Roy, Sudeshna
Citation
Roy, S. (2008, November 25). Synergy of intercalation and coordination binding to design novel DNA-targeting antineoplastic metallodrugs. Retrieved from
https://hdl.handle.net/1887/13281
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/13281
Note: To cite this publication please use the final published version (if applicable).
| 7
Mono- and dinuclear ruthenium compounds coordinated to terpyridine as a carrier ligand †
Chapter
Terpyridine, a widely-used versatile heterocyclic ligand can be synthetically modified to generate useful, new ligands. In the search for DNA-targeting antitumour agents, three terpy-based ligands (i.e., 2,2':6',2"- terpyridine (terpy), 4'-Chloro-2,2':6',2"-terpyridine (Cl-terpy) and bis[4'- (2,2':6',2"-terpyridyl)]-diethyleneglycol ether (dtdeg)) and five ruthenium compounds have been studied. Two planar intercalators 1,10- phenanthroline (phen) and dipyrido[3,2-a: 2′,3′-c]phenazine (dppz) have been used to synthesise dinuclear compounds, which act as carrier ligands.
The activities of some free ligands and four ruthenium compounds have been tested against seven human tumour cell lines. It is generally noticed that a synergistic effect is evident when the combination of toxic metal ion coordinated to an active organic moiety is used depending on the nature of the metal (labile, inert) and where it is bound. In this study it has been observed that the ruthenium compounds, [Ru(terpy)Cl3] and [Ru(Cl- terpy)Cl3], display almost no anticancer activities but the free ligands are active in both cases. The Ru(II) compound, [Cl(dppz)Ru(dtdeg)Ru(dppz)Cl]Cl2, was found active against some cancer cell lines when compared with cisplatin whereas the other dinuclear compound, [Cl(phen)Ru(dtdeg)Ru(phen)Cl]Cl2 was devoid of any activity.
The spectral changes were recorded by CD spectroscopy using Calf Thymus DNA as substrate. The activity profile could be related to the DNA binding mode; however, any linear correlation between cytotoxicity and DNA- interactions has not been observed as yet.
7.1. Introduction
The search for better metallodrug than cisplatin has been focused to achieve the three goals (a) wider range of activity, (b) overcoming the resistance (intrinsic or acquired) and (c) oral administration. To fulfil these features, the need to look beyond platinum or
“cisplatin-like” compounds has been accepted by the bioinorganic research community. At that point, searching through transition metals leads the focus to ruthenium. Ruthenium has an octahedral geometry, stable but easily accessible oxidation states, favourable kinetics and well-established coordination compounds, has easily accessible synthetic routes, and therefore is a suitable candidate for a search towards broader medicinal applications. The bioavailability, biodistribution and mode of action of ruthenium compounds are expected to be completely different than platinum compounds, therefore these compounds comprise an entirely distinct class of antitumour agents.1
Terpyridine (L10) (vide Figs. 7.1 and 7.2, for the structure and those of other ligands and ruthenium compounds) has been a well-known heterocyclic aromatic ligand, which has been mostly used in synthetic inorganic chemistry to design supramolecular motifs.2, 3 This ligand can be synthetically modified to generate different derivatives which are also reported extensively in literature.4-8 This class of ligands containing multiple pyridine rings, is commonly known as ‘polypyridines’ and has been used in coordination chemistry on large scale. These M-terpy compounds (where M = transition metal) have been under extensive investigations due to their unique electrochemical, optical and photophysical properties.9-11 M-terpys’ interact with different biological substrates either by intercalation12-14 or by coordinative binding.15-20 The multidirectional applications include use as antitumour agents,21-23, protein probing agents15, 19 and radiotherapeutics24-26.
The number of publications based on terpy, modified terpys’ and coordination compounds has been growing extensively in the last decade. However, in the literature the coordinatively saturated cationic ruthenium compounds [Ru(N-N)3]n+ are frequently reported. Among this plethora of articles, a very limited number of articles deal with biological aspects of this group of ligands or compounds. Several substitutions at the 4'- position include use of small groups (like Cl, OH, Ph, Me), or a fluorescent anthracene moiety. In fact, when searched for citations with Cl-terpy (L11) and [Ru(Cl-terpy)Cl3] (C11), by October 2008 only 51 and 11 articles were found, respectively. Some derivatives of terpy and their respective coordination compounds with ruthenium have been synthesised but DNA interaction studies, or cell viability assays are not done. It therefore,
N N
L5
N CH3
OH N H
L8
N
N N
L10
N
N N
Cl
L11
N N N
O
O O
N N N
L12
Figure 7.1. Schematic structures of terpy-based ligands (L10-L12). Some other ligands (L5, L8) have been added for comparison.
appeared worthy to investigate the biological activities of substituted terpys’.
There are some reports in literature about anticancer-active dinuclear ruthenium compounds, including synthesis and biological studies.31-47 The extensively studied, and interesting compounds are NAMI-analogues, which have been synthesised by Alessio et al.32, 48 These compounds modify the cell cycle distribution of human and murine carcinoma cell lines, form interstrand crosslinks with linear plasmid DNA and inhibit gelatinase MMP-9 to reduce tumour cell invasion. Other dinuclear ruthenium compounds are mainly groove binders, with partial intercalation interaction through DNA base stacks.
It has been established that the ligands around the ruthenium centre, linker rigidity (or flexibility) and oxidation states influence the biological activity.49
The redox-active ligand, L8 (Fig. 7.1) has already been mentioned in Chapters 5 and 6 with its conversion to ligand L9 (Fig. 7.1) upon metal coordination and will not be repeated in this chapter. Addition of L8 to [Ru(terpy)Cl3] (C10) forms a diamagnetic mononuclear compound, C12 [Ru(terpy)(Hpyrimol)Cl], which helps to shed light on the effect of the structural modification of C10.
The ligand, dtdeg (L12, bis-[4’-(2,2’:6’,2”-terpyridyl)]-diethyleneglycol ether) has been another modification of the terpy motif via coupling of two parts through a flexible ligand.27 The ligand is capable to form symmetric or asymmetric heterometallic or homometallic compounds.28 Therefore, this ligand might assist to form long-range M-DNA adducts, which will be definitely different than cisplatin-DNA adducts.29, 30 These dinuclear
Cl N
N N
Ru Cl Cl
C10
Cl N
N N
Ru Cl Cl Cl
C11
N
N OH
N
N N
Ru Cl
C12
N N N
O O O
N N N N
N
N N
Ru Ru N
N
N N Cl Cl
Cl2
C13
N N N
O O O
N N N
N N Ru
Ru N N Cl Cl
Cl2
C14
Figure 7.2. Ruthenium compounds (of the ligands shown in Fig. 7.1) synthesised and studied in this chapter (C10-C14).
compounds are expected to have enhanced activity compared to mononuclear terpy compounds, because a single molecule can load the double amount of active metal centres to the target (cancer cells). After coordination to two ruthenium atoms, the formed compound [Cl3Ru(dtdeg)RuCl3] can be further modified by addition of monodentate (imidazole, pyridine, chloride), bidentate (bipyridine, phenanthroline, dipyridophenazine), or tridentate (terpyridine) ligands, to produce a number of dinuclear compounds with
changes in the outer ligand sphere (i.e., changes in aromaticity, hydrophilicity, electronic property).
In this chapter, five ruthenium compounds are either reproduced (following synthetic routes from literature) or synthesised for the first time from the known precursor, Cl3Ru(dtdeg)RuCl3. Among them three compounds are mononuclear viz., [RuIII(terpy)Cl3] (C10), [RuIII(Cl-terpy)Cl3] (C11), [Ru(terpy)(Hpyrimol)Cl] (C12) and other two compounds are dinuclear diamagnetic, [Cl(dppz)RuII(dtdeg)RuII(dppz)Cl]Cl2 (C13) and [Cl(phen)RuII(dtdeg)RuII(phen)Cl]Cl2 (C14).
The DNA-binding studies have been performed for all the ligands (L10-L12, including L5) and ruthenium compounds (C10-C14) in an attempt to correlate the DNA- binding mode with the in vitro cytotoxicity. The antitumour activities of terpy derivatives have been analysed and the results have been summarised in this chapter. The key point of this chapter reveals that free ligands may have a surprisingly high antitumour activity. The coupling of two terpy fragments to synthesise a flexible ligand, dtdeg, has been important for the synthesis of dinuclear compounds. The addition of an extended aromatic molecule (dppz) as bidentate ligand by substitution of chloride ligands, on the both metal centres, induces significant antitumour activity. The cytostatic activity can be related to the denaturation of cellular DNA caused by the metal compounds.50
7.2. Experimental 7.2.1. Materials
RuCl3·3H2O was generously provided as a loan from Johnson-Matthey (Reading, U.K.). The ligands dppz (L2), Hpyramol (L8), Cl-terpy (L11) and dtdeg (L12) were synthesised following the published synthetic routes.4, 27, 49, 51 The solvents used for synthesis were purchased from Biosolve (AR grade) and used without any further purification. Deuterated solvents (for NMR experiments) and ligands phenanthroline (L5) and terpyridine (L10) were purchased from Sigma-Aldrich (Germany). The sources of other materials required for biological studies were mentioned previously in Chapter 2 (vide section 2.2.1).
7.2.2. Chemical syntheses of ruthenium compounds (a) C10: [RuIII(terpy)Cl3]
This compound was synthesised following a reported procedure.52 An absolute ethanolic solution (150 mL) of the ligand L10 (233.27 mg, 1 mmol) was prepared after
gentle heating on constant stirring. To the colourless ligand solution, RuCl3·3H2O (261.47 mg, 1 mmol) was added and refluxed for 3 h at 80 °C. The reaction mixture was cooled down slowly to room temperature to yield a deep-brown precipitate. After filtration, this precipitate was washed with ice-cold ethanol (3×5 mL) and finally with diethyl ether (3×5 mL) and dried in air. The yield was 343.59 mg, 78% for C10. Elemental analysis (%) for RuC15H11N3Cl3: expt.(calcd.) C 40.10 (40.88), N 9.45 (9.53), H 2.48 (2.52).
(b) C11: [RuIII(Cl-terpy)Cl3]
This compound was synthesised following a report from literature.27 An absolute ethanolic solution (150 mL) of the ligand L11 (267.72 mg, 1 mmol) was prepared after gentle heating on constant stirring. To the pale-brown ligand solution, RuCl3·3H2O (261.47 mg, 1 mmol) was added and refluxed for 3 h at 80 °C. The reaction mixture was cooled down slowly to room temperature to yield a deep-brown precipitate. After filtration, this precipitate was washed with ice-cold ethanol (3×5 mL) and finally with diethyl ether (5×5 mL). The air-dried product was collected and yield was 342.5 mg, 72%. Elemental analysis (%) for RuC15H10N3Cl4: expt.(calcd.) C 38.12 (37.92), N 8.91 (8.84), H 2.10 (2.12).
(c) C12: [RuII(terpy)(Hpyrimol)Cl]
This compound was synthesised following the typical synthetic route as described in literature.53 The ruthenium starting material, [Ru(terpy)Cl3](100 mg, 0.2269 mmol) was dissolved with heating in 30 mL absolute ethanol while stirring. To the resulting brownish solution, an ethanolic yellow solution of the ligand L8 (97.23 mg, 0.4538 mmol) was added dropwise. The reaction mixture was heated to reflux for 6 h and a deep purple solution was obtained. This reaction solution was filtered in warm condition to exclude any unreacted material and the deep purple filtrate was reduced in volume by evaporation to 5 mL. A deep purple crystalline powder was precipitated upon slow addition of 100 mL of diethyl ether and collected by suction filtration. The yield was 95.4 mg, 68% and elemental analysis (%) for RuC28H22N5OCl: expt.(calcd.) C 57.96 (57.88), N 12.40 (12.05), H 3.55 (3.82).
(d) C13: [Cl(dppz)RuIIdtdegRuII(dppz)Cl]Cl2
This compound was synthesised following the synthetic way described in literature.27, 49 In brief, [Cl3RuIIIdtdegRuIIICl3] (100 mg, 0.1016 mmol), dppz (57.36 mg, 0.2032 mmol), LiCl (30.15 mg, 0.7112 mmol) and triethylamine (46.2 mg, 0.4572 mmol)
were dissolved in 45 mL of ethanol/water (4/1) with gentle heating. The reaction mixture (reddish brown) was refluxed for 24 h under aerobic conditions and was filtered warm to exclude any undissolved starting material. The filtrate (deep maroon coloured) was reduced in volume and this solution was loaded on an alumina column. The middle band (reddish- brown) was collected by elution with dichloromethane-hexane (3:1 v/v) and reduced in volume by evaporation. To this concentrated solution, diethyl ether (100 mL) was added very slowly, while stirring. A deep reddish-brown solid was collected and washed with ice- cold ethanol (3×3 mL) and diethyl ether (5×5 mL). The yield was (94.62 mg, 62%) and elemental analysis (%) for Ru2C70H48N14Cl4O3 (+3H2O+3HCl) [water and acid molecules have been added to fit the experimental values]: expt.(calcd.) C 51.16 (51.25), N 12.40 (11.95), H 3.55 (3.50).
(c) C14: [Cl(phen)RuIIdtdegRuII(phen)Cl]Cl2
This compound was synthesised following the reported synthetic method.27, 49 In brief: Cl3RuIIIdtdegRuIIICl3 (100 mg, 0.1016 mmol), phen (36 mg, 0.2032 mmol), LiCl (24.9 mg, 0.59 mmol) and triethylamine (46.2 mg, 0.4572 mmol) were dissolved in 45 mL of ethanol/water (4/1) with gentle heating. The reaction mixture (maroon coloured) was refluxed for 24 h under aerobic condition and the reaction mixture was filtered warm to exclude any undissolved starting material. The filtrate (deep maroon coloured) was reduced in volume and diethyl ether (100 mL) was added very slowly while stirring. A deep reddish-brown solid was collected and washed with ice-cold ethanol (3×3 mL) and diethyl ether (5×5 mL) thoroughly. The yield was (90.68 mg, 70%) and elemental analysis (%) for Ru2C58H44N10Cl4O3 expt.(calcd.) C 54.28 (54.72), N 10.88 (11.00), H 3.36 (3.48).
7.2.3. Analysis
The one-dimensional or two-dimensional COSY spectra were recorded on a 300 MHz Bruker spectrometer with a 5 mm multi-nucleus probe at ambient temperature (25 °C) in dmso-d6 or methanol-d4 solution. The ESI-MS spectra were recorded on a Finnigan AQA instrument equipped with Electro Spray Interface (ESI). Elemental analysis of the samples was performed in a Perkin Elmer Series II CHNS/O Analyser 2400. Typically 2 mg to 2.2 mg of sample was used for the C, H, N and S analyses.
The ligands (L10, L11 and L12) after recrystallisation have been characterised by
1H NMR, ESI-MS and elemental analysis. All the data matched with the reported data in literature. The ruthenium compounds (C10 and C11) are in the +III oxidation state,
therefore, the scan in the paramagnetic region (-50 to +50 ppm) has been performed and the peak positions are in agreement with reported values.27 The other three Ru(II) compounds (C12, C13 and C14) do not show any peak in the paramagnetic region and the spectra are sharp and occur in the normal proton range (0-11 ppm). The peaks match with similar dinuclear diamagnetic compounds.49 The peak positions for free ligands and ruthenium compounds in NMR spectroscopy have been mentioned in Table 7.1. The ESI- MS spectra with a typical ruthenium isotopic pattern are shown in Fig. 7.3. The elemental analysis data fits well with the expected values and the purity of the samples has been confirmed. No suitable single crystals for these compounds could be obtained yet. For the compound C12 the mass spectrum shows the M and [M-Cl]+ at m/z = 583.96 and 545.91, respectively. For compound C13 the major peaks at m/z = 1440.51 and 702.85 correspond to [M-Cl]+ and [M-2Cl-]/2 species, respectively.
N
N N
L10 3
4
5 6
5' 4' 3'
5"
6"
3"
4" N
N N
Cl
L11 3
4
5 6
3' 5'
5"
6"
3"
4"
N N L5 5
2 3
4
6 7
8 9
N N
N N
L2
1 2 4 3
5
N N
O O N
N N N
O
L12 1 2
5
6 3
4 5' 5"
3' 3"
4"
6"
Figure 7.3. Proton numbering scheme for the ligands and the same numbers are used for compounds.
7.2.4. Biological studies
The following studies were undertaken: (a) CD studies: the experimental details have been mentioned previously in Chapter 2 (vide section 2.2.7) and are not repeated in this section. (b) Cell culture studies: the experimental details along with the origin of human cancer cell lines have been provided in Chapter 2 (vide section 2.2.5) and are
Table 7.1. Proton resonating positions (in ppm) for the ruthenium compounds in dmso-d6.
Samples Proton resonances (ppm)
C10 -9.1
(3,3") -3.4
(4,4") -8.2
(5,5") -35.8
(6,6") 5.3
(3',5') -23.4
(4') -- --
C11 -9.4 (3,3")
-3.2 (4,4")
-8.6 (5,5")
-35.3 (6,6")
6.4
(3',5') -- -- --
C13 7.65 (6,6")
8.42 (3,3")
8.14
(3',5') 9.29 (1) 7.95 (2) 8.96 (3) 8.36 (4) 7.98 (5) C14 7.58
(6,6")
8.48 (3,3")
8.24 (3',5')
10.29
(2) 7.16 (3) 8.91 (4) 8.62 (5) --
Figure 7.4. Selected part of the ESI-MS spectra of compounds C12 (a) and C13 (b).
therefore not repeated in this section.(c) Cytotoxicity Assays: the experimental details have been described in Chapter 2 (vide section 2.2.6) and therefore, in this section have been omitted.
7.3. Results 7.3.1. Syntheses
Terpyridine is a useful ligand for ruthenium, and only some derivatives of the parent ligand (L10, 2,2':6',2"-terpyridine) have been used in literature, with varying electronic properties. The 4' position has been utilised to modify the ligand structurally.
Another method of modification has been carried out by coupling two terpy units via a rigid or flexible linker and this structural change influences the overall electronic properties of the ligands.
The ruthenium derivatives were synthesised by coordinating the ligands to different ruthenium containing precursors (RuCl3·3H2O, or [Cl3Ru(dtdeg)RuCl3]). The starting material contained ruthenium in the 3+ oxidation state, but the tuning of reaction conditions facilitated the formation of either Ru(III) or Ru(II) compounds. These compounds have been easily differentiated by standard paramagnetic scans along with other physical/analytical techniques. The ESI-MS spectra show typical isotopic patterns, which further confirm the formation and purity of the compounds.
7.3.2. DNA interaction followed by CD spectroscopy
All the ligands and compounds have been studied for their DNA interaction modes.
Typically three different R values (R is the concentration ratio of DNA per Nucleotide phosphate to Ru compound) have been taken to follow the spectral change along with the time-dependent recording. Some of the selected spectra have been redrawn in Figs. 7.5(a)- (f) and 7.6 (a)-(d).
The spectral changes on the conservative DNA conformation induced by three ligands, L10, L11 and L5 follow similar trends. The typical B-DNA canonical form has been retained; however, the absorption intensity is decreased around the positive band near 280 nm and increased around the negative band of 245 nm. The decrease in positive band intensity indicates some denaturation of DNA, as observed in case of some trans-platinum compounds.54 In case of L12, the conformation of DNA appears to be severely distorted.
The positive band has been started to split in two peaks at R = 1, along with helicity deformation (prominent from the changes in negative band). The negative band has been
Figure 7.5. CD spectral changes in CT DNA upon addition of (a) L10, (b) L11, (c) L5, (d) L12, (e) C10, (f) C11 at 37 °C with R=1.
red-shifted by 3 nm with the appearance of the new ellipticity at around 230 nm. The base stacking framework has been still retained, but the right-handedness of the helix has been seriously distorted.
The Ru(III) compounds C10 and C11 exhibit different behaviours, in spite of their structural similarity. Both of them have three chloride ligands with a tridentate N,N,N-
Figure 7.6. CD spectral changes in CT DNA upon addition of (a) C12, (b) C14, (c) C13 with R=1 and (d) free ruthenium compound (C13) at 37 °C.
donor ligand. C10 preserves the H-bonding base-stacking stability, but the right-handed helicity has been disturbed significantly. A new band appears at around 220 nm with increases in intensity. The negative band (at 245 nm) has been slightly increased. These changes indicate the induction of a conformation change, i.e., from the B form to A form.55,
56 C10 has been reported to bind to DNA in vitro and forms DNA-interstrand crosslinks similar, as known for transplatin.57, 58 It has been also reported that crosslink-induced changes were concentrated to base pairs surrounding the metallated nucleosides and these adducts neither disrupted Watson-Crick H-bonding, nor altered the global B-like helical parameters of the DNA duplex.59 On the contrary, C11 induced a major detrimental effect of the DNA base stacking, while retaining the helical property with a 4 nm blue shift. A small new positive band starts appearing at around 235 nm. Therefore, C11 denatures the DNA in a much higher extent than C10 does.
The Ru(II) compounds cause different conformational changes depending on the nuclearity of the compound. The mononuclear compound C12 induces very slight changes in the conservative bands of Calf Thymus DNA. However, a new intense positive band at around 215 nm appears. The dinuclear compounds, C13 and C14 have the same linker albeit with different ancillary ligands (dppz and phen) in the outer face. The spectral changes, therefore, exhibit almost an identical trend, indicating a quite insignificant effect from the ancillary ligands. These two compounds clearly changed the B form to the A form with an intense positive band at 220 nm (accompanied by the disappearance of the negative band at 240 nm). These compounds can have dual binding modes via partial intercalation and coordinative binding with formation of long-range adducts as shown in similar series of compounds.49 In addition, these compounds themselves may self-aggregate, as deduced from the chirality depicted in Fig. 7.6(d). The compounds then behave as a right-handed helix, as is evident from intense positive and negative bands at 280 and 240 nm, respectively. This aggregate can arrange parallel or anti-parallel to the DNA strand and consequently influence the DNA global change.
7.3.3. Cell viability assay
The IC50 values for the free ligands were found either comparable to, or even better than those for cisplatin (vide Table 7.2). Cytotoxicity values of L10 and L11 were found significantly higher in all cases than that of cisplatin, except against the ovarian cancer cell line IGROV, which was found in the same range of cisplatin (0.56 μM).The cytostatic activity of L5 was also found comparable with the cisplatin activity. However, upon coordination to Ru, the ligands L10 and L11 (so compounds C10 and C11, respectively) led to a significant decrease (on average 80 to 100-fold) of its cytotoxic activity. In case of the dinuclear compound C13, the activity to inhibit the cellular growth is comparatively higher than the mononuclear compounds. This compound does not show any activity against renal cancer, A498, and ovarian cancer, IGROV. Other cell lines experience proliferation inhibition by C13 in a similar concentration as cisplatin.
The other dinuclear compound C14 shows some activity in H226 and A498 cell lines when compared to cisplatin. The activity of C14 is increased by 2-fold when compared to the dppz analogue, C13. But it could be argued that cisplatin is not the best reference compound for these dinuclear compounds as the structures are significantly different. This major difference may probably induce different mode of absorption
(uptake), distribution and cellular processing. The selective activity in two cell lines and lack of activity in other cell lines are at this point can not be explained in details.
Table 7.2. IC50 values (μM) for some ligands and their respective ruthenium compounds obtained by SRB assay. For abbreviations and structures Figs. 7.1 and 7.2 are referred to.
Cisplatin is included for comparison.
Cell lines
Samples A498 EVSA-T H226 IGROV M19 MCF-7 WIDR
L10 0.38 0.23 0.25 0.57 0.47 0.23 0.29
C10 79.6 66.9 63.7 90.1 68.5 63.9 78.9
L11 0.38 0.54 0.41 0.59 0.49 0.51 0.34
C11 92.7 >100 84.6 >100 >100 95.6 >100
L5 2.93 5.89 3.44 4.10 5.14 5.85 2.34
C13 43.5 3.25 11.7 21.9 8.58 4.96 5.77
C14 27.6 32.1 11.4 49.1 31.1 25.8 27.5
cisplatin 7.50 1.40 10.9 0.56 1.85 2.32 3.22
7.4. Discussion
The tridentate ligand terpy allows multiple ways of compound or ligand design.
This ligand can be modified easily, both sterically and electronically by substituting at the 4' position. Being a tridentate ligand, it is very useful in ruthenium chemistry as it allows to vary the other three coordination sites. The synthesis of Ru(III) compounds has been straightforward in good yield with almost no work-up steps. This fact is very beneficial for the synthesis of a series of compounds. The single limitation of these compounds is their often poor solubility. Retaining the terpy moiety intact the solubility profile can be improved by binding the anion of cbdca (cyclobutane dicarboxylic acid) to the metal, or by sulfonating the 4' position of the terpy itself.
It has been extensively reported that metal compounds can bind to DNA either coordinatively, or non-coordinatively, and this DNA-targeting property in some cases leads to explanation of the strong or weak anticancer activity of the metal compounds.60 However, coordinative interaction takes place when the ruthenium compounds form monodentate, intra- or interstrand adducts with DNA bases after hydrolysis of one or more
of the labile ligands. The coupling of cytotoxic ligands and labile chloride ligands via coordination to the toxic ruthenium centre, might lead to a different antiproliferative profile. The DNA conformation changes induced by ruthenium compounds not only depend on oxidation states, but also the nuclearity and the overall geometry of the compounds. The ligands (L10 and L11) show significant denaturation of DNA, whereas the effect of Ru(III) compounds has been lower in case of (C10 and C11). The dinuclear compounds (C13 and C14) can be self-stacked and thereby influence the binding and folding of this compound around DNA, and possibly influencing the stability and conformation of the DNA.
The design and synthesis of potential new anticancer metallodrugs therefore may originate from terpyridine derivatives. All three free ligands (L10-L12) with the terpy framework have better or comparable cytotoxicities against cancer cell lines, when comparing to the world-wide clinically used cisplatin. The in vitro cytotoxicity can be induced by a potent toxic agents and not necessarily always correlate to DNA-binding or interactions. In some cases, this activity may be related to the denaturation of the helix, as the irreversible form changes could lead the cells to apoptosis. On the other hand, addition of a toxin (metal) to an active agent (free ligands) does not necessarily always increase the activity. The Ru(III) compounds are assumed as ‘prodrugs’ and hypothesised to be activated by reduction, especially in hypoxic cancer cells. The synthesised Ru(III) compounds contain some labile chloride ligands, which presumably undergo hydrolysis in vivo and in vitro. If ‘activation by solvolysis’ takes place, the positively charged compounds can form several adducts with cellular components and therefore be hindered to reach DNA selectively. This phenomenon leads to design hydrolysis-inert compounds using bidentate carboxylic acids, which can prevent the attack by multiple ruthenophiles intracellularly.
Another modification of the original metal coordination sphere can be executed by adding two more terpy ligands. The fact that terpy ligands are highly promising; the optimisation of the terpy-bearing ruthenium compounds to reach a similar activity profile remains a challenge. Additionally, the cellular processing studies for Ru(III) compounds demand a detailed study to rationalise the low cytotoxicity of the compounds C10 and C11.
This low activity, however, may lead to testing these compounds against metastatic cancer.
The antimetastatic compound, NAMI, is also devoid of this cytotoxicity, even though it has been promisingly useful to treat solid tumours.
The dinuclear compounds C13 and C14 have been synthesised without any complications. The two aromatic ligands (dppz and phen) differ in the aromaticity and are therefore helpful to compare the effects of aromatic face on the biological activity. The dinuclear compounds with bpy and terpy ligands have been synthesised previously and the new compounds (C13 and C14) assist to assume their activity against cancer cells. The dinuclear compound of formula [Cl(bpy)Ru(dtdeg)Ru(bpy)Cl]Cl2 bearing bpy has been tested by van der Schilden49 to be active in some cell lines (HBL-100, L1210 and L1210cis) with IC50 value of average 33 μM, whereas the terpy bearing compound is not active. The dppz-bearing compound, C13 is very promising and displays a high activity and C14 exhibits selective activity. This observation can be explained by the addition of planar dppz (L2) or phen (L5) which, in contrast to the sterically rigid terpy, is easier to intercalate in DNA base pairs.
7.5. Conclusions
In conclusion, it has been shown that terpy-based ligands display strong cytotoxic activity. This activity possibly originates from the larger extent of DNA denaturation, though the detailed research to elucidate the exact reasons is crucial. Upon coordinating the ligands to ruthenium, in most cases the resulting compounds show dramatically reduced antitumour activity, with an overall 200-fold diminished activity against all seven cancer cells. Modification of ligands, metal coordination sphere, or even adding two terpy ligands might be an efficient way to increase the anticancer activity. The most probable mode of DNA binding of the ruthenium compounds appears to be coordinative binding, but the reactivity of Ru(II) in situ could be controlled by changing the chlorides to other, less easily leaving ligands. For some compounds presented in this chapter no test results have become available as yet, therefore a comparison or profiling the biological activity of all new compounds is not feasible at this point. The high activity of the dinuclear Ru(II) compound C13 underlines the interesting prospect for polynuclear metal compounds as a novel class of anticancer drugs. Several possibilities to fine-tune the ruthenium compounds are available, e.g., just by applying different substituents keeping the linker fixed or by using rigid, semi-rigid or flexible linkers. Therefore, terpy-based ligands may direct the anticancer ruthenium research in a significant and different pathway.
7.6. References
1. Ang, W. H.; Dyson, P. J., Eur. J. Inorg. Chem. 2006, 4003-4018.
2. Constable, E. C., Chem. Soc. Rev. 2007, 36, 246-253.
3. Eryazici, I.; Moorefield, C. N.; Newkome, G. R., Chem. Rev. 2008, 108, 1834- 1895.
4. Heller, M.; Schubert, U. S., Eur. J. Org. Chem. 2003, 947-961.
5. Hofmeier, H.; Schubert, U. S., Chem. Soc. Rev. 2004, 33, 373-399.
6. Potts, K. T., Bull. Soc. Chim. Belg. 1990, 99, 741-768.
7. Potts, K. T.; Konwar, D., J. Org. Chem. 1991, 56, 4815-4816.
8. Thompson, A. M. W. C., Coord. Chem. Rev. 1997, 160, 1-52.
9. Uma, V.; Elango, M.; Nair, B. U., Eur. J. Inorg. Chem. 2007, 3484-3490.
10. Uma, V.; Kanthimathi, M.; Weyhermuller, T.; Nair, B. U., J. Inorg. Biochem. 2005, 99, 2299-2307.
11. Uma, V.; Vaidyanathan, V. G.; Nair, B. U., Bull. Chem. Soc. Jpn. 2005, 78, 845- 850.
12. Jennette, K. W.; Lippard, S. J.; Vassilia.Ga; Bauer, W. R., Proc. Natl. Acad. Sci. U.
S. A. 1974, 71, 3839-3843.
13. Lippard, S. J., Acc. Chem. Res. 1978, 11, 211-217.
14. McFadyen, W. D.; Wakelin, L. P. G.; Roos, I. A. G.; Hillcoat, B. L., Biochem. J.
1987, 242, 177-183.
15. Brothers, H. M.; Kostic, N. M., Inorg. Chem. 1988, 27, 1761-1767.
16. Bugarcic, Z. D.; Heinemann, F. W.; van Eldik, R., Dalton Trans. 2004, 279-286.
17. Lowe, G.; Vilaivan, T., J. Chem. Soc., Perkin Trans. 1 1996, 1499-1503.
18. Ratilla, E. M. A.; Brothers, H. M.; Kostic, N. M., J. Am. Chem. Soc. 1987, 109, 4592-4599.
19. Ratilla, E. M. A.; Kostic, N. M., J. Am. Chem. Soc. 1988, 110, 4427-4428.
20. Strothkamp, K. G.; Lippard, S. J., Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 2536- 2540.
21. Becker, K.; Herold-Mende, C.; Park, J. J.; Lowe, G.; Schirmer, R. H., J. Med.
Chem. 2001, 44, 2784-2792.
22. Lowe, G.; Droz, A. S.; Vilaivan, T.; Weaver, G. W.; Park, J. J.; Pratt, J. M.;
Tweedale, L.; Kelland, L. R., J. Med. Chem. 1999, 42, 3167-3174.
23. Ma, D. L.; Shum, T. Y. T.; Zhang, F. Y.; Che, C. M.; Yang, M. S., Chem. Commun.
2005, 4675-4677.
24. Kobayashi, K.; Frohlich, H.; Usami, N.; Takakura, K.; Le Sech, C., Radiat. Res.
2002, 157, 32-37.
25. Le Sech, C.; Takakura, K.; Saint-Marc, C.; Frohlich, H.; Charlier, M.; Usami, N.;
Kobayashi, K., Radiat. Res. 2000, 153, 454-458.
26. Usami, N.; Furusawa, Y.; Kobayashi, K.; Frohlich, H.; Lacombe, S.; Le Sech, C., Int. J. Radiat. Biol. 2005, 81, 515-522.
27. Velders, A. H., Ph. D. Thesis, Leiden University, Leiden, 2001.
28. van der Schilden, K.; Garcia, F.; Kooijman, H.; Spek, A. L.; Haasnoot, J. G.;
Reedijk, J., Angew. Chem. Int. Ed. 2004, 43, 5668-5670.
29. Brodie, C. R.; Aldrich-Wright, J. R., Eur. J. Inorg. Chem. 2007, 4781-4793.
30. van der Schilden, K.; Velders, A. H.; Haasnoot, J. G.; Reedijk, J., J. Inorg.
Biochem. 2001, 86, 466.
31. Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G., Ruthenium anticancer drugs, Metal Ions in Biological Systems, Vol 42: Metal Complexes in Tumor Diagnosis and as Anticancer Agents, 2004.
32. Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G., Curr. Top. Med. Chem. 2004, 4, 1525-1535.
33. Bergamo, A.; Stocco, G.; Casarsa, C.; Cocchietto, M.; Alessio, E.; Serli, B.; Zorzet, S.; Sava, G., Int. J. Oncol. 2004, 24, 373-379.
34. Bergamo, A.; Stocco, G.; Gava, B.; Cocchietto, M.; Alessio, E.; Serli, B.; Iengo, E.;
Sava, G., J. Pharmacol. Exp. Ther. 2003, 305, 725-732.
35. Bratsos, I.; Birarda, G.; Jedner, S.; Zangrando, E.; Alessio, E., Dalton Trans. 2007, 4048-4058.
36. Bratsos, I.; Serli, B.; Zangrando, E.; Katsaros, N.; Alessio, E., Inorg. Chem. 2007, 46, 975-992.
37. Hotze, A. C. G.; Kariuki, B. M.; Hannon, M. J., Angew. Chem. Int. Ed. 2006, 45, 4839-4842.
38. Huxham, L. A.; Cheu, E. L. S.; Patrick, B. O.; James, B. R., Inorg. Chim. Acta 2003, 352, 238-246.
39. Iengo, E.; Mestroni, G.; Geremia, S.; Calligaris, M.; Alessio, E., J. Chem. Soc., Dalton Trans. 1999, 3361-3371.
40. Iengo, E.; Zangrando, E.; Mestroni, S.; Fronzoni, G.; Stener, M.; Alessio, E., J.
Chem. Soc., Dalton Trans. 2001, 1338-1346.
41. Magennis, S. W.; Habtemariam, A.; Novakova, O.; Henry, J. B.; Meier, S.; Parsons, S.; Oswald, I. D. H.; Brabec, V.; Sadler, P. J., Inorg. Chem. 2007, 46, 5059-5068.
42. McDonnell, U.; Kerchoffs, J.; Castineiras, R. P. M.; Hicks, M. R.; Hotze, A. C. G.;
Hannon, M. J.; Rodger, A., Dalton Trans. 2008, 667-675.
43. Mendoza-Ferri, M. G.; Hartinger, C. G.; Eichinger, R. E.; Stolyarova, N.; Severin, K.; Jakupec, M. A.; Nazarov, A. A.; Keppler, B. K., Organometallics 2008, 27, 2405-2407.
44. Mendoza-Ferri, M. G.; Hartinger, C. G.; Nazarov, A. A.; Kandioller, W.; Severin, K.; Keppler, B. K., Appl. Organomet. Chem. 2008, 22, 326-332.
45. Pascu, G. I.; Hotze, A. C. G.; Sanchez-Cano, C.; Kariuki, B. M.; Hannon, M. J., Angew. Chem. Int. Ed. 2007, 46, 4374-4378.
46. Ravera, M.; Gabano, E.; Baracco, S.; Sardi, M.; Osella, D., Inorg. Chim. Acta 2008, 361, 2879-2886.
47. Serli, B.; Zangrando, E.; Iengo, E.; Alessio, E., Inorg. Chim. Acta 2002, 339, 265- 272.
48. Alessio, E.; Iengo, E.; Zorzet, S.; Bergamo, A.; Coluccia, M.; Boccarelli, A.; Sava, G., J. Inorg. Biochem. 2000, 79, 173-177.
49. van der Schilden, K., Ph. D. Thesis, Leiden University, Leiden, 2006.
50. Brabec, V.; Nováková, O., Drug Resist. Update 2006, 9, 111-122.
51. Pachón, L. D.; Golobic, A.; Kozlevčar, B.; Gamez, P.; Kooijman, H.; Spek, A. L.;
Reedijk, J., Inorg. Chim. Acta 2004, 357, 3697-3702.
52. Adcock, P. A.; Keene, F. R.; Smythe, R. S.; Snow, M. R., Inorg. Chem. 1984, 23, 2336-2343.
53. Hotze, A. C. G., Ph. D. Thesis Leiden University, Leiden, 2003.
54. Brabec, V., DNA modifications by antitumor platinum and ruthenium compounds:
their recognition and repair. Progress in Nucleic Acid Research and Molecular Biology, Academic Press: 2002.
55. Johnson, W. C., CD of Nucleic Acids, Circular Dichroism: Principles and Applications, VCH Publishers Inc.: New York, 1994.
56. Volosov, A.; Woody, R. W., Circular Dichroism: Principles and Applications.
VCH: 1994.
57. van Vliet, P. M., Ph. D. Thesis, Leiden University, Leiden, 1996.
58. van Vliet, P. M.; Toekimin, S. M. S.; Haasnoot, J. G.; Reedijk, J.; Nováková, O.;
Vrána, O.; Brabec, V., Inorg. Chim. Acta 1995, 231, 57-64.
59. Poklar, N.; Pilch, D. S.; Lippard, S. J.; Redding, E. A.; Dunham, S. U.; Breslauer, K. J., Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 7606-7611.
60. Richards, A. D.; Rodger, A., Chem. Soc. Rev. 2007, 36, 471-483.
