Synthesis, structure, properties and biological behaviour of the complex [RuIV (H2L) CI2] 2H2O (H4L- 1,2-cyclohexanediamminetetraacetic acid)

Synthesis, Structure, Properties And Biological Behaviour Of The Complex [Ru iv (HL) CI] 2HO (H4L1,2-Cyclohexanediamminetetraacetic Acid) Rosario A. Vilaplana," A. Castifleiras b and Francisco Gonzb.lez-Vilchez aDepartamento de Quimica Inorgdnica, Secci6n de Quimica Bioinorgdnica, Facultad de Quimica, Aptdo. Correos 553, E-41071 Sevilla, Spain. hDepartamento de Quhnica Inorgdnica, Facultad de Farmacia, Universidad de Santiago de Compostela, E15 782 Santiago de Compostela, Spain GRAPHICAL ABSTRACT The octahedral complex [Ru(Hzcdta)CI2]’2H20 contains Ru(IV) surrounded by two nitrogen atoms, N(I) and N(2), and two chloride ions c/s to each other, all in the equatorial plane. Two oxygen atoms, O(11) and O(21) are located in trans axial octahedral positions. The complex shows remarkable in vitro and in vivo activity against different carcinomas. Cll OI 1 to whom correspondence should be addressed; phone: +34 95 4557159; fax: +34 95 4557081; e-mail: [email protected] 275 Vol. 2, Nos. 3-4, 2004 Synthesis, Structure, Properties and Biological Behaviour Of the Complex ABSTRACT The highly water-soluble ruthenium complex [Ru(H2L)CI2]’2H20, 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)CIz].2HzO 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 against the human adenocarcinoma ADLD, indicates that [Ru(HL)CI].2HzO 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(N H3)2C12], carboplatin [cis-diammineI, -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 276 Rosario A. Vilaplana et al. Bio&organic Chem&try and Applications 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)/ have been reported /37,38/. Research on the formation constants and dissociation kinetic of cdta/lanthanide(lll)/ complexes /39,40/ as well as solution studies and determination of the crystal structures of cdta complexes formed with the trivalent ions of AI, Ga, In and Sc /41/have been also published. The research developed so far on platinum-group metal complexes formed with cdta concerns first the the X-ray crystallographic study of the diprotonated ligand and its complexes with [PdCI4] 2and [PtCI4] 2 salts /42a/. Additionally, the reaction kinetic processes involved in the fo rmation of Pd(ll) and Pt(ll) complexes of different composition with amminepolycarboxylate ligands (i.e., {(H6L)[Pd/PtCI4]} or [Pd(H2L)]) were also studied/42b/. Dinitrogen complexes formed between Ru(ll) and cdta were isolated and characterized/42c/. On the other hand, promising results were obtained in the first study developed on the antitumour activity of new Pd(ll)(cdta) complexes/42d/. Different Ru(lll) complexes such as [Ru(H4L)CI] 3-n and [Ru(HL)Ci]3"-I), were synthesized and characterized by analytical and electrochemical techniques/43/. Finally, reduction of molecular nitrogen to ammonia in aqueous solution under ambient conditions occurs in the presence of an illuminated RuO2/Pt/CdS system; this reaction is catalysed by several Ru(ll) complexes as, for example, [Ru(cdta)N] 2-/44/. 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 277 Vol. 2, Nos. 3-4, 2004 Synthesis, Structure, Properties and Biological Behaviour 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 cm - range, 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 278 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 Table presents a summary of the relevant crystal data and refinement results for complex I. The 279 Vol. 2, Nos. 3-4, 2004 Synthesis, Structure, Properties and Biological Behaviour Of the Complex Table 1 Crystal data and structure refinement for [Ru(H2cdta)Cl2].2H20 Identificat ion code Empirical formula Formula weight Temperature Waelengt h Crystal system, space group Unit cell dimensions Volume Z, Calculated density bsorpt ion coefficient F (000) Crystal size T..heta range for data collection Limiting indices Reflections collected / unique Completeness to theta 27.44 ?cbsorpt ion correction Max.. and rain. transmission Refinement method Data / restraints / parameters Goodness-of-fit on Final R indices [I>2sigma(1)] R ndices (all data) _bsolute structure parameter Largest diff. peak and hole rudcta Cl4 H24 C12 .|2 O10 Ru 552.32 293 (2) K 0.71073 A Hexagonal, P6(5) (No. 170) a 13. 567 (2) A b 13. 567 (2) A c 22. 286(6) A 3552.5(12) A^3 alpha 90 deg. beta 90 deg. gamma 120 deg. 6, i. 549 .Mg/m" 3 0. 935 mm"-I 1680 0.40 x 0.34 x 0.20 mm 1.73 to 27.44 deg. -14<=h<=0, 0<=k<=17, 0<=!<=28 3123 / 2779 [R(int) 0.0103] i00.0 % Psi .-scans 0.8350 and 0.7061 Full-matrix least-squares on 2779 / ! / 271 0.943 R1 0.0498, wR2 0.0518 R1 0.0498, wR2 0.0518 0.05(6) 0.515 and -0.380 e.A-3 molecular structure obtained by X-ray diffraction analysis is shown in Fig. along with the numbering of atoms in the molecule. The tetradentate chelating ligand (cdta) contains two free carboxylic groups and is bonded to the central ion by two nitrogen atoms, N(l) and N(2), and two chloride anions cis to each other, sharing the equatorial plane with the nitrogen atoms. The remaining coordination positions are occupied by two oxygen atoms, O(11) and O(21), from coordinated carboxylate groups, situated in trans axial octahedral positions that complete in this way the octahedral coordination sphere around ruthenium atom. 280 Rosario A. Vilaplana et al. .Bioinorganic Chemistry and Applications CI1 O12 O11 Rul Cl2 N2 O 22, N1 Fig. 1" O14 0,13 View of the molecular structure of complex showing the octahedral environment of the ruthenium central ion. Table 2 presents selected bonds lengths and angles for complex 1. As expected, the chloride coordinated ions are located much further from the ruthenium atom (2.37 A) than the two nitrogen atoms (2.11 / and 2.13 A). A possible explanation is related to the notable nucleophilic character of the chelating ligand that induces a notable increasing of the distance Ru-CI. Furthermore, the bulky chelating ligand might be the origin of the slight reduction observed in the CI-Ru-CI angle value (91.4), in comparison with the same angle in cisplatin (91.9). These two effects have also been found in similar complexes with other aminopolycarboxylic ligands, i.e., Ru(edta)CI2 [22] and Ru(pdta)Cb, [23]. Clearly, the distance Ru-O(I 1) is shorter (2.03 /) than the distance Ru-O(21) (2.07 /). As a consequence, the octahedral configuration around ruthenium atom is slightly distorted (Fig. I) with angles 281 Vol. 2, Nos. 3-4, 2004 Synthesis, Structure, Properties and Biological Behaviour Of the Complex Table 2 Selected bond lengths [A] and angles [deg] for [Ru(H2cdta)Cl2].2H20 Ru (I) -O(II) 2. 028 (3) Ru (i) -O (21) 2.070 (3) Ru (i) -I., (I) 2.108 (2) Ru (i) -l.I (2) 2. 128 (2) Ru (!) -CI (2) 2.3658(9) Ru (I) -el (I) 2.3723(8) 0 (ll)-Ru (I)-O (21) o (11) -Ru (I) -t, (i) o (21) -tu ([) -u O (II) -Ru (l) -I.-| (’2) 0 (21 -Ru ! -n (2) I- (i) -Ru (i) -l,I (2) o (I l -Ru (I) -cl. (2) O (2.) -Ru (I) -CI {2) I.i (I) -Ru (I) -el (2) I.I (2) -Ru (I) -CI (2) O (ll)-Ru (1)-Cl (i) O(21)-Ru (i) -CI (i) I..I ()-Ru ()-Cl () II(2)-Ru (1)-Cl (I) C1 (2) -Ru (1)-CI (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) 9.1.39(3) significantly reduced from ideal octahedral values, i.e., N(I)-Ru-N(2), 84.5; O(1 I)-Ru-N(I), 80.7 and 0(2 I)-Ru-N(2), 79.1 . 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 bonding that involves two pairs of oxygen atoms, O(13)...O(24), 2.65 /l and O(23)...O(14z), 2.67 A (symmetry transformations; 1: 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 {O(1)...O(213), 2.89/l and O(2)...O(113), 2.87 A (symmetry transformations; 3: y, -x+y, z+l/6)}. This Hbonded pattern can be described as a network of supramolecular helices. In the c direction the packing generates a sixfold screw axis and parallel channels (Fig. 3), with cavities of radius 2.15 A. These channels are hydrophilic because the oxygen atoms of carboxylic moieties and those of water molecules point towards the cavities. 282 Rosario A. Vilaplana et al. Bioinorganic Chemistr), and Applications Fig. 2: Packing drawing view of showing the assembling of molecules in a 3D-network by intennolecular hydrogen bonding involving two pairs of oxygen atoms. 283 VoL 2. Nos. 3-4, 2004 Synthesis, Structure, Properties and Biological Behaviour ()./’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. ACKNOWLEDGEMENTS We greatly appreciate financial support from MCYT, Spain (Grants PPQ2000-0035-P4 and BQU20012455) and EC (COST Chemistry Projects D20/005 and D20/009). The authors are grateful to Prof. A. Cervilla and V. Folgado, from Valencia University (Spain), for the help provided in the cyclovoltammetric study. REFERENCES 1. N. Farrell, in: Platinum-based Drugs’ in Cancer Chemotherapy, L.R. Kelland and N. Farrell (Eds.), Humana Press, Totowa, N.J., 2000; p. 321. 2. E.R. Jamieson and S.J. Lippard, Chem.. Rev., 99, 2467 (1999). 3. M.J. Bloemink and J. 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