Synergy of intercalation and coordination binding to design novel DNA-targeting antineoplastic metallodrugs

Roy, Sudeshna

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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

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| 6

Synthesis, crystal structure and biological studies of a mononuclear ruthenium(II)-Hpyrimol compound ^†

Chapter

A new Ru(II) compound of formula, trans-[Ru^II(Hpyrimol)2Cl2] has been synthesised starting from a redox-active ligand, Hpyramol, via ligand dehydrogenation to the ligand, Hpyrimol. This mononuclear compound has two coordinated labile chloride ligands at axial positions and unlike other metal compounds (that give a tridentate motif with this N,N,O- donor) the coordination motif of Hpyrimol is bidentate N,N-donor. The phenol group does not coordinate and forms a stable hydrogen bond with coordinated chloride anions. The compound has been fully characterised and evaluated for biological activities. Combined effects of coordinative binding and partial intercalation to DNA are evident from spectroscopic studies. The high in vitro antitumour activity makes this compound interesting to be a starting point for a new library of Ru(II) antitumour compounds.

6.1. Introduction

The anticancer metallodrug research, in the post-cisplatin era, has been steered by several transition metals. Among the other metals, ruthenium has acquired special attention due to three chemical properties, i.e., multiple, stable oxidation states (+1 to +8 and -2) among which +2 and +3 are easily accessible in aqueous solution,^{1, 2} slow rate of ligand exchange similar to Pt(II) and Pt(IV), and the iron-mimetic property of ruthenium (with selective binding affinity to transferrin and albumin).^3-5 Ruthenium has been in medicinal practice already for over twenty years. Ruthenium red¹ is the polycationic oxido-bridged trinuclear species [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]⁶⁺, which has not only been utilised as a botanical stain, but also recently been adopted by microbiologist for clinical specimen staining.⁶ This water-soluble complex cation is also a potential agent for several other applications.^7-9 Ruthenium can be exploited for multifaceted applications such as (a) radiodiagonistic imaging agent (isotopes ⁹⁷Ru and ¹⁰⁷Ru), (b) antimicrobials e.g., [RuCl2(chloroquine)]2, (c) immunosuppressants e.g., cis- [Ru(NH3)4(Him)2]³⁺, (d) NO-scavenger or delivery tools e.g., Ru(III) polyaminocarboxylates (AMD6245 and AMD1226) for the medication of diabetes, arthritis, epilepsy, septic shock and stroke, (e) antibiotics e.g., Ru(III) thiosemicarbazone, and (f) anticancer agents e.g., NAMI-A and KP1019.^10-12

The search for new metallodrugs with improved anticancer activity, lower systemic toxicity and overcoming cisplatin resistance has been continued to yield several potential ruthenium compounds. Unlike Pt(II), ruthenium forms stable octahedral compounds and has a strong affinity towards both DNA and cellular transport proteins.

Depending on the nature of metal compounds, often monodentate adducts are formed.

Ruthenium compounds often form interstrand crosslinks with DNA strands in vitro; for steric reasons intrastrand crosslinks are not favourable. Therefore, the mode of action, biodistribution, activation and cellular processing are expected to be different from cisplatin-like platinum drugs. Consequently, the activity and probability of surpassing resistance might be enhanced effectively.³ Two different approaches for the synthesis of new ruthenium compounds are known, viz., (a) trial and error methods to build an expanded library of known anticancer agents analogues followed by cytotoxicity assays and (b) stepwise exploration of mode of action and cellular processing for a particular compound followed by the application of the same idea for the design new compounds.¹¹ Ruthenium drugs can be useful via multiple ways namely (a) DNA-targeting and binding

agents, (b) photodynamic therapy, (c) antimetastatic agents, (d) antimitochondrial agents and (e) radiosensitiser agents.

Ruthenium compounds are additionally interesting due to the (a) reliable synthetic routes with predictable structures, (b) facile tuning of chemical and biological properties via ligand modification and (c) extended knowledge of the chemical, biological and photophysical properties of ruthenium.^{2, 13} Ruthenium compounds being generally stable in octahedral geometry with six possible coordination sites, a wide choice of ligands (monodentate or polydentate) is feasible to synthesise either coordinatively saturated cationic compounds (such as, [Ru(phen)3]²⁺, [Ru(phen)2(dppz)]²⁺ and [Ru(azpy)3]²⁺, where phen is 1,10-phenanthroline, dppz is dipyrido[3,2-a:2′,3′-c]phenazine and azpy is 2-phenylazopyridine), or compounds (neutral or charged) with comparatively labile chloride ligands (such as, [Ru(terpy)Cl3], [Ru(azpy)2Cl2] or [Ru(terpy)(apy)Cl]ClO4

where terpy is 2,2':6',2"-terpyridine and apy is 2,2'-azobispyridine).^{10, 12, 14} The first series of compounds bind to DNA non-covalently (electrostatic and intercalative), whereas the second type of compounds form guanine adducts on DNA strands similar to cisplatin. In some cases the cytotoxic activity is reasonably correlated to DNA-binding affinity, however, no clear-cut structure–activity relationship has emerged yet.

The chloridoruthenium compounds can be classified into several groups according to the other ligands (ammine, dimethyl sulfoxide, heterocyclic, arene). The Ru(II) cationic compound ([RuCl(NH3)5]⁺) with ammine-chloride ligands is not cytotoxic,^15-17, though the Ru(III) analogues cis-[RuCl2(NH3)4]⁺ and fac-[RuCl3(NH3)3] exhibit antitumour activity comparable to cisplatin for some selected cell lines.^{2, 18} In the cases of (dimethyl sulfoxide)chloridoruthenium(II) compounds (cis- and trans-[RuCl2(dmso)4]) the trans-isomer displays a better activity, both in vitro and in vivo.¹⁹ This isomer eventually overcomes the cisplatin resistance in P388 leukemia cell lines and also has emerged as a better antimetastatic agent.²⁰ For the compounds containing monodentate heterocyclic ligands e.g., imidazole or indazole (NAMI, NAMI-A, KP1019) the heterocyclic (or dmso) ligands are in trans-geometry; they are very promising anticancer drugs. NAMI-A entered clinical trials, being the first ruthenium drug and KP1019 followed thereafter.^21-25

The ruthenium polypyridyl compounds with chloride as hydrolysable ligand are devoid of any straightforward structure-activity trend.²⁶ The compounds cis- [Ru(bpy)2Cl2] and cis-[Ru(phen)2Cl2] are lacking any cytotoxic activity in vitro.^{14, 27} On the contrary, cis-[Ru(azpy)2Cl2] shows antitumour activity comparable to cisplatin and to

fluorouracil.^28-30 Therefore, the compounds Ru^IIL2Cl2 (where L stands for a bidentate heterocyclic N-donor ligand) cannot be easily predicted to be anticancer active depending on the geometry and the presence of labile chloride ligands.

The ligand Hpyramol (4-methyl-2-N-(2-pyridylmethyl)aminophenol, L8), which has been mentioned already in Chapter 5 was used to synthesise a mononuclear Ru(II) compound. Hpyarmol undergoes metal-mediated (copper, platinum and zinc) oxidative dehydrogenation to the “pyrimol” form via the ‘acid’ “Hpyrimol” [4-methyl-2-N-(2- pyridylmethylene)aminophenol, L9]. The aim of this chapter is to follow the trend of biological activity of metal compounds varying from inert to redox-active ones. In addition, a main focus of the ruthenium compound (C9) synthesis has been to study DNA-targeting and DNA-cleaving species, which eventually leads to irreparable damage to DNA, followed by programmed suicide of the cell (apoptosis). The copper compound of this ligand cleaves phage DNA catalytically.³¹ This finding leads to the idea that if the ruthenium compound bearing the same ligand can cleave DNA efficiently, it might be a potential cytotoxic agent. The expected binding motif along with the experimentally observed coordination mode has been depicted in Fig. 6.1 along with two forms of ligands.

N CH₃

N N OH

CH₃

OH N H

N CH₃

N

O M ^N

CH₃

N

OH M

Hpyramol L8 Hpyrimol L9

M-pyrimol M-Hpyrimol

(a) (b)

(c) (d)

Figure 6.1. Schematic structures of (a) ligand L8, (b) after dehydrogenation forming L9, (c) after metallation and (d) with different coordination motif.

6.2. Experimental 6.2.1. Chemicals

Hpyramol L8, was synthesised in a single-step reaction as reported in literature.³² The ruthenium precursor, RuCl3·3H2O was generously provided as a loan by Johnson- Matthey (Reading, U.K.). The second ruthenium precursor, K2[RuCl5(H2O)] was synthesised following a reported route.³³ The supercoiled plasmid DNA used for cleavage studies has been purchased from Invitrogen Life Technology (California, U.S.A.) as ΦX174 phage DNA (0.25 µg/µL). Deuterated solvents used for NMR experiments were also purchased from Sigma-Aldrich B.V. (The Netherlands). The solvents used for synthesis were purchased from Biosolve (The Netherlands) (AR grade) and used without further purifications. Sodium chloride and sodium dihydrogenphosphate (NaH2PO4) were purchased from Merck, Germany. Disodium hydrogenphosphate (Na2HPO4) was obtained from ACF Chemiefarma NV (The Netherlands) and the water was of MilliQ quality from Millipore, U.S.A. Calf Thymus DNA (CT DNA) was purchased from Sigma-Aldrich B.V.

(The Netherlands) and purified following a typical method of size-exclusion chromatography and lyophilisation. The buffer used was 10 mM phosphate buffered saline (PBS) with 50 mM NaCl.

6.2.2. Synthesis of Ru^II(Hpyrimol)2Cl2, C9 (a) Method 1

The ruthenium starting material, K2[RuCl5(H2O)], (352 mg, 0.93 mmol) was dissolved in absolute methanol (50 mL) at room temperature. LiCl (120 mg, 2.82 mmol) was added to the ruthenium solution and stirred in the dark to generate a clear solution.

L8 (200 mg, 0.93 mmol) was dissolved in absolute methanol (40 mL) and added dropwise to the ruthenium solution. The reaction mixture was stirred at room temperature for 24 h under aerobic condition and the colour of the reaction solution changed gradually from reddish-brown via blue to deep purple. After filtration (to get rid off any undissolved starting material), the volume of the reaction mixture was reduced to ∼10 mL and 20 mL acetone was slowly added. This solution was kept standing at 4 °C overnight to yield deep brown crystals. The crystals were filtered and washed with diethyl ether to yield a shiny deep brown solid. The crystals suitable for X-ray diffraction were taken directly from the reaction solution mixture. Yield of the product was 347.2 mg.

(b) Method 2

The ruthenium precursor RuCl3·3H2O (300 mg, 1.15 mmol) was dissolved in methanol (50 mL) and L8 (246 mg, 1.15 mmol) was dissolved in methanol (50 mL) stirring at room temperature. The L8 solution was added dropwise to the ruthenium solution. This mixture was stirred for 48 h at room temperature. After filtration (to get rid off any undissolved starting material) and volume reduction to ∼10 mL, 25 mL of acetone was added to the reaction mixture. This solution was stored at 4 °C overnight to yield bright shiny brown crystalline solid. This solid was filtered out, washed with cold acetone and finally with diethyl ether. The solid was air-dried and the yield was 403.5 mg.

6.2.3. Characterisation and analysis of C9

The ruthenium compound C9 has been characterised by various analytical methods (NMR, mass spectroscopy, elemental analysis). Detailed descriptions of the analytical techniques and instrumentation have been explained in Chapters 2 and 3.

Elemental analysis (%) for RuCl2C26H24N4O2·2CH3COCH3 (acetone from crystallisation):

expt. (calcd.) C 53.50 (53.93), N 7.84 (7.86), H 5.54 (5.90). ESI-MS measurements of a freshly prepared solution of crystalline compound in methanol-water (80:20 v/v) showed three major peaks at m/z = 597.88, 560.88 and 524.91 with ruthenium isotopic pattern, corresponding to C9 + H⁺, C9 – Cl^- and [C9 – 2Cl^-]+H⁺ species, respectively.

6.2.4. Structural determination of C9

The single crystal suitable for X-ray diffraction was collected directly from mother liquor (reaction mixture). Single-crystal X-ray diffraction data were collected with a Nonius Kappa CCD diffractometer using graphite monochromated Mo-Kα 281 radiation (λ = 0.71073 Å). The data were processed by using the software package, DENZO.³⁴ The structures were solved by direct methods implemented in SHELXS-97³⁵ and refined by a full-matrix least-squares procedure based on F² with SHELXL-98.³⁶ All non-hydrogen atoms were refined anisotropically, while the hydrogen atoms were included in the model at geometrically calculated positions and refined by using a riding model. Details of the crystal data, data collection, and refinement parameters are listed in Table 6.1. Selected bond lengths and bond angles are presented in Table 6.2. The figure of the structure was drawn with ORTEP.³⁷ These data are available from the Cambridge Crystallographic Data Centre (CCDC number CCDC 704761) via the link www.ccdc.cam.ac.uk/data_request/cif.

Table 6.1. Crystallographic details of compound C9.

Empirical formula C26H24Cl2N4O2Ru•2C3H6O

Formula weight 712.62

Crystal size [mm³] 0.06 × 0.14 × 0.18

Crystal colour brown

Temperature 293 K

Crystal system triclinic

Space group P-1 (No. 2)

a [Å] 8.8760(2)

b [Å] 9.0154(2)

c [Å] 10.4403(3)

α [°] 91.625(1)

β [°] 107.003(1)

γ [°] 92.203(1)

V [ų] 797.65(3)

Z 1

Density (calculated) [g/cm³] 1.484

μ [mm^-1] 0.701

Absorption correction range 0.88 – 0.96 (sinθ/λ)max [Å^-1] 0.65 Reflections (integrated/total) 13273 / 3593

No. of reflections 3541

No. parameters / restraints 260 / 0 R1/wR2 [all data] 0.0280 / 0.0240

S 1.05 Min/max residual density [e/ų] -0.72 / 0.30

Table 6.2. Selected bond lengths and bond angles of C9, as well as the N=C double bond.

Bond lengths (Å) Bond angles (°)

Ru(1) – Cl(1) 2.4045(4) Cl(1) – Ru(1) – N(1) 85.56(4) Ru(1) – N(1) 2.0773(13) Cl(1) – Ru(1) – N(2) 92.23(3) Ru(1) – N(2) 2.0799(12) N(1) – Ru(1) – N(2) 77.66(5) N(1) – C(8) 1.291(2) Cl(1) – Ru(1) – Cl(2) 180.00

6.2.5. Biological studies

For CD and UV-titration studies the experimental details for conformation changes of CT DNA have been provided in Chapter 2. DNA-cleavage studies have been performed and the experimental details for electrophoretic mobility in agarose gel have been described in Chapter 5. The cell viability assays have been done using SRB assay in vitro and the experimental details have been mentioned in Chapter 2. These experimental details will not be discussed again in this chapter.

6.3. Results and discussion 6.3.1. Syntheses and characterisations

The synthesis of the compound was found successful starting from two different Ru(III) precursors under aerobic condition. Generally the bis-substitution takes place when two equivalents of ligand are added to the ruthenium precursor. Unexpectedly, in this case addition of molar equivalent of ligand yielded the bis-substituted compound, but can be explained by more thermodynamic stability of the product than the mono- substituted product. The yield of the product C9, trans-dichloridobis[4-methyl-2-N-(2- pyridylmethylene)aminophenol]ruthenium(II)-bis-(acetone) did not vary significantly upon changing the precursor and post-synthesis work up. This compound is highly soluble in acetone, acetonitrile, methanol and dimethyl sulfoxide. The oxidation of the starting ligand L8 to form L9 is evident from all the measurements. The elemental analysis clearly indicates formation of this compound. The mass spectrum of the compound is shown in Fig. 6.2. The ESI-MS shows the presence of ionic species after dissociation of chloride ligands along with the molecular ion peak. The isotopic pattern typical to a single ruthenium centre is shown in the figure below.

Figure 6.2. Partial ESI-MS spectra of the ruthenium compound C9 with a typical ruthenium isotopic distribution pattern.

6.3.2. Crystal structure

The Ru(II) compound C9 crystallises in octahedral geometry with the Ru-N4 in the equatorial plane and the chlorides are at axial position perpendicular to the Ru-N4 plane. The crystal structure shows that the chloride groups are stabilised via hydrogen bonding to non-coordinating phenol groups. The ligand L9 does not possess a planar structure, rather the half part of the ligand bearing 4-methyl aminophenol moiety remains almost perpendicular to the pyridylmethylene portion. Unlike other transition metal compounds (copper, platinum and zinc) prepared from the same starting ligand L8, ruthenium forms a bis-substituted compound, where the L9 behaves as a bidentate N,N- donor and phenol does not transform to phenolate ligand. Therefore, the expected tridentate coordination motif is absent and the 4-methyl aminophenol moiety is dangling parallel to the chloride groups. The Hpyrimol groups are trans to each other around the ruthenium centre. The There are two reciprocal orientations of the ligands L9 possible (geometrical isomers, cis- and trans-) but only trans- isomer is possible to isolate due to thermodynamic stability. ORTEP perspective view of the compound C9 is presented in Fig. 6.3. The solvent of crystallisation, acetone and the hydrogen atoms are omitted for clarity. The packing diagram of the compound is shown in Fig. 6.4, which depicts an array with intramolecular hydrogen bonding network. The crystallographic details and the selected crystal parameters are summarised in Tables 6.1 and 6.2, respectively. The

[M-Cl^-]⁺ [M-2Cl^-]+H⁺

excellent R value (0.03) indicates the quality of the crystal and accuracy in the measurements.

Figure 6.3. ORTEP representation of C9, as determined by X-ray crystallography. The solvent of crystallisation and the hydrogen atoms are excluded for clarity except for the phenolic -OH. The structure is presented with 50% displacement probability and some atomic labelling.

6.3.3. DNA-interaction studies (a) Circular dichroism

The CD spectra were recorded at 37 °C and the concentration of the DNA was kept fixed at 100 μM throughout the experiments. When R (the ratio of concentration of DNA per nucleotide upon concentration of compound) is less than 1, the amount of compound is higher than the amount of DNA. Multiple binding possibilities are more feasible in this case. The changes are very prominent when compared to compound-free DNA. The conservative positive band intensity at 280 nm is decreased to become almost negative, whereas the intensity of negative band at 240 nm is increased. The new ellipticity is observed at 220 nm and this peak has been red-shifted by 10 nm with increasing amount of compound. With R = 1 this change in the spectrum continues. The

Figure 6.4. 3D-packing diagram of C9, as derived from X-ray crystallography. The prominent intramolecular H-bonds are presented by blue dotted lines.

positive peak is decreased and negative peak is increased in intensity when compared to native free DNA. The right handed B-canonical form is retained; however, the base- stacking is significantly impaired. The spectra of C9 after interaction with CT DNA are shown in Fig. 6.5.

Figure 6.5. CD spectral changes on CT DNA upon addition of different amounts of C9 with arrows indicating gradual changes of the B-DNA right-handed helical form (enlarged scale for R=1 on the right).

The intensity of the positive band is consistently decreasing with time (from 0 h to 4 h) with the constant increase of the negative band (data not shown). After this time period, the DNA starts precipitating from the solution mixture and the spectra hereafter could not be recorded.

(b) UV-vis spectroscopy

The absorption spectral changes of C9 after interaction with CT DNA have been recorded and are presented in Fig. 6.6. The compound absorbs in the UV-vis range at wavelength 556 nm and 277 nm strongly with shoulders at 447 nm and 322 nm, respectively [Fig. 6.6(a)]. The 556 nm peak can be assigned as MLCT band and the 277 nm peak originates from a ligand centred π-π^∗ transition.^{38, 39} The hyperchromism is gradual with decreasing amounts of DNA. In other words, hypochromism is observed with increasing amount of DNA in the reaction mixture. This change is an indication of intercalative binding.^{40, 41} C9 is not a planar molecule, but it may intercalate partially using the ligand surface to slide through the DNA base pairs. The assumable binding mode might be coordination if solvolysis of chloride ligands takes place. This neutral compound might form a cationic intermediate followed by solvolysis, which could be attracted to phosphate backbone of DNA via electrostatic interaction and subsequently coordinate to guanine or adenine. The spectrum of C9 in phosphate buffer [Fig. 6.6(a)]

(diluted from a stock solution prepared fresh in dmso) and the changes in the spectra after DNA interaction are shown in Fig. 6.6(b).

Figure 6.6. UV-vis spectra of C9 (a) in buffer and (b) spectral changes of C9 upon addition of increasing amount of CT DNA. Arrows indicate the direction of gradual increase in absorption with decreasing R.

6.3.4. DNA-cleavage studies

The agarose gel electrophoresis technique has been used to investigate the DNA- cleaving ability of C9. The gel pictures are shown in Figs. 6.7 and 6.8, respectively.

Supercoiled or closed circular (Form I) phage DNA has been used to study the cleavage property. In all cases some open circular DNA (Form II) can be seen, but in no case the linear (Form III) has been observed. This compound C9 thus differs in cleaving property compared to the copper analogue C8 (vide Chapter 5).³¹

Figure 6.7. Gel electrophoretic picture of C9 after interaction with supercoiled φX174 phage DNA with lane 1 DNA blank (20 μM), lane 2 DNA + 20 μM C9, lane 3 DNA + 100 μM C9, lane 4 DNA + 5 μM C9 + 20 μM AsĀ, lane 5 DNA + 5 μM C9 + TEMPO, lane 6 DNA + 20 μM Ascorbic acid, lane 7 DNA + 20 μM RuCl3 solution.

Figure 6.8. Gel electrophoretic picture of C9 after interaction with supercoiled φX174 phage DNA with lane 1 DNA blank (20 μM), lane 2 DNA + 10 μM C9, lane 3 DNA + 20 μM C9, lane 4 DNA + 50 μM C9, lane 5 DNA + 100 μM C9, lane 6 DNA + 200 μM C9, lane 7 DNA + 10 μM C9+ Ascorbic acid, lane 8 DNA + Ascorbic acid, lane 9 DNA + C9+ TEMPO, lane 10 DNA + TEMPO, lane 11 DNA + L8, lane 12 DNA + 20 μM C9+

TEMPO, lane 13 DNA + 200 μM RuCl3 solution.

The amount of open circular or nicked (Form II) form increases with increasing amount of C9 in presence of a constant concentration of DNA (20 µM per base pairs). In addition, a retardation of the mobility of supercoiled form is observed (lane 2-6). This phenomenon clearly indicates that the binding of C9 is to the DNA strand, rather than resulting in cleaving. The cleavage is single stranded, as Form III is absent. The reducing agent, ascorbic acid or the stable radical (TEMPO) do not have any effect on cleavage process. The stoichiometric cleavage and several controls are shown in Fig. 6.8.

1 2 3 4 5 6 7

Form I Form II

1 2 3 4 5 6 7 8 9 10 11 12 13

Form I Form II

6.3.5. Cytotoxicity assay

Cell viability tests have been performed to evaluate in vitro cytotoxicity of C9 and compared with the starting ligand, L8, using a SRB assay against several human cancer cell lines after 120 h incubation of multiple concentrations of the compound. To compare the activity with some active or inactive reference compounds, data has also been collected from a recent theoretical study.⁴² The IC50 values are summarised in Table 6.3.

The starting ligand, L8 is not active, whereas C9 is active and even comparable to cisplatin (except for the IGROV and WIDR cell lines). From the values, it is evident that the cytostatic activity of C9 is similar to that of β-[Ru(azpy)₂Cl₂].

Table 6.3. IC50 values (μM) for C9 and some selected active/inactive cis- dichloridoruthenium(II) compounds against human cancer cell lines, including some data from literature.

6.4. Conclusions

An interesting Ru(II) compound with two trans-chloride ligands has been synthesised. This compound may react in a mixed-binding mode with DNA. A prediction based on a simple hypothesis without any experimental evidence could be the conversion of the compound to different species. Under physiological condition, the chloride groups might be hydrolysed and there are possibilities of formation of cationic bis- or monoaqua intermediate, [RuL2(H2O)2]²⁺ or [RuL2Cl(H2O)]⁺. By external electrostatic interaction

Cell lines

Samples A498 EVSA-T H226 IGROV M19 MCF7 WIDR

cisplatin 7.50 1.40 10.9 0.56 1.85 2.32 3.22 L8

Hpyramol 37.4 13.7 46.6 22.7 38.6 21.1 67.9

C9

trans-[Ru(L9)2Cl2] 3.15 6.46 12.1 12.2 6.26 6.65 15.7 cis-[Ru(bpy)2Cl2] 114.7 > 129.3 73.0 105.3 112.5 100.8 122 α-[Ru(azpy)2Cl2]²⁸ 0.74 0.056 0.35 0.08 0.013 0.11 0.29 β-[Ru(azpy)2Cl2]²⁸ 8.8 0.960 13.3 3.40 0.75 6.23 11.4 α-[Ru(azpy)(bpy)Cl2]¹⁰ 55.3 13.3. 27.1 5.23 5.22 17.3 22.0

towards polyanionic phosphate backbone of DNA, this intermediate would be attracted.

Consequently, this compound also may partially intercalate via the aromatic ligand moiety. These assumptions are further demonstrated by interaction studies with calf- thymus DNA. The stability of base-stacking is clearly disturbed and in spite of a retained right-handed helicity, the B form has been modified. This compound not only binds to DNA, it additionally cleaves DNA via single-strand cutting. An in vitro cytotoxicity assay shows a very promising behaviour, as this compound shows activity against several cancer cell proliferations.

Only a few trans-chloridoruthenium(II) compounds have been reported in literature. The only trans compounds reported are mainly NAMI analogues with Ru(III) centres. In these types of compounds, the monodentate heterocyclic ligands are trans and at axial positions with chlorides occupying the equatorial coordination sites. These compounds therefore cannot be suitable compound to compare with C9. The better comparison can be done with trans-[Ru^IICl2(dmso)4]. This compound is not only antitumour active but also possess a strong anti-metastatic property. The active species in vivo has been identified to be trans, cis, cis-[Ru^IICl2(dmso)2(H2O)2] after substitution of coordinated dmso molecules by aqua ligands.^{43, 44}

In the present compound the chlorides are trans and at axial positions. No structure-activity relationship could be deduced so far, but this observation may open up a new synthetic design. More Ru(II) compounds with similar ligand architecture are needed to be synthesised. The compound is different in structural framework, therefore the mode of activation and biodistribution are required to be established and then compared to well- known antimetastatic ruthenium compounds. Additionally as ruthenium compounds are better antimetastatic agents, this compound may also be interesting for antimetastatic activity evaluation.

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