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Structural Determinants of p53-Independence in Anticancer Ruthenium-Arene Schiff-Base Complexes.

PMID: 27174050
Subscriber access provided by UNIV OF PITTSBURGH Article 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 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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Babak1, Daniel Yuan Qiang Wong1, Giorgia Pastorin2, 3, Christian 15 16 Gaiddon*4, 5 and Wee Han Ang*,1, 2 17 18 19 20 1Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore 21 22 23 2NUS Graduate School for Integrative Sciences and Engineering 24 25 26 3Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 Singapore 27 28 29 4U1113 INSERM, 3 Avenue Molière, Strasbourg 67200, France 30 31 5Oncology section, FMTS, Université de Strasbourg, Strasbourg, France 32 33 34 35 36 37 38 39 40 KEYWORDS 41 42 43 Ruthenium Arene Schiff-Base Complexes, Anticancer, Structure-Activity Relationship Studies, 44 45 46 p53-independent activity. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1 ACS Paragon Plus Environment Molecular Ph armaceutics Page 2 of 40 1 2 3 ABSTRACT 4 5 6 p53 is a key tumor supressor gene involved in key cellular processes and implicated in cancer 7 8 9 therapy. However, it is inactivated in more than 50% of all cancers due to mutation or 10 11 overexpression of its negative regulators. This leads to drug resistance and poor 12 13 chemotherapeutic outcome as most clinical drugs act via a p53-dependent mechanism of action. 14 15 16 An attractive strategy to circumvent this resistance would be to identify new anticancer drugs 17 18 that act via p53-independent mode-of-action. In the present study, we identified 9 Ru (II)-Arene 19 20 Schiff-base (RAS) complexes able to induce p53-independent cytotoxicity and discuss structural 21 22 23 features that are required for their p53-independent activity. Increasing hydrophobicity led to an 24 25 increase in cellular accumulation in cells with a corresponding increase in efficacy. We further 26 27 28 showed that all 9 complexes demonstrated p53-independent activity. This was despite significant 29 30 differences in their physicochemical properties suggesting that the iminoquinoline ligand, a 31 32 common structural feature for all the complexes, is required for the p53-independent activity. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 ACS Paragon Plus Environment Page 3 of 40 Molecular Ph armaceutics 1 2 3 INTRODUCTION 4 5 6 p53 is a key tumor supressor gene involved in the mediation of cellular DNA repair, cell cycle 7 8 9 arrest, apoptosis-induction and neurotoxicity of anticancer drugs.1-2 It is inactivated in more than 10 11 50% of all cancers due to mutation or overexpression of p53 negative regulators.3 As a result, 12 13 these cancers are more resistant to drugs that act via p53-dependent pathways. For instance, p53- 14 15 16 dependent drugs such as oxaliplatin (OXP) or doxorubicin (DOX) are less effective in inducing 17 18 apoptosis in these cancer types even after causing significant DNA damage.4-5 Various strategies 19 20 have been explored for more effective treatment of cancers with defective p53 function. One 21 22 23 such strategy involves the resensitization of these cancers to chemotherapy by restoring p53 24 25 activity. This could be done through (i) the inhibition of the interaction between p53 and its 26 27 28 negative regulators or (ii) restoring misfolded mutant p53, using targeted small molecules or 29 30 selective peptide sequences.6 However, such treatment is often limited by the off-target effects, 31 32 low efficacy or development of resistance towards these ‘p53-activators’.6-7 A more attractive 33 34 35 strategy would be to identify new anticancer candidates that are able to bypass this resistance 36 37 mechanism entirely by inducing p53-independent antiproliferation. 38 39 40 The success of Ru (III)-containing compounds such as KP1019, which received favorable 41 42 43 evaluations in preclinical studies,8-9 has lead to recent interest in the development of more active 44 45 Ru (II) complexes as anticancer agents.10-12 Some prominent examples of cytotoxic Ru (II) 46 47 complexes that have undergone preclinical studies are RAPTA-C, RM175 and RDC11.13-16 48 49 50 However, many of these Ru (II) complexes have p53-dependent activity and are subjected to the 51 52 same resistance mechanism of p53-mutated cancers. Previous studies indicated that RAPTA-C, 53 54 RM175 and RDC11 induced p53 and its target genes to various degrees.17-19 Many other reported 55 56 57 Ru (II) complexes also exert their antiproliferation effect via p53-dependent pathways.20-26 In 58 59 60 3 ACS Paragon Plus Environment Molecular Ph armaceutics Page 4 of 40 1 2 3 contrast, only a limited number of Ru (II) complexes have been reported to demonstrate p53- 4 5 independent activity (Fig 1). Several Ru (II)-arene complexes bearing azopyridine, 6 7 8 iminopyridine or chloroquine ligand, and a imidazole-bearing phenanthroline Ru (II) complex 9 10 demonstrated similar toxicity in both wild type colorectal carcinoma HCT116 and p53-null 11 12 13 lineage.27-29 Another Ru (II)-arene complex bearing iminophosphorane ligand induced apoptotic 14 15 cell death without p53-induction.30 Other metal complexes of Pt and Os have also been shown to 16 17 act via p53-independent mechanism.27, 31-33 In all cases, the metal centre has little or no influence 18 19 20 on the p53-dependence of these complexes; the p53-independence of these complexes could 21 22 often be ‘switched on’ or ‘switched off’ by structural modification to the ligands.27, 33 In light of 23 24 this, a greater understanding of the structural determinents of p53-independence could lead to the 25 26 27 identification of new organoruthenium compounds with p53-independent activity, adding to the 28 29 limited pool of drugs for the effective treatment of resistant cancers with mutated p53 status. 30 31 32 Previously, a Ru (II)-Arene Schiff-base (RAS) complex bearing 1,3,5-triisopropylbenzene 33 34 35 (TPB) and iminoquinoline ligands, RAS-1T, was identified as a promising lead cytotoxic 36 37 compound with an unique mode-of-action.34 Preliminary investigations suggested that RAS-1T 38 39 acted via a mechanism distinct from classical alkylating agents including cisplatin (CDDP) and 40 41 42 other reported anticancer Ru (II) complexes. RAS-1T did not induce p53 overexpression and 43 44 was equipotent in epithelial cells BJwt (p53+/+) and BJshp53 (p53-/-). Further investigation of 45 46 47 RAS-1T and its hexamethylbenzene (HMB) analogue, RAS-1H, revealed that both compounds 48 49 induced non-apoptotic cell death via ER-stress pathways but the pathways were different for 50 51 each compound.35 52 53 54 In this work we report a structure-activity relationship study on an expanded set of RAS 55 56 57 complexes that are structurally related to RAS-1H (4) and RAS-1T (5), aimed to (i) investigate 58 59 60 4 ACS Paragon Plus Environment Page 5 of 40 Molecular Ph armaceutics 1 2 3 if p53-independent activity is a general feature of this class of iminoquinoline-containing Ru (II) 4 5 complexes and (ii) identify the key structural features that lead to their p53-independence. We 6 7 8 further discuss their activity against a panel of colorectal (HCT116 and SW480) and gastric 9 10 (AGS and KATOIII) cancer cells, the influence of the arene and chelating ligands on RAS 11 12 13 complexes’ stability to aquation and biological nucleophiles, as well as intracellular 14 15 accumulation. 16 17 18 19 20 21 RESULTS & DISCUSSION 22 23 24 25 Synthesis and Characterization of RAS Complexes 26 27 28 RAS complexes 1 – 9 (Fig 2) were synthesized using the general reaction route as shown in 29 30 Scheme 1. Recently, we reported the synthesis and characterization of 4, 5 and 9.34-35 2- 31 32 33 Quinolinecarboxaldehyde and different aniline derivatives were reacted in dry EtOH or MeOH to 34 35 give the bidentate imine ligand with varying degree of purity. Most of the imine ligands could 36 37 not be isolated as pure compounds due to the facile hydrolysis of the imine bond and the crude 38 39 40 product was used directly for synthesis of the RAS complexes by addition of stoichiometric 41 42 amount of the corresponding [Ru(η6-arene)Cl ] in MeOH. Complexes 1 – 9 were further 2 2 43 44 45 purified and isolated via flash column chromatography. In addition, we report an improved 46 47 synthetic protocol of 8 and 9 that did not require purification by column chromatography. 48 49 Briefly, the imine ligand was prepared from 2-quinolinecarboxaldehyde and excess 3- 50 51 52 chloroaniline in toluene using a Dean-Stark apparatus and the crude ligand treated directly with 53 54 [Ru(η6-arene)Cl ] in MeOH. The product was filtered through a short plug of neutral alumina 2 2 55 56 57 58 59 60 5 ACS Paragon Plus Environment Molecular Ph armaceutics Page 6 of 40 1 2 3 and recrystallized using vapour diffusion of diethyl ether into a saturated CH Cl solution at 4 2 2 4 5 ºC. 6 7 8 9 The 1H NMR spectra of 1 – 9 showed resonances typical of RAS complexes.34 The 10 11 disappearance of the singlet peak corresponding to quinolinecarboxaldehyde at ca. 10.2 ppm and 12 13 the appearance of the peak corresponding to the imine proton at ca. 8.5-9.5 ppm indicated the 14 15 16 formation of the chelate ligand. The desymmetrization of the Ru complex due to chelation 17 18 resulted in additional splitting pattern of the arene proton signals (arene = toluene, cymene, 19 20 TPB). ESI-MS spectra of these compounds also showed the characteristic [M]+ molecular ion 21 22 23 peaks with Ru and Cl isotopic pattern. The purity of the complexes was confirmed by either 24 25 elemental analysis (EA) or RP-HPLC to be >95%. 26 27 28 Solid-state structural information was obtained for 4 and 9 via single crystal X-ray diffraction 29 30 31 studies (Fig 3) and selected structural data are given in Table 1 and Table 2. Single crystals 32 33 were obtained via slow diffusion of diethyl ether into saturated CH Cl solution at 4 ºC. RAS 34 2 2 35 36 complexes 4 and 9 adopted the classical ‘piano-stool’ structure similar to most reported Ru(II)- 37 38 arene complexes with some notable deviations.26, 30, 36 The Ru-C bond ranges for 4 and 9 of arene 39 40 2.196-2.283 Å and 2.175-2.280 Å, respectively, are longer than RM175 (biphenyl) and RAPTA- 41 42 43 C (cymene) of 2.161-2.231 Å and 2.180-2.262 Å, respectively, presumably due to the sterically 44 45 encumbered HMB ligand. All corresponding bond length and angles between 4 and 9 were 46 47 similar, suggesting that changing the 4-OMe group to a 3-Cl group did not alter the core RAS 48 49 50 structure. The conformation adopted by the iminoquinoline ligands could be described by the 51 52 torsion angle θ of 37.6(9)˚ and 34.6(6)˚ for 4 and 9, respectively, disrupting the 53 C22-N2-C23-C24 54 55 extended conjugation within the chelate ligand. This twisted conformation was presumably due 56 57 58 59 60 6 ACS Paragon Plus Environment Page 7 of 40 Molecular Ph armaceutics 1 2 3 to the steric bulk of the arene ligand. The same structural feature was observed in the recently 4 5 reported RAS complexes.34 6 7 8 9 10 11 12 Effect of structural variation on stability and Log P OW 13 14 15 Aqueous stability and Log P are important factors that affect a potential drug’s 16 OW 17 bioavailability and cell permeability,37 which can potentially influence the molecular target and 18 19 20 mode-of-action. To determine the stability of the 9 RAS complexes under investigation towards 21 22 aquation and biological nucleophiles, we monitored their UV-Vis profile in double distilled H O 23 2 24 25 (ddH 2 O) and Hanks Balanced Salt Solution (HBSS) containing 10% fetal bovine serum (FBS) 26 27 over 24 h. Any reaction would manifest as a shifts in the UV-Vis profile, producing isosbestic 28 29 point(s) in the overlapping spectrums at different time points. The investigated RAS complexes 30 31 32 were generally stable in water (Fig S1). Slight shifts in the spectrums and the formation of 33 34 several isosbestic points were observed for 6, 7 and 8 after 24 h in water, which suggests that the 35 36 aquation of the Ru-Cl bond in 6, 7 and 8 occurred very slowly in the absence of other competing 37 38 39 nucleophiles at r. t.. 40 41 42 Although all RAS complexes could be considered to be resistant to aquation in water, only 43 44 some could be considered stable towards interfering nucleophiles as seen in their stability in 45 46 47 serum-containing HBSS (Fig S2). This is particularly well illustrated by focusing on the slight 48 49 variations implemented when comparing 4, 5, 8 and 9 (Fig 4). In general, compounds with more 50 51 52 π-donating arene (HMB, TPB) and less π-acidic iminoquinoline (-OMe) ligands stabilized the 53 54 Ru-Cl bonds making it less susceptible towards substitution. For instance, 5 was stable in HBSS 55 56 with 10% FBS. However, changing the 4-OMe to the more π-acidic 3-CF or 3-Cl group (7 and 57 3 58 59 60 7 ACS Paragon Plus Environment Molecular Ph armaceutics Page 8 of 40 1 2 3 8) labilised the Ru-Cl bond. Replacing the TPB arene with the more π-donating HMB in 9 4 5 6 resulted in a more stable complex resistant to Ru-Cl aquation. Furthermore, it cannot be ruled out 7 8 that compounds bearing labile benzene or toluene arene ligands (1 and 2) decomposed in serum- 9 10 containing buffer due to the displacement of arene ligand by biological nucleophiles (Fig S2). 11 12 13 14 Log P OW of 1 – 5 and 9 were determined using the shake-flask method.38 Log P OW of 6, 7 and 8 15 16 could not be determined as their spectra were significantly shifted after the partitioning and 17 18 phase separation (data not shown), presumably due to non-specific interactions with the n- 19 20 21 octanol phase. The trend in the Log P studies were as expected, where RAS complexes with OW 22 23 more hydrophobic ligands displayed higher Log P values (Table 3). OW 24 25 26 27 28 29 Effects of ligand structural variations on cytotoxicity profile 30 31 32 33 To determine the effects of structural variation to cytotoxicity, we obtained the IC values of 50 34 35 the nine RAS complexes in colorectal cancer cell lines HCT116 and SW480, and gastric cancer 36 37 cell lines AGS and KATOIII (Table 3). In general, RAS complexes bearing the more 38 39 40 hydrophobic HMB and TPB arene ligands demonstrated the highest cytotoxicity in the cell lines, 41 42 with IC values in the low µM range. Complex 5 displayed the greatest efficacies with IC ca. 43 50 50 44 45 1.0 µM in HCT116, AGS and KATOIII and 4.1 µM in SW480, which was 34- and 7-fold more 46 47 cytotoxic than CDDP in AGS and KATOIII, respectively. For RAS complexes with 4-OMe (1 – 48 49 5), IC values decreased with the increasing hydrophobicity of the arene ligand, from the least 50 50 51 52 hydrophobic benzene in 1 to the most hydrophobic TPB in 5. Interestingly, starting from 53 54 complex 3 in the reverse order, the IC became higher in HCT116 and AGS versus SW480 and 50 55 56 57 58 59 60 8 ACS Paragon Plus Environment Page 9 of 40 Molecular Ph armaceutics 1 2 3 KATOIII, suggesting that hydrophobicity also affected selectivity between different cancer 4 5 types. 6 7 8 9 Both 4 and 9 demonstrated biphasic dose-response curve in AGS and KATOIII cells, 10 11 respectively. AGS cell viability decreased with increasing concentration of 4 to reach a plateau 12 13 of 40% between 5 µM and 20 µM before dropping drastically to 0% above 20 µM (Fig 5a).34 A 14 15 16 similar biphasic profile was seen in KATOIII cells treated with 9 (Fig 5b). In contrast, the TPB 17 18 analogues (5 and 8) did not display such biphasic dose-response profiles. The basis for this 19 20 unique concentration-dependent mechanism of antiproliferative activity induced by HMB- 21 22 23 bearing RAS complexes (4 and 9) remains to be elucidated. This biphasic dose-response was not 24 25 observed in benzene, toluene, cymene or TPB RAS analogues. Few reported anticancer 26 27 28 compounds exhibit such biphasic dose-response profiles. One such compound is anti-tubule 29 30 taxane Docetaxal, which induced mitotic catastrophe at lower doses and p53-independent 31 32 apoptosis at higher doses in prostate cancer (PC3, DU145) and breast cancer cell lines (MCF7, 33 34 35 MDA-MB-231).39-42 A similar concentration-dependent dual mode-of-action for 4 and 9 would 36 37 be highly conceivable. 38 39 40 41 42 43 44 RAS complexes accumulate in cells mainly by passive diffusion 45 46 47 One possibility for the higher cytotoxicity of compounds bearing hydrophobic arene ligands 48 49 could be attributed to better cellular uptake via passive diffusion.43 More efficient cellular 50 51 accumulation of the RAS compounds may result in a higher efficacy. The RAS complexes 52 53 54 evaluated were considered hydrophilic compounds by virtue of their Log P values. We ow 55 56 investigated whether varying the hydrophobicity of the arene ligands (1 – 5) affected cellular 57 58 59 60 9 ACS Paragon Plus Environment Molecular Ph armaceutics Page 10 of 40 1 2 3 uptake in HCT116 cells, with 6 and OXP for comparison. Cell samples were centrifuged, 4 5 washed, and digested separately in concentrated HNO before their Ru/Pt contents were analyzed 6 3 7 8 by ICP-MS (Table S1). 9 10 11 There was a positive correlation between hydrophobicity, cellular accumulation and 12 13 cytotoxicity for 1 – 5 (Fig 6a-b). When treated at the same concentration of 1.5 µM, the most 14 15 16 hydrophobic complex 5 accumulated 7.5-times more than 1, the least hydrophobic complex. 17 18 Interestingly, complex 6 accumulated 3 times less than complex 4 but had a higher cytotoxicity. 19 20 Hence, with the exception of complex 6, the higher uptake also corresponded with lower IC 21 50 22 23 suggesting that uptake was partially responsible for the higher cytotoxicity of the more 24 25 hydrophobic RAS complexes. In addition, there was an interesting abrupt improvement of drug 26 27 28 intake at a Log P ow just above -1.5, suggesting that this value might be considered as a goal for 29 30 designing cytotoxic Ru (II) arene compound. 31 32 33 Since the RAS complexes are cationic and bear hydrophobic ligands, we investigated if 34 35 36 organic cation transporters (OCT) could be responsible for their activity in colorectal cancer in a 37 38 similar manner as OXP. OXP was effective in the treatment of colorectal cancer compared to 39 40 CDDP and attributed to the fact that OXP was a substrate for OCT1/2 and would be more 41 42 43 efficiently accumulated in OCT-overexpressing colorectal cancer cells.44 As the RAS complexes 44 45 are positively charged and contained hydrophobic ligands, they could possibly be substrates of 46 47 OCTs in a similar fashion to OXP. Hence HCT116 cells were treated with 5 and 6 for 7 h in the 48 49 50 presence and absence of OCT2-inhibitor cimetidine and compared to OXP as a positive control. 51 52 Cimetidine did not affect the viability of cells treated with 5 and 6, suggesting that both are not 53 54 substrates of OCT2 (Fig 7a-b). In contrast, the viability of cells treated with OXP increased in 55 56 57 the presence of cimetidine (Fig 7c), indicating reduced uptake of the cytotoxic OXP. Taken 58 59 60 10 ACS Paragon Plus Environment Page 11 of 40 Molecular Ph armaceutics 1 2 3 together, the data suggested that RAS complexes accumulated in the cell mainly via passive 4 5 diffusion. A general trend indicated that increasing hydrophobicity resulted in increased cellular 6 7 8 uptake and higher toxicity, which was characteristic of entry by passive diffusion. However, 9 10 exception such as in complex 6 indicated that additional mechanisms and physicochemical 11 12 13 determinants might exist and could participate in the uptake process. Although, OCT transporters 14 15 were not involved in the uptake of RAS complexes, the possibility that other energy-dependent 16 17 uptake pathways are involved in the cellular accumulation of RAS complexes cannot be 18 19 20 excluded. 21 22 23 24 25 26 Structural determinant of p53-Independent mode-of-action 27 28 29 We determined previously that 4 and 5 induced cell death in AGS and HCT116 cells without 30 31 32 concomitant induction of p53.34-35 To further verify the p53-independence of the RAS 33 34 complexes, we evaluated the expression of p53 as well as downstream, cell cycle-arresting and 35 36 apoptosis-associated biomarkers in treated HCT116 cells. Cells were exposed to 4, 5, 8 and 9 for 37 38 39 2 h, 6 h and 24 h at their IC and IC values. In addition, their activities were probed in the 50 75 40 41 absence and presence of p53-inhibitor pifithrin-α. OXP was used as a positive control. 42 43 44 Western blot analysis was performed against p53 and its targets, cell cycle-regulating cyclin 45 46 47 D1 and p21 (Fig 8a).45-47 In general, there was a lack of induction of these biomarkers by 4, 5, 8 48 49 and 9 although p53-independent induction of p21 was observed with 4, 5 and 9 after 24 h 50 51 exposure.48 In contrast, OXP exhibited a concentration and time-dependent upregulation of p53, 52 53 54 cyclin D1 and p21. RT-qPCR analyses were performed on p53-regulated anti-apoptotic gene 55 56 BCL2 and pro-apoptotic genes NOXA and BAX (Fig 8b).49-51 Complexes 4 and 9 induced a slight 57 58 59 60 11 ACS Paragon Plus Environment Molecular Ph armaceutics Page 12 of 40 1 2 3 increase in NOXA expression at the later time points (6 h and 24 h) while 5 and 8 did not cause 4 5 significant change in NOXA expression. Cells treated with 4, 5, 8 and 9 also induced negligible 6 7 8 change in expression of BAX and BCL2. In contrast, OXP treatment at IC led to 3-fold 75 9 10 induction of NOXA, 5.5-fold increase in BAX and a significant reduction in BCL2 after 24 h 11 12 13 exposure. To demonstrate functionally that the cytotoxic activities of 4, 5, 8 and 9 were 14 15 independent of p53, cells were co-incubated with pifithrin-α. Co-treatment of 4, 5, 8 and 9 with 16 17 pifithrin-α improved the efficacies of RAS complexes while in the case of OXP, co-treatment 18 19 20 incurred a 3-fold increase in IC suggesting that OXP exerted its cytotoxicity via a p53- 50 21 22 dependent pathway. 23 24 25 The lack of significant induction of p53 and its downstream targets implied that these RAS 26 27 28 complexes exerted their antiproliferative effects via p53-independent pathways. On the other 29 30 hand, OXP significantly increased the expression of p53, cyclin D1, p21, NOXA and BAX and 31 32 decreased the expression of BCL2. This was in agreement with previous studies, which showed 33 34 35 that oxaliplatin induced p53-mediated cycle arrest and (to a smaller degree) p53-mediated 36 37 apoptosis in HCT116 cells.52-53 Furthermore, p53 inhibition with pifithrin-α did not adversely 38 39 affect RAS cytotoxicities compared to OXP. This further confirmed that the RAS complexes 40 41 42 acted via a p53-independent mode-of-action. 43 44 45 To ascertain that the p53-independent activity is a general characteristic of this class of 46 47 iminoquinoline-containing RAS complexes, we measured p53 expression following 24 h 48 49 50 treatment at IC concentrations for the remaining complexes 1, 2, 3, 6 and 7 as well as measured 50 51 52 viabilities of treated cells after 48 h in the absence and presence of pifithrin-α. Likewise, OXP 53 54 was used as a positive control. There was a lack of p53 induction by these RAS complexes and 55 56 57 their activities not negatively impacted by the presence of pifithrin-α, as seen by the similar or 58 59 60 12 ACS Paragon Plus Environment Page 13 of 40 Molecular Ph armaceutics 1 2 3 lower cell viabilities in cells co-treated with pifithrin-α (Fig S3), in keeping with the other RAS 4 5 complexes. 6 7 8 9 In general, all RAS complexes tested in this studies demonstrated p53-independent activity, 10 11 even though their physicochemical properties such as stability to nucleophiles and 12 13 hydrophobicity differed significantly. A similar class of Ru (II) arene complexes bearing imino- 14 15 16 and azo-pyridine ligands (Fig 1; top-right) has been shown to demonstrate p53-independent 17 18 activity only when the chloride ligand was substituted with iodide.27 It was reported that the 19 20 choice of halide ligand strongly influenced the p53-dependence on their class of compound. The 21 22 23 key difference with RAS complexes was that they demonstrated p53-independent activities 24 25 without the need for iodide substitution. The iminoquinoline and Ru (II)-arene functionalities 26 27 28 common to all 9 RAS complexes were crucial for the observed p53-independence cytotoxicity 29 30 and structural tuning at the non-halide sites could be used to fine-tune their antiproliferative 31 32 modes-of action. 33 34 35 36 CONCLUSION 37 38 39 The current study gives insights to the structural determinants of cytotoxicity and p53- 40 41 independence in Ru (II)-arene complexes through a detailed chemical and in vitro SAR study of 42 43 44 9 RAS complexes. In general, the more hydrophobic complexes displayed higher cellular 45 46 accumulation and cytotoxicity across a panel of cancer cell lines. In addition, the lack of OCT 47 48 involvement in the uptake of RAS complexes suggested that entry occurred mainly via passive 49 50 51 diffusion, despite the fact that these complexes were hydrophilic in nature. RAS complexes also 52 53 displayed tolerance towards aquation, although analogues with benzene or toluene ligands were 54 55 significantly less stable towards biological nucleophiles as these arenes were much more prone 56 57 58 59 60 13 ACS Paragon Plus Environment Molecular Ph armaceutics Page 14 of 40 1 2 3 to displacement. These complexes also exerted their anticancer activities via p53-independent 4 5 pathways as seen in the lack of activation of p53 and downstream targets, and their unchanged 6 7 8 activity in the presence of small molecule p53-inhibitors. Compounds such as this may have 9 10 important implication in the treatment of resistant cancers with mutated p53 or p53-null status. 11 12 13 14 15 16 17 EXPERIMENTAL 18 19 20 Materials. All experimental procedures were carried out without additional precautions to 21 22 exclude air or moisture unless otherwise specified. All chemicals and solvents were used as 23 24 25 received unless otherwise specified. Dry ethanol and methanol were obtained by drying them 26 27 over molecular sieves 3-4 Å 24 h before use. RuCl .xH O was purchased from both Precious 3 2 28 29 30 Metals Online. [(η6-benzene)RuCl ] , [(η6-toluene)RuCl ] , [(η6-cymene)RuCl ] , [(η6- 2 2 2 2 2 2 31 32 hexamethylbenzene)RuCl ] and [(η6-1,3,5-triisopropylbenzene)RuCl ] were synthesized 2 2 2 2 33 34 according to previously reported protocols.43, 54-56 Thiazolyl blue tetrazolium bromide (MTT), 35 36 37 Trizma® Base BioUltra, Nonidet P-40, DL-Dithiothreitol, Non-fat Dried Milk Bovine, 38 39 TWEEN® 20, Ponceau S and Cimetidine, were purchased from Sigma-Aldrich. All other 40 41 chemicals used were purchased from Sigma-Aldrich (Singapore). Ultrapure water used was 42 43 44 purified by a Milli-Q UV purification system (Sartorius Stedim Biotech SA). Gibco® Versene 45 46 solution, Gibco® Trypsin/EDTA solution, 10% SDS solution, Penicillin-Streptomycin (10 000 47 48 49 U/mL), Dulbecco's Phosphate-Buffered Saline (10x), TRIzol® Reagent and Applied 50 51 Biosystem® High Capacity cDNA Reverse Transcription Kit were purchased from Life 52 53 Technologies. HycloneTM RPMI 1640, DMEM medium and Fetal bovine serum (FBS) was 54 55 56 purchased from Thermo Fisher Scientific Inc. Bio-rad Protein Assay Dye Reagent Concentrate, 57 58 59 60 14 ACS Paragon Plus Environment Page 15 of 40 Molecular Ph armaceutics 1 2 3 40% Acrylamide/Bis solution, 10x Tris/glycine buffer, TEMED, 4x Laemmli Sample Buffer, 4 5 Nitrocellulose Membrane, 0.2 µm and 0.45 µm were purchased from Bio-rad Laboratories. 6 7 8 cOmplete, mini Protease Inhibitor Cocktail Tablets, RNase A, FastStart Universal Probe Master 9 10 (Rox) were purchased from Roche Diagnostics. LuminataTM Classico Crescendo Western HRP 11 12 13 Substrate were purchased from Merck Millipore Corporation. 14 15 16 Instrumentation. 1H NMR spectrums were obtained using either a Bruker AMX 300, Avance 17 18 400 or AMX 500 spectrometer and the chemical shifts (δ) were reported in parts per million with 19 20 reference to residual solvent peaks. Electrospray-ionization Mass Spectrometry (ESI-MS) spectra 21 22 23 were obtained using Thermo Finnigan MAT ESI-MS System. UV-vis spectra were obtained 24 25 using the Shimadzu UV-1800 UV Spectrophotometer with a TCC-240A Temperate Controlled 26 27 28 Cell Holder. Ru concentrations were determined using the Optima ICP-OES (Perkin-Elmer) 29 30 operated by CMMAC, NUS. Elemental analyses of selected Ru complexes were carried out 31 32 using a Perkin-Elmer PE 2400 elemental analyzer by CMMAS, NUS. 33 34 35 36 HPLC analysis of compound purity. Determination of the purity of the RAS Complexes were 37 38 done using analytical HPLC on a Shimadzu Prominence System equipped with a DGU-20A 3 39 40 Degasser, two LC-20AD Liquid Chromatography Pump, a SPD-20A UV/Vis Detector and a 41 42 43 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 44 45 rate of 1.0 mL/min with detection at both 214 nm and 254 nm. The gradient elution conditions 46 47 were as follows: 20-80% solvent B over 30 min, where solvent A is 10 mM NH OAc pH 7.0 and 48 4 49 50 solvent B is CH CN. 3 51 52 53 Synthesis of 1, [(η 6 -benzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 2- 54 55 Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and p-anisidine (61.5 mg, 0.5 mmol) was added 56 57 58 59 60 15 ACS Paragon Plus Environment Molecular Ph armaceutics Page 16 of 40 1 2 3 to dry EtOH (10 mL) and and stirred at r.t. over 24 h. The solvent was removed in vacuo and 4 5 dried. The resultant crude 4-methoxy-N-(2-quinolinylmethylene)-aniline (94 mg, approx. 0.36 6 7 8 mmol) was treated with [(η6-benzene)RuCl ] (89.6 mg, 0.18 mmol) in MeOH (20 mL) and 2 2 9 10 stirred at r.t. over 12 h. The solvent was removed in vacuo and the resulting solid purified by 11 12 13 gradient elution column chromatography (1:1 v/v EtOH/CHCl 3 , R f = 0.2; EtOH, R f = 0.5). The 14 15 purified product was redissolved in CHCl and filtered through a syringe column to remove trace 3 16 17 amount of dissolved silica, before the solvent was removed in vacuo. The final product was then 18 19 20 dried in vacuo for 1 h to give a dark red solid. Yield: 40 mg (22%). 1H NMR (400 MHz, D O): δ 2 21 22 8.68 (d, 3J= 9 Hz, 1H, quinolinyl), 8.57 (s, 1H, PhN=CH), 8.35 (d, 3J = 8 Hz, 1H, quinolinyl), HH 23 24 8.07 (t, 3J = 8 Hz, 1H, quinolinyl), 8.01 (d, 3J = 8 Hz, 1H, quinolinyl), 7.87 (t, 3J = 8 Hz, 25 HH HH HH 26 27 1H, quinolinyl), 7.81 (d, 3J = 8 Hz, 1H, quinolinyl), 7.74 (d, 3J = 9 Hz, 2H, C H OMe), 7.05 HH HH 6 4 28 29 (d, 3J = 9 Hz, 2H, C H OMe), 5.83 (s, 6H, C H ), 3.88 (s, 3H, OCH ) ppm. ESI-MS (+ve 30 HH 6 4 6 6 3 31 mode): m/z = 477 [M]+. ). Purity of the complex was determined to be >95% pure by RP-HPLC 32 33 34 (% Purity): 95.8% at 214 nm and 96.4% at 254 nm; t = 12.5 min. r 35 36 37 Synthesis of 2, [(η 6 -toluene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 2 was 38 39 obtained with [(η6-toluene)RuCl ] (94.6 mg, 0.18 mmol) and crude 4-methoxy-N-(2- 40 2 2 41 42 quinolinylmethylene)-aniline (94 mg, approx. 0.36 mmol) using the same protocol as used for 1 43 44 (Gradient elution: 1:4 v/v EtOH/CHCl , R = 0.2; EtOH, R = 0.7). The final product was then 45 3 f f 46 47 dried in vacuo for 1 h to give a dark red solid. Yield: 65 mg (35%). 1H NMR (400 MHz, DMSO- 48 49 d6): δ 9.16 (s, 1H, PhN=CH), 8.89 (d, 3J = 8 Hz, 1H, quinolinyl), 8.76 (d, 3J = 9 Hz, 1H, HH HH 50 51 quinolinyl), 8.31 (t, 3J = 8 Hz, 2H, quinolinyl), 8.15 (t, 3J = 7 Hz, 1H, quinolinyl), 7.99 (m, 52 HH HH 53 54 3H, quinolinyl /C H OMe), 7.22 (d, 3J = 9 Hz, 2H, C H OMe), 6.07 (t, 3J = 6 Hz, 1H, 6 4 HH 6 4 HH 55 56 C H ), 6.01 (d, 3J = 6 Hz, 1H, C H ), 5.81 (t, 3J = 6 Hz, 1H, C H ), 5.71 (t, 3J = 6 Hz, 1H, 6 5 HH 6 5 HH 6 5 HH 57 58 59 60 16 ACS Paragon Plus Environment Page 17 of 40 Molecular Ph armaceutics 1 2 3 C H ), 5.37 (d, 3J = 6 Hz, 1H, C H ), 3.90 (s, 3H, ArOCH ), 2.17 (s, 3H, CH ) ppm. ESI-MS 6 5 HH 6 5 3 3 4 5 (+ve mode): m/z = 491 [M]+. Purity of the complex was determined to be >95% pure by RP- 6 7 8 HPLC (% Purity): 98.1% at 214 nm and 95.8% at 254 nm; t = 14.4 min. r 9 10 11 Synthesis of 3, [(η 6 -cymene)RuCl(4-methoxy-N-(2-quinolinylmethylene)-aniline)]Cl. 3 was 12 13 obtained with [(η6-cymene)RuCl ] (109.72 mg, 0.179 mmol) and crude 4-methoxy-N-(2- 14 2 2 15 16 quinolinylmethylene)-aniline (94 mg, approx. 0.358 mmol) using the same protocol as used for 1 17 18 (Gradient elution: 1:4 v/v EtOH/CHCl , R = 0.3; 1:1 v/v EtOH/CHCl , R = 0.5). The final 19 3 f 3 f 20 product was then dried in vacuo for 1 h to give a dark orange solid. Yield: 125 mg (61%). 1H 21 22 23 NMR (400 MHz, DMSO-d6): δ 9.15 (s, 1H, PhN=CH), 8.91 (d, 3J = 8 Hz, 1H, quinolinyl), HH 24 25 8.73 (d, 3J = 9 Hz, 1H, quinolinyl), 8.32 (t, 3J = 7 Hz, 2H, quinolinyl), 8.17 (t, 3J = 7 Hz, 26 HH HH HH 27 28 1H, quinolinyl), 8.01 (t, 3J HH = 7 Hz, 1H, quinolinyl), 7.96 (d, 3J HH = 9 Hz, 2H, C 6 H 4 OMe), 7.23 29 30 (d, 3J = 9 Hz, 2H, C H OMe), 6.11 (d, 3J = 6 Hz, 1H, i-PrC H Me), 5.89 (d, 3J = 6 Hz, 1H, HH 6 4 HH 6 4 HH 31 32 i-PrC H Me), 5.75 (d, 3J = 6 Hz, 1H, C H ), 5.39 (d, 3J = 6 Hz, 1H, C H ), 3.91 (s, 3H, 33 6 4 HH 6 4 HH 6 4 34 35 ArOCH ), 2.32 (sept, 1H, 3J = 7 Hz, CH(Me) ), 2.24 (s, 3H, i-PrArCH ), 0.90 (d, 3J = 7 Hz, 3 HH 2 3 HH 36 37 3H, CH(CH ) ), 0.74 (d, 3J = 7 Hz, 3H, CH(CH ) ) ppm. ESI-MS (+ve mode): m/z = 533 3 2 HH 3 2 38 39 [M]+. Purity of the complex was determined to be >95% pure by RP-HPLC (% Purity): 99.4% at 40 41 42 214 nm and 96.0% at 254 nm; t = 19.0 min. r 43 44 45 Synthesis of 4, [(η 6 -hexamethylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)- 46 47 aniline)]Cl. Complex 4 was synthesized according to published procedure.35 48 49 50 51 Synthesis of 5, [(η 6 -1,3,5-triisopropybenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)- 52 53 aniline)]Cl. Complex 5 was synthesized according to published procedure.34 54 55 56 57 58 59 60 17 ACS Paragon Plus Environment Molecular Ph armaceutics Page 18 of 40 1 2 3 Synthesis of 6, [(η 6 -1,3,5-triisopropybenzene)RuCl(N-(2-quinolinylmethylene)-1-naphthyl- 4 5 amine)]Cl. 2-Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and 1-napthylamine (53.2 mg, 0.5 6 7 8 mmol) was added to dry EtOH (10 mL) and and stirred at r.t. over 24 h. The solvent was 9 10 removed in vacuo and dried. The resultant crude N-(2-quinolinylmethylene)-1-naphthylamine 11 12 13 (141 mg, approx. 0.5 mmol) was treated with [(η6-1,3,5-triisopropylbenzene)RuCl 2 ] 2 (188 mg, 14 15 0.25 mmol) in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo 16 17 and the resulting solid purified by column chromatography (1:4 v/v EtOH/CHCl , R = 0.6). The 18 3 f 19 20 purified product was redissolved in CHCl and filtered through a syringe column to remove trace 3 21 22 amount of dissolved silica, before the solvent was removed in vacuo. The final product was then 23 24 dried in vacuo for 1 h to give a dark red solid. Yield: 198 mg (62%). 1H NMR (400 MHz, 25 26 27 CD OD): δ 9.12 (s, 1H, PhN=CH), 8.91 (d, 3J = 12 Hz, 1H, quinolinyl), 8.86 (d, 3J = 11 Hz, 3 HH HH 28 29 1H, quinolinyl), 8.18 (m, 8H, quinolinyl/napthyl), 7.72 (m, 3H, quinolinyl/napthyl), 5.86 (s, 3H, 30 31 C H ), 2.18 (sept, 3J = 9 Hz, 3H, CHMe ), 1.07 (d, 3J = 9 Hz, 9H, CH(CH ) ), 0.98 (d, 3J 32 6 3 HH 2 HH 3 2 HH 33 34 = 9 Hz, 9H, CH(CH ) ) ppm. ESI-MS (+ve mode): m/z = 623 [M]+. Purity of the complex was 3 2 35 36 determined to be >95% pure by elemental analysis. Anal. Calcd for C H Cl N Ru·H O (%): C 37 35 38 2 2 2 38 39 62.12, H 5.96, N 4.14; Found: C 62.29, H 5.59, N 4.18. 40 41 42 Synthesis of 7, [(η 6 -1,3,5-triisopropybenzene)RuCl(3-trifluoromethyl-N-(2-quinolinyl- 43 44 methylene)aniline)]Cl. 2-Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and 3- 45 46 47 (trifluoromethyl)aniline (62.5 µl, 0.5 mmol) was added to dry EtOH (10 mL) and and stirred at 48 49 r.t. over 24 h. The solvent was removed in vacuo and dried. The resultant crude 3- 50 51 trifluoromethyl-N-(2-quinolinylmethylene)aniline (150 mg, approx. 0.5 mmol) was treated with 52 53 54 [(η6-1,3,5-triisopropylbenzene)RuCl ] (188 mg, 0.25 mmol) in MeOH (20 mL) and stirred at r.t. 2 2 55 56 over 12 h. The solvent was removed in vacuo and the resulting solid purified by gradient elution 57 58 59 60 18 ACS Paragon Plus Environment Page 19 of 40 Molecular Ph armaceutics 1 2 3 column chromatography (1:4 v/v EtOH/CHCl , R = 0.4; 1:1 v/v EtOH/CHCl , R = 0.7). The 3 f 3 f 4 5 purified product was redissolved in CHCl and filtered through a syringe column to remove trace 6 3 7 8 amount of dissolved silica, before the solvent was removed in vacuo. The final product was then 9 10 dried in vacuo for 1 h to give a light brown solid. Yield: 156 mg (46%). 1H NMR (400 MHz, 11 12 13 DMSO-d6): δ 9.23 (s, 1H, PhN=CH), 8.96 (d, 3J HH = 9 Hz, 1H, quinolinyl), 8.91 (d, 3J HH = 8 Hz, 14 15 1H, quinolinyl), 8.53 (s(broad), 1H, C H ), 8.43 (d, 3J = 8 Hz, 1H, C H ), 8.31 (t, 3J = 8 Hz, 6 4 HH 6 4 HH 16 17 2H, quinolinyl), 8.16 (t, 3J = 9 Hz, 1H, quinolinyl/C H ), 8.01 (t, 3J = 8 Hz, 2H, 18 HH 6 4 HH 19 20 quinolinyl/C H ), 7.91 (t, 3J = 8 Hz, 1H, quinolinyl/C H ), 5.60 (s, 3H, C H ), 2.37 (sept, 3J 6 4 HH 6 4 6 3 HH 21 22 = 7 Hz, 3H, CHMe ), 1.16 (d, 3J = 7 Hz, 9H, CH(CH ) ), 0.84 (d, 3J = 7 Hz, 9H, CH(CH ) ) 2 HH 3 2 HH 3 2 23 24 ppm. ESI-MS (+ve mode): m/z = 641 [M]+. Purity of the complex was determined to be >95% 25 26 27 pure by elemental analysis. Anal. Calcd for C H Cl F N Ru·3H O (%): C 52.60, H 5.66, N 32 35 2 3 2 2 28 29 3.83; Found: C 52.43, H 5.46, N 3.84. 30 31 32 Synthesis of 8, [(η 6 -1,3,5-triisopropybenzene)RuCl(3-chloro-N-(2-quinolinylmethylene)- 33 34 35 aniline)]Cl. 2-Quinolinecarboxaldehyde (78.6 mg, 0.5 mmol) and 3-chloroaniline (52.9 mg, 0.5 36 37 mmol) was added to dry EtOH (10 mL) and stirred at r.t. over 24 h. The solvent was removed in 38 39 vacuo and dried. The resultant crude 3-chloro-N-(2-quinolinylmethylene)aniline (133 mg, 40 41 42 approx. 0.5 mmol) was treated with [(η6-1,3,5-triisopropylbenzene)RuCl ] (188 mg, 0.25 mmol) 2 2 43 44 in MeOH (20 mL) and stirred at r.t. over 12 h. The solvent was removed in vacuo and the 45 46 47 resulting solid purified by column chromatography (1:4 v/v EtOH/CHCl 3 , R f = 0.6). The purified 48 49 product was redissolved in CHCl and filtered through a syringe column to remove trace amount 3 50 51 of dissolved silica, before the solvent was removed in vacuo. The final product was then dried in 52 53 54 vacuo for 1 h to give a dark red solid. Yield: 89 mg (28%). 1H NMR (400 MHz, MeOD-d4): δ 55 56 9.02 (d, 3J = 9 Hz, 1H, quinolinyl), 9.00 (s, 1H, PhN=CH), 8.79 (d, 3J = 8 Hz, 1H, HH HH 57 58 59 60 19 ACS Paragon Plus Environment Molecular Ph armaceutics Page 20 of 40 1 2 3 quinolinyl), 8.25 (m, 3H, quinolinyl/C H ), 8.15 (t, 3J = 8 Hz, 1H, quinolinyl/C H ), 8.06 (dt, 6 4 HH 6 4 4 5 3J = 5 Hz, 1H, quinolinyl/C H ), 7.99 (t, 3J = 8 Hz, 1H, quinolinyl/C H ), 7.65 (d, 3J = 5 6 HH 6 4 HH 6 4 HH 7 8 Hz, 2H, quinolinyl/C H ), 5.60 (s, 3H, C H ), 2.49 (sept, 3J = 7 Hz, 3H, CHMe ), 1.23 (d, 3J 6 4 6 3 HH 2 HH 9 10 = 7 Hz, 9H, CH(CH ) ), 0.97 (d, 3J = 7 Hz, 9H, CH(CH ) ) ppm. ESI-MS (+ve mode): m/z = 11 3 2 HH 3 2 12 13 607 [M]+. Purity of the complex was determined to be >95% pure by elemental analysis. Anal. 14 15 Calcd for C H Cl N Ru·3H O (%): C 53.41, H 5.93, N 4.02; Found: C 53.02, H 5.66, N 3.87. 31 35 3 2 2 16 17 18 Synthesis of 9, [(η 6 -hexamethylbenzene)RuCl(3-chloro-N-(2-quinolinylmethylene)- 19 20 21 aniline)]Cl. Complex 9 was synthesized according to published procedure.34 22 23 24 Alternative synthetic route for 8 and 9. 2-Quinolinecarboxaldehyde (314 mg, 2 mmol) and 3- 25 26 chloroaniline (268 mg, 4 mmol) were placed to a 50 mL round-bottom flask equipped with a 27 28 Dean-Stark apparatus. 25 mL of toluene was added to dissolve the reactants and the resulting 29 30 31 brown solution was refluxed overnight. The mixture was allowed to cool and concentrated under 32 33 reduced pressure to give the dark brown oil which was dissolved in a minimal amount of diethyl 34 35 36 ether. The black insoluble residue was filtered and the solution was again concentrated under 37 38 reduced pressure and dried in vacuo to give a brown semi-solid. The crude product containing 39 40 small amount of unreacted 3-chloroaniline was used without additional purification. 1H NMR 41 42 43 (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 44 45 = 8.6 Hz, 1H, quinolinyl), 8.12 (dd, J = 15.9, 7.9 Hz, 2H, quinolinyl), 7.90 – 7.83 (m, 1H, 46 47 quinolinyl), 7.76 – 7.69 (m, 1H, quinolinyl), 7.54 – 7.47 (m, 2H, quinolinyl), 7.43 – 7.35 (m, 2H, 48 49 50 quinolinyl). The corresponding [(η6-arene)RuCl ] (0.15 mmol) and 3-chloro-N-(2- 2 2 51 52 quinolinylmethylene)-aniline (180 mg, 0.675 mmol) were dissolved in 10 mL of dry MeOH 53 54 under N atmosphere and stirred for 48 h. A resulting brown solution was evaporated under 55 2 56 57 reduced pressure and the brown residue was washed with copious amounts of diethyl ether. The 58 59 60 20 ACS Paragon Plus Environment Page 21 of 40 Molecular Ph armaceutics 1 2 3 solid was subsequently dissolved in minimal amount of CH Cl and eluted through a short layer 2 2 4 5 of neutral Al O (5 cm) by CH Cl to remove unreacted 3-chloro-N-(2- 6 2 3 2 2 7 8 quinolinylmethylene)aniline and 3-chloroaniline. The product was eluted with CHCl /MeOH 3 9 10 mixture (9:1) and the red filtrate was evaporated to dryness under reduced pressure. The red solid 11 12 13 was redissolved in a minimal amount of dichloromethane and centrifuged (13,000 rpm for 7 min) 14 15 to remove the undissolved residues. The red microcrystals (8) or needle-shaped X-ray quality 16 17 single crystals (9) were produced by slow diffusion of diethyl ether into the CH Cl solutions. 18 2 2 19 20 Crystals were washed with diethyl ether and dried in vacuo. 21 22 23 Complex 8. Yield: 38 mg (20%). Anal. Calcd, C H Cl N Ru·0.25 CH Cl (%): C, 56.50; H, 31 35 3 2 2 2 24 25 5.39; N, 3.22. Found: C, 56.09; H, 5.04; N, 4.46. 26 27 28 Complex 9. Yield: 40 mg (22%). Anal. Calcd, C H Cl N Ru·2CH Cl ·0.25 H O (%): C, 50.45, 29 28 29 3 2 2 2 2 30 31 H, 4.60, N, 4.06. Found: C, 50.36, H, 5.01, N, 4.21. 32 33 34 Tissue culture. The human colorectal carcinoma cells HCT116, human colorectal 35 36 adenocarcinoma cells SW480, human gastric adenocarcinoma AGS, human gastric carcinoma 37 38 39 KATOIII were acquired from ATCC® (Manassa,VA). HCT116 and SW480 were cultured in 40 41 DMEM medium containing 10% FBS and 1% Penicillin/Steptomycin (complete DMEM). AGS 42 43 44 was cultured in RPMI 1640 medium containing 10% FBS and 1% Penicillin/Steptomycin. 45 46 KATOIII was cultured in RPMI 1640 medium containing 20% FBS and 1% 47 48 Penicillin/Steptomycin. All cell lines were grown at 37 °C in a humidified atmosphere of 95% 49 50 51 air and 5% CO . Experiments were performed on cells within 20 passages. 2 52 53 54 Determination of Log P . Log P of the RAS complexes were determined using the shake ow ow 55 56 flask method.38 The RAS complex were predissolved in ddH O that was presaturated with n- 57 2 58 59 60 21 ACS Paragon Plus Environment Molecular Ph armaceutics Page 22 of 40 1 2 3 octanol (for 24 h and left to stand until phase separation occurs). The UV-vis spectrum for each 4 5 samples was obtained and the absorbances at the λ of each compound were determined. Equal 6 max 7 8 volume of n-octanol was added to each sample solution and the heterogeneous mixtures shaked 9 10 for 2 h before centrifuging at 4000 rpm for 1 min to achieve phase separation. The final 11 12 13 absorbance of the aqueous phase at the λ max of each compound were determined and their water- 14 15 octanol partition coefficient were calculated. All experiments were done in triplicate. 16 17 18 Inhibition of cell viability assay. The anti-proliferation activity the RAS Complexes on 19 20 exponentially growing cancer cells were determined using MTT assay as described previously.57 21 22 23 HCT116, SW480, AGS and KATOIII were seeded at 10 000 cells per well (100 µL) in Cellstar® 24 25 96-well plates (Greiner Bio-One) and incubated for 24 h. Thereafter, cancer cells were exposed 26 27 28 to drugs at different concentration in media for 48 h. The final concentration of DMSO in 29 30 medium was < 1% (v/v) at which cell viability was not significantly inhibited. The medium was 31 32 removed and replaced with MTT solution (100 µL, 0.5 mg/mL) in media and incubated for an 33 34 35 additional 45 min. Subsequently, the medium was aspirated, and the purple formazan crystals 36 37 dissolved in DMSO (100 µL). The absorbance due to the dissolved purple formazan was then 38 39 obtained at 565 nm. Inhibition to cell viability was evaluated with reference to the IC value, 40 50 41 42 which is defined as the concentration needed for a 50% reduction of survival based on the 43 44 survival curves. IC values were calculated from the dose - response curves (cell viability vs 45 50 46 47 drug concentration) obtained in repeated experiments and adjusted to actual [Ru] administered, 48 49 which was determined using ICP-OES. The experiments were performed in 4 replicates for each 50 51 drug concentration and were carried out at least three times independently. 52 53 54 For cell viability assays involving inhibitors, Cimetidine (1.5 mM) were added together with test 55 56 57 compounds and incubated for 7 h. Thereafter, the drugs were removed and replace with fresh 58 59 60 22 ACS Paragon Plus Environment Page 23 of 40 Molecular Ph armaceutics 1 2 3 media for the remainder of the 48 h. Pilfithrin-α (10 µM) was incubated with drugs for the entire 4 5 48 h duration. Cell viability in the absence and presence of inhibitor was normalized against 6 7 8 untreated control. Experiments were performed in 3 replicates and carried out at least three times 9 10 independently. 11 12 13 Drug uptake studies. HCT116 cells were grown on Cellstar® 6-well plates (Greiner Bio-One) 14 15 16 at a density of 500 000 cells/well for 24 h before being treated with complexes 1 – 5, 6 and 17 18 oxaliplatin at 1.5 µM for 24 h. Thereafter, the drug-containing media was removed and washed 19 20 once with EDTA/PBS followed by trypsinization. The cells were collected, counted and digested 21 22 23 separately with 69% nitric acid. The samples were diluted to a final concentration of 2% nitric 24 25 acid and the Ru or Pt content quantified via ICP-MS. Experiments were performed three times 26 27 28 independently. 29 30 31 Antibodies and Western blot protocol. HCT116 cells were grown on Cellstar® 6-well plates 32 33 (Greiner Bio-One) at a density of 500 000 cells/well for 24 h before being treated with 34 35 36 complexes 4, 5, 8 and 9 at IC 50 and IC 75 for 2 h, 6 h and 24 h. Oxaliplatin treatment at IC 50 and 37 38 IC was used as a positive control for several experiments. The cells were lysed with lysis buffer 75 39 40 [100 µL, 1% NP40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), protease inhibitor]. The cell 41 42 43 lysate were transferred to separate 2 mL tubes and sonicated for 10 s. The samples were then 44 45 centrifuged at 13000 rpm, 4˚C for 15 min. The supernatant liquid containing the proteins were 46 47 collected and total protein content of each sample was quantified via Bradford’s assay. 50 µg of 48 49 50 proteins from each sample were reconstituted in loading buffer [5% DDT, 1x Protein Loading 51 52 Dye] and heated at 95˚C for 5 min. The protein mixtures were resolved on a 10% SDS-PAGE gel 53 54 by electrophoresis and transferred to a nitrocellulose membrane. The proteins bands were 55 56 57 visualized via enhanced chemiluminescence imaging (PXi, Syngene) after treatment with the 58 59 60 23 ACS Paragon Plus Environment Molecular Ph armaceutics Page 24 of 40 1 2 3 primary antibodies and the appropriate secondary antibodies. Equal loading of protein was 4 5 confirmed by comparison with actin. The following antibodies were used: p53 (FL-393), p21 (F- 6 7 8 5) and Cyclin D1 (H-295) from Santa Cruz Biotechnologies. β-Actin (ab75186) from Abcam. 9 10 ECL Anti-rabbit IgG (NA934V) and ECL Anti-mouse IgG (NA931) from GE Healthcare Life 11 12 13 Sciences. All antibodies were used at 1:1000 dilutions except for actin (1:10000), anti-mouse and 14 15 anti-rabbit (1:5000). 16 17 18 Primers and qPCR protocol. Treatment conditions for HCT116 cells were similar to the 19 20 protocol in western blot. RNA was extracted using TRIzol® Reagent and reverse transcription 21 22 23 was performed with 2 µg of the extracted RNA using Applied Biosystem® High Capacity cDNA 24 25 Reverse Transcription Kit with an Applied Biosystem® 2720 Thermal Cycler. Quantitative PCR 26 27 28 was done on the resulting cDNA using FastStart Universal Probe Master (Rox) with Applied 29 30 Biosystem® 7500 Real Time PCR System. The relative starting quantities of genes of interest 31 32 were normalized against the housekeeping genes TBP and samples were done in duplicates. The 33 34 35 specificity of the amplification was controlled by a melting curve. The gene and Assay ID of 36 37 TaqMan probes are as follows: NOXA (Hs00560402_m1), BAX (Hs00180269_m1), BCL2 38 39 (Hs00608023_m1) and TBP (Hs00427620_m1). 40 41 42 43 44 45 ACKNOWLEDGEMENTS 46 47 48 The authors thank staff of CMMAC, NUS for performing elemental analysis and ICP-OES 49 50 51 analysis. 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