-N-(2-

quinolinylmethylene)aniline and 3-chloroaniline. The product was eluted with CHCl3/MeOH mixture (9:1) and the red filtrate was evaporated to dryness under reduced pressure. The red solid was redissolved in a minimal amount of dichloromethane and centrifuged (13,000 rpm for 7 min) to remove the undissolved residues. The red microcrystals (8) or needle-shaped X-ray quality single crystals (9) were produced by slow diffusion of diethyl ether into the CH2Cl2 solutions. Crystals were washed with diethyl ether and dried in vacuo. Complex 8. Yield: 38 mg (20%). Anal. Calcd, C31H35Cl3N2Ru·0.25 CH2Cl2 (%): C, 56.50; H, 5.39; N, 3.22. Found: C, 56.09; H, 5.04; N, 4.46. Complex 9. Yield: 40 mg (22%). Anal. Calcd, C28H29Cl3N2Ru·2CH2Cl2·0.25 H2O (%): C, 50.45, H, 4.60, N, 4.06. Found: C, 50.36, H, 5.01, N, 4.21. Tissue culture. The human colorectal carcinoma cells HCT116, human colorectal adenocarcinoma cells SW480, human gastric adenocarcinoma AGS, human gastric carcinoma KATOIII were acquired from ATCC® (Manassa,VA). HCT116 and SW480 were cultured in DMEM medium containing 10% FBS and 1% Penicillin/Steptomycin (complete DMEM). AGS was cultured in RPMI 1640 medium containing 10% FBS and 1% Penicillin/Steptomycin. KATOIII

was

cultured

in

RPMI

1640

medium

containing

20%

FBS

and

1%

Penicillin/Steptomycin. All cell lines were grown at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Experiments were performed on cells within 20 passages. Determination of Log Pow. Log Pow of the RAS complexes were determined using the shake flask method.38 The RAS complex were predissolved in ddH2O that was presaturated with n-



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Page 22 of 40

octanol (for 24 h and left to stand until phase separation occurs). The UV-vis spectrum for each samples was obtained and the absorbances at the λmax of each compound were determined. Equal volume of n-octanol was added to each sample solution and the heterogeneous mixtures shaked for 2 h before centrifuging at 4000 rpm for 1 min to achieve phase separation. The final absorbance of the aqueous phase at the λmax of each compound were determined and their wateroctanol partition coefficient were calculated. All experiments were done in triplicate. Inhibition of cell viability assay. The anti-proliferation activity the RAS Complexes on exponentially growing cancer cells were determined using MTT assay as described previously.57 HCT116, SW480, AGS and KATOIII were seeded at 10 000 cells per well (100 µL) in Cellstar® 96-well plates (Greiner Bio-One) and incubated for 24 h. Thereafter, cancer cells were exposed to drugs at different concentration in media for 48 h. The final concentration of DMSO in medium was < 1% (v/v) at which cell viability was not significantly inhibited. The medium was removed and replaced with MTT solution (100 µL, 0.5 mg/mL) in media and incubated for an additional 45 min. Subsequently, the medium was aspirated, and the purple formazan crystals dissolved in DMSO (100 µL). The absorbance due to the dissolved purple formazan was then obtained at 565 nm. Inhibition to cell viability was evaluated with reference to the IC50 value, which is defined as the concentration needed for a 50% reduction of survival based on the survival curves. IC50 values were calculated from the dose - response curves (cell viability vs drug concentration) obtained in repeated experiments and adjusted to actual [Ru] administered, which was determined using ICP-OES. The experiments were performed in 4 replicates for each drug concentration and were carried out at least three times independently. For cell viability assays involving inhibitors, Cimetidine (1.5 mM) were added together with test compounds and incubated for 7 h. Thereafter, the drugs were removed and replace with fresh


Page 23 of 40

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media for the remainder of the 48 h. Pilfithrin-α (10 µM) was incubated with drugs for the entire 48 h duration. Cell viability in the absence and presence of inhibitor was normalized against untreated control. Experiments were performed in 3 replicates and carried out at least three times independently. Drug uptake studies. HCT116 cells were grown on Cellstar® 6-well plates (Greiner Bio-One) at a density of 500 000 cells/well for 24 h before being treated with complexes 1 – 5, 6 and oxaliplatin at 1.5 µM for 24 h. Thereafter, the drug-containing media was removed and washed once with EDTA/PBS followed by trypsinization. The cells were collected, counted and digested separately with 69% nitric acid. The samples were diluted to a final concentration of 2% nitric acid and the Ru or Pt content quantified via ICP-MS. Experiments were performed three times independently. Antibodies and Western blot protocol. HCT116 cells were grown on Cellstar® 6-well plates (Greiner Bio-One) at a density of 500 000 cells/well for 24 h before being treated with complexes 4, 5, 8 and 9 at IC50 and IC75 for 2 h, 6 h and 24 h. Oxaliplatin treatment at IC50 and IC75 was used as a positive control for several experiments. The cells were lysed with lysis buffer [100 µL, 1% NP40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), protease inhibitor]. The cell lysate were transferred to separate 2 mL tubes and sonicated for 10 s. The samples were then centrifuged at 13000 rpm, 4˚C for 15 min. The supernatant liquid containing the proteins were collected and total protein content of each sample was quantified via Bradford’s assay. 50 µg of proteins from each sample were reconstituted in loading buffer [5% DDT, 1x Protein Loading Dye] and heated at 95˚C for 5 min. The protein mixtures were resolved on a 10% SDS-PAGE gel by electrophoresis and transferred to a nitrocellulose membrane. The proteins bands were visualized via enhanced chemiluminescence imaging (PXi, Syngene) after treatment with the



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Page 24 of 40

primary antibodies and the appropriate secondary antibodies. Equal loading of protein was confirmed by comparison with actin. The following antibodies were used: p53 (FL-393), p21 (F5) and Cyclin D1 (H-295) from Santa Cruz Biotechnologies. β-Actin (ab75186) from Abcam. ECL Anti-rabbit IgG (NA934V) and ECL Anti-mouse IgG (NA931) from GE Healthcare Life Sciences. All antibodies were used at 1:1000 dilutions except for actin (1:10000), anti-mouse and anti-rabbit (1:5000). Primers and qPCR protocol. Treatment conditions for HCT116 cells were similar to the protocol in western blot. RNA was extracted using TRIzol® Reagent and reverse transcription was performed with 2 µg of the extracted RNA using Applied Biosystem® High Capacity cDNA Reverse Transcription Kit with an Applied Biosystem® 2720 Thermal Cycler. Quantitative PCR was done on the resulting cDNA using FastStart Universal Probe Master (Rox) with Applied Biosystem® 7500 Real Time PCR System. The relative starting quantities of genes of interest were normalized against the housekeeping genes TBP and samples were done in duplicates. The specificity of the amplification was controlled by a melting curve. The gene and Assay ID of TaqMan probes are as follows: NOXA (Hs00560402_m1), BAX (Hs00180269_m1), BCL2 (Hs00608023_m1) and TBP (Hs00427620_m1).

ACKNOWLEDGEMENTS The authors thank staff of CMMAC, NUS for performing elemental analysis and ICP-OES analysis. They also thank Ligue contre le cancer, CNRS, European COST action CM1105, Ministry of Education and the National University of Singapore (R143-000-638-112) for funding the research presented in this study.


Page 25 of 40

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[Pt(BDI(QQ))]Cl.

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Figures and Figure Captions

Figure 1. p53-dependent RAPTA-C, RM175 and the limited number of cytotoxic p53independent Ru (II) complexes.

Figure 2. Molecular structures of 1 – 9.



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Figure 3. Molecular representation of cations of 4 (top) and 9 (bottom). Hydrogen atoms, chloride anions and co-crystallized solvent molecules have been omitted for clarity.


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Page 33 of 40

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Figure 4. Change in UV-Vis spectra of 4, 5, 8 and 9 in HBSS with 10% FBS over 24 h. Varying the functional groups on the chelate ligand and arene ring affects the stability of the Ru-Cl bond.



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Figure 5. Dose-response curve of 4, 5, 8 and 9 in gastric cancer (a) AGS and (b) KATOIII. HMB analogues 4 and 9 elicited an unexpected biphasic response in the respective cell line, suggesting a different mode of action as compared to their respective analogous compounds.


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Figure 6: Drug uptake studies. (a) A plot of IC50 of complexes 1 - 5 in 4 different cell lines against their respective Log POW. (b) A plot of cellular [Ru] accumulation of each complex against their respective Log POW when treated at a fixed concentration of 1.5 µM. Ru amount determined by digesting known number of treated cells with concentrated HNO3 followed by measurement with ICP-MS. Mean ± s.d. of three independent experiments.

Figure 7: OCT2 is not involved in the cellular uptake of 5 or 6. Cell viability of colorectal cancer HCT116 after treatment with different concentrations of (a) 5, (b) 6 and (c) OXP were determined, in the presence or absence of OCT2-inhibitor cimetidine (1.5 mM). Cells were treated for 7 h before the drug solution was removed and replaced with drug-free media. Mean ± s.e.m of four replicates. Experiments were done independently for three times.



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Figure 8. (a) Protein expression of p53 and its cell-cycle-arresting targets Cyclin D1 and p21 in HCT116 cells were analysed by Western blot (actin as loading control), after treatment with RAS compounds at the specified conditions. (b) Gene expression of pro-apoptotic p53-target NOXA and BAX and anti-apoptotic BCL2 were quantified by RT-qPCR (normalized against TBP) after treatment. mRNA levels are presented as fold induction relative to untreated control. OXP was used as positive control. (c) IC50 of 4, 5, 8 and 9 in the absence and presence p53-inhibitor pilfithrin-α (10 µM). Mean ± s.e.m of three independent experiments.


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Schemes

Scheme 1. General synthetic route for 1 – 9.



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Page 38 of 40

Tables Table 1. Selected X-ray crystallographic data for 4 and 9. Complex Formula Formula weight Temperature [K] Wavelength [Å] Crystal size [mm3] Crystal system Space group a [Å] b [Å] c [Å] α [deg] β [deg] γ [deg] V [Å3] Z Dc [Mg/m3] µ [mm-1] θ range [deg] no. of unique data max., min. transmn final R indices [I>2σ(I)] R indices (all data) goodness-of-fit on F2 peak/hole [e Å-3]

4 C29H32Cl2N2ORu.2CH3OH 660.56 100(2) 0.71073 0.450 x 0.430 x 0.130 Monoclinic P21/c 17.548(2) 8.8135(12) 20.148(3) 90 106.579(4) 90 2986.5(7) 4 1.469 0.739 2.540 to 28.282 31886 0.7457 and 0.5969 R1 = 0.0771, wR2 = 0.1235 R1 = 0.1800, wR2 = 0.1516 1.022 2.766 and -1.396

9 C28H29 Cl3N2Ru.2CH2Cl2 770.80 100(2) 0.71073 0.304 x 0.225 x 0.074 Triclinic Pī 8.2070(3) 12.9134(6) 15.5705(7) 95.336(2) 104.273(2) 96.960(2) 1574.44(12) 2 1.626 1.117 2.230 to 28.281 24879 0.7458 and 0.6526 R1 = 0.0520, wR2 = 0.1042 R1 = 0.0836, wR2 = 0.1139 1.047 1.133 and -1.682

a

R = Σ||Fo| - |Fc||/Σ|Fo|, wR2 = {Σ[w(Fo2 - Fc2)2]/Σ[w(Fo2)2]}1/2. Goodness-of-fit (GooF) = [Σ[w(Fo2 - Fc2)2]/(n - p)]1/2, where n is the number of data and p is the number of parameters refined.


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Table 2. Selected bond lengths [Å], angles [°] and torsion angles [°] for 4 and 9. Complex

4

9

Ru1-Carene

2.196-2.283

2.175-2.280

Ru1-N1

2.100(5)

2.103(3)

Ru1-N2

2.070(5)

2.081(3)

Ru1-Cl1

2.4122(16)

2.4124(10)

N2-C22

1.293(8)

1.291(5)

N2-C23

1.428(8)

1.437(5)

N1-Ru1-N2

76.4(2)

75.97(12)

N1-Ru1-Cl1

86.36(14)

89.02(9)

N2-Ru1-Cl1

89.61(15)

90.06(9)

-37.6(9)

34.6(6)

C22-N2-C23-C24

Table 3. Log POW and cytotoxicity data for 1 – 9. IC50

[µM] b

Complex

Log Powa

HCT116

SW480

AGS

KATOIII

1

-2.32 ± 0.22

288 ± 44

155 ± 4

300 ± 10

213 ± 24

2

-2.01 ± 0.21

175 ± 22

62.1 ± 17.9

117 ± 10

103 ± 21

3

-1.58 ± 0.12

25.5 ± 4.0

27.0 ± 12.0

12.4 ± 1.7

14.2 ± 2.5

415

-1.40 ± 0.15

5.76 ± 1.22

34.7 ± 19.3

3.04 ± 0.91

13.7 ± 5.0

515

-0.85 ± 0.02

1.19 ± 0.12

4.07 ± 1.35

1.01 ± 0.07

1.22 ± 0.24

6

n.d.

3.48 ± 0.36

5.32 ± 2.09

1.16 ± 0.28

1.86 ± 0.24

7

n.d.

6.87 ± 1.11

13.9 ± 5.5

3.07 ± 0.72

3.89 ± 0.15

8

n.d.

5.76 ± 0.13

11.0 ± 4.7

2.76 ± 0.82

3.28 ± 1.12

9

-1.07 ± 0.03

3.05 ± 0.26

16.0 ± 6.1

1.95 ± 0.40

2.22 ± 0.35

Oxaliplatin

n.d.

1.22 ± 0.18

1.08 ± 0.36

n.d.

n.d.

Cisplatin15

n.d.

n.d.

n.d.

34.7 ± 0.8

8.64 ± 0.11

a

b

Log Pow values determined via the shake-flake method against 1:1 n-octanol:H2O partitioning. IC50 values is the concentration of Ru complexes required to inhibit 50% of cell growth with respect to control groups, measured by MTT assay after 48 h of incubation. Data obtained are based on the average of three independent trials, and the reported errors are the corresponding standard deviations. The IC50 were corrected using actual [Ru] determined using ICP-OES.



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TOC GRAPHICS


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Structural Determinants of p53-Independence in Anticancer

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