Martina
Gobec‡
a,
Jakob
Kljun‡
bc,
Izidor
Sosič‡
a,
Irena
Mlinarič-Raščan
a,
Matija
Uršič
c,
Stanislav
Gobec
*a and
Iztok
Turel
*bc
aFaculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia. E-mail: stanislav.gobec@ffa.uni-lj.si
bEN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia. E-mail: iztok.turel@fkkt.uni-lj.si
cFaculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia
First published on 4th April 2014
In this study, we present the synthesis, biological characterization, and first crystal structure of an organometallic–clioquinol complex. Combining ruthenium with the established apoptotic agent and 8-hydroxyquinoline derivative, clioquinol, resulted in a complex that induces caspase-dependent cell death in leukaemia cells. This activity is copper independent and is improved compared to the parent compound, clioquinol. The study of the mode of action reveals that this clioquinol–ruthenium complex does not intercalate between DNA base pairs. Additionally, this clioquinol–ruthenium complex shows proteasome-independent inhibition of the NFκB signalling pathway, with no effects on cell-cycle distribution. These data suggest a mechanism of action that involves a target profile that is different from that for clioquinol alone.
Ruthenium-based compounds have also attracted increasing interest as potential antitumor agents. The ruthenium ion can be either reactive, when it bears halide ligands that can dissociate in aqueous solution to confer multifunctional potential to the target complexes, or act solely as a building block without being involved in direct interactions with biological target(s), thus acting as a scaffold to organize various well-established bioactive organic compounds around its metal center.6 Indeed, these compounds show promise for significant advances due to their high delivery to cancer cells, as well as the low occurrence of side effects and the favourable toxicological profile.5 It is known that some ruthenium compounds bind to DNA more strongly and are less affected by cell repair mechanisms compared to cisplatin.7 However, as recently described, some ruthenium-based compounds can induce cell death through other mechanisms.8
Clioquinol (CqH) shows a wide range of biological activities. It has been used as an antimicrobial agent for many years, and recently, very encouraging data have been reported for clioquinol use in the treatment of Alzheimer's9 and Parkinson's diseases.10 Clioquinol was studied up to a pilot phase 2 study, but due to difficulties in preventing diiodo 8-hydroxyquinoline contamination upon large scale chemical synthesis no further phase II/III studies have been carried out.9,11 Although the mechanism of action has not been fully elucidated, the biological activity of clioquinol has been ascribed to its ability to cross the blood–brain barrier and to its chelation of metal ions, such as Cu(II) and Zn(II), which are associated with protein aggregation and degeneration processes in the brain.12 Moreover, it has been shown that in the presence of copper(II) ions, clioquinol inhibits the proteasomal activity and proliferation in cultured human cancer cells.13–15 In a similar study, clioquinol was fully characterized as an inducer of cell death in leukaemia and myeloma cell lines, where its actions are copper dependent and are also due to copper-dependent inhibition of the proteasome.16 With transition metal ions, such as zinc(II) and copper(II), clioquinol forms stable [M(Cq)2] complexes, however the active copper(II)–clioquinol species and its mechanism of action have not yet been determined.12,17,18 Nowadays, topical formulations of clioquinol are still available for the treatment of topical fungal and parasitic infections.16
A way to extend or improve the biological activity of known bioactive compounds is to bind them to a metal centre, which can result in synergistic/improved activity.19 The most advanced example of this concept relates to ferroquine, a ferrocenyl analogue of the antimalarial drug chloroquine.20 Ferroquine is an antimalarial candidate that completed a phase IIb clinical trial in 2011, and it is active against parasites that have acquired resistance to chloroquine and other antimalarial agents.21 This concept has been extended to ruthenium–arene derivatives of chloroquine, which inhibit the growth of colon cancer cells.22
We recently reported on our studies of ruthenium complexes with the quinolone antibacterial agents23–26 that show increased toxicities against selected cancer cell lines and enhanced inhibition of the cathepsins, the enzymes of the cysteine protease family that are involved in the development and progression of many diseases, including cancer and arthritis.27 Herein, we present the synthesis and biological evaluation of a clioquinol–ruthenium complex (1) (Fig. 1). In our efforts to ‘teach an old dog new tricks’, we combined the potential of an established apoptotic agent, clioquinol, with an organoruthenium moiety. The main goal was to obtain an agent with improved anticancer properties in comparison with the parent compound, clioquinol. Even though the synthesis of this clioquinol–ruthenium complex was reported recently, we have considerably improved the synthetic procedure in terms of the yield and the synthesis time, and we have also proceeded with its full physicochemical characterization in the solid state and in solution.28 The stability of the clioquinol–ruthenium complex in dimethylsulfoxide (DMSO) and in aqueous solution was studied, and its crystal structure was also determined.
Interestingly, the Cambridge structural database currently reports crystal structures of only nine clioquinol metal complexes: [ReOCl2(Cq)(PPh3)],29trans-[Zn(Cq)2(OH2)], trans-[Cu(Cq)2],17trans-[Ni(Cq)2], (Me2NH2)[Ni(Cq)3],30 (CqH2)[PdCl2(Cq)],31trans-[Pd(Cq)2],32 [PtCl(Cq)(dmso)]33 and [Ce(Cq)4].34 In all cases, clioquinol acts as a bidentate ligand in its deprotonated form, as is usual for simple 8-hydroxyquinoline ligands. It is a very versatile ligand for the construction of novel molecules, as among the above listed compounds we can find square-planar, pyramidal, octahedral and dodecahedral complexes. Here, we report the first crystal structure of an organometallic clioquinol complex. Moreover, more than 60 crystal structures of ruthenium coordination compounds with 8-hydroxyquinoline ligands are reported, most of which contain the parent ligand 8-hydroxyquinoline;35–38 however, the crystal structure of the 2-methyl-5,7-dichloro-8-hydroxyquinoline and 5,7-dimethyl-8-hydroxyquinoline complexes with an analogous chemical structure to the clioquinol–ruthenium complex (1) is also reported.39
Complex 1 links into supramolecular dimers about a centre of inversion (Fig. 2). The inter-ruthenium distance Ru1–Ru1′ in the dimers is 5.409(4) Å, which is in line with the observations from our previous study on organoruthenium complexes with β-diketonate ligands.40
Intrigued by the findings of Gasser et al.,41 who reported deleterious effects of the commonly used bioassay co-solvent DMSO on the stability of organoruthenium compounds, we explored the stability of the clioquinol–ruthenium complex (1) in DMSO-d6 and in 10% DMSO-d6/90% H2O solution using 1H NMR (see ESI†). Monitoring these solutions along the time, no changes were observed in the NMR signals, and the integrity of the complex in DMSO solution as well as the aqua species in 10% DMSO aqueous solution was intact within the time-frame of the biological experiments. The stability of the complex in the presence of phosphate buffers in solutions with pH values 8.0, 8.5, and 9.0 was monitored by UV-Vis spectroscopy (see ESI†). The spectra remain unchanged after 24 hours. Both experiments show a remarkable stability of complex 1, as no release of ligand Cq was observed.
Agent | IC50 against selected cancer cell lines (μM) | |||||
---|---|---|---|---|---|---|
MCF-7 | PC-3 | HOS | Raji | Thp-1 | Jurkat | |
a Cells were treated with different concentrations of clioquinol or 1 for 24 h. Cell viability was determined using the MTS assay. IC50 values are presented as the means ± SD from three independent experiments. | ||||||
Clioquinol | >50 | 45 ± 7 | 28 ± 4 | 25 ± 5 | 24 ± 6 | 22 ± 5 |
1 | >50 | 42 ± 10 | 27 ± 2 | 6 ± 3 | 6 ± 5 | 5 ± 2 |
Clioquinol is known to intercalate between DNA base pairs30 therefore we investigated whether 1 has the same properties. A ctDNA intercalation assay was performed with a fixed concentration of 1 (15 μM) to which five varying concentrations of the ctDNA were added until the concentration ratio between ctDNA and 1 reached 10. The UV–visible spectra were recorded from 230 nm to 400 nm. For the absorption maxima, determined at 285 nm, no decreased intensity or shift to higher wavelengths could be observed (see ESI†). This suggests that the clioquinol–ruthenium complex (1) most probably does not act as a DNA intercalator.
Very recently, it was shown that clioquinol inhibits histone deacetylase activity, which leads to the arrest of leukaemic cells in the G0/G1 phase of the cell cycle.42 The analysis of the cell cycle of Raji cells treated for 24 h with either clioquinol or 1 showed that unlike clioquinol, the clioquinol–ruthenium complex (1) had no effects on the cell-cycle distribution (Fig. 5).
Moreover, the complex 1 shows copper-independent reduction of cell viability. It has previously been shown that clioquinol-methoxy derivatives that cannot chelate copper have no anticancer activity.15 It has also been established that copper ions are a prerequisite for the proteasome inhibition of clioquinol and the consequent induction of apoptosis. The clioquinol–ruthenium complex presented here shows these cytotoxic effects in the absence of copper, with concentration-dependent cell death by apoptosis that is mediated through caspase activation. Additionally, the concentration needed to initiate apoptosis is significantly lower for 1 (5 μM) than for clioquinol (25 μM) (Fig. 4). The mechanism of cell death induced by 1 does not appear to require reactive oxygen species, as these did not show increased production (see ESI†).
When we addressed the mode of action of the clioquinol–ruthenium complex, we hypothesized that it maintains similar targets to the parent clioquinol. First, possible intercalation-based interactions of 1 with ctDNA were investigated, as it was recently shown that clioquinol can intercalate between DNA base pairs.22 The absorption pattern of the 15 μM clioquinol–ruthenium complex was followed upon addition of increasing amounts of ctDNA (0–150 μM). The absorption maximum at 285 nm was not shifted to higher wavelengths, and the intensity was not decreased (see ESI†), which suggests that the clioquinol–ruthenium complex (1) does not act as a DNA intercalator. The results of this preliminary experiment, however, do not absolutely exclude the possibilities of other types of interactions of 1 with DNA (especially the possibility of covalent interactions).
It has recently been shown that clioquinol inhibits histone deacetylase activity, which leads to the arrest of leukaemic cells in the G0/G1 phase of the cell cycle.42 Surprisingly, while clioquinol resulted in severe arrest of Raji cells in the G0/G1 phase of the cell cycle, complex 1 had no effect on the cell-cycle distribution (Fig. 5), which indicates that it has a different target profile. This was further demonstrated in a cell-free proteasome assay. Here, clioquinol is known to have an antiproliferative action that is mediated through copper-dependent inhibition of the proteasome.13–15 The clioquinol–ruthenium complex (1) does not inhibit the chymotrypsin-like activity of the proteasome; however, as we showed in the NFκB transcriptional activity assay, complex 1 appears to modulate the NFκB pathway, as it can reduce TNF-α-induced activation of NFκB by >50% (Fig. 6). Moreover, lower concentrations of the clioquinol–ruthenium complex are needed to observe this effect, compared to clioquinol (2.5 μM vs. 10 μM, respectively).
Taken together, these data indicate that the clioquinol–ruthenium complex (1) has different targets in comparison with clioquinol; it does, however, inhibit the NFκB pathway, although not via proteasome inhibition. Constitutive NFκB activation is a common feature of many haematological malignancies, and is believed to be crucial for the survival of these malignant leukaemia cells.43 Therefore, targeting any cascade in the NFκB signalling pathway is a valid anticancer strategy.
NMR: 1H NMR (500 MHz, CDCl3): δ 8.91 (d, J = 4.1 Hz, 1H, C2H), 8.36 (dd, J = 8.5, 0.9 Hz, 1H, C4H), 7.80 (s, 1H, C6H), 7.46 (dd, J = 8.6, 4.9 Hz, 1H, C3H), 5.66 (d, J = 6.0 Hz, 1H, Ar-H cym), 5.44 (d, J = 6.0 Hz, 2H, Ar-H cym), 5.38 (d, J = 5.9 Hz, 1H, Ar-H cym), 2.88 (hept, J = 6.8 Hz, 1H, Ar-CH(CH3)2 cym), 2.34 (s, 3H, Ar-CH3 cym), 1.30 (d, J = 6.9 Hz, 3H, Ar-CH(CH3)2 cym), 1.18 (d, J = 6.9 Hz, 3H, Ar-CH(CH3)2 cym).
1H NMR (500 MHz, DMSO-d6): δ 9.30 (dd, J = 4.9, 1.0 Hz, 1H, C2H), 8.36 (dd, J = 8.6, 1.0 Hz, 1H, C4H), 7.79 (s, 1H, C6H), 5.93 (d, J = 6.0 Hz, 1H, Ar-H cym), 5.78 (d, J = 5.9 Hz, 1H, Ar-H cym), 5.71 (d, J = 5.9 Hz, 1H, Ar-H cym), 5.64 (d, J = 6.0 Hz, 1H, Ar-H cym), 2.71 (hept, J = 6.9 Hz, 1H, Ar-CH(CH3)2 cym), 2.19 (s, 3H, Ar-CH3 cym), 1.17 (d, J = 6.9 Hz, 3H, Ar-CH(CH3)2 cym), 1.09 (d, J = 6.9 Hz, 3H, Ar-CH(CH3)2 cym).
13C NMR (126 MHz, CDCl3): δ 167.08 (C8), 149.24 (C2), 141.82 (C9), 137.15 (C6), 135.09 (C4), 127.42 (C10), 122.64 (C3), 112.57 (C5), 102.16 (Ar-C cym), 97.63 (Ar-C cym), 82.73 (Ar-C cym), 81.98 (Ar-C cym), 81.63 (Ar-C cym), 80.97 (C7), 80.69 (Ar-C cym), 77.26 (CDCl3), 77.01 (CDCl3), 76.76 (CDCl3), 31.00 (Ar-C cym), 22.34 (Ar-C cym), 22.18 (Ar-C cym), 18.52 (Ar-C cym).
15N (through 1H–15N HMBC, CDCl3): δ 238.17 (N1).
Selected IR resonances (cm−1, ATR): 1572, 1542, 1481, 1441, 1369, 1360, 1250, 1109, 877, 751.
CHN: Calc. for C19H18Cl2INORu: C, 39.67; H, 3.15; N, 2.44. Found C, 39.67; H, 2.90; N, 2.25.
UV/Vis (λ) [nm] (ε) [L mol−1 cm−1], c = 1 × 10−4 mol L−1, MeOH: 285 (22500), 341 (3200), 355 (3300), 418 (2600), 467 (2200).
ESI-HRMS (CH3CN) m/z (found for [M − Cl]+ (calcd)): 539.9155 (539.9165).
Footnotes |
† Electronic supplementary information (ESI) available: Materials and methods, crystallographic and spectroscopic data (Table S1 and Fig. S1–S9), bioassays (Fig. S10–S13). CCDC 977981. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4dt00463a |
‡ These authors contributed equally. |
This journal is © The Royal Society of Chemistry 2014 |