Coordination of Hydroxyquinolines to a Ruthenium Bis-dimethyl

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Coordination of hydroxyquinolines to a ruthenium bisdimethyl-phenanthroline scaffold radically improves potency for potential as antineoplastic agents David Kayvon Heidary, Brock Howerton, and Edith Caroline Glazer J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 14, 2014

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Journal of Medicinal Chemistry

Coordination of Hydroxyquinolines to a Ruthenium Bisdimethyl-phenanthroline Scaffold Radically Improves Potency for Potential as Antineoplastic Agents David K. Heidary, Brock S. Howerton, Edith C. Glazer* University of Kentucky Department of Chemistry, 505 Rose Street, Lexington, Kentucky 40506 Corresponding author: Tel: (859) 257-2198 email: [email protected] Received Date Abbreviations Used MCR, multi-cellular resistance; HQ, 8-hydroxyquinoline; SAR, structure-activity relationships; GFP, green fluorescent protein; IVTT, in vitro transcription and translation; IC50, inhibitory concentration for 50% reduction; TLC, thin layer chromatography; MeOH, methanol; dmbpy, 6,6’-dimethyl-2,2’bipyridine; dmphen, 2,9-dimethyl-1,10-phenanthroline; PARP-1, poly ADP-ribose polymerase-1; pERK, phosphorylated extracellular signal-regulated kinase; GAPDH, gyceraldehyde-3-phosphate dehydrogenase, FBS, fetal bovine serum.

Abstract

A series of ruthenium coordination complexes containing hydroxyquinoline ligands were synthesized that exhibited radically improved potencies up to 86-fold greater than clioquinol, a known cytotoxic compound. The complexes were also >100-fold more potent than clioquinol in a tumor spheroid model, ACS Paragon Plus Environment

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with values similar to currently used chemotherapeutics for the treatment of solid tumors. Cytotoxicity occurs through rapid processes that induce apoptosis, but appear to be mediated by cell-cycle independent mechanisms. The ruthenium complexes do not inhibit the proteasome at concentrations relevant for cell death, and contrary to previous reports, clioquinol and other hydroxyquinoline compounds do not act as direct proteasome inhibitors to induce cell death.

Introduction Hydroxyquinoline (HQ; 1; see Table 1) and its derivatives, particularly clioquinol (5; see Figure 1), have been investigated for decades for various biological and medical applications. These compounds have demonstrated activity as pesticides, antifungal, antibiotic, and anticancer agents, along with rescuing Aβ toxicity in cells1 and restoring cognitive function in mouse models of Alzheimer’s disease.2, 3

The diversity of these biological activities suggests that the compounds interfere with different cellular

processes, likely through different mechanisms. HQ is a nitrogen-containing heterocyclic compound that is able to chelate several metal cations, including Cu(II),2 Fe(II),4 Zn(II),5 Al(III),6 and Ru(II).7, 8 In general, the biological effects of hydroxyquinoline compounds are attributed to the chelation of an endogenous metal, such as Cu(II), Zn(II), and Fe(II), acting as an ionophore9 and altering metal homeostasis,10 metal-dependant protein aggregation1 or degradation processes,11 or inhibiting metaldependant enzymes.12 We are interested in developing light-activated metal complexes, and due to the significant biological activity of hydroxyquinoline derivatives, we anticipated that they could be useful as photo-triggered agents.13 Our goal was to incorporate these ligands into strained ruthenium compound scaffolds14, 15 to create pro-drugs that would be biologically active only upon irradiation-induced ligand release from the metal center. However, we have discovered that the complexes are not particularly photoreactive, complicating any goals for developing light-activated hydroxyquinoline systems. Rather, the Ru(II) complexes containing hydroxyquinoline ligands exhibit significant potential as cytotoxic agents in their intact coordination complex form. In this report we explore the structure-activity relationships (SAR) ACS Paragon Plus Environment

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that mediate this biological activity, and investigate the mechanism of cytotoxicity and mechanism of cell death for the coordinatively inert Ru(II) complexes. In contrast to the vast majority of other reports on such biologically active hydroxyquinoline systems, the kinetically inert Ru(II) center irreversibly occupies the metal coordination sites of the HQ ligands, precluding any interactions with other metal centers. Thus, the complex is not capable of acting through the chelation mechanism previously accepted for the free HQ ligands. In a complimentary manner, unlike cisplatin and other promising biologically active ruthenium complexes such as NAMI-A (Figure 1), the complexes do not have any ligands that are subject to substitution. In addition to revealing remarkable improvements in potency through coordination, this study also clearly indicates that both hydroxyquionoline derivatives and their Ru(II) complexes are not proteasome inhibitors, contrary to reports on the free ligands. In addition, the compounds do not bind or damage nucleic acids, in contrast to most substitutionally inert, biologically active octahedral Ru(II) complexes.16 Instead, these compounds act through some other, currently unknown, mechanism of action.

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Figure 1. Structures of biologically active ruthenium complexes NAMI-A and Ru(bpy)2dppz (dppz = dipyrido[3,2-a:2’,3’-c]phenazine; bpy = 2,2’-bipyridine), peptide based proteasome inhibitors MG132 and Bortezomib (Velcade), and clioquinol and compound 12 described in this study. Note that racemic mixtures containing both enantiomers of Ru(bpy)2dppz and 12 are generated in the synthesis of these compounds, though only the Λ enantiomer is shown here for simplicity.

Results and Discussion Synthesis In order to explore structure-activity relationships (SAR), a small family of heteroleptic Ru(II) complexes were synthesized that contained either two methylated 1,10-phenanthroline ligands (2,9dimethyl-1,10-phenanthroline) or two 2,2’-bipyridine ligands (bpy) and one hydroxyquinoline type ligand. Hydroxyquinoline (1) and 7 different hydroxyquinoline derivatives were investigated, including clioquinol, as shown in Table 1. The ligands chosen contained substituents at the 2, 5 and 7 positions in order to investigate the impact of modification on both the coordination face and the back side of the ligand. The Ru(II) complexes were synthesized from a racemic mixture of the Δ and Λ enantiomers of Ru(dmphen)2Cl2 or Ru(bpy)2Cl2, and form a mixture of enantiomers upon coordination of the hydroxyquinoline ligand. While the hydroxyquinoline ligand is asymmetric, the coordination of this ACS Paragon Plus Environment

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ligand in two different orientations to each enantiomer of the Ru(LL)2Cl2 backbone (LL = a general bidentate ligand that is symmetric) produces only two structures which are non-superimposable mirror images, as illustrated in Figure 2. The compounds in this study were tested as racemic mixtures, but the different enantiomers may have different affinities for their particular target, which could affect the cytotoxic potency. This will be explored in the future. All complexes were exhaustively purified to ensure no contamination of either free ligands or coordinatively unsaturated Ru(II) centers. As the hydroxyquinoline is deprotonated and coordinates the Ru(II) as an anion, the complexes carry a +1 charge, except in the case of hydroxyquinoline ligands containing ionizable groups such as sulfonic acids.

Cytotoxicity Studies The potency of the free hydroxyquinoline ligands 1-8 were determined against a panel of solid tumor and leukemic cell lines (Table 1; A549 human non-small cell lung carcinoma, H226 squamous cell carcinoma, BT-549 human breast carcinoma, K562 chronic myelogenous leukaemia, HL60 human promyelocytic leukemia, Jurkat human acute T cell leukemia) and compared to the Ru(II) hydroxyquinoline coordination compounds (Tables 2 and 3). For compound 2, addition of a methyl group at the 2 position of the hydroxyquinoline (R3; adjacent to the nitrogen), improved potency by > 2– 5-fold in several cell lines compared to the parent compound, but diminished it by ~3-fold in two other cell lines. Incorporation of halogens at the 5- and 7- positions, as with 5,7-dichloro-hydroxyquinoline (4) and clioquinol (5), also improved potency relative to the parent hydroxyquinoline ligand. Somewhat surprisingly, the hydroxyquinoline containing only one chlorine at the 5-position (3) was more potent across all cells lines than the disubstituted systems, with an average IC50 value of 1.52 µM. The greatest effect was observed in the A549 and H226 cell lines, where the monosubstituted system was 3–8-fold more potent than 5,7-dichloro-hydroxyquinoline or clioquinol. Addition of a nitro or an amine group at the 5-position (6 and 8) reduced potency slightly, to an average of 2.41 and 2.86 µM. The potency of


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clioquinol was similar to that of 6, with an average IC50 value of 2.86 µM. The only free ligand that was essentially inactive in all cell lines up to 30 µM was the 8-hydroxyquinoline-5-sulfonic acid (7). Figure 2. Addition of the asymmetric HQ ligand to the two enantiomers of the Ru(bpy)2 backbone produces only one pair of enantiomers. Each of the mirror image molecules with the HQ ligand (shown in blue) can be rotated about the horizontal axis to give a different orientation of the HQ ligand (shown in red). The different representations (top and bottom) are non-superimposable by simple translation, but only represent different orientations of the same molecule. σ indicates the mirror plane.


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Table 1. Cytotoxicity IC50 values (µM) for various hydroxyquinoline ligands in cancer cell lines R2 R1 HO

N R3

Compound

R1

R2

R3

A549

H226

BT549

K562

HL60

Jurkat

1

H

H

H

>30

>30

>30

1.21 ± 0.04

11.20 ± 1.05

3.22 ± 0.14

2

H

H

CH3

14.70 ± 1.31

5.03 ± 0.38

14.10 ± 1.28

4.15 ± 0.21

6.15 ± 0.74

9.32 ± 0.54

3

H

Cl

H

1.50 ± 0.03

0.26 ± 0.08

1.60 ± 0.02

1.93 ± 0.50

1.11 ± 0.13

2.73 ± 0.33

4

Cl

Cl

H

13.10 ± 0.02

1.71 ± 0.11

2.20 ± 0.28

1.25 ± 0.05

3.60 ± 0.25

3.38 ± 0.07

5 / clioquinol

I

Cl

H

4.30 ± 2.52

2.28 ± 0.81

3.70 ± 0.75

1.05 ± 0.03

3.00 ± 0.31

4.15 ± 1.12

6

H

NO2

H

2.30 ± 0.24

1.10 ± 0.42

7.27 ± 1.13

1.35 ± 0.16

2.31 ± 1.17

2.68 ± 0.05

7

H

SO3

H

>30

>30

>30

>30

>30

29.80 ± 4.6

8

H

NH2

H

3.90 ± 1.87

2.50 ± 3.01

4.02 ± 0.42

2.24 ± 0.14

0.80 ± 0.09

2.27 ± 0.15

Unexpectedly, coordinating the compounds to the ruthenium scaffold radically increased potencies across all cell lines. Previous SAR studies have indicated that the nitrogen and hydroxyl groups of the quinolines are required for activity, as functionalizing these groups renders the compound inactive.11 Contrary to expectations, coordination of these groups to ruthenium did not reduce, but instead significantly improved potencies for all HQ compounds. Compound 12, containing the clioquinol ligand, was the most potent, with IC50 values ranging from 0.04 – 0.31 µM. This represents an increase in cytotoxicity by an average of 42-fold compared to the free clioquinol ligand. The enhancement in cytotoxicity was cell line dependant, with BT549 cells exhibiting a 20-fold improvement in potency ACS Paragon Plus Environment

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with 12, while an 86-fold increase was seen in A549 cells. This improvement in cytotoxicity was observed even for those compounds that did not show any activity, such as 7 (with no activity observed to 30 µM), where the Ru(II) complex (compound 14) exhibited IC50 values ranging from 1.34 – 8.95 µM. This ligand, which was effectively inactive, became more potent than clioquinol when coordinated to the ruthenium center. Clearly, addition of the ruthenium backbone greatly enhanced the cytotoxic activity of the entire hydroxyquinoline family of compounds. Table 2. Cytotoxicity IC50 values (µM) for various hydroxyquinoline ligands coordinated to the Ru(dmphen)2 scaffold in cancer cell lines R2 R1 O

N

Ru (dmphen)2

R3

Compound

R1

R2

R3

A549

H226

BT549

K562

HL60

Jurkat

9

H

H

H

1.30 ± 0.16

0.35 ± 0.06

0.10 ± 0.01

0.098 ± 0.007

0.52 ± 0.06

0.60 ± 0.08

10

H

H

CH3

0.37 ± 0.01

1.10 ± 0.06

0.80 ± 0.29

0.30 ± 0.05

0.49 ± 0.07

0.66 ± 0.06

11

Cl

Cl

H

0.12 ± 0.006

0.50 ± 0.17

0.60 ± 0.05

0.36 ± 0.04

0.11 ± 0.006

0.18 ± 0.02

12

I

Cl

H

0.05 ± 0.01

0.078 ± 0.009

0.18 ± 0.009

0.031 ± 0.003

0.04 ± 0.004

0.17 ± 0.004

13

H

NO2

H

3.72 ± 0.09

4.30 ± 0.26

0.70 ± 0.06

2.17 ± 0.14

5.70 ± 0.52

3.04 ± 0.11

14

H

SO3

H

1.55 ± 0.23

1.34 ± 0.08

1.44 ± 0.12

1.83 ± 0.11

8.95 ± 2.99

1.52 ± 0.08

To determine if the chemical features of co-ligands on the ruthenium scaffold played a role in the potencies observed, the two dmphen coordinating ligands were replaced with two 2,2’-bipyridine (bpy) ligands. Surprisingly, the incorporation of bpy ligands greatly reduced the potencies of the compounds. The average IC50 value for the bpy-scaffold appended to clioquinol (16) shifted to 1.83 µM against the panel of cell lines, which is only slightly more potent than the values observed for the uncoordinated ACS Paragon Plus Environment

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hydroxyquinoline ligand. This loss of potency was particularly striking between compound 14 and 17, where the IC50 values shifted from an average of 3.3 µM with the dmphen scaffold to >100 µM with the bpy-containing scaffold. Even more surprising was the behavior in the BT549 cell line. While the potency of the clioquinol complex 12 fell off radically when the dmphen ligands were replaced with bpy (compound 16; e.g., 56-fold in the A549 cell line, 51-fold in the HL60 cell line), there was only a 2-fold change in potency in the BT549 cell line. Table 3: Cytotoxicity IC50 values (µM) for various hydroxyquinoline ligands coordinated to the Ru(bpy)2 scaffold in cancer cell lines R2 R1 O

N Ru (bpy)2

Compound

R1

R2

A549

H226

BT549

K562

HL60

Jurkat

15

Cl

Cl

19.74 ± 3.81

14.80 ± 0.44

11.10 ± 1.14

3.94 ± 0.01

14.90 ± 2.91

5.84 ± 0.54

16

I

Cl

2.79 ± 1.47

3.37 ± 0.17

0.38 ± 0.13

0.57 ± 0.14

2.04 ± 0.58

4.16 ± 1.46

17

H

SO3

>100

>100

>100

>100

>100

18.38 ± 0.65

Potency in 3-D Tumor Spheroids Compounds that were potent in 2-D cell culture were assayed for efficacy in 3-D tumor spheroids. The 3-D tumor spheroid model better reflects the complexity of in vivo tumors, including features such as hypoxia, slowed growth, and increased resistance to cytotoxic agents (an effect termed mutlicellular resistance, MCR).17-19 A549 cells were seeded in agarose-coated 96-well plates and incubated for 7–10 days, producing spheroids of 580 ± 5 µM in diameter. The ruthenium compounds were screened in dose response and compared to the potency of clioquinol in the tumor spheroids. As shown in Figure 3 and Table 4, both compounds 11 and 12 were highly effective, with IC50 values of ~1 and 0.4 µM. Once ACS Paragon Plus Environment

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again, this compares favorably with the potency of the free ligands, as the IC50 for clioquinol was 42.9 µM. While there was a 9-fold decrease in potency for 12 against a tumor spheroid compared to 2-D tissue culture, the ruthenium complex is 107-fold more potent than clioquinol. Interestingly, one of the less potent compounds, 13, exhibited the lowest MCR (2.9). While still 10- or 25-fold less potent than 11 or 12, this compound appears promising in this more complex model. Therefore, it is advisable not to eliminate compounds early due to poor potency in the 2-D model. Instead, it is best to obtain the MCR values from screening in the 3-D tumor spheroids. Figure 3. Dose response curves for cell cytotoxicity of Velcade (orange triangles), clioquinol (black circles) and 12 (blue squares) in A549 tumor spheroids. 100

% Viable

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GL109 Velcade Clioquinol

80 60 40 20 0

-3

-2

-1

0

1

2

Log concentration (µM) Table 4: Efficacy of compounds (µM) in 3-D A549 tumor spheroids

Compound

2-D

3-D

MCR

5 / clioquinol

4.3 ± 2.52

42.93 ± 8.58

10

9

1.3 ± 0.16

2.43 ± 0.45

1.9

11

0.12 ± 0.006

1.02 ± 0.22

8.5

12

0.05 ± 0.01

0.39 ± 0.04

7.8

13

3.72 ± 0.09

10.90 ± 1.67

2.9

MG132

0.11 ± 0.07

4.68 ± 0.85

43

Velcade

0.01 ± 0.0003

0.086 ± 0.016

8.6


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Bortezomib (Velcade, Millennium Pharmaceuticals, Cambridge, MA, Figure 1), an FDA approved chemotherapeutic which acts through inhibition of the proteasome,20, 21 was also assayed, along with the proteasome inhibitor MG132.22,

23

The IC50 values for these two compounds demonstrate far better

potency than clioquinol (0.01 and 0.11 µM in 2-D, 0.086 and 4.68 µM in 3-D for Velcade and MG132, respectively). From this data, both 11 and 12 are more potent that MG132 in the spheroid and both are within 4-fold of the potency of Velcade, one of the most potent chemotherapeutics. In addition, both 11 and 12 were more potent than the standard chemotherapeutics doxorubicin (2.2 µM), taxol (>10 µM), or mitoxantrone (4.8 µM) against these tumor spheroids (Figure S12). Effect of Copper It has long been accepted that clioquinol acts as a chelating agent, and its specific biological effects are a function of the endogenous metal that the clioquinol coordinates. Dou and coworkers have shown that the addition of 10 µM CuCl2 improves the cytotoxicity of clioquinol by approximately 10-fold, and Schimmer has also found CuCl2 enhanced the ability of clioquinol to inhibit the activity of the proteasome.11, 24-26 As a result, we explored the effect of CuCl2 alone and in combination with clioquinol or 12.

Several of the cell lines displayed dose-dependent cytotoxicity in the presence of CuCl2,

indicating that copper alone is sufficient to induce cell death. The HL60 and A549 cell lines were the most susceptible to CuCl2, with IC50 values of 1.1 and 4.5 µM respectively. Notably, the inorganic copper salt had no effect on Jurkat cells, as previously reported.24 The addition of 10 µM CuCl2 did improve potencies for clioquinol, where submicromolar values were observed across the panel of cell lines (see Table 5). In contrast, no significant changes in IC50 values were seen with the ruthenium complexes in the presence of copper, highlighting the fact that this compound can not act as a copper ionophore. Despite the significant improvement in the cytotoxic activity of clioquinol due to copper chelation, 12 was still >10-fold more potent than clioquinol even in the presence of copper. The only exception to this was the behavior in the BT549 cell line. In this case, a 60-fold increase in potency of clioquinol was observed when it was dosed with CuCl2, despite the fact that this cell line does not


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appear to have intrinsic sensitivity to copper (IC50=100 µM; 30% cell death observed at the experimental condition of 10 µM CuCl2; see Table 5). Interestingly, co-dosing of copper with 12 actually decreased the activity of the ruthenium compound. The reason for the unusual sensitivity of the BT549 cell line is currently unknown, but may relate to the mechanism of action, as addressed below. Table 5. Effect of CuCl2 on the potency (µM) of clioquinol (5) and 12

a

Cell Line

5 + 10 µM CuCl2

Sensitizati a on Ratio 5

12 + 10 µM CuCl2

Sensitization a Ratio 12

CuCl2 IC50 µM

% Cell Death 10 µM CuCl2

A549

0.54 ± 0.028

8

0.029 ± 0.005

1.7

4.50 ± 0.57

90

H226

0.56 ± 0.022

4

0.041 ± 0.011

1.9

100 ± 11

22

BT549

0.06 ± 0.006

62

0.29 ± 0.065

0.6

100 ± 19

30

K562

1.39 ± 0.006

0.8

0.011 ± 0.009

2.8

>100

0

HL60

0.93 ± 0.010

3

0.048 ± 0.005

0.8

1.13 ± 0.13

100

Jurkat

3.74 ± 0.017

1.1

0.16 ± 0.003

1

>100

0

The sensitization ratio is the ratio of the IC50 value in the absence and presence of CuCl2.

Effect of glutathione Glutathione (GSH) is known to inactivate platinum containing chemotherapeutics,27 and it also has the potential to affect the stability or activity of metal complexes as it acts as a biological reducing agent. In order to test complex 12 under challenging biologically conditions that would mimic features of the tumor environment, a dose response was performed in the presence of 10 mM GSH. The IC50 value obtained for HL60 cells under these conditions was 0.046 µM, essentially unchanged from the value in the absence of GSH (0.040 µM). In addition, the compounds were found to be stable in PBS and cell media over a 72 hour period, as no changes in the absorption spectra were observed (see SI Figure 11). These experiments support the premise that the Ru(II) complexes are stable in the presence of thiols and media, and are interacting with their cellular targets in their intact form.


12

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Mechanism of cell death The mechanism of cell death was explored for 12 in comparison to clioquinol and the proteasome inhibitors MG132 and Velcade. HL60 cells were analyzed 8 hours after treatment with compound. As shown in Figure 4, compound 12 induced cleavage of Caspase 3 and 9, which coincided with activation of PARP, indicating the executionary phase of apoptosis. Interestingly, clioquinol and the proteasome inhibitor MG132 exhibited less Caspase 9 cleavage at the concentrations assayed, and clioquinol, MG132 and Velcade all displayed no Caspase 3 cleavage. The activation of PARP did occur with MG132, and clioquinol and Velcade to a lesser degree. Significantly more PARP activation was observed for these compounds after 16 hours (Figure S1). Clioquinol is known to induce caspasedependant apoptosis in a concentration and time dependant manner,5 with maximal signal apparent at 32 hours. These studies indicate that 12 induces the executionary phase of apoptosis far more rapidly than clioquinol or the validated proteasome inhibitors. The phosphoylation of p38 and c-Jun were also evaluated, as these proteins are subject to increased phosphorylation during apoptosis. As shown in Figure 4, both clioquinol and 12 increased phosphorylation levels of p38 (Thr180, Tyr182), compared to the untreated control. However, this effect was at a lower level that observed with the proteasome inhibitors MG132 and Velcade. The level of phosphorylated Jun (Ser 73) was unaffected for treatment with clioquinol and 12, but was slightly increased with MG132. The phosphorylation of Akt (Ser 473) was suppressed only with 12, indicating that this compound may induce apoptosis through suppressing the activity of the phosphoinositol-3kinase pathway. Taken together, it appears that both 12 and clioquinol induce apoptosis through caspase and cell signaling mediated events. The reduction in Akt signaling by 12 may be required for the rapid onset of apoptosis, or could indicate an upstream biological target for 12 that is not shared by clioquinol or the proteasome inhibitors.


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Figure 4. Activation of apototosis pathways with 12 (1 µM), clioquinol (20 µM), MG132 (1 µM) and Velcade (0.1 µM) in HL60 cell lysates 8 hours after compound addition. N.C. is the no compound control.

Mechanism of action studies The cytotoxic mechanism of action for hydroxyquinolines has been stated to occur through proteasome inhibition.11,

25, 28, 29

To determine if complex 12 acted through interfering with protein

degradation in this manner, proteasome inhibition assays were carried out using a fluorescent proteasome functional assay. While 12 was found to inhibit the proteasome, this occurred with an IC50 value of 1.65 µM, a concentration 33 times higher than is required for cytotoxicity. Furthermore, as shown in Figure 5A, no effect was seen with clioquinol at concentrations 10 times above the IC50 for cytotoxicity. To confirm the accuracy of the IC50 values for proteasome inhibition assay, the proteasome inhibitor MG132 was tested as a positive control. The IC50 value for MG132 for proteasome inhibition was found to be 0.025 µM, which was ca. 4-fold more potent than the IC50 value of 0.110 µM for cytotoxicity. Thus, the disconnect between the activity in the proteasome inhibition assay and cell cytotoxicity suggested that both clioquinol and 12 do not exert their biologically relevant, cytotoxic effects through proteasome inhibition.


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In order to provide verifying evidence of normal proteasome function in cells at concentrations near the IC50, HL60 cells were treated with the compounds for 8 hrs and then immunoblotted for the presence of ubiquitinated proteins. The characteristic smear corresponding to increased levels of ubiquitinated protein occurred with cells dosed with either MG132 or Velcade (Figure 5B). However, no increase in ubiquitinated protein was seen with either 12 or clioquinol at concentrations well above their cytotoxic IC50 values. This supports the results from the fluorescent proteasome inhibition assay and indicates that the proteasome is not inhibited either in vitro or in cells at concentrations capable of inducing cell death. Taken together, it is apparent that proteasome inhibition does not occur at concentrations near the IC50 value for cytotoxicity for either clioquinol or its ruthenium complex 12. While clioquinol has long been explored as a potential chemotherapeutic and is believed to be a proteasome inhibitor,26 its biological relevant mechanism of action appears to be through some other mechanism where it is more potent. In fact, in the previous reports of the proteasome inhibition activity of clioquinol, concentrations between 1 and 10 mM were required for inhibition,24, 25 in contrast to the 10-20 µM IC50 values for cytotoxicity, indicating that reports of the compound’s biological mechanism of action are inconsistent with proteasome inhibition. Recently, this feature has been addressed with studies that show that clioquinol effects potent inhibition of the proteasome when combined with equalmolar copper, with a potency that is the same as copper alone (for assays performed in cell lysates).25, 29 Clioquinol is a far less potent inhibitor of the proteasome in the absence of copper.26 The hypothesis is that the clioquinol acts as an ionophore, facilitating uptake of the copper before it is likely liberated and inhibits the proteasome. It is possible that the enhanced sensitivity of the BT549 cell line to clioquinol in the presence of CuCl2 (where a 60-fold enhancement in the cytotoxicity IC50 value was observed) may be due to proteasomal inhibition, as this and other triple negative breast cancer cell lines have been shown to be particularly vulnerable to proteasome inhibitors.30 Indeed, this cell line was sensitive to MG132, with an IC50 of 0.011 µM; the average of the IC50 values for MG132 in the other cell lines was 0.062 µM.


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Figure 5. Inhibition of proteasome. A) Dose response in the proteasomal chymotrypsin-like inhibition assay with MG132 (orange triangles), clioquinol (black cicles), and 12 (blue squares). B) Western Blot for polyubiquitinated proteins for 12 (1 µM), clioquinol (20 µM), MG132 (1 µM) and Velcade (0.1 µM) in HL60 cell lysates. N.C. is the no compound control.

Cell Cycle Analysis To investigate possible effects on the cell cycle, HL60 cells were incubated with 12, clioquinol, MG132 and Velcade from 8 to 24 hours followed by propidium iodide staining and analysis by flow cytometry. While both clioquinol and MG132 showed an increase in S phase over time associated with an increase in apoptotic cells, 12 did not show a consistent trend in cell cycle population change over time (Figure 6). However, a different pattern of cell cycle arrest was observed in the K562 cell line (Figure S2). Here, 12 appeared to induce G1 arrest, while the proteasome inhibitor MG132 induces G2/M arrest. No clear arrest point was observed for clioquinol.


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Figure 6. Cell cycle analysis for A) 12 (1 µM), B) clioquinol (20 µM), C) MG132 (1 µM) and D) Velcade (0.1 µM) in HL60 cells.

Effects on Transcription and Translation Given the results demonstrating that both clioquinol and its ruthenium complex are not proteasome inhibitors, alternative mechanisms of action were explored. Many ruthenium complexes have been shown to bind DNA, which could affect transcription and translation. In addition, metal complexes of hydroxyquinoline ligands have been described as DNA binders, likely through intercalation.31,

32

To

determine if the mechanism of 12 was due to any interaction with DNA or RNA, a coupled in vitro transcription and translation (IVTT) assay, using green fluorescent protein (GFP) as a reporter,33 was performed. This assay allows for identification of agents that interfere with DNA or RNA function, rather than simply reporting on nucleic acid damage that induces large structural changes, as are revealed by DNA gel electrophoresis. The assay mixture contains all the components that are required for transcription and translation, and is generated from HeLa cell extract. The IVTT mixture was incubated for 10 minutes in the presence of increasing concentrations of 12 or with 10 µM clioquinol, and then a GFP encoding plasmid was introduced into the IVTT mixture. No change in GFP production ACS Paragon Plus Environment

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was observed over a range of concentrations of 12, from 0.15 µM (where significant cytotoxicity occurred) to 10 µM (Figure 7).

In addition, no inhibition of GFP synthesis was observed with

clioquinol, indicating that neither the free ligand nor the ruthenium complex appears to have any functional impact on DNA or the processes of transcription or translation.34

Figure 7. Effect of clioquinol (10 µM) and 12 on transcription and translation.

Selectivity for cancer cells In order to assess if the compounds possessed sufficient toxicity against malignant cell lines in comparison to healthy cells, additional screening was performed in human peripheral blood mononuclear cells (PBMCs). PBMCs where chosen as a healthy cell control and also to probe for potential adverse effects such as neutropenia, which is a common side effect of chemotherapeutics. The IC50 values for select compounds in PBMCs are contrasted with the potency in the HL60 cell line in Table 6. The two lead coordination compounds 11 and 12 exhibited 32–47-fold increased toxicity in the cancer cell line relative to this healthy cell control. This degree of selectivity compares favorably with several other chemotherapeutics tested, especially the proteasome inhibitors Velcade and MG132, where the compounds are equally toxic to cancer cells and PBMCs.


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Table 6. Cytotoxicity values (µM) for various compounds in PBMCs and the HL60 cell lines

Compound

PBMC

HL60

Ratio

5 / clioquinol

17.05 ± 1.72

3.0 ± 0.21

5.7

11

5.17 ± 1.10

0.11 ± 0.006

47.0

12

1.31 ± 0.2

0.04 ± 0.006

32.8

Cisplatin

120 ± 20.8

3.1 ± 0.35

38.7

Doxorubicin

0.20 ± 0.02

0.02 ± 0.003

10

MG132

0.64 ± 0.05

0.96 ± 0.11

0.66

Velcade

0.007 ± 0.0004

0.007 ± 0.004

1

Comparison to other metal complexes containing hydroxyquinoline ligands Recently, there has been interest in creating coordination complexes containing hydroxyquinoline ligands10,

35-38

or other coordinating groups39 that have shown potential as drugs. This approach has

generally resulted in only modest improvements in potency. Marchio and coworkers demonstrated that 6–7-fold increases in potency were observed in two cell lines when the cells were treated with hydroxyquinoline ligands in the presence of equalmolar copper.10 They identified a good correlation between the IC50 of the complex and the logP of the free ligand, and hypothesized that the ligands act as ionophores for the copper. Gobec, Turel and coworkers made a ruthenium cymene complex containing clioquinol as a ligand, and observed similar potencies for the complex and free ligand in adherent cell lines with a 4-fold improvement in the IC50 in leukemia and lymphoma cell lines.35 The behavior of this complex was independent of copper. Aleman and coworkers made platinum complexes from hydroxyquinoline ligands, where the metal center contained other labile ligands that would allow for covalent bond formation to DNA.37 While the cytotoxicity for the free ligands is not reported, there were variations in the IC50 values of the platinum complexes as a function of the substituents on the hydroxyquinoline ligand (from 2 to 20-fold), indicating that the activity is mediated at least in part by the chemical features of the ligand. Very recently, Liu and coworkers reported two Ru(II) complexes ACS Paragon Plus Environment

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containing the 8-hydroxyquinoline ligand and bpy or phen coligands (forming complexes analogous to 9), and demonstrated inhibition of angiogenesis and tumor growth in vivo.38 This report highlights the promise of coordinatively inert Ru(II) complexes with these HQ ligands as anti-cancer agents. The ruthenium compounds reported here are not capable of acting as ionophores for other metals, as there are no free coordinating groups on the complex. Furthermore, the compounds are stable, so the biological activity observed is related solely to the properties of the intact coordination complex. As there are no reactive sites on the complex or the ligands, reactions are not feasible such as those observed with the platinum hydroxyquinoline complexes containing labile ligands. There is no correlation between the logP of the hydroxyquinoline complex and the cytotoxicity IC50 values (see Figure S3), so variations in cellular uptake or partitioning appear unlikely to be the cause of the SAR observed. As the biologically relevant target for the coordination compounds remains to be identified, it is challenging to rationalize the SAR. However, incorporation of small substituents at the 2, 5 and 7positions of the hydroxyquinoline compound increase potency, as does the 2,9-dimethyl-1,10phenanthroline containing backbone. Additional studies are underway to attempt to identify the cellular targets of these complexes and to further refine the SAR. Conclusions A small library of 9 cytotoxic ruthenium complexes containing 7 different hydroxyquinoline ligands was synthesized to explore structure-activity relationships. The presence of halogens at the 5- and 7positions resulted in the most potent compounds, and incorporation of electron rich substituents such as nitro groups and sulfonic acids at the 5-position of the hydroxyquinoline reduced potency up to 220fold. Coordination of the hydroxyquinoline ligands to the ruthenium center improved potencies up to 86-fold, with an average improvement of 42-fold for complex 12 compared to clioquinol. The coligands of the ruthenium complex play an important role as well, with up to 50-fold differences in potency with 2,9-dimethyl-1,10-phenanthroline ligands vs. 2,2’-bipyridine ligands. The choice of the 2,9-dimethyl-1,10-phenanthroline backbone appears quite important to this class of compounds. ACS Paragon Plus Environment

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Despite the previous literature reports on clioquinol and other metal complexes containing this ligand acting as proteasome inhibitors, it is clear that this is not the biologically important cellular effect that eventually induces cytotoxicity. Clioquinol is cytotoxic at doses 10-fold lower than those required to observe proteasome inhibition in two different assays, and compound 12 required doses 30-fold higher than the IC50 for cytotoxicity to induce proteasome inhibition. While both the ligand and the metal complex are capable of inhibiting this process for protein degradation at high concentrations, it seems quite unlikely that this is responsible for their cytotoxic effect.

Experimental Materials and Methods All materials were obtained from commercial sources and were used without further purification. The ligands purchased were 8-hydroxyquinoline (Alfa Aesar, 99%), 8-hydroxy-2-methyl quinoline (Alfa Aesar, 98%), 5,7-dichloro-8-hydroxyquinoline (Alfa Aesar, 99%), 5-chloro-8-hydroxy-7-iodoquinoline (Alfa Aesar, 98%), 8-hydroxy-5-nitoquinoline (Alfa Aesar, 96%), 8-hydroxyquinoline-5-sulfonic acid monohydrate (Acros, 98%). All 1H NMR spectra were obtained on a Varian Mercury Spectrometer (400 MHz) with chemical shifts reported relative to the residual solvent peak of acetonitrile at δ 1.93. Electrospray ionization mass spectra were obtained on a Varian 1200L mass spectrometer. Absorption spectra were recorded on an Agilent 8453 Diode Array spectrophotometer. FT-IR spectra were acquired on a Thermo Scientific Nicolet 6700 FT-IR Spectrometer. All synthesized compounds were isolated in >95% purity, as determined by analytical HPLC. For HPLC analysis, the chloride salts of the ruthenium complexes were injected on an Agilent 1100 Series HPLC equipped with a model G1311 quaternary pump, G1315B UV diode array detector and ChemStation software version B.01.03. Chromatographic conditions were optimized on a Column Technologies Inc. C18, 120 Å (250 mm x 4.6 mm inner diameter, 5 μM) fitted with a Phenomenex C18 (4 mm x 3 mm) guard column. Injection volumes of 15 μL of 100 μM solutions of the complex were used. The detection wavelength was 280 nm. Mobile phases were: Mobile Phase A: 0.1% formic acid in dH20; Mobile Phase B: 0.1% formic acid in HPLC ACS Paragon Plus Environment

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grade acetonitrile. The mobile phase flow rate was 1.0 mL/min. The following mobile phase gradient was used: 98%-95% A (containing 2%-5% B) from 0-5 min; 95%-70% A (5%-30% B) from 5-15 min; 70%-40% A (30%-60% B) from 15-20 min; 40%-5% A (60%-95% B) from 20-30 min; 5%-98% A (95%-2% B) from 30-35 min; reequilibration at 98% A (2% B) from 35-40 min. Note: Ruthenium complexes containing sulfonic acids were not amenable to characterization by 1H NMR or HPLC analysis. Partition coefficients were determined by the “shake flask” method using pre-saturated solvents pH 7.4 phosphate buffer (0.129 M NaCl) and n-octanol.39 The complexes were dissolved in the solvent they were most soluble in at 100 µM concentrations, and the measurements were performed in triplicate. The containers were subjected to 100 rotations performed by hand, followed by one hour of settling time. To ensure complete separation, the solutions were centrifuged at 1500 rpm for 10 minutes. Each phase was analyzed for the presence of compound by UV-Vis spectroscopy.

Preparation of the Ru(dmphen)2Cl2 scaffold: A solution of RuCl3 (3.8 mmol, 1 g), 2,9-dimethyl-1,10-phenanthroline (7.6 mmol, 1.59 g), and LiCl (45.9 mmol, 1.95 g) were added to 25 ml dry DMF and refluxed at 150 ºC for 12 hours. The solution was then allowed to cool to room temperature, and the purple product was precipitated with a mixture of acetone/ether (50 mL each). The precipitate was collected by vacuum filtration, washed with acetone/ether, and dried under vacuum. The yield of Ru(dmphen)2Cl2•2H2O was 71%. General Preparation of HQ Complexes: Note: The synthesis and purification of the complexes were performed under low ambient light in order to avoid any photo-decomposition. The synthesis of compound 9 is presented as an example. Ru(dmphen)2-8HQ (9) Ru(dmphen)2Cl2 (100 mg, 0.160 mmol) and 8-hydroxyquinoline (25.6 mg, 0.176 mmol) were added to 4 mL of degassed ethylene glycol in a 15 mL pressure tube. The mixture was heated at 150 ºC for 4 hours while protected from light. The deep purple solution was allowed to cool to room temperature and ACS Paragon Plus Environment

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poured into 25 mL of dH2O. Addition of a saturated aq. KPF6 solution (ca. 1 mL) produced a deep purple precipitate that was collected by vacuum filtration. The purification of the purple solid was carried out by flash chromatography (silica gel, loaded in 0.1% KNO3, 2% H2O in MeCN). A gradient was run, and the pure complex eluted at 0.8% KNO3, 16% H2O in MeCN. The product fractions were concentrated under reduced pressure, and a saturated aq. solution of KPF6 was added, followed by extraction of the complex into CH2Cl2. The solvent was removed under reduced pressure to give a purple solid to yield 124.4 mg (96%) of the pure complex. 1H NMR (CD3CN): δ 8.45 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 8.6 Hz, 1H), 8.28 (d, J = 8.2 Hz, 1H), 8.18 – 7.99 (m, 5H), 7.79 (dd, J = 8.6, 1.5 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.34 (dd, J = 8.6, 3.1 Hz, 2H), 7.08 (t, J = 7.8, 1H), 6.71-6.62 (m, 3H), 6.23 (dd, J = 7.8, 0.8 Hz, 1H), 2.74 (s, 3H), 2.18 (s, 3H), 1.98 (s, 3H) 1.83 (s, 3H). Purity by HPLC = 97%. ESI MS calcd for C37H30N5ORu [M]+ 662.2; found 662.1 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 497 (8,800). Ru(dmphen)2-2-Me-8-HQ (10) Yield: 96 mg (49%). 1H NMR (CD3CN): δ 8.40 (d, J = 8.2 Hz, 2H), 8.32 (d, J = 8.2 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.11-8.04 (m, 4H), 7.77 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.02 (t, J = 8.2 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 6.62 (d, J = 7.8 Hz, 1H), 6.17 (d, J = 7.8 Hz, 1H), 2.77 (s, 3H), 2.28 (s, 3H), 2.25 (s, 3H) 1.55 (s, 3H), 1.20 (s, 3H). Purity by HPLC = 98.1%. ESI MS calcd for C87H32N5ORu [M]+ 676.2; found 676.2 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 500 (8,800). Ru(dmphen)2-5,7-dichloro-8-HQ (11) Yield: 60 mg (43%). 1H (CD3CN): δ 8.48 (dd, J = 8.2, 2.9 Hz, 2H), 8.32 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 8.12-8.01 (m, 4H), 7.69 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 8.2 Hz), 1H), 7.38-7.34 (m, 3H), 6.87-6.83 (m, 1H), 6.77 (dd, J = 8.2, 1.4 Hz, 1H), 2.64 (s, 3H), 2.18 (s, 3H), 1.82 (s, 3H). Purity by HPLC = 95.3%. ESI MS calcd for C37H28Cl2N5ORu [M]+ 730.1; found 730.1 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 490 (13,400). Ru(dmphen)2-5-Cl-7-I-8-HQ (12) ACS Paragon Plus Environment

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Yield: 112 mg (72%). 1H (CD3CN): δ 8.48 (dd, J = 8.3, 3.2 Hz, 2H), 8.30 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 8.6 Hz, 1H), 8.05-8.02 (m, 3H), 7.70 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.57 (s, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 6.88 (dd, J = 8.6, 8.6 Hz, 1H), 6.75 (dd, J = 5.1, 1.2 Hz, 1H), 2.58 (s, 3H), 2.23 (s, 3H), 1.90 (s, 3H), 1.82 (s, 3H). Purity by HPLC = 98%. ESI MS calcd for C37H28ClIN5ORu [M]+ 822.0; found 822.1 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 455 (17,400). Ru(dmphen)2-5-NO2 8-HQ (13) Yield: 130 mg (95%). 1H (CD3CN): δ 9.03 (dd, J = 8.9, 1.1 Hz, 1H), 8.50 (d, J = 8.2 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.34 (d, J = 8.5 Hz, 1H), 8.26-8.23 (m, 2H), 8.19-8.04 (m, 4H), 7.71 (d, J = 8.2 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.02 (dd, J = 9, 8.9 Hz, 1H), 6.84 (dd, J = 5.0, 1.1 Hz, 1H), 6.20 (d, J = 9.3 Hz, 1H), 2.59 (s, 3H), 2.18 (s, 3H), 1.94 (s, 3H), 1.81 (s, 3H). Purity by HPLC = 98%. ESI MS calcd for C37H29N6O3Ru [M]+ 706.8; found 707.2 [M]+. UV/Vis (dH2O): λmax nm (ε M-1cm-1) 520 (17,700). Ru(dmphen)2-5-sulfonic acid-8-HQ (14) Mass spectra alone were obtained for this compound, as it did not produce any 1NMR data and was not retained at all on the HPLC column. ESI MS calcd for C37H30N5O4RuS [M]+ 742.1; found 742.1 [M]+.

General preparation of Ru(bpy)2L complexes: Ru(bpy)2Cl2 (0.192 mmol, 100 mg) and the different HQ ligands (0.211 mmol) were added to 4 mL of 50:50 EtOH:H20 in a 15 mL pressure tube. The mixture was heated at 100 ºC for 4 hours, after which the deep purple solution was allowed to cool to room temperature. Addition of a saturated aq. KPF6 solution resulted in precipitation of the complex, which was extracted into methylene chloride. Purification of the purple solid was carried out by flash chromatography (silica gel, loaded in 0.1% KNO3, 2% H2O in MeCN). The pure complex eluted at 0.8% KNO3, 16% H2O in MeCN, and the product fractions were concentrated under reduced pressure. A saturated aq. solution of KPF6 was ACS Paragon Plus Environment

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added, and the complex was extracted into CH2Cl2, followed by removal of the solvent under reduced pressure to give a purple solid. Ru(bpy)2-5,7-dichloro-8-HQ (15) Yield: 132 mg (89%). 1H (CD3CN): δ 8.65 (d, J = 5.0 Hz, 1H), 8.53-8.42 (m, 4H), 8.28 (dd, J = 8.6, 1.2 Hz, 1H), 8.00 (tt, J = 7.8, 1.9 Hz, 2H), 7.93-7.85 (m, 4H), 7.72 (d, J = 6.2 Hz, 1H), 7.58 (s, 1H), 7.537.49 (m, 2H), 7.35 (td, J = 6.2, 1.2 Hz, 1H), 7.29-7.22 (m, 3H). Purity by HPLC = 98%. ESI MS calcd for C29H20Cl2N5ORu [M]+ 626.0; found 626.0 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 495 (14,000). Ru(bpy)2-5-chloro-7-iodo-8-HQ (16) Yield: 62 mg (37%). 1H (CD3CN): δ 8.63-8.61 (m, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.45-8.42 (m, 2H), 8.38 (d, J = 7.8 Hz, 1H), 8.27 (dd, J = 8.6, 1.2 Hz, 1H), 7.99 (tt, J = 7.8, 1.6 Hz, 2H), 7.93-7.82 (m, 5H), 7.71 (d, J = 5.1 Hz, 1H), 7.53-7.50 (m, 2H), 7.34-7.21 (m, 4H). Purity by HPLC = 95.4%. ESI MS calcd for C29H20ClIN5ORu [M]+ 717.9; found 717.9 [M]+. UV/Vis (CH3CN): λmax nm (ε M-1cm-1) 495 (14,000). Ru(bpy)2-5-sulfonic acid-8-HQ (17) ESI MS calcd for C29H22N5O4RuS [M]+ 638.04; found 638.1 [M]+.

Cell Lines and Cell Cytotoxicity Assay. All cell lines were purchased from ATCC, cultured in their recommended media, and incubated at 37 0C with 5% CO2. For cytotoxicity studies, the cell lines were cultured in Opti-MEM supplemented with 1% FBS and 50 U/ml Penicillin-Streptomycin (Pen/Strep) and plated in 96 well Greiner tissue culture treated plates. The number of cells added per well was adjusted based on growth rates with A549, BT549, and NIH-H226 cells seeded at 2,000 cells/well. The Jurkat and K562 cells were seeded at 10,000 cells/well, while HL60 were seeded at 30,000 cells/well. The cells were incubated with the compounds for 72 hours prior to the addition of resazurin as previously described.14 A further incubation time of between 4 and 8 hours was carried out to allow for oxidation of the dye by viable cells. Viability was quantified by measuring the fluorescence emission at 590 nm, with excitation at 485 nm. The data was subtracted from wells that did not contain cells to


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establish the background fluorescence from resazurin. IC50 values were determined by fitting the data to a sigmoidal dose response using the Prism software package.

Cytotoxicity in the presence of Copper Chloride. A 10 mM solution of CuCl2 was prepared in sterile diH20, filtered and stored at 4 0C. For CuCl2 cytotoxicity determination, cell lines were seeded in 96 well plates in a 50 µl volume of Opti-MEM 1% FBS, followed by the addition of an equal volume of CuCl2 in Opti-MEM 1% FBS, with 100 µM being the highest concentration assayed. For cytotoxicity studies of compounds in the presence of 10 µM CuCl2, the cells were seeded in Opti-MEM with 1% FBS containing 20 µM CuCl2 followed by the addition of 50 µl of compound in Opti-MEM with 1% FBS. The cells were incubated for 72 hrs followed by the addition of resazurin, and data collected as described above.

Cytotoxicity in the presence of GSH. A 100 mM stock solution of glutathione (GSH) was prepared in Opti-MEM containing 1% FBS and 50 U/ml Penn-Strep. HL60 cells were seeded in 96-well plates at 30,000 cells/well in the presence or absence of 20 mM GSH, followed by the addition of an equal volume of compound. Viability was measured after 72 hrs using resazurin, as described above.

PBMC cytotoxicity assay. Human PBMC’s (Stem Cell Research) isolated from a 27 year old female non-smoker were rapidly thawed, washed with, and resuspended in DMEM media containing 10% FBS and 50 U/ml Penn/Strep. Initial cell viability was determined after a 1:2 dilution with trypan blue followed by manual counting with a hemacytometer. Cells were added at 150,000 cells/well to 96-well plates containing compounds in dose response and incubated at 37 0C with 5% CO2. Viability was determined after 72 hrs using Cell Titer Glo (Promega) and luminescence measured with a SpectraFluor Plus (Tecan). All data was measured in triplicate and analyzed using Prism.


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Immunoblotting. HL60 cells at a density of 4.5x105 cells/ml were seeded at 2 ml/well in 6 well Greiner tissue culture plates with Opti-MEM supplemented with 1% FBS and Pen/Strep. The cells were incubated for 8 hours with 1 µM GL109, 20 µM clioquinol, 1 µM MG132 or 0.1 µM Velcade, then collected and washed with ice cold PBS followed by the addition of ice cold lysis buffer (20 mM Tris pH 7.5, 1% Triton X100, 5 mM EDTA, 5 mM Sodium Pyrophosphate, 5 mM Sodium Fluoride, 150 mM NaCl, 2 mM Sodium Vanadate, 1 mM PMSF, and 1x Roche complete protease inhibitor cocktail). The cells were lysed on ice for 15 min followed by centrifugation for 10 min at 20,800 xg at 4 0C. The supernatant was transferred to a 1.5 ml tube, an aliquot removed for protein concentration determination by BCA, with SDS sample buffer added to the remaining lysate and boiled at 95 0C for 5 min then stored at -80 0C. A volume of 4.5 µg of HL60 cell lysate for each condition was resolved on a 4-12% Bis-Tris gel, followed by transfer to a nitrocellulose membrane. The membrane was blocked for 1 hour in PBST (PBS with 0.1% Tween 20) with 5% non-fat milk, followed by the addition of primary antibodies (Santa Cruz Biotechnology Inc., Cell Signaling Technology) at a 1:1000 dilution (Caspase 3, sc-7272; Caspase 9, sc-8355; Parp-1, sc-7150; Ubiquitin, sc-8017; GAPDH, sc-166574) and incubated overnight at 4 0C. For phospho-specific antibodies, membranes were blocked in PBST with 2.5% BSA followed by the addition of antibodies at a 1:1000 dilution (phospho-p38, #4155; phospho-Jun, #3270; phospho-Akt, sc7985R; phospho-ERK, sc-7383) and incubated overnight at 4 0C. The membranes were washed for 5 min with PBST, and repeated for a total of 4 washes, then incubated with secondary antibody (Jackson ImmunoResearch) for 1 hour at room temperature, washed 4x with PBST followed by the addition of luminal (Clarity, Bio-Rad) and imaged on a Chemi-Doc system (Bio-Rad).

Spheroid Viability Assay. The spheroids were generated as previously described14 and the process is summarized briefly here. A549 cells were plated at 7000 cell/well on agarose coated 96 well plates in a ACS Paragon Plus Environment

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50 µl volume and allowed to coalesce for 24 hours. Another 50 µl volume of media was added gently as to not disturb the forming spheroids. The cells were allowed to grow for 7 – 10 days followed by the addition of compound. The compounds were prepared in Opti-MEM supplemented with 1% FBS and 50 U/ml Penicillin-Streptomycin and 100 µl added to each well of spheroids. Viability was determined after 72 hours, where 100 µl of media was aspirated, followed by the addition of 100 µl of CellTiter Glo (Promega). Luminescence was measured on a SpectraFluoro Plus plate reader (Tecan).

Cell cycle analysis. HL60 cells were seeded in Opti-MEM with 1% FBS, followed by the addition of either 1 µM 12, 1 µM MG132, 0.1 µM Velcade, or 20 µM clioquinol. Cells were transferred to 12x75 mm polystyrene tubes (Becton-Dickenson) at 8, 16, and 24 hrs, centrifuged at 440 xg for 5 min, resuspended in 0.25 ml PBS, vortexed briefly, followed by the addition of 2.3 ml of 70% ethanol that was at -20 0C. The cells were placed at 4 0C overnight and then centrifuged at 770 xg for 5 min, aspirated, rehydrated with 1 ml of PBS for 5 min, centrifuged at 770 xg for 5 min, aspirated, and resuspended with the addition of 0.5 ml propidium iodide solution (20 µg/ml propidium iodide, 0.2 mg/ml RNAse A, 0.1% Triton X-100). The cells were incubated at room temperature for 30 min then analyzed on a BD FACS Calibur at the University of Kentucky Flow Cytometry Facility.

In vitro transcription and translation (IVTT) assay. IVTT assays were carried out according the manufacturers protocol with minor modifications to allow for compound screening.33 Reactions were carried out in 12.5 µl volumes where clioquinol and 12 were incubated with the IVTT reaction mixture for 10 min. prior to addition of the GFP encoding plasmid, pCFE-GFP. The mixture was gently mixed and incubated at 30 0C for 2 hrs prior to measurement of GFP fluorescence using a SpectraFluor Plus plate reader (Tecan) with an excitation of 485 nm and emission of 530 nm.

Supporting Information.


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Additional figures for cell cytotoxicity studies, cell cycle analysis, apoptosis induction, and relationship of logP to IC50 values. This material is available free of charge via the Internet at http://pubs.acs.org. Acknowledgements. We thank Greg Bowman (University of Kentucky Flow Cytometry Facilty) for assistance with cell cycle data collection and analysis, and John May (University of Kentucky Environmental Research and Training Laboratory; ERTL) for mass spectrometry analysis of ruthenium complexes. This work was supported by the American Cancer Society (RSG-13-079-01-CDD).

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Table of Contents graphic.


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Coordination of Hydroxyquinolines to a Ruthenium Bis-dimethyl

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