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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,Sixto Domnguez a, Erasmo Chinea a, Matas Lopez Rodrguez b, Marta Feliz c,

Rosa Llusar c, Felipe Brito d, 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, Spainb Instituto Universitario de Bioorganica A. Gonzalez, Universidad de La Laguna, Tenerife, Canary Islands, Spainc 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, Venezuelae 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 2006Available 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 beendetermined using X-ray diffraction methods and they are dimeric. The coordination sphere of copper(II) ions is described as a distortedsquare-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-(dithio-carboxy)sarcosine]copper(II), Cu(Sdtc)2 (4), and bis[(dicarboxymethyl)dithiocarbamato]copper(II), Cu(Idadtc)2 (5), with nitric oxide inaqueous solution at pH 7.4 and 20 C was spectrophotometrically studied and the experimental data were analysed by means of theSPEFO-LETAGROP and HYPERQUAD programs, respectively. The stability constants of the complexes CuL2(NO) and CuL2(NO)2 [L = dithio-carbamate] 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 pro-duced 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 radicalmolecule known [1,2]. This molecule is synthesized in vivoin mammalian cells and it acts as a short-lived intercellularmessenger in important physiological processes [13]. In

0277-5387/$ - see front matter 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.poly.2006.06.008

* Corresponding author. Tel.: +34 922318457; fax: +34 922318461.E-mail address: [email protected] (A. Mederos).

www.elsevier.com/locate/poly

Polyhedron 25 (2006) 33663378
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addition to regulate smooth muscle relaxation, neurotrans-mission, and immune response alterations in NO produc-tion and metabolism. The NO participates in multiplepathological circumstances such as hypertension, cardiomy-opathy, pulmonary disorders, arteriosclerosis and endotox-emia [46]. Three different heme-containing NO-synthases

(NOS) encoded by different genes mediate a common reac-tion, which converts L-arginine and molecular oxygen to cit-rulline and NO. Nevertheless, while two NOS proteins areconstitutive enzymes first characterized in neurons (nNOSor Type I NOS) and vascular endothelium (eNOS or TypeIII NOS) in macrophages and other cell types the NO gen-eration occurs through a normally absent inducible (iNOSor Type II NOS) isoform [46]. Under normal physiologicalconditions the widely distributed nNOS and eNOS isoformsrelease low levels of NO required for neural transmission,regulation of the vascular tone and normal tissue homeosta-sis. In contrast, the expression of the iNOS enzyme in mac-rophages and other cell types exposed to bacterial

lipopolysaccharide (LPS), interferon-c (IFNc) and otheragents generates large and sustained quantities of NO, ahallmark of such critical situations as septic shock [7], renaldamage, adult respiratory distress syndrome, diabetes, neu-rodegeneration and chronic inflammatory disease [5]. Inthiscontext while NO donor drugs are currently used tocompensate the reduced L-arginineNO pathway of patientssuffering from hypotension and angina, two different thera-peutical strategies have been used to counteract the damag-ing effect of NO overproduction [7,8]. While the firstapproach is based on aminoacid and non-amino acidNOS inhibitors with pharmacologically useful isoform

selectivity (particularly for iNOS), the second alternativemethod involves the use of other molecules endowed withthe ability to scavenge or remove the excess of NO generatedduring the septic shock or chronic inflammatory diseases[7,8]. Despite the fact that some selective NOS inhibitorsare useful in animal models of septic shock, treatment withthese agents is frequently accompanied by adverse sideeffects such as pulmonary hypertension and mortality [8].Therefore the search for NO scavenger molecules withdemonstrated pharmacological activity represents a truealternative to the use of NOS inhibitors in a broad spectrumof disease models [46].

Among these scavenger molecules are some metal-dith-iocarbamates. Recent reviews have focused on novel aspectsof the chemistry of dithiocarbamates including topics asdiverse as electrochemistry, photoelectron spectroscopyand analytical applications (e.g., for the determination ofmetals in foodstuff, water and environmental samples)and highlighted the potential value of these compounds asuseful NO trapping agents [9].

In this respect kinetic studies of the interaction of irondithiocarbamate complexes with NO have been reportedand a mechanism for the reaction proposed [1013]. Never-theless, the studies of thermodynamic equilibria betweeniron dithiocarbamate complexes with NO in aqueous solu-

tion are still scarce.

Reported data [9,14,15] have shown the stability of thebonds between copper(II) dithiocarbamates and nitricoxide. We have carried out for first time the thermody-namic studies in aqueous solution [16,17]. In a previouswork [16], we have published the first report on the stablecoordination in aqueous solution of NO to copper(II)

bis(dithiocarbamates). Coordinated NO in complexes suchas CuL2(NO) and CuL2(NO)2 is not removed by purgingthe solution with Ar or air. Only when the solution con-taining both complexes is heated up to 333 K, theCuL2(NO)2 complex is dissociated to CuL2(NO), which isstable. NO coordinates instantaneously to copper(II) inCuL2(NO) and then a slow electron transfer of theunpaired 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 confirmed in the following work[17]. In this last work [17], the theoretical studies by meansof SPARTAN02 programs were carried out also to take into

account the possibility of the direct attack of NO on Satom of the dithiocarbamate. However, the isomer withSNO bond was found to be by 12.29 kcal/mol less exoter-mic than the CuNO bonded isomer in complex CuL2(NO)thus confirming the experimental data in the first work [16].

This paper deals with the thermodynamic study of cop-per(II) dithiocarbamates complexes with NO. These studiesallowed us to determine the stability constants of the com-plex species presented in our system. From the speciationdiagrams we can deduce that copper(II) dithiocarbamatesis more effective to trap NO.

In this work, we have prepared new copper(II) dithio-

carbamates: bis(4-piperidonedithiocarbamato)copper(II)[Cu(Pdtc)2]2 (1), bis(piperidinedithiocarbamato)copper(II)[Cu(Ppdtc)2]2 (2) and bis[(2-piperidinecarboxy)dithiocarb-amato]copper(II) Cu(Ppidtc)2 (3), which have been charac-terized. The X-ray structure of 1 and 2 were determined.Complexes 1 and 2 are not soluble in water so their inter-action with nitric oxide has been carried by theoreticalmethods. Complex 3 is soluble in water, which allowedus to study their interaction with nitric oxide in aqueoussolution and analyse the chemical speciation of the com-plexes formed.

Furthermore, in this work we have also studied spectro-photometrically the interaction of the previously preparedbut not characterized, bis[N-(dithiocarboxy)sarcosine]-copper(II), Cu(Sdtc)2 (4) [18] and bis[(dicarboxym-ethyl)dithiocarbamato]copper(II), Cu(Idadtc)2 (5) [19] withnitric oxide in aqueous solution. The analyses of the experi-mental data were carried out by means of the LETAGROP-SPEFOand HYPERQUAD programs [2022] and the stability constantsof 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 effectsof these dithiocarbamates. We report herein the ability of3, 4, 5 and the previously studied [17] bis[(dihydroxy-ethyl)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 macro-phages J774 cells activated by the simultaneous treatmentwith lipopolysaccharide(LPS) and interferon IFNc.

2. Experimental

2.1. General remarks

Elemental analyses for carbon, hydrogen, nitrogen andsulfur were performed on Fisons EA 1108 CHNS-O instru-ment. The IR spectra were recorded in the 4000450 cm1

region using KBr Pellets on Bruker IF S55 spectrophotome-ter.The electronic spectrawere recorded on a Cary50 Varianand Shimadzu UV-2101PC spectrophotometer (spectro-photometric studies). The electrospray ionization (ESI) massspectral measurement of the complex was carried out on aMicromass QUATTRO LC spectrometer. A solution ofthe complex (0.606 mM) was prepared in dimethylsulfox-ideacetonitrile 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 Kwith Quantum Design SQUID magnetometer. Diamag-netic corrections of the constituent atoms were estimatedfrom Pascals constants. Experimental susceptibilities werealso corrected for the temperature-independent paramag-netism and the magnetization of the sample holder.

2.2. Materials

4-Piperidone monohydrate hydrochloride, piperidine, 2-

piperidinecarboxylic acid, sarcosine, iminodiacetic acid,N,N-dihydroxyethylamine, carbon disulfide and coppermetal were purchased from SigmaAldrich. Cupric acetatemonohydrate was purchased from Fluka. Cupric chloridedehydrate, ammonia solution, nitric acid and sodiumhydroxide were obtained from Merck. Potassium hydrox-ide was obtained from Panreac.

For the preparation of all aqueous solution bidistilledwater was used.

Lipopolysaccharide (LPS) was obtained from Boehrin-ger. The NO donnor sodium nitroprusside (NPS), Inter-feron c (IFNc), L-glutamine, sodium nitrite, sulfanilamideand N-(1-naphtyl)ethylendiamine were purchased fromSigmaAldrich (Barcelona, Spain). Fetal bovine serum(FBS), Dulbecos modified Eagles medium (DMEM), anti-biotics (penicillin, streptomycin), phosphate-buffered saline(PBS) and other tissue culture reagents were obtained fromGibco (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-piperi-

done monohydrate hydrochloride (3 g, 0.02 mol) was pre-

pared. To this solution cooled down to 0 C, 3 ml

(0.05 mol) of carbon disulfide dissolves in 10 ml of ethanolwas added dropwise under stirring. Then, an aqueous solu-tion of 7 g of Cu(NO3)2 3H2O (0.03 mol) was added and abrown precipitate appeared. After collection by filtration,the precipitate was washed with water and dried by repea-ter washing with ether. The complex was crystallized by

slow evaporation at room temperature from acetonitrileas solvent. Yield: 60%.

2.3.2. [Cu(Ppdtc)2]2 (2)

To an aqueous solution of piperidine (7.3 mmol), potas-sium hydroxide (0.8 g) and carbon disulfide (0.4 ml) wereadded. A yellow solution appeared to which an aqueoussolution of cupric acetate monohydrate was added. Theresulting brown precipitate was filtered off and washed withwater and dried with ether. Single crystals were obtained byslow evaporation at room temperature using a mixture ofethanol 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-piperi-

dinecarboxylic acid and 5.6 g (0.1 mol) of potassiumhydroxide (1:2), 3 ml (0.05 mol) of CS2 was added drop-wise. To this solution an aqueous solution of 6.25 g(0.025 mol) of CuSO4 5H2O (2:1) was added dropwiseand a brown precipitate appeared. The resulting brownprecipitate was filtered off and washed with water and driedin a dessicator. Yield: 60%.

Elemental analyses for [Cu(Pdtc)2]2, [Cu(Ppdtc)2] andCu(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] andCu(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 werecollected with a Bruker Smart CCD and Bruker NoniusKAPPA CCD diffractometers, respectively, using mono-chromatic Mo Ka radiation (k = 0.71073 A). For 1, thedata were processed with SAINT [24] and corrected forabsorption using SADABS [25]. The structure was solved bydirect methods and refined by full-matrix least-squarestechniques against F2 using SHELXTL-NT 5.10 [26]. Data

Table 1Elemental analyses of the copper(II) complexes

Complex Elemental analysis: found (calculated %)

C H N S

[Cu(Pdtc)2]2 34.99 (34.99) 3.89 (3.89) 6.80 (7.24) 31.11 (30.11)[Cu(ppdtc)2]2 37.62 (37.55) 5.33 (5.22) 7.75 (7.30) 33.83 (33.37)Cu(Ppidtc)2 40.24 (40.88) 4.62 (4.90) 5.94 (5.75) 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 refinements for 2 were car-ried out with the programs COLLECT [27] and DENSO [28].The structure was solved using SIR-97 [29] and refined withSHELXL-97 [30] using full-matrix least-squares with aniso-tropic thermal parameters for all non-H atoms. Hydrogenatoms 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 crystallographicinformation is given in Table 2. For 1, selected bond dis-tances and angles are listed in Table 3. For 2, due to thehigh value of R, the bond distances and angles are shownin Table S1.

2.5. Molecular modeling

In order to obtain the most stable conformation, calcu-lations of molecular modeling for different compounds

were carried out using molecular mechanics force fieldMM+, which is an extension of MM2 developed by Allin-ger and Burker [32]. The geometries were optimized usingthe UFF force field [33]. Both MM+ and UFF providequalitative accounts of molecular geometry conformation

and strain energy. The MM+ calculations were made withthe HYPERCHEM program [34] and conformations of all com-pounds were generated using molecular dynamics for eachone with the following options: starting 0 K, simulationtemperature 300 K, heat time 0.1 ps, run time 1 ps. Theseconformations are optimized using a FletcherReeves (con-jugate gradient) protocol with the standard tight convergecriterion (RMS gradient = 0.01 kcal A1 in vacuo).

Molecular mechanics calculations were made also usingthe UFF force field implemented in GAUSSIAN-03 W pack-

age [35].

Table 2Summary of the crystallographic results for the complexes 1 and 2

Complex [Cu(Pdtc)2]2 (1) [Cu(ppdtc)2]2 (2)

Empirical formula C24H32Cu2N4O4S8 C24H40Cu2N4S8Formula weight 824.20 768.26Temperature (K) 297(2) 293(2)Wavelength (A) 0.71073 0.71069Crystal system triclinic triclinicSpace group P1 P1a (A) 8.658(3) 9.036(5)

b (A) 10.319(3) 9.973(5)c (A) 11.064(4) 13.465(5)a () 62.811(1) 91.630(5)b () 77.880(7) 109.210(5)c () 65.953(7) 114.340(9)

V (A3) 802.6(5) 1024.8(9)Z 1 1Dcalc (Mg m

3) 1.705 1.244Absorption coefficient (mm1) 1.884 1.461

F(000) 422 398Crystal size (mm) 0.36 0.27 0.26 0.15 0.15 0.20h Range () 2.0730.51 5.0026.12Limiting indices 7 6 h 6 12,

14 6 k6 14,15 6 l6 15

9 6 h 6 11,

12 6 k6 12,16 6 k6 15

Reflections collected/unique (Rint)

6532/4604 (0.0209) 8106/3913 (0.108)

Observed data [I> 2.0r(I)] 3891 1672Number of reflections

and parameters4606 and 191 3840 and 172

R, wR2, S 0.0296, 0.0820,1.02

0.1012, 0.2808,0.85

Maximum and averageshift/error

0.01/0.00 0.77/0.12

Minimum and maximum

residual density (e A 3

)

0.54 and 1.03 0.35 and 0.46

Table 3Selected bond distances (A) and bond angles () of [Cu(Pdtc)2]2

Bond lengths (A)

Cu(1)S(4) 2.3067(9)Cu(1)S(3) 2.3080(8)Cu(1)S(5) 2.3352(8)Cu(1)S(2) 2.3732(9)

Cu(1)S(2)#1 2.7036(10)S(2)C(1) 1.7347(17)S(2)Cu(1)#1 2.7036(10)S(3)C(7) 1.7177(18)S(4)C(1) 1.7108(18)S(5)C(7) 1.7168(18)N(2)C(7) 1.328(2)N(2)C(8) 1.463(2)N(2)C(9) 1.465(2)N(1)C(1) 1.327(2)N(1)C(2) 1.459(2)N(1)C(3) 1.464(2)

Bond angles ()

S(4)Cu(1)S(3) 153.82(3)S(4)Cu(1)S(5) 99.51(2)S(3)Cu(1)S(5) 76.52(2)S(4)Cu(1)S(2) 76.260(19)S(3)Cu(1)S(2) 100.963(18)S(5)Cu(1)S(2) 165.37(2)S(4)Cu(1)S(2)#1 99.42(2)S(3)Cu(1)S(2)#1 106.72(3)S(5)Cu(1)S(2)#1 102.36(3)S(2)Cu(1)S(2)#1 92.20(2)C(1)S(2)Cu(1) 83.43(6)C(1)S(2)Cu(1)#1 102.04(6)Cu(1)S(2)Cu(1)#1 87.80(2)C(7)S(3)Cu(1) 85.01(6)C(1)S(4)Cu(1) 86.02(6)C(7)S(5)Cu(1) 84.18(6)C(7)N(2)C(8) 122.67(16)C(7)N(2)C(9) 123.62(15)C(8)N(2)C(9) 113.55(15)N(2)C(7)S(5) 123.48(13)N(2)C(7)S(3) 122.84(13)S(5)C(7)S(3) 113.68(10)N(2)C(9)C(10) 111.71(18)N(2)C(8)C(11) 109.45(18)C(1)N(1)C(2) 124.71(15)C(1)N(1)C(3) 122.53(15)C(2)N(1)C(3) 112.57(14)N(1)C(1)S(4) 123.27(13)N(1)C(1)S(2) 122.70(13)S(4)C(1)S(2) 114.02(9)N(1)C(3)C(4) 108.96(16)N(1)C(2)C(6) 109.94(16)

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2.6. Equilibrium studies by electronic spectroscopy

The electronic spectra were recorded on a ShimadzuUV-2101 PC spectrophotometer.

A solution of copper(II) dithiocarbamate complexes andNO saturated aqueous solution were prepared by generat-

ing the gas from the reaction of metallic copper with 5 Mnitric acid [2] using Schlenk techniques:

8HNO3 +3Cu! 3Cu(NO3)2 + 4H2O+2NO

The gas was purified by passing it through five traps contain-ing 50% NaOH solutions. The purified gas was bubbled in aSchlenk flask containing 20 ml of cold water. A thin Teflonhose was connected into the NO aqueous solution throughthe side arm of the flask. Teflon stopcocks were used to iso-late each part of the Schlenk system when necessary. Thewhole system, tightly isolated from air, was previously deox-ygenated by passing pure Ar for approximately 1 h [16,17].Once the NO aqueous solution was obtained, the Schlenk

flask was disconnected and then installed in a thermostatedbath at 20 C and the Teflon hose was connected to an Orionautomatic microburet and purged with pure Ar. The con-centrations of NO used for the calculations were made con-sidering the reported concentration for saturated aqueoussolution of NO (2.057 mM) at 20 C [36]. The solutions ofthe bis(dithiocarbamato)copper(II) complexes 3, 4 and 5,respectively, were prepared in a 7.4 phosphate buffer solu-tion (0.06 M). A number of 610 ll aliquots of the NOsaturated aqueous solution thermostated at 20 C wereadministered to the air tight flask containing the Cu(II) com-plex solution (3, 4 and 5, respectively) using an Orion auto-

matic microburette. The temperature of the cell was held at20 C with a Peltier controller system attached to the spec-trophotometer. The same apparatus was used to administerthe studied solution to the optical cell.

For a solution of Cu(Ppidtc)2 (3) (48.5 lM) the elec-tronic spectral data (19 absorbance values for each addi-tion of NO solution within the range 420476 nm at20 C) were processed using HYPERQUAD software [22].

For a solution of Cu(Sdtc)2 (4) (76 lM) the electronicspectral data (16th absorbance values for each additionof NO solution within the range 348436 nm at 20 C) wereprocessed using SPEFO version [20] of LETAGROP software[21].

For a solution of Cu(Idadtc)2 (5) (52.1 lM) the elec-tronic spectral data (33 absorbance values for each addi-tion of NO solution within the range 348436 nm at20 C) were processed using HYPERQUAD software [22].

2.7. Effect of dithiocarbamates on NO release in cell-free

experiments

Stock solutions (30 mM) of copper(II) dithiocarbamates3, 4, 5 and 6, respectively, were prepared in PBS (pH 7.4)and sterilized using 0.2 lm Millipore filters. Sterile solu-tions of the NO donor SNP were prepared in DMEM (final

concentration 30 mM) just before use. Aliquots of the SNP

stock solution were dispensed into 96 well tissue cultureplates containing either DMEM alone (to yield final con-centrations of the donor in the range of 30100 lM) ormedium supplemented with copper(II) dithiocarbamatesat final concentrations 10 lM. Culture plates containingthe appropriate concentrations of SNP alone or in combi-

nation 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 nitritedetermination (see below).

2.8. Effect of copper(II) dithiocarbamates on NO released

by J774 murine macrophages stimulated with LPS

and IFNc

The J774 murine macrophage cell line characterized bythe ability to express iNOS and generate large quantitiesof NO in response to the simultaneous stimulation withLPS and IFNc [7,8,37,38] was donated by Dr. M. Fresno

(CBM, Universidad Autonoma de Madrid). Cells weregrown in 100 mm Falcons tissue culture plates containingDMEM/10% fetal bovine serum (FBS) supplemented withL-glutamine (2 mM) and antibiotics (100 U/ml penicillinand 100 lg/ml streptomycin) and maintained at 37 C ina 5% CO2 incubator (Haerus). Cells from confluent cul-tures (6575%) were detached form the plates, resuspendedin 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). Cellswere left untreated (controls) or stimulated (24 h) with acombination of LPS and IFNc (final concentration 1 lg/ml

and 100U/ml, respectively) alone or in the presence of theindicated concentrations of the different copper(II) dithioc-arbamates. After this period of time media were aspiratedand used to measure total nitrite levels (see below) andthe lack of cell toxicity determined by previously describedmethods (Mosman) [39].

2.9. Nitrite determinations

The ability of copper(II) dithiocarbamates to trap NOgenerated by chemical donors (NPS) or cytomix(LPS + IFNc)-stimulated macrophages was evaluated bychanges in the concentration of stable NO metabolites(nitrite + nitrate) in culture medium as determined by theGriess reaction [40]. Suitable aliquots (100 ll) of culturemedium were mixed with an equal volume of Griess reagent(a 1:1 mixture of 1% sulfanilamide and 0.1% naphthylethy-lenediamine dihydrochloride in 2.5% H3PO4), incubated for10 min at room temperature [40] and concentrations weredetermined at 550 nm in an EL-312 Microplate reader (Bio-tek Instruments, Winooski, VT). Sterile-filtered standardsolutions of sodium nitrite (050 lM) were freshly preparedin culture medium and incubated in parallel with eachexperiment and comparable results were obtained using anitrate reductase assay kit provided by Alexis Biochemicals

(Laufelfingen, Switzerland).



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2.10. Statistical methods

Results for nitrite concentrations are expressed as themeans SEM from triplicate or quadruplicate cultures,and each experiment was repeated at least three times.The minimal effective doses and ED50 were determined

with a software program based on a four-parameter logisticequation as previously described [40]. Statistical differenceswere examined using ANOVA and, as indicated, Studentst-test for comparison of the means. P< 0.5 was consideredsignificant.

3. Results and discussion

3.1. Description of the structures of the complexes 1 and2

The crystallographic data of the complexes 1 and 2 aregiven in Table 2.

The structure of 1 is built up by centre-symmetric

dimeric entities. Fig. 1 shows a perspective view of thedimeric unit showing the atomic numbering scheme ofthe complex.

For this compound, the distances CuS and CuS 0 are2.373 A and 2.704 A, respectively, in good agreement withthe 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 CuS 0: 2.322 A and 2.740 A, respectively).

The coordination sphere of copper(II) ions is describedas a distorted square-pyramid. The basal coordinationpositions are occupied by four sulfur atoms belonging to

two dithiocarbamate ligands.

Each bridging sulfur atom simultaneously occupies anequatorial coordination site on one copper(II) ion and anapical site on the other copper(II).

The axial CuS bond distance (2.7036(10) A) is longerthan the equatorial CuS ones. The CuS(2) bond lengthin [(2.373(10) A] are longer in the CuS equatorial bond

lengths because S(2) is a bridging atom. The bond anglesS(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 planarowing to the inversion centre. These results are in goodagreement with earlier determinations [4143].

The overall geometry of the complex 2 (Fig. 2) is similarto complex 1. However, because of the high values of R(R = 0.1012), the bond parameters (Table S1) are not accu-rate and should be treated with caution.

3.2. IR spectra

The most significant bands for the 1, 2 and 3 complexesare given in Table 4. The bands in the range 14801510 cm1 are attributed to the m(CN) (partial doublebond) stretching vibration [17,41,44]. This position corre-sponds to a partial double bond character, and is recordedin the range previously reported for similar compounds[4547]. The band in the range 9401050 cm1, belongingto the m(SC) (partial double bond) stretching is consideredindicative of dithiocarbamate acting as a bidentate ligand[44,48]. The band at 1588 cm1 corresponds to antisym-metric carboxyl stretching frequencies of the non-com-plexed 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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C@O stretching vibration of the no complexed ketonicgroup CH2COCH2 of the ligand [47,50].

3.3. Electronic spectra

The most significant bands of the copper(II) complexes1 and 3 are presented in Table 5. For complex 2 in DMSO,the values are similar to complex 1. The most intense bandat 273 nm is probably a band of the ligand; the band at438 nm is a r(S) ! Cu(II) ligand to metal charge transferin 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 (mono-mers) or pyc CuS5 geometry (dimers).

3.4. Magnetic properties

The magnetic behaviour of complexes 1 and 3 is shownin Figs. S1 and S2 by means of a vMT versus T plot (vMbeing the molar magnetic susceptibility). At room temper-ature both compounds give a value for vMTthat is slightlylower 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 isindicative of the occurrence of weak antiferromagneticinteractions in 1 and 3. The pattern of the curve for bothcompounds is very similar, thus although the structure of3 is not known in solid state it must be very similar to thatof1. Other polymerizations also are possible: [Cu(Et2dtc)]4is tetramer [55]. The structure of 1, Fig. 1, consist of dinu-clear copper(II) entities bridged by l-S atoms. The antifer-romagnetic coupling for these complexes is due to spincancellation through the S bridge atoms [56]. However,we have not been able to fit the magnetic data to thenumerical expression for copper(II) dinuclear complexes.

May be some paramagnetic impurities or intermolecularinteractions are present in the polycrystalline sampleswhich were measured, that make the data to deviate fromthe ideal model.

3.5. Mass spectrometry

Complex 1 was further characterized by electrosprayionization (ESI) mass spectrometry. The ESI spectrum ofthe monomeric complex [Cu(Pdtc)2]2 obtained atDV= 20 V is presented in Fig. 3. The main peak observedat m/z 411.5 is corresponding to [Cu(Pdtc)2]

+. The presenceof three ions is not in good agreement with the isotropicdistribution of the Cu: Cu63 (69.2%); Cu65 (30.8%) [57].Probably the other two peaks can be assigned to proton-ated ions, Table 6.

This result implies that the dimer in dimethylsulfoxideacetonitrile solution is broken showing only the monomericcomplex.

3.6. Molecular modeling

In Ref. [17], we have studied the interaction between NOand the bis[1-(2-hydroxyethyl)piperazinedithiocarbamato]-copper(II) by means of molecular mechanics and semi-

empirical methods.

Fig. 2. Perspective view of 2 with thermal ellipsoids at the 50% probability level and showing the selective atom numbering scheme.

Table 4

Most significant IR bands (cm1) of [Cu(Rdtc)2]2

Compound [Cu(Pdtc)2]2 [Cu(ppdtc)2]2 Cu(Ppidtc)2

m(CN) 1508.84 s 1508.65 s 1474 sm(CS) 1005.16 sa 1000.9 sa 970 mm(C@O) 1716.12 sm(COO) 1588 sm(OH) 3414 s

a Singlet.

Table 5Electronic data of the complexes 1 and 3

Complex k/nm (e/M1 cm1) Solvent

1 273 (50.000) 438 (24.450) 650 (2.400) DMSO

3 273 (505) 433 (184) 630 (13.4) water

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

405 406 407 408 409 410 411 412 413 4140

10 0

%

MS123 1 (0.526) Sm (Mn, 2x0.60)411.5

413.5

414.5412.5

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

Table 6Principal ions in the ESI spectra of the monomeric complex of[Cu(Pdtc)2]2

Complex m/z Relative abundance (%) Assignment

Cu(Pdtc)2 411.5 100 [Cu(Pdtc)2]+

412.5 20 [Cu(Pdtc)2 + H]2+

413.5 70 [Cu(Pdtc)2 + 2H]3+

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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mations in gaseous phase for the compounds M (4a),M(NO) (4b), M(NO)2 (4c), D (5a), D(NO) (5b) andD(NO)2 (5c), respectively, obtained by theoretical calcula-tions. The most symmetrical conformations correspondto M(NO)2 and D(NO)2. It was found that in the dithiocar-bamate 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 thedithiocarbamate was also taken into account. For themonomeric complex M, the isomer with SNO bond wasfound to be 12.29 kcal/mol less exothermic than theCuNO bond isomer M(NO). Also the complexes withSNO bond are more distorted than the complexes con-taining CuNO bond. It was found in all cases, both formonomer and dimer that the bond angle CuNO is 120,which indirectly shows that the charge transfer is metalto ligand which agree well with the previous experimentalinformation [16].

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 maxi-mum decreased in intensity and blue-shifted to 425 nm.At the same time a new band appears at 356 nm. Bothbands correspond to LMCT transitions between the copperion and dithiocarbamate and NO, respectively [16,51]. Anisosbestic point is observed at 388 nm (Fig. 6), indicatingthat the reversible process is taking place. The blue shiftvariation 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 (curve3, Fig. 6) give two inflection points at 1:1 and 1:2 Cu:NOmolar ratios, 388 and 390 nm, with formation of the com-plexes Cu(Sdtc)2(NO) and Cu(Sdtc)2(NO)2, respectively(Fig. 7). The experimental data were analysed by meansof the SPEFO version [20] of the LETAGROP program [21].The calculations indicate that 4 reacts stepwise with twomoles of NO (Scheme 1, Table 7). The values of the stabil-ity constants and the species distribution diagram (Fig. 7)indicate that the complex corresponding to equilibrium(1) is thermodynamically more stable than the complexcorresponding to equilibrium (2). The diagram also indi-cates that the complex 1:2 is formed from complex 1:1(Fig. 7, Scheme 1 and Table 7). With excess of nitric oxidethe complex Cu(Sdtc)2(NO)2 forms quantitatively (Fig. 7).The dithiocarbamate complex Cu(Sdtc)2 is a versatile agentto capture the NO under physiological conditions (pH 7.4).

A Cu(Ppidtc)2 (3) solution presents a maximum at432 nm (log e = 4.01). With addition of a NO solution themaximum decreased in intensity and it is blue-shifted. Atthe same time a new band appears at 356 nm. An isosbesticpoint is observed at %382 nm, indicating that a reversibleprocess is taking place. The experimental data were ana-lysed using the HYPERQUAD program [22]. The stability con-

stants for Cu(Ppidtc)2NO and Cu(Ppidtc)2(NO)2 complexes

are given in Table 7. That values for log K1 indicate that thecomplex Cu(Ppidtc)2NO2 is weaker than the Cu(Sdtc)2NOcomplex.

Complex 5 presents a maximum at 432 nm (log e = 4.09)which decreased in intensity with the addition of a NOsolution and is blue-shifted (curve 3, Fig. 8). This decrease

was accompanied by the simultaneous appearance of a new

0

0.2

0.4

0.6

0.8

300 350 400 450 500

wavelength (nm)

A

bsorbance

1

2

3

R

1)1:0

2)1:1

3)1:2

Fig. 6. UV spectra at 293 K for Cu(Sdtc)2 (76 lM) with different additionsof 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.

Fig. 7. Species distribution for 3 NO system at pH 7.4 and 293 K.

Concentration of complex 3: 76 lM.

Cu(Dtc)2 + NO Cu(Dtc)2NOK1

Cu(Dtc)2NO + NO Cu(Dtc)2(NO)2K2

A

B

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

Table 7Stability constants of the Cu(Dtc)2NO and Cu(Dtc)2(NO)2 complexes

Complex log K1 log K2 ReferenceCu(Ppidtc)2 3.16(1) 2.61(1) this workCu(Sdtc)2 4.7(5) 3.9(6) this workCu(Idadtc)2 8.5(1) 2.4(1) this workCu(Deadtc)2 10.3(1) 2.2(1) [17]



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band at 352 nm which corresponds to a charge transfertransition between the copper ion of the dithiocarbamate

complex and NO [16]. An isosbestic point is observed at%383 nm, batochromically shifted, by successive additionsof NO to the Cu(Idadtc)2 solution indicating that reversible

processes are taking place. The experimental data analysedusing the HYPERQUAD program [22] indicated that 5 reactsstepwise with two moles of NO (Scheme 1 and Table 7).

Calculation of the stability constants for Cu(Idadtc)2NOand Cu(Idadtc)2(NO)2 complexes allowed to obtain thevalues for log K1, complex 1:1, equilibrium A in Scheme

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

300 350 400 450 500

wavelength (nm)

A

1

2

3

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 different 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 7.4and 20 C. Concentration of the Cu(Idadtc)2 complex: 5.21 lM.

Cu(Deadtc)2

[NPS] (mM)

0.01 0.03 0.10 0.30

5

10

15

20

25NPS

0.03 mM

0.01 mM

Cu

S

SCN

CH2OH

CH2OH 2

NPS

0.03 mM

0.01 mM

Cu(Ppidtc)2

[NPS] (mM)

0.01 0.03 0.10 0.30

[NO2

-](M)

[NO2

-](M)

5

10

15

20

25

CuS

S

CN

HOOC

2

0.01 mM

(a)

(b)

(c)

(d)

Cu(Idadtc)2

[NPS] (mM)

0.01 0.03 0.10 0.30

[NO2

-](M)

5

10

15

20

25NPS

0.03 mM

0.01 mM

Cu

S

SCN

COOH

COOH2

Cu(Sdtc)2

[NPS] (mM)

0.01 0.03 0.10 0.30

[N

O2

-](M)

5

10

15

20

25NPS

0.03 mM

0.01 mM

CuS

S

CN

COOH 2

CH3

Fig. 10. Effect of copper(II) dithiocarbamates (at final concentrations of 10 and 30 lM) on nitrite levels present in culture media containing the NO-donnor NPS (final concentration). Each data point represents nitrite concentrations (means SEM) three different experiments using triplicate or

quadruplicate wells per data point.



11/13

1, and log K2, complex 1:2, equilibrium B in Scheme 1 indi-cating that the complex 1:1 is much stronger than the com-plex 1:2. These data, together with the species distributiondiagram (Fig. 9) indicate a stepwise process in which for-mation of the 1:2 complex requires that the complex 1:1is practically formed in accordance with previous studies

of our laboratory [16,17]. Similar behaviour with NO hasthe 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] trapNO in aqueous solution, in the next set of experimentswe explored their ability to trap NO generated by increas-ing concentrations (10300 lM) of the specific NO-donorcompound NPS (Fig. 10). Total nitrite levels were extre-

mely low in wells containing DMEM alone, augmentedin a concentration-dependent manner in cultures supple-mented with NPS and significantly decreased in the pres-ence of two different doses (10 lM o r 3 0 lM) of thedifferent copper(II) dithiocarbamates.

3.9. Effect of the copper(II) dithiocarbamates on theconcentration of NO generated by cultured macrophages

stimulated with LPS and IFNc

Since the above-mentioned data demonstrate that cop-per(II) dithiocarbamate derivatives trap NO under cell-freeconditions we performed additional experiments to testwhether a similar activity was exerted in cytomix(LPS + IFNc)-stimulated murine macrophages (Fig. 11).As expected nitrite concentrations were negligible inunstimulated cells (see control in Fig. 11) or culturesexposed only to the highest dose (300 lM) of copper(II)dithiocarbamate derivatives. In contrast, activation of

[Cu(Ppidtc)2] (M)

CONTROL + C3 + LPS + IFN 30 100 300

[NO2

-](M)

[NO2

-](M)

0

20

40

60

80

Cu(Deadtc)2

(M)

CONTROL + C4 + LPS + IFN 30 100 3000

20

40

60

80

(a)

(b)

(c)

(d)

[Cu(Sdtc)2] (M)


[NO2

-](M)

0

20

40

60

80

[Cu(Idadtc)2] (M)


[NO2-

](M)

0

20

40

60

80

Fig. 11. Effect of increasing concentrations of copper(II) dithiocarbamates on NO production in control or cytomix (LPS and IFNc) stimulated J774murine macrophages. Macrophages were cultured (24 h) in the absence or presence of medium alone (controls), with citomix (LPS + IFN c) alone or in thepresence of three different concentrations (30300lM) of each Cu(II)dithiocarbamate. Each data point represents nitrite concentrations (means SEM)of three different 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.



12/13

macrophage iNOS by the simultaneous treatment (24 h)with cytomix (LPS and IFNc) dramatically increasedNO2

concentration (up to 65 lM) being these reactiondose-dependently reduced by the addition of cultures sig-nificantly reduced in the presence of graded doses of thesecopper(II) dithiocarbamates.

When considered together the data derived from exper-iments using chemical donors (SNP) or cytomix(LPS + IFNc)-activated macrophages demonstrate thatthe different copper(II) dithiocarbamates studied are effec-tive NO scavengers, which rapidly react with NO and pre-vent the formation of its stable oxidation product NO2

in aqueous solution.

This results confirm previous studies with Cu(ProDT-C)2 3H2O [16].

4. Conclusions

Single crystals of1 and 2 were prepared for the first time

and characterized by X-ray diffraction. They are dimeric.For complex 2, selected bond distance and bond anglesare given in Supplementary material because of the highvalue of R (Table S1).

Complex 3 was also prepared for the first time.The stability constants and speciation diagrams in

aqueous solution of the Cu(Deadtc)2NO [17] and Cu-(Idadtc)2 NO systems indicate that these copper(II)dithiocarbamates have a great ability to trap NO underin physiologic conditions. The formation of the complexesCuL2(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 complexesCuL2(NO) and CuL2(NO)2 is simultaneous and thereforethis dithiocarbamate copper(II) complex is more versatileto uptake and release NO in physiological conditions.

The species distribution diagrams in aqueous solution atphysiological pH (7.4) also indicate that these copper(II)dithiocarbamates help to know the chemistry to regulatethe NO in the tissues and to control its biological function.

Since NO yields stable oxidation products (mainlyNO2

and lower levels of NO3) in aqueous solution we

also demonstrate that the copper(II) dithiocarbamatesstudied are able to decrease total nitrite concentrationsproduced by the model NO-donor NPS. Of further interesta similar decrease in the total nitrite concentrations wasobserved when the copper(II) dithiocarbamates were addedto murine macrophages challenged with LPS and IFNc toinduce iNOS expression. These data support the potentialapplication of copper(II) dithiocarbamates as therapeuticagents in animal models of septic shock. Verification of thispossibility is currently under investigation in ourlaboratories.

5. Abbreviations

DMEM Dulbecos modified eagle medium

IFNc interferon gamma

iNOS inducible nitric oxide sinthaseLPS lypopolyssacarideNOS nitric oxide sinthaseSNP sodium nitroprussidePGE2 prostaglandin type E2FBS fetal bovine serum

Acknowledgements

Agostinho Francisco Cachapa is very grateful to theAgencia Espanola de Cooperacion Internacional (AECI)and University of La Laguna for a predoctoral fellowshipat Departamento de Qumica Inorganica, Universidad deLa Laguna, Spain. Also thanks are due to the Departa-mento de Ciencias Experimentales, Universitat Jaume I,Spain where he carried out some of the experimental workof the present paper.

Financial support from MEC-Spain (research

projects PM98-0148 and BQU2002-02794) is gratefullyacknowledged.

We thank the Servicio General de Medidas Magneticasof La Laguna University for the magnetic measurements.

Appendix A. Supplementary material

Supplementary crystallographic data for complex 1 havebeen deposited with the CCDC No. 256287. These data canbe obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by e-mailing [email protected], 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 crystallo-graphic data (Tables S2S10) are includes as Supplemen-tary material in this paper.

Figs. S1 and S2 corresponding to the magnetic behav-iour of 1 and 3 complexes are also included as Supplemen-tary material. Supplementary data associated with thisarticle can be found, in the online version, atdoi:10.1016/j.poly.2006.06.008.

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