Polyhedron 25 (2006) 33663378 www.elsevier.

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Studies of the interaction between bis(dithiocarbamato)copper(II) complexes with nitric oxide in aqueous solution and biological applications
Agostinho Cachapa a, Alfredo Mederos a,*, Pedro Gili a, Rita Hernandez-Molina a, a a b Sixto Domnguez , Erasmo Chinea , Matas Lopez Rodrguez , Marta Feliz c, c d Rosa Llusar , Felipe Brito , Carlos M. Ruiz de Galarreta e, Carlos Tarbraue e, German Gallardo e
a

Departamento de Qumica Inorganica, Facultad de Farmacia, Universidad de La Laguna, 38200 La Laguna, Tenerife, Canary Islands, Spain b Instituto Universitario de Bioorganica A. Gonzalez, Universidad de La Laguna, Tenerife, Canary Islands, Spain c Departamento de Ciencias Experimentales, Universitat Jaime I, Castellon, Spain d Centro de Equilibrios en Solucion, Escuela de Qumica, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela e Departamento de Bioqumica, Biologa Molecular y Fisiologa, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Gran Canaria, Canary Islands, Spain Received 27 February 2006; accepted 12 June 2006 Available online 16 June 2006

Abstract Three new bis(dithiocarbamato)copper(II) complexes have been prepared and characterized by elemental analysis, UVVis, and IR spectroscopy: bis(4-piperidonedithiocarbamato)copper(II), [Cu(Pdtc)2]2 (1), bis(piperidinedithiocarbamato)copper(II), [Cu(Ppdtc)2]2 (2), and bis[(2-piperidinecarboxy)dithiocarbamato]copper(II), Cu(Ppidtc)2 (3). The crystal structures of the complexes 1 and 2 have been determined using X-ray diraction methods and they are dimeric. The coordination sphere of copper(II) ions is described as a distorted square-pyramid: the axial CuS bond distances 2.7 and 2.8 A for 1 and 2, respectively, are longer than the equatorial CuS ones (2.3 A). Complexes 1 and 2 are not soluble in water. In contrast, 3 is water soluble. The interaction of 3, the previously prepared bis[N-(dithiocarboxy)sarcosine]copper(II), Cu(Sdtc)2 (4), and bis[(dicarboxymethyl)dithiocarbamato]copper(II), Cu(Idadtc)2 (5), with nitric oxide in aqueous solution at pH 7.4 and 20 C was spectrophotometrically studied and the experimental data were analysed by means of the SPEFO-LETAGROP and HYPERQUAD programs, respectively. The stability constants of the complexes CuL2(NO) and CuL2(NO)2 [L = dithiocarbamate] were determined. The interaction of 1 and 2 with nitric oxide was theoretically studied by molecular mechanics methods. Complexes 3, 4 and 5 and the previously studied bis[(dihydroxyethyl)dithiocarbamato]copper(II), Cu(Deadtc)2 (6), decrease the NO produced in vitro either by sodium nitroprusside (NPS) or by cultured murine macrophages J774 stimulated with lipopolysaccharide (LPS) and interferon-c (IFNc). Inducible nitric oxide synthase (iNOS) activity was determined by measuring the stable NO end product, nitrite, using a colorimetric assay. 2006 Elsevier Ltd. All rights reserved.
Keywords: Copper(II) dithiocarbamates; Nitric oxide; Stability constants; Speciation; Murine macrophages

1. Introduction Nitric oxide is the simplest thermally stable free radical molecule known [1,2]. This molecule is synthesized in vivo in mammalian cells and it acts as a short-lived intercellular messenger in important physiological processes [13]. In

Corresponding author. Tel.: +34 922318457; fax: +34 922318461. E-mail address: amederos@ull.es (A. Mederos).

0277-5387/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2006.06.008

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addition to regulate smooth muscle relaxation, neurotransmission, and immune response alterations in NO production and metabolism. The NO participates in multiple pathological circumstances such as hypertension, cardiomyopathy, pulmonary disorders, arteriosclerosis and endotoxemia [46]. Three dierent heme-containing NO-synthases (NOS) encoded by dierent genes mediate a common reaction, which converts L-arginine and molecular oxygen to citrulline and NO. Nevertheless, while two NOS proteins are constitutive enzymes rst characterized in neurons (nNOS or Type I NOS) and vascular endothelium (eNOS or Type III NOS) in macrophages and other cell types the NO generation occurs through a normally absent inducible (iNOS or Type II NOS) isoform [46]. Under normal physiological conditions the widely distributed nNOS and eNOS isoforms release low levels of NO required for neural transmission, regulation of the vascular tone and normal tissue homeostasis. In contrast, the expression of the iNOS enzyme in macrophages and other cell types exposed to bacterial lipopolysaccharide (LPS), interferon-c (IFNc) and other agents generates large and sustained quantities of NO, a hallmark of such critical situations as septic shock [7], renal damage, adult respiratory distress syndrome, diabetes, neurodegeneration and chronic inammatory disease [5]. In this context while NO donor drugs are currently used to compensate the reduced L-arginineNO pathway of patients suering from hypotension and angina, two dierent therapeutical strategies have been used to counteract the damaging eect of NO overproduction [7,8]. While the rst approach is based on aminoacid and non-amino acid NOS inhibitors with pharmacologically useful isoform selectivity (particularly for iNOS), the second alternative method involves the use of other molecules endowed with the ability to scavenge or remove the excess of NO generated during the septic shock or chronic inammatory diseases [7,8]. Despite the fact that some selective NOS inhibitors are useful in animal models of septic shock, treatment with these agents is frequently accompanied by adverse side eects such as pulmonary hypertension and mortality [8]. Therefore the search for NO scavenger molecules with demonstrated pharmacological activity represents a true alternative to the use of NOS inhibitors in a broad spectrum of disease models [46]. Among these scavenger molecules are some metal-dithiocarbamates. Recent reviews have focused on novel aspects of the chemistry of dithiocarbamates including topics as diverse as electrochemistry, photoelectron spectroscopy and analytical applications (e.g., for the determination of metals in foodstu, water and environmental samples) and highlighted the potential value of these compounds as useful NO trapping agents [9]. In this respect kinetic studies of the interaction of iron dithiocarbamate complexes with NO have been reported and a mechanism for the reaction proposed [1013]. Nevertheless, the studies of thermodynamic equilibria between iron dithiocarbamate complexes with NO in aqueous solution are still scarce.

Reported data [9,14,15] have shown the stability of the bonds between copper(II) dithiocarbamates and nitric oxide. We have carried out for rst time the thermodynamic studies in aqueous solution [16,17]. In a previous work [16], we have published the rst report on the stable coordination in aqueous solution of NO to copper(II) bis(dithiocarbamates). Coordinated NO in complexes such as CuL2(NO) and CuL2(NO)2 is not removed by purging the solution with Ar or air. Only when the solution containing both complexes is heated up to 333 K, the CuL2(NO)2 complex is dissociated to CuL2(NO), which is stable. NO coordinates instantaneously to copper(II) in CuL2(NO) and then a slow electron transfer of the unpaired electron of the NO reduces the Cu(II) to Cu(I). The thermodynamic stability of the complexes CuL2(NO) and CuL2(NO)2 (L = (dihydroxyethyl)dithiocarbamate), in aqueous solution was conrmed in the following work [17]. In this last work [17], the theoretical studies by means of SPARTAN02 programs were carried out also to take into account the possibility of the direct attack of NO on S atom of the dithiocarbamate. However, the isomer with SNO bond was found to be by 12.29 kcal/mol less exotermic than the CuNO bonded isomer in complex CuL2(NO) thus conrming the experimental data in the rst work [16]. This paper deals with the thermodynamic study of copper(II) dithiocarbamates complexes with NO. These studies allowed us to determine the stability constants of the complex species presented in our system. From the speciation diagrams we can deduce that copper(II) dithiocarbamates is more eective to trap NO. In this work, we have prepared new copper(II) dithiocarbamates: bis(4-piperidonedithiocarbamato)copper(II) [Cu(Pdtc)2]2 (1), bis(piperidinedithiocarbamato)copper(II) [Cu(Ppdtc)2]2 (2) and bis[(2-piperidinecarboxy)dithiocarbamato]copper(II) Cu(Ppidtc)2 (3), which have been characterized. The X-ray structure of 1 and 2 were determined. Complexes 1 and 2 are not soluble in water so their interaction with nitric oxide has been carried by theoretical methods. Complex 3 is soluble in water, which allowed us to study their interaction with nitric oxide in aqueous solution and analyse the chemical speciation of the complexes formed. Furthermore, in this work we have also studied spectrophotometrically the interaction of the previously prepared but not characterized, bis[N-(dithiocarboxy)sarcosine]copper(II), Cu(Sdtc)2 (4) [18] and bis[(dicarboxymethyl)dithiocarbamato]copper(II), Cu(Idadtc)2 (5) [19] with nitric oxide in aqueous solution. The analyses of the experimental data were carried out by means of the LETAGROP-SPEFO and HYPERQUAD programs [2022] and the stability constants of the complexes CuL2NO and CuL2(NO)2 were determined. In addition and based on the above-mentioned results, we have also carried out studies on the biological eects of these dithiocarbamates. We report herein the ability of 3, 4, 5 and the previously studied [17] bis[(dihydroxyethyl)dithiocarbamato]copper(II), Cu(Deadtc)2 (6) to trap NO released in vitro either by the model donor agent

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sodium nitroprusside (NPS) or produced by murine macrophages J774 cells activated by the simultaneous treatment with lipopolysaccharide(LPS) and interferon IFNc. 2. Experimental 2.1. General remarks Elemental analyses for carbon, hydrogen, nitrogen and sulfur were performed on Fisons EA 1108 CHNS-O instrument. The IR spectra were recorded in the 4000450 cm1 region using KBr Pellets on Bruker IF S55 spectrophotometer. The electronic spectra were recorded on a Cary 50 Varian and Shimadzu UV-2101PC spectrophotometer (spectrophotometric studies). The electrospray ionization (ESI) mass spectral measurement of the complex was carried out on a Micromass QUATTRO LC spectrometer. A solution of the complex (0.606 mM) was prepared in dimethylsulfoxideacetonitrile 50:50 v/v. The FAB-MS were recorded using VGAutoSpec Micromass spectrophotometer. Magnetic susceptibility measurements on polycrystalline samples were carried out in the temperature range 5300 K with Quantum Design SQUID magnetometer. Diamagnetic corrections of the constituent atoms were estimated from Pascals constants. Experimental susceptibilities were also corrected for the temperature-independent paramagnetism and the magnetization of the sample holder. 2.2. Materials 4-Piperidone monohydrate hydrochloride, piperidine, 2piperidinecarboxylic acid, sarcosine, iminodiacetic acid, N,N-dihydroxyethylamine, carbon disulde and copper metal were purchased from SigmaAldrich. Cupric acetate monohydrate was purchased from Fluka. Cupric chloride dehydrate, ammonia solution, nitric acid and sodium hydroxide were obtained from Merck. Potassium hydroxide was obtained from Panreac. For the preparation of all aqueous solution bidistilled water was used. Lipopolysaccharide (LPS) was obtained from Boehringer. The NO donnor sodium nitroprusside (NPS), Interferon c (IFNc), L-glutamine, sodium nitrite, sulfanilamide and N-(1-naphtyl)ethylendiamine were purchased from SigmaAldrich (Barcelona, Spain). Fetal bovine serum (FBS), Dulbecos modied Eagles medium (DMEM), antibiotics (penicillin, streptomycin), phosphate-buered saline (PBS) and other tissue culture reagents were obtained from Gibco (Grand Island, NY). 2.3. Preparation of the bis(dithiocarbamato)copper(II) complexes 2.3.1. [Cu(Pdtc)2]2 (1) An ammonia solution (20%, 4 ml, 0.04 mol) of 4-piperidone monohydrate hydrochloride (3 g, 0.02 mol) was prepared. To this solution cooled down to 0 C, 3 ml

(0.05 mol) of carbon disulde dissolves in 10 ml of ethanol was added dropwise under stirring. Then, an aqueous solution of 7 g of Cu(NO3)2 3H2O (0.03 mol) was added and a brown precipitate appeared. After collection by ltration, the precipitate was washed with water and dried by repeater washing with ether. The complex was crystallized by slow evaporation at room temperature from acetonitrile as solvent. Yield: 60%. 2.3.2. [Cu(Ppdtc)2]2 (2) To an aqueous solution of piperidine (7.3 mmol), potassium hydroxide (0.8 g) and carbon disulde (0.4 ml) were added. A yellow solution appeared to which an aqueous solution of cupric acetate monohydrate was added. The resulting brown precipitate was ltered o and washed with water and dried with ether. Single crystals were obtained by slow evaporation at room temperature using a mixture of ethanol and ether (50:50) as solvent. Yield: 54%. 2.3.3. Cu(Ppidtc)2, probably [Cu(Ppidtc)2]2 (3) In an aqueous solution of 6.46 g (0.05 mol) of 2-piperidinecarboxylic acid and 5.6 g (0.1 mol) of potassium hydroxide (1:2), 3 ml (0.05 mol) of CS2 was added dropwise. To this solution an aqueous solution of 6.25 g (0.025 mol) of CuSO4 5H2O (2:1) was added dropwise and a brown precipitate appeared. The resulting brown precipitate was ltered o and washed with water and dried in a dessicator. Yield: 60%. Elemental analyses for [Cu(Pdtc)2]2, [Cu(Ppdtc)2] and Cu(Ppidtc)2 complexes are presented in Table 1. The compound Cu(Sdtc)2 was reported by Sakay [18], Cu(Idadtc)2 by Hulanicki and Minczewska [19] and Cu(Deadtc)2 was synthesized according to Radha et al. [23]. 2.4. X-ray structure determination X-ray data for single crystal of 1 and 2 complexes were collected with a Bruker Smart CCD and Bruker Nonius KAPPA CCD diractometers, respectively, using mono chromatic Mo Ka radiation (k = 0.71073 A). For 1, the data were processed with SAINT [24] and corrected for absorption using SADABS [25]. The structure was solved by direct methods and rened by full-matrix least-squares techniques against F2 using SHELXTL-NT 5.10 [26]. Data

Table 1 Elemental analyses of the copper(II) complexes Complex Elemental analysis: found (calculated %) C [Cu(Pdtc)2]2 [Cu(ppdtc)2]2 Cu(Ppidtc)2 34.99 (34.99) 37.62 (37.55) 40.24 (40.88) H 3.89 (3.89) 5.33 (5.22) 4.62 (4.90) N 6.80 (7.24) 7.75 (7.30) 5.94 (5.75) S 31.11 (30.11) 33.83 (33.37) 27.15 (27.37)

[Cu(Pdtc)2]2 = C24H32O4N4S8Cu2. [Cu(ppdtc)2]2 = C24H40N4S8Cu2. Cu(Ppidtc)2 = C14H20O4N2S4Cu.

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reduction and cell parameters renements for 2 were carried out with the programs COLLECT [27] and DENSO [28]. The structure was solved using SIR-97 [29] and rened with SHELXL-97 [30] using full-matrix least-squares with anisotropic thermal parameters for all non-H atoms. Hydrogen atoms were placed at idealized positions. The PLATON [31] package of programs was used for absorption corrections (MultiScan option) and for molecular graphics generation. Both crystals 1 and 2 are laminar dark brown. For 1 and 2, a summary of the key crystallographic information is given in Table 2. For 1, selected bond distances and angles are listed in Table 3. For 2, due to the high value of R, the bond distances and angles are shown in Table S1. 2.5. Molecular modeling In order to obtain the most stable conformation, calculations of molecular modeling for dierent compounds were carried out using molecular mechanics force eld MM+, which is an extension of MM2 developed by Allinger and Burker [32]. The geometries were optimized using the UFF force eld [33]. Both MM+ and UFF provide qualitative accounts of molecular geometry conformation

Table 3 Selected bond distances (A) and bond angles () of [Cu(Pdtc)2]2 Bond lengths (A) Cu(1)S(4) Cu(1)S(3) Cu(1)S(5) Cu(1)S(2) Cu(1)S(2)#1 S(2)C(1) S(2)Cu(1)#1 S(3)C(7) S(4)C(1) S(5)C(7) N(2)C(7) N(2)C(8) N(2)C(9) N(1)C(1) N(1)C(2) N(1)C(3) Bond angles () S(4)Cu(1)S(3) S(4)Cu(1)S(5) S(3)Cu(1)S(5) S(4)Cu(1)S(2) S(3)Cu(1)S(2) S(5)Cu(1)S(2) S(4)Cu(1)S(2)#1 S(3)Cu(1)S(2)#1 S(5)Cu(1)S(2)#1 S(2)Cu(1)S(2)#1 C(1)S(2)Cu(1) C(1)S(2)Cu(1)#1 Cu(1)S(2)Cu(1)#1 C(7)S(3)Cu(1) C(1)S(4)Cu(1) C(7)S(5)Cu(1) C(7)N(2)C(8) C(7)N(2)C(9) C(8)N(2)C(9) N(2)C(7)S(5) N(2)C(7)S(3) S(5)C(7)S(3) N(2)C(9)C(10) N(2)C(8)C(11) C(1)N(1)C(2) C(1)N(1)C(3) C(2)N(1)C(3) N(1)C(1)S(4) N(1)C(1)S(2) S(4)C(1)S(2) N(1)C(3)C(4) N(1)C(2)C(6) 2.3067(9) 2.3080(8) 2.3352(8) 2.3732(9) 2.7036(10) 1.7347(17) 2.7036(10) 1.7177(18) 1.7108(18) 1.7168(18) 1.328(2) 1.463(2) 1.465(2) 1.327(2) 1.459(2) 1.464(2) 153.82(3) 99.51(2) 76.52(2) 76.260(19) 100.963(18) 165.37(2) 99.42(2) 106.72(3) 102.36(3) 92.20(2) 83.43(6) 102.04(6) 87.80(2) 85.01(6) 86.02(6) 84.18(6) 122.67(16) 123.62(15) 113.55(15) 123.48(13) 122.84(13) 113.68(10) 111.71(18) 109.45(18) 124.71(15) 122.53(15) 112.57(14) 123.27(13) 122.70(13) 114.02(9) 108.96(16) 109.94(16)

Table 2 Summary of the crystallographic results for the complexes 1 and 2 Complex Empirical formula Formula weight Temperature (K) Wavelength (A) Crystal system Space group a (A) b (A) c (A) a () b () c () V (A3) Z Dcalc (Mg m3) Absorption coecient (mm1) F(000) Crystal size (mm) h Range () Limiting indices [Cu(Pdtc)2]2 (1) C24H32Cu2N4O4S8 824.20 297(2) 0.71073 triclinic P 1 8.658(3) 10.319(3) 11.064(4) 62.811(1) 77.880(7) 65.953(7) 802.6(5) 1 1.705 1.884 422 0.36 0.27 0.26 2.0730.51 7 6 h 6 12, 14 6 k 6 14, 15 6 l 6 15 6532/4604 (0.0209) 3891 4606 and 191 0.0296, 0.0820, 1.02 0.01/0.00 0.54 and 1.03 [Cu(ppdtc)2]2 (2) C24H40Cu2N4S8 768.26 293(2) 0.71069 triclinic P 1 9.036(5) 9.973(5) 13.465(5) 91.630(5) 109.210(5) 114.340(9) 1024.8(9) 1 1.244 1.461 398 0.15 0.15 0.20 5.0026.12 9 6 h 6 11, 12 6 k 6 12, 16 6 k 6 15 8106/3913 (0.108) 1672 3840 and 172 0.1012, 0.2808, 0.85 0.77/0.12 0.35 and 0.46

Reections collected/ unique (Rint) Observed data [I > 2.0r(I)] Number of reections and parameters R, wR2, S Maximum and average shift/error Minimum and maximum residual density (e A3)

and strain energy. The MM+ calculations were made with the HYPERCHEM program [34] and conformations of all compounds were generated using molecular dynamics for each one with the following options: starting 0 K, simulation temperature 300 K, heat time 0.1 ps, run time 1 ps. These conformations are optimized using a FletcherReeves (conjugate gradient) protocol with the standard tight converge criterion (RMS gradient = 0.01 kcal A1 in vacuo). Molecular mechanics calculations were made also using the UFF force eld implemented in GAUSSIAN-03 W package [35].

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2.6. Equilibrium studies by electronic spectroscopy The electronic spectra were recorded on a Shimadzu UV-2101 PC spectrophotometer. A solution of copper(II) dithiocarbamate complexes and NO saturated aqueous solution were prepared by generating the gas from the reaction of metallic copper with 5 M nitric acid [2] using Schlenk techniques: 8HNO3 + 3Cu ! 3Cu(NO3 )2 + 4H2 O + 2NO The gas was puried by passing it through ve traps containing 50% NaOH solutions. The puried gas was bubbled in a Schlenk ask containing 20 ml of cold water. A thin Teon hose was connected into the NO aqueous solution through the side arm of the ask. Teon stopcocks were used to isolate each part of the Schlenk system when necessary. The whole system, tightly isolated from air, was previously deoxygenated by passing pure Ar for approximately 1 h [16,17]. Once the NO aqueous solution was obtained, the Schlenk ask was disconnected and then installed in a thermostated bath at 20 C and the Teon hose was connected to an Orion automatic microburet and purged with pure Ar. The concentrations of NO used for the calculations were made considering the reported concentration for saturated aqueous solution of NO (2.057 mM) at 20 C [36]. The solutions of the bis(dithiocarbamato)copper(II) complexes 3, 4 and 5, respectively, were prepared in a 7.4 phosphate buer solution (0.06 M). A number of 610 ll aliquots of the NO saturated aqueous solution thermostated at 20 C were administered to the air tight ask containing the Cu(II) complex solution (3, 4 and 5, respectively) using an Orion automatic microburette. The temperature of the cell was held at 20 C with a Peltier controller system attached to the spectrophotometer. The same apparatus was used to administer the studied solution to the optical cell. For a solution of Cu(Ppidtc)2 (3) (48.5 lM) the electronic spectral data (19 absorbance values for each addition of NO solution within the range 420476 nm at 20 C) were processed using HYPERQUAD software [22]. For a solution of Cu(Sdtc)2 (4) (76 lM) the electronic spectral data (16th absorbance values for each addition of NO solution within the range 348436 nm at 20 C) were processed using SPEFO version [20] of LETAGROP software [21]. For a solution of Cu(Idadtc)2 (5) (52.1 lM) the electronic spectral data (33 absorbance values for each addition of NO solution within the range 348436 nm at 20 C) were processed using HYPERQUAD software [22]. 2.7. Eect of dithiocarbamates on NO release in cell-free experiments Stock solutions (30 mM) of copper(II) dithiocarbamates 3, 4, 5 and 6, respectively, were prepared in PBS (pH 7.4) and sterilized using 0.2 lm Millipore lters. Sterile solutions of the NO donor SNP were prepared in DMEM (nal concentration 30 mM) just before use. Aliquots of the SNP

stock solution were dispensed into 96 well tissue culture plates containing either DMEM alone (to yield nal concentrations of the donor in the range of 30100 lM) or medium supplemented with copper(II) dithiocarbamates at nal concentrations 10 lM. Culture plates containing the appropriate concentrations of SNP alone or in combination with copper(II) dithiocarbamates were placed (24 h) in a Haereus tissue culture incubator (5% CO2 and 37 C) and thereafter media were aspirated and used for nitrite determination (see below). 2.8. Eect of copper(II) dithiocarbamates on NO released by J774 murine macrophages stimulated with LPS and IFNc The J774 murine macrophage cell line characterized by the ability to express iNOS and generate large quantities of NO in response to the simultaneous stimulation with LPS and IFNc [7,8,37,38] was donated by Dr. M. Fresno (CBM, Universidad Autonoma de Madrid). Cells were grown in 100 mm Falcons tissue culture plates containing DMEM/10% fetal bovine serum (FBS) supplemented with L-glutamine (2 mM) and antibiotics (100 U/ml penicillin and 100 lg/ml streptomycin) and maintained at 37 C in a 5% CO2 incubator (Haerus). Cells from conuent cultures (6575%) were detached form the plates, resuspended in serum depleted medium (DMEM/2% FBS) and aliquots (200 ll) containing equal number of cells ($25 104 cells) inoculated into 96-well tissue culture plates (Falcon). Cells were left untreated (controls) or stimulated (24 h) with a combination of LPS and IFNc (nal concentration 1 lg/ml and 100 U/ml, respectively) alone or in the presence of the indicated concentrations of the dierent copper(II) dithiocarbamates. After this period of time media were aspirated and used to measure total nitrite levels (see below) and the lack of cell toxicity determined by previously described methods (Mosman) [39]. 2.9. Nitrite determinations The ability of copper(II) dithiocarbamates to trap NO generated by chemical donors (NPS) or cytomix (LPS + IFNc)-stimulated macrophages was evaluated by changes in the concentration of stable NO metabolites (nitrite + nitrate) in culture medium as determined by the Griess reaction [40]. Suitable aliquots (100 ll) of culture medium were mixed with an equal volume of Griess reagent (a 1:1 mixture of 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% H3PO4), incubated for 10 min at room temperature [40] and concentrations were determined at 550 nm in an EL-312 Microplate reader (Biotek Instruments, Winooski, VT). Sterile-ltered standard solutions of sodium nitrite (050 lM) were freshly prepared in culture medium and incubated in parallel with each experiment and comparable results were obtained using a nitrate reductase assay kit provided by Alexis Biochemicals (Laufelngen, Switzerland).

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2.10. Statistical methods Results for nitrite concentrations are expressed as the means SEM from triplicate or quadruplicate cultures, and each experiment was repeated at least three times. The minimal eective doses and ED50 were determined with a software program based on a four-parameter logistic equation as previously described [40]. Statistical dierences were examined using ANOVA and, as indicated, Students t-test for comparison of the means. P < 0.5 was considered signicant. 3. Results and discussion 3.1. Description of the structures of the complexes 1 and 2 The crystallographic data of the complexes 1 and 2 are given in Table 2. The structure of 1 is built up by centre-symmetric dimeric entities. Fig. 1 shows a perspective view of the dimeric unit showing the atomic numbering scheme of the complex. For this compound, the distances CuS and CuS 0 are 2.373 A and 2.704 A, respectively, in good agreement with the values found for the dimeric dithiocarbamates: [CuS2 CN(Et)2]2 [41] (CuS and CuS 0 : 2.327 A and 2.844 A, respectively) and [CuS2CN(CH2Et)2]2 [41] (CuS and Cu S 0 : 2.322 A and 2.740 A, respectively). The coordination sphere of copper(II) ions is described as a distorted square-pyramid. The basal coordination positions are occupied by four sulfur atoms belonging to two dithiocarbamate ligands.

Each bridging sulfur atom simultaneously occupies an equatorial coordination site on one copper(II) ion and an apical site on the other copper(II). The axial CuS bond distance (2.7036(10) A) is longer than the equatorial CuS ones. The CuS(2) bond length in [(2.373(10) A] are longer in the CuS equatorial bond lengths because S(2) is a bridging atom. The bond angles S(2)Cu(1)S(4), S(3)Cu(1)S(5), 76.26(2), 76.52(2), respectively, are nearly the same. The bridging network S(2)Cu(1)S(2)a is strictly planar owing to the inversion centre. These results are in good agreement with earlier determinations [4143]. The overall geometry of the complex 2 (Fig. 2) is similar to complex 1. However, because of the high values of R (R = 0.1012), the bond parameters (Table S1) are not accurate and should be treated with caution. 3.2. IR spectra The most signicant bands for the 1, 2 and 3 complexes are given in Table 4. The bands in the range 1480 1510 cm1 are attributed to the m(CN) (partial double bond) stretching vibration [17,41,44]. This position corresponds to a partial double bond character, and is recorded in the range previously reported for similar compounds [4547]. The band in the range 9401050 cm1, belonging to the m(SC) (partial double bond) stretching is considered indicative of dithiocarbamate acting as a bidentate ligand [44,48]. The band at 1588 cm1 corresponds to antisymmetric carboxyl stretching frequencies of the non-complexed ionic COO in the ligand of the complex 3 [49]. The band at 1716 cm1 in the complex 1 is assigned to

Fig. 1. Perspective view of 1 with thermal ellipsoids at the 50% probability level and showing the selective atom numbering scheme.

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Fig. 2. Perspective view of 2 with thermal ellipsoids at the 50% probability level and showing the selective atom numbering scheme. Table 4 Most signicant IR bands (cm1) of [Cu(Rdtc)2]2 Compound m(CN) m(CS) m(C@O) m(COO) m(OH)
a

[Cu(Pdtc)2]2 1508.84 s 1005.16 sa 1716.12 s

[Cu(ppdtc)2]2 1508.65 s 1000.9 sa

Cu(Ppidtc)2 1474 s 970 m 1588 s 3414 s

Singlet.

C@O stretching vibration of the no complexed ketonic group CH2COCH2 of the ligand [47,50]. 3.3. Electronic spectra The most signicant bands of the copper(II) complexes 1 and 3 are presented in Table 5. For complex 2 in DMSO, the values are similar to complex 1. The most intense band at 273 nm is probably a band of the ligand; the band at 438 nm is a r(S) ! Cu(II) ligand to metal charge transfer in a planar CuS4 (CuS5 in pyc geometry) chromophore, and the band at 630650 nm (shoulder) is a dd band [5154], in agreement with a planar CuS4 geometry (monomers) or pyc CuS5 geometry (dimers). 3.4. Magnetic properties The magnetic behaviour of complexes 1 and 3 is shown in Figs. S1 and S2 by means of a vMT versus T plot (vM being the molar magnetic susceptibility). At room temperature both compounds give a value for vMT that is slightly lower then expected for two non-interacting Cu(II) ions

(0.9 cm3 mol1 K), and upon cooling vMT continuously decreases also for both compounds. This behaviour is indicative of the occurrence of weak antiferromagnetic interactions in 1 and 3. The pattern of the curve for both compounds is very similar, thus although the structure of 3 is not known in solid state it must be very similar to that of 1. Other polymerizations also are possible: [Cu(Et2dtc)]4 is tetramer [55]. The structure of 1, Fig. 1, consist of dinuclear copper(II) entities bridged by l-S atoms. The antiferromagnetic coupling for these complexes is due to spin cancellation through the S bridge atoms [56]. However, we have not been able to t the magnetic data to the numerical expression for copper(II) dinuclear complexes. May be some paramagnetic impurities or intermolecular interactions are present in the polycrystalline samples which were measured, that make the data to deviate from the ideal model. 3.5. Mass spectrometry Complex 1 was further characterized by electrospray ionization (ESI) mass spectrometry. The ESI spectrum of the monomeric complex [Cu(Pdtc)2]2 obtained at DV = 20 V is presented in Fig. 3. The main peak observed at m/z 411.5 is corresponding to [Cu(Pdtc)2]+. The presence of three ions is not in good agreement with the isotropic distribution of the Cu: Cu63 (69.2%); Cu65 (30.8%) [57]. Probably the other two peaks can be assigned to protonated ions, Table 6. This result implies that the dimer in dimethylsulfoxide acetonitrile solution is broken showing only the monomeric complex. 3.6. Molecular modeling

Table 5 Electronic data of the complexes 1 and 3 Complex 1 3 k/nm (e/M1 cm1) 273 (50.000) 273 (505) 438 (24.450) 433 (184) 650 (2.400) 630 (13.4) Solvent DMSO water

In Ref. [17], we have studied the interaction between NO and the bis[1-(2-hydroxyethyl)piperazinedithiocarbamato]copper(II) by means of molecular mechanics and semiempirical methods.

A. Cachapa et al. / Polyhedron 25 (2006) 33663378

MS123 1 (0.526) Sm (Mn, 2x0.60) 10 0

3373

411.5

413.5

414.5 412.5

0 4 05

4 06

407

408

409

4 10

411

412

413

4 14

Fig. 3. ESI mass spectra of the monomeric complex of [Cu(Pdtc)2]2 obtained in 50:50 acetonitriledimethylsulfoxide; DV = 20 V.

Table 6 Principal ions in the ESI spectra of the monomeric complex of [Cu(Pdtc)2]2 Complex Cu(Pdtc)2 m/z 411.5 412.5 413.5 Relative abundance (%) 100 20 70 Assignment [Cu(Pdtc)2]+ [Cu(Pdtc)2 + H]2+ [Cu(Pdtc)2 + 2H]3+

Analogously, in this work we have studied in gaseous phase the interaction between NO and the monomeric complex Cu(Pdtc)2, M, and the dimeric complex [Cu(Pdtc)2]2, D. Figs. 4 and 5 show the most stable confor-

Fig. 4. Most stable conformations in gaseous phase for: (a) monomer M; (b) M(NO); and (c) M(NO)2.

Fig. 5. Most stable conformations in gaseous phase for: (a) dimeric D; (b) D(NO); and (c) D(NO)2.

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A. Cachapa et al. / Polyhedron 25 (2006) 33663378

0.8
1

mations in gaseous phase for the compounds M (4a), M(NO) (4b), M(NO)2 (4c), D (5a), D(NO) (5b) and D(NO)2 (5c), respectively, obtained by theoretical calculations. The most symmetrical conformations correspond to M(NO)2 and D(NO)2. It was found that in the dithiocarbamate complexes, the [CuS4] groups become more planar and they are coordinated to two molecules of nitric oxide. A possibility of direct NO attack on S atom of the dithiocarbamate was also taken into account. For the monomeric complex M, the isomer with SNO bond was found to be 12.29 kcal/mol less exothermic than the CuNO bond isomer M(NO). Also the complexes with SNO bond are more distorted than the complexes containing CuNO bond. It was found in all cases, both for monomer and dimer that the bond angle CuNO is 120, which indirectly shows that the charge transfer is metal to ligand which agree well with the previous experimental information [16].

0.6

Absorbance

R 1)1:0 2)1:1 3)1:2

0.4

0.2

0 300

350

400

450

500

wavelength (nm)

Fig. 6. UV spectra at 293 K for Cu(Sdtc)2 (76 lM) with dierent additions of NO saturated aqueous solution: (1) Cu(Sdtc)2:NO = 1:0. (2) Cu(Sdtc)2:NO = 1:1. (3) Cu(Sdtc)2:NO = 1:2.

3.7. Solution studies A Cu(Sdtc)2 (4) solution presents a maximum at 432 nm (log e = 3.92). With addition of a NO solution the maximum decreased in intensity and blue-shifted to 425 nm. At the same time a new band appears at 356 nm. Both bands correspond to LMCT transitions between the copper ion and dithiocarbamate and NO, respectively [16,51]. An isosbestic point is observed at 388 nm (Fig. 6), indicating that the reversible process is taking place. The blue shift variation of the absorbance from 432 nm (ratio Cu:NO = 1:0) to 425 nm (ratio Cu:NO = 1:2, curve 3, Fig. 6) and the red shift of the band at 356360 nm (curve 3, Fig. 6) give two inection points at 1:1 and 1:2 Cu:NO molar ratios, 388 and 390 nm, with formation of the complexes Cu(Sdtc)2(NO) and Cu(Sdtc)2(NO)2, respectively (Fig. 7). The experimental data were analysed by means of the SPEFO version [20] of the LETAGROP program [21]. The calculations indicate that 4 reacts stepwise with two moles of NO (Scheme 1, Table 7). The values of the stability constants and the species distribution diagram (Fig. 7) indicate that the complex corresponding to equilibrium (1) is thermodynamically more stable than the complex corresponding to equilibrium (2). The diagram also indicates that the complex 1:2 is formed from complex 1:1 (Fig. 7, Scheme 1 and Table 7). With excess of nitric oxide the complex Cu(Sdtc)2(NO)2 forms quantitatively (Fig. 7). The dithiocarbamate complex Cu(Sdtc)2 is a versatile agent to capture the NO under physiological conditions (pH 7.4). A Cu(Ppidtc)2 (3) solution presents a maximum at 432 nm (log e = 4.01). With addition of a NO solution the maximum decreased in intensity and it is blue-shifted. At the same time a new band appears at 356 nm. An isosbestic point is observed at %382 nm, indicating that a reversible process is taking place. The experimental data were analysed using the HYPERQUAD program [22]. The stability constants for Cu(Ppidtc)2NO and Cu(Ppidtc)2(NO)2 complexes

Fig. 7. Species distribution for 3NO system at pH 7.4 and 293 K. Concentration of complex 3: 76 lM.

Cu(Dtc)2 + Cu(Dtc)2NO

NO + NO

K1 K2

Cu(Dtc)2NO

Cu(Dtc)2(NO)2 B

Scheme 1. A: equilibrium (1); B: equilibrium (2).

Table 7 Stability constants of the Cu(Dtc)2NO and Cu(Dtc)2(NO)2 complexes Complex Cu(Ppidtc)2 Cu(Sdtc)2 Cu(Idadtc)2 Cu(Deadtc)2 log K1 3.16(1) 4.7(5) 8.5(1) 10.3(1) log K2 2.61(1) 3.9(6) 2.4(1) 2.2(1) Reference this work this work this work [17]

are given in Table 7. That values for log K1 indicate that the complex Cu(Ppidtc)2NO2 is weaker than the Cu(Sdtc)2NO complex. Complex 5 presents a maximum at 432 nm (log e = 4.09) which decreased in intensity with the addition of a NO solution and is blue-shifted (curve 3, Fig. 8). This decrease was accompanied by the simultaneous appearance of a new

A. Cachapa et al. / Polyhedron 25 (2006) 33663378

0.7 0.6 0.5 0.4

3375

1 2 3

A
0.3 0.2 0.1 0 300

350

400

450

500

wavelength (nm)

Fig. 8. Experimental UVVis spectrophotometric data for the Cu(Idadtc)2/NO system at pH 7.4 and 20 C. Concentration of the Cu(Idadtc)2 complex (5.21 lM) and dierent ratios R = Cu(Idadtc)2:NO (1), Cu(Idadtc)2:NO 1:0; (2), Cu(Idadtc)2:NO 1:1 (3); and Cu(Idadtc)2:NO 1:2.

Fig. 9. Species distribution diagram for Cu(Idadtc)2NO system at pH 7.4 and 20 C. Concentration of the Cu(Idadtc)2 complex: 5.21 lM.

band at 352 nm which corresponds to a charge transfer transition between the copper ion of the dithiocarbamate complex and NO [16]. An isosbestic point is observed at %383 nm, batochromically shifted, by successive additions of NO to the Cu(Idadtc)2 solution indicating that reversible

processes are taking place. The experimental data analysed using the HYPERQUAD program [22] indicated that 5 reacts stepwise with two moles of NO (Scheme 1 and Table 7). Calculation of the stability constants for Cu(Idadtc)2NO and Cu(Idadtc)2(NO)2 complexes allowed to obtain the values for log K1, complex 1:1, equilibrium A in Scheme

25

Cu(Ppidtc)2
S Cu S CN 2 HOOC

NPS

25
CH3 S

NPS

Cu(Sdtc) 2
COOH

20

20

Cu S

CN 2

[NO2-] (M)

[NO2 ] (M)

15 0.03 mM 10 0.01 mM 5

15 0.03 mM 10 0.01 mM 5

0.01

0.03

0.10

0.30

0.01

0.03

0.10

0.30

(a)

[NPS] (mM)

(c)

[NPS] (mM)

25
S CH2OH CN CH2OH

Cu(Deadtc)2

NPS

Cu(Idadtc) 2
25
S Cu S CN COOH 2

NPS
COOH

20

Cu

20

[NO2-] (M)

15
0.03 mM

[NO2 ] (M)

0.03 mM 15

10
0.01 mM

10

0.01 mM

0.01

0.03

0.10

0.30

0.01

0.03

0.10

0.30

(b)

[NPS] (mM)

(d)

[NPS] (mM)

Fig. 10. Eect of copper(II) dithiocarbamates (at nal concentrations of 10 and 30 lM) on nitrite levels present in culture media containing the NOdonnor NPS (nal concentration). Each data point represents nitrite concentrations (means SEM) three dierent experiments using triplicate or quadruplicate wells per data point.

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A. Cachapa et al. / Polyhedron 25 (2006) 33663378

1, and log K2, complex 1:2, equilibrium B in Scheme 1 indicating that the complex 1:1 is much stronger than the complex 1:2. These data, together with the species distribution diagram (Fig. 9) indicate a stepwise process in which formation of the 1:2 complex requires that the complex 1:1 is practically formed in accordance with previous studies of our laboratory [16,17]. Similar behaviour with NO has the Cu(Deadtc)2 complexes, with a higher value for log K1 [17] (Table 7). 3.8. The copper(II) dithiocarbamates reduce nitrite concentrations derived from the nitric oxide producing agent NPS Since the complexes 3, 4, 5 (this work) and 6 [17] trap NO in aqueous solution, in the next set of experiments we explored their ability to trap NO generated by increasing concentrations (10300 lM) of the specic NO-donor compound NPS (Fig. 10). Total nitrite levels were extre-

mely low in wells containing DMEM alone, augmented in a concentration-dependent manner in cultures supplemented with NPS and signicantly decreased in the presence of two dierent doses (10 lM or 30 lM) of the dierent copper(II) dithiocarbamates. 3.9. Eect of the copper(II) dithiocarbamates on the concentration of NO generated by cultured macrophages stimulated with LPS and IFNc Since the above-mentioned data demonstrate that copper(II) dithiocarbamate derivatives trap NO under cell-free conditions we performed additional experiments to test whether a similar activity was exerted in cytomix (LPS + IFNc)-stimulated murine macrophages (Fig. 11). As expected nitrite concentrations were negligible in unstimulated cells (see control in Fig. 11) or cultures exposed only to the highest dose (300 lM) of copper(II) dithiocarbamate derivatives. In contrast, activation of

80

80

60

60

[NO2 ] (M)

20

40

[NO2 ] (M)

40

20

0 CONTROL + C3 + LPS + IFN 30 100 300

0
CONTROL + C2 + LPS + IFN 30 100 300

(a)

[Cu(Ppidtc)2] (M)

(c)
80

[Cu(Sdtc)2] (M)

80
60

60

[NO2 ] (M)

[NO2 ] (M)

40

40

20

20

0 CONTROL + C4 + LPS + IFN 30 100 300

0 CONTROL + C1 + LPS + IFN 30 100 300

(b)

Cu(Deadtc)2 (M)

(d)

[Cu(Idadtc)2] (M)

Fig. 11. Eect of increasing concentrations of copper(II) dithiocarbamates on NO production in control or cytomix (LPS and IFNc) stimulated J774 murine macrophages. Macrophages were cultured (24 h) in the absence or presence of medium alone (controls), with citomix (LPS + IFNc) alone or in the presence of three dierent concentrations (30300 lM) of each Cu(II)dithiocarbamate. Each data point represents nitrite concentrations (means SEM) of three dierent experiments using triplicate or quadruplicate wells per data point. Additional groups of cells were treated with the highest dose (300 lM) of copper(II) dithiocarbamates alone.

A. Cachapa et al. / Polyhedron 25 (2006) 33663378

3377

macrophage iNOS by the simultaneous treatment (24 h) with cytomix (LPS and IFNc) dramatically increased NO2 concentration (up to 65 lM) being these reaction dose-dependently reduced by the addition of cultures signicantly reduced in the presence of graded doses of these copper(II) dithiocarbamates. When considered together the data derived from experiments using chemical donors (SNP) or cytomix (LPS + IFNc)-activated macrophages demonstrate that the dierent copper(II) dithiocarbamates studied are eective NO scavengers, which rapidly react with NO and prevent the formation of its stable oxidation product NO2 in aqueous solution. This results conrm previous studies with Cu(ProDTC)2 3H2O [16]. 4. Conclusions Single crystals of 1 and 2 were prepared for the rst time and characterized by X-ray diraction. They are dimeric. For complex 2, selected bond distance and bond angles are given in Supplementary material because of the high value of R (Table S1). Complex 3 was also prepared for the rst time. The stability constants and speciation diagrams in aqueous solution of the Cu(Deadtc)2NO [17] and Cu(Idadtc)2NO systems indicate that these copper(II) dithiocarbamates have a great ability to trap NO under in physiologic conditions. The formation of the complexes CuL2(NO) and CuL2(NO)2 is successive. The results for the Cu(Sdtc)NO and Cu(Ppidtc)2NO systems indicate that the formation of the complexes CuL2(NO) and CuL2(NO)2 is simultaneous and therefore this dithiocarbamate copper(II) complex is more versatile to uptake and release NO in physiological conditions. The species distribution diagrams in aqueous solution at physiological pH (7.4) also indicate that these copper(II) dithiocarbamates help to know the chemistry to regulate the NO in the tissues and to control its biological function. Since NO yields stable oxidation products (mainly NO2 and lower levels of NO3 ) in aqueous solution we also demonstrate that the copper(II) dithiocarbamates studied are able to decrease total nitrite concentrations produced by the model NO-donor NPS. Of further interest a similar decrease in the total nitrite concentrations was observed when the copper(II) dithiocarbamates were added to murine macrophages challenged with LPS and IFNc to induce iNOS expression. These data support the potential application of copper(II) dithiocarbamates as therapeutic agents in animal models of septic shock. Verication of this possibility is currently under investigation in our laboratories. 5. Abbreviations DMEM Dulbecos modied eagle medium IFNc interferon gamma

iNOS LPS NOS SNP PGE2 FBS

inducible nitric oxide sinthase lypopolyssacaride nitric oxide sinthase sodium nitroprusside prostaglandin type E2 fetal bovine serum

Acknowledgements Agostinho Francisco Cachapa is very grateful to the Agencia Espanola de Cooperacion Internacional (AECI) and University of La Laguna for a predoctoral fellowship at Departamento de Qumica Inorganica, Universidad de La Laguna, Spain. Also thanks are due to the Departamento de Ciencias Experimentales, Universitat Jaume I, Spain where he carried out some of the experimental work of the present paper. Financial support from MEC-Spain (research projects PM98-0148 and BQU2002-02794) is gratefully acknowledged. We thank the Servicio General de Medidas Magneticas of La Laguna University for the magnetic measurements. Appendix A. Supplementary material Supplementary crystallographic data for complex 1 have been deposited with the CCDC No. 256287. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, by e-mailing data_request@ccdc.cam. ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336 033). For complex 2, Table S1 and supplementary crystallographic data (Tables S2S10) are includes as Supplementary material in this paper. Figs. S1 and S2 corresponding to the magnetic behaviour of 1 and 3 complexes are also included as Supplementary material. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.poly.2006.06.008. References
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