Synthesis, Structure, Properties And Biological Behaviour Of The Complex [RuIV (H2L) Cl2].2H2O (H4L= 1,2-Cyclohexanediamminetetraacetic Acid)

The highly water-soluble ruthenium complex [Ru(H2L)Cl2]2H2o, in which H4L is the sequestering ligand trans-l, 2-cyclohexanediamminetetraacetic acid (cdta) has been synthesized, structurally characterized and its properties studied. The X-ray crystallographic study shows that the chelating coordinated ligand is tetradentate while the ruthenium environment is octahedral and slightly distorted, with two chloride anions coordinated in cis positions. Potentiometric, conductimetric and infrared studies confirm the presence of two free carboxylic groups, while electronic and voltammetric studies show that the central ion is Ru(IV). The testing of the cytotoxic activity of this complex against three different human cancer cell lines indicates that [Ru(H2L)Cl2].2H2O shows a remarkable and selective antiproliferative effect against the human uterine neck carcinoma HeLa and the malign adenocarcinoma ADLD, showing only a discrete turnout cell inhibition activity against colon adenocarcinoma HT-29. The important antiprotiferative behaviour of complex 1 against the human adenocarcinoma ADLD, indicates that [Ru(H2L)Cl2].2H2O might be considered as potential antineoplastic compound.


INTRODUCTION
Metal-based antitumour drugs constitute in our days a broad research area of increasing interest /1-4/. Concretely, cisplatin, cis-[Pt(NH3)2C12], carboplatin [cis-diammine-I, -cyclobutanedicarboxylatoplatinum(II)] and oxaliplatin [trans-(R,R)-l,2-diamminecyclohexaneoxalatoplatinum (II)], are currently being used clinically /5-10/. However, the problem of both acquired and inherent resistance of tumour cells presents a main limitation to the more widespread clinical use of platinum complexes /3,7,9/. In the hope of overcoming these limitations, other platinum-based antitumour drugs have been synthesized and tested for antitumour activity/11-15/. Furthermore, new anticancer drugs containing transition metal ions other than platinum /16/have been also assayed. Possible advantages may involve different coordination geometries, several metal-ion oxidation states and biological targets other than DNA.
In the design of these new drugs, ruthenium complexes have raised great interest/17-24/. As an example, the antitumour activity of the highly water soluble complex H[Ru(H2L)CI]-4H20 (H4L: 1,2propylenediammine-N,N,N',N'-tetraacetic acid, pdta) has been evaluated in vivo and in vitro/23,25/. This complex rapidly binds to serum proteins producing stable adducts in which the core Ru(lII)(pdta) is probably bound to histidines on the protein surface/26-28/. On the other hand, the complex damages nuclear DNA, inhibits DNA recognition and stimulates NADPH oxidase and a respiratory burst in phagocytic neutrophils and elicits phosphorylation of tyrosine residues/25,29/.
Another interesting ligand is the potential hexadentate sequestering agent trans-l,2cyclohexanediamminetetraacetic acid (cdta), in which the ethylenediammine group of edta has been replaced by the heterocyclic moiety trans-l,2-cyclohexanediammine. The ligand cdta is widely used nowadays in varied applications, i.e., as detoxifier of heavy metals that contaminate patients/30/, for use in endodontics /31/, for extraction of pectin polymers /32/ and for separation and quantitation by several techniques of inorganic ions and anionic metal complexes/33/. Although the metal complexes formed by cdta are known from time ago/34/, recent literature has shown new synthesis procedures and important properties of new isolated complexes. Concretely, those formed by cdta with Cu (ll) and Ni (ll) have been studied by X-ray photoelectron spectroscopy and X-ray crystal diffraction studies /35,36/ and interesting results on the structure, electrochemistry, kinetics, pKa values and influence of chelate effects on the water-exchange mechanisms of complexes cdta/Fe(lll)/Fe (ll) As a result of the attractive research in progress on biological properties of complexes formed by Ru(lll) with amminepolycarboxylic ligands, as well as the absence of structural and antitumour activity of Ru(cdta) complexes, we present in this paper the synthesis, characterization by chemical and spectroscopic techniques, x-ray crystal structure and biological behaviour of the water-soluble complex [Ru(HzL)CI].2H:O (I), in which the ligand cdta (H4L) is acting as tetradentate molecule/45/. Our results demonstrate that the complex contains Ru(IV) and shows activity against several in vitro and in vivo tumours. This research open a new way for the medical and pharmaceutical development of potential antineoplastic complexes formed by the chelating agent cdta with platinum metals. EXPERIMENTAL X-ray crystallography A prismatic yellow-amber crystal of compound was mounted (glass fiber) on an Enraf Nonius CAD4 automatic diffractometer /46/ and 2388 unique reflections were measured. Cell constants and orientation matrix for data collection were obtained by least-squares refinement of the 20 values of 25 reflections. Intensities of 3123 reflections within the range 2 <20 <50 were measured and collected at 293 K using monochromatic MoK, radiation (2 0.71073 A) and the a/20 scan technique. Intensities were corrected for Lorentz and polarization effects/47/and 2388 {(1>2o->(I)} were considered as observed. A semiempirical Of the Complex absorption correction (q-scans) was made/48/.
The structure was solved by the Patterson method/49/ and subsequent difference Fourier maps, and refined on F by a full matrix least-squares procedure using anisotropic displacement parameters/50/. All hydrogen atoms were located in difference map and included as fixed contributions riding on attached atoms with isotropic thermal parameters 1.2 times those of their carrier.atoms. The H atoms of two water molecules, O(1) and O(2), were not located. Therefore, the contribution of the density of a disordered water molecule was subtracted from the measured structure factors with use of the SQUEEZE option /51/. Subsequent refinement then converged with R factors and parameter errors significantly better than for all attempts to model the solvent disorder. The Flack x parameter (absolute structure parameter) was calculated to be 0.05 (6) for the present structure and 0.95 (6) for the inverted structure, thus providing strong evidence that the absolute structure has been assigned correctly/52/. Criteria of a satisfactory complete analysis were the ratios of rms shift to standard deviation less than 0.001 and no significant features in final difference maps. Atomic scattering factors, from "International Tables for Crystallography"/53/. Molecular graphics, from PLATON /51/and SCHAKAL/54/. Chemicals Hydrated ruthenium (III) chloride (Sigma) was dissolved in ethanol and refluxed for 30 min. After concentration to dryness, the compound was stored under CaCI_ (RuCI3). The ligand cdta was used as purchased (Sigma). All other chemicals and solvents were analytical grade reagent products.
Analytical, potentiometric and conductimetric studies Elemental microanalyses were performed at the Microanalytical Laboratory of the Barcelona University. Metal content was determined by atomic absorption spectroscopy using a Perkin Elmer 2380 model, at 10 mA and 349.9 rim. Hydration water molecules were determined by thermal analysis. Potentiometric and conductimetric studies were carried out with a Crison MicroTT 2022 titrimeter, provided with autoburette Microbur 2030. Aqueous solutions of the complex (50-100 mg/100 ml) were titrated against a 30 mM NaOH solution. Electrical conductimetry of the same solution was performed on a Crison 525 conductimeter.

Electronic and infrared spectroscopy
The electronic spectra of solutions were recorded on a Jasco V550 spectrometer interfaced with a PC. IR spectra were recorded on an FT-IR Jasco 300E instrument in the 200-4000 cmrange, using either Nujol mulls supported between polyethylene plates or KBr pellets.

Voltammmetric studies
Cyclic voitammetry measurements were carried out using a Princeton Applied Research analyser. A Rosario A. l,71al)lana et aL Bioinol2,anic Chem&try and Applications glassy carbon electrode was used as working electrode on aqueous solutions (NaCIO4 0.15 M) of complex at concentrations ranging between 1.0 and 6.0 mM. Potentials were measured against a saturated (NaC1) calomel electrode (SCE) as reference electrode. Voltammograms (CV) were obtained at potential values between +1.5 and -1.5 V. Scan rate was equal to 50 m V/s.

Biological in vitro and in vivo assays
Complex was dissolved in a mixed 1:1 DMSO:H20 solution and used as concentrated stock solution from which diluted solutions (I/10 dilt/tion factor) were prepared and added to growing culture medium of each one of the cellular types included in the study. The in vitro assays were done against three different human cancer cell cultures: malignant melanoma (ADLD), uterine neck carcinoma (HeLa) and colon adenocarcinoma (HT-29). For determination of plating efficiency and colorimetric reading, we prepared plates of 24 and 96 wells, respectively, that were then inoculated with 5,000 to 30,000 cells/well. After 24 h from the inoculation, different concentrations of the testing product were added to the cancer cells. After 48 h of the named addition, the cells were washed and then fixed. Finally, the coloring and reading of the plates were carried out. In all cases we determined the number of cells at the beginning (Tot), just before the addition of the testing products (To) and 48 h later (T controls and T tests), according to Scheme 1.
The antitumour activity of was tested in vivo against Ehrlich ascitic tumour (EAT), the intraperitoneally-implanted P388 lymphocytic leukemia and the subrenal capsule (transplanted human mammary carcinoma) MX-I xenograft. The experiments were performed according to the NIH protocols at ONI Centre, Madrid, Spain. CD2F female mice with weights within a 3 g value range and a minimum weight of 18 g were used for all experiments except for MX-1 carcinoma, where athymic swiss mice were used (4 g value range and weight of 17 g). The test groups was formed by 6 animals and control group by 12.
Synthesis of the complex [RuV(HL)CI].2HO [1] Solid cdta (228 mg, 1.0 mmol) was added to a clear solution of RuCI3 (0.230 mg, 1.1 retool) dissolved in a 50 ml of HCI 0.1 M, while stirring. The deep red mixture was introduced into a sealed pressure reactor and heated for 16 h at 120C in electric oven. After cooling, the pink-red obtained solution was slowly concentrated to about 5 ml by evaporation at room temperature. Yellow-amber prismatic crystals suitable for X-ray diffraction studies were recovered and the solid analysed. Two hydration water molecules per mol of compound were found by thermal analysis. Anal. (%), Calc. for C4H2008N2CI2Ru'2H20: C,30.4;H,4.5;N,5.1;CI,12.8;Ru,18.3. Found: C,30.6;H,4.1;N,5.3;CI,12.2;Ru,17.9. RESULTS AND DISCUSSION X-ray structure  (6) 0.515 and -0.380 e.A-3 molecular structure obtained by X-ray diffraction analysis is shown in Fig. along (2) Ru (i) -l.I (2) 2. 128 (2) Ru (!) -CI (2) 2.3658 (9) (1) 173.48 (8) 80.72 (9) 94.89 (8) 95.57 79.14(i0) 84.54 (8) 93.05 (7) 91.97 (7) 92.69 (7) 170.39 (8) 91.83 (6) 92.21 (6) 171.69 (8) 92.57 (6)  It is important to remark that the nonbonding CI...C1 distance (bite) in compound (3.35 A), is directly correlated with the distance between adjacent or proximal coordination sites in biological targets such as DNA. This bite distance is identical to that for cisplatin, and corresponds to the separation between two appropriate DNA-nucleobase donor atoms, this fact enabling cross-linking formation after interaction of complex with DNA inside the cell/29/. in the packing, the molecules of complex are assembled in a 3D-network by intermolecular hydrogen x-y+ 1, x, z-l/6 and 2: y, -x+y+ 1, z+ 1/6), as found in similar cdta complexes /42a/, i.e., the hydroxyl groups of the non-coordinated carboxylic moiety act as hydrogen-atom donors while the carbonyl groups are the hydrogen-atom acceptors in these associations (Fig. 2). In addition, the two water molecules located between neighbouring molecular units are also H-bonded to oxygen carboxylato groups  Potentiometric and conductimetric study The potentiometric titration of an aqueous solution of the complex (Fig. 4a) shows a sharp increase of pH for the consumption of 2 g-equiv, of alkali with an inflection point registered at pH 6.9. Furthermore, a change in the electrical conductivity of the solution is also observed at A equal to 168 S cm (Figure 4b these facts indicating the simultaneous neutralization of two free-COOH groups of the same strength. These results supported the tetradentate character for the cdta, which contains two coordinated carboxylate moieties and two free carboxylic groups. Molecular weight deduced from the titration corresponds closely with the expected for the dehydrated complex (MW: 516).

Ultraviolet and visible spectroscopy
In the electronic spectrum (not shown) three different absorptions bands at 28410 cm 1 (352 nm; 11310 dm mol cm), 31056 cm (322 nm; 15600 dm mol cm) and 35336 cm (283 rim; 4070 dm toolcm) are observed. The first two main bands could be attributed to the electronic transitions 2Eg --3Tlg and 3T2g 3T -g, respectively, while the smaller band (shoulder) registered at 283 nm could be due to the transition 3A g *--Tg. This behaviour suggests a low spin d system, normally presenting two close bands of similar intensities originated by transitions from unpaired basal states.

Infrared spectroscopy
The FT-infrared spectrum of compound 1 (not shown) presents a broad, split signal with main peaks at 2940 cm and 2902 cm 1 corresponding to the symmetric stretching vibrations (vs) of the methylene groups (C-H bonds) contained in the heterocyclic ligand and of the same groups next to coordinated and free carboxylic groups. The O-H stretching vibrations of the free COOH groups are responsible for the broad absorptions registered between 3350 cm and 2600 cm. The complex shows strong characteristic bands registered at 1735 cm (vas, C=O bond of free carboxylic groups) and 1610 cm (Vas COOcoordinated carboxylate groups) whereas the Vs of coordinated carboxylate groups appeared at 1400 cm-. The difference of 210 cm between v, and vs of coordinated carboxylate groups indicates the predominantly ionic character of complex 1. -1 Water normally gives broad absorption at about 3400 cm Complex shows indeed a broad and intense -I band around 3350 cm that might be attributed to the hydration water molecules most likely bonded to anionic coordinated moieties through hydrogen bonds/55/. Table 3 presents the main bands observed in the infrared spectrum of complex I.

Cyclic voltammetry
The cyclic voltammogram (CV) of just prepared 4.0 mM aqueous solutions (pH 2.0) of complex (NaCIO, 0.15 M) is presented in Fig. 5. At electric potentials between +0.5 V and -0.5 V, the CV show a cathodic wave (A) immediately followed by the anodic wave (A'), both clearly constituting a coupled pair, AA'. If the potential varies between +1.5 V and -1.5 V, an intense cathodic peak is observed at about-0.8 V, while an anodic prominent peak appears at about 1.0 V. These features could be attributed to redox processes affecting the ligands/58/. Table 4 presents the cyclovoltammetric parameters corresponding to the coupled pair AA', characteristic  of a reversible one-electron process (diffusive control), in agreement with the values of the cathodic current function, in the order of 300 A cm mol 1 s I/2 V"/z. The pair AA' of just prepared solutions of complex is always preceded by another less intense pair (BB', Ef 50 mV ) coupled with a reversible third pair CC' at positive potential values (i.e., Ef--+ 535 mV; see Fig.5). Different coexisting Ru(lll) species seem to be responsible for the processes AA' and BB', as suggested by the invariance of the used scanning speed interval. However, the process BB' is the origin of the coupled process CC' (initial scanning in anodic direction), the last probably due to the oxidation Ru(IIi)-Ru(IV). In fact, two reversible/quasi-reversible couples at 0.25 V and 0.54 V are observed, characteristic of the one-electron reduction processes Ru(lll)/Ru (ll) and Ru(IV)/Ru(III), respectively, in agreement with previous structural results shown in this research.    Fig. 6, which presents the evolution with time of the total number of culture cells in the absence (control curve) or presence of increased amounts of the tested compound (curves a and b  Fig. 6: Antiproliferative effects exerted by the [Ru(H2L)CI2]-2H20 complex on the grow kinetics of the human uterine neck carcinoma HeLa. Cells were exposed to several doses of the ruthenium complex for a total of five days, as indicated: control curve (untreated HeLa cells); curve a (25.4 IaM); curve b (40.8 luM).
To determine if this cytotoxic behaviour holds true for other human tumours, the inhibition assay was repeated against the human malign melanoma ADLD (Fig. 7) whose proliferation rate (control curve) is higher than that observed for HeLa cells. A substantial increase of the antiproliferative properties was detected against this melanoma at the same doses assayed against HeLa carcinoma. At a dose of 25.4 IuM, (curve a) the inhibition of ADLD cells is not only more important that in the previous assays but increases with increasing time (inhibition of about 33% after 72 h; 42.4% on the fourth day, and 46.4% on the last day). If the dose is increased (40.8 M) the same general pattern is observed (increasing cytotoxicity at increasing time) and the complex shows important antiproliferative response next to 70% during the last two days of treatment (curve b). Fig. 8 shows the antiproliferative behaviour of complex against the colon adenocarcinoma cell line HT-29. As can be seen, the cell growth observed in this culture (control curve) is slightly slower than HeLa cell culture while the cytotoxic study indicates that ruthenium complex exhibits discrete cell inhibition activity at both doses assayed. The analysis of the antiproliferative curve at the low dose (25. 4 IuM, curve a) shows that ruthenium complex behaves in a similar way to that analyzed for HeLa cells (the inhibition remains constant after three days of treatment), but the antiproliferative activity decreased to about 20%, if compared to that observed at this dose for HeLa cells (27%). When the dose is increased (40.8 IuM, curve b), the observed cell inhibition after 72 h of treatment is enhanced to 25%. Interestingly, this inhibition also remains constant during the treatment. Fi. 8: Antiproliferative effects exerted by complex on the grow kinetics of the human colon adenocarcinoma cancer HT-29. Cells were exposed to several doses of the ruthenium complex for a total of five days, as indicated: control curve (untreated HT-29 cells); curves a and b correspond to the treatment of cells at same doses as Fig. 6.

()./'the Complex
The in vivo antitumour activity of complex against EAT and P388 tumours was evaluated at several treatment doses in the range 20--240 mg/kg body weight. The therapeutic activity of the complex was obtained from the T/C percentage which is described as T/C% (100) x mean life span of treated mice/mean life span of untreated mice; turnout free survivors were excluded. The minimum value of T/C for moderate activity is 120 (EAT and P388 tumours); if T/C > 125 the complex is considered a candidate for further antitumour assays. T/C values required for activity in MX-1 xenograft tumour must be lower than 20 (T/C <20).
For EAT, a T/C (%) of 350 was obtained (25 mg/kg) and the complete remission of the turnout observed at a dose of 50 mg/kg. The toxic .dose (LDs0) was equal to 100 mg/kg. In the case of the lymphocytic leukemia P388, a T/C(%) value of 140 was obtained (60-120 mg/kg) while in MX-I xenograft carcinoma, this parameter was equal to 16 at a dose of 240 mg/kg. These results evidence a notable and specific antiproliferative effect of complex [Ru(HzL)CI2]'2H20 against the human tumour cell lines ADLD and HeLa, with selective and important cytotoxic properties in both cases. The in vivo antitumour activity is also remarkable in the studied turnouts. A further investigation of the antineoplastic activity of this new potential ruthenium drug against other types of turnout is advisable.