Immunomodulatory Effects of Palladium ( II ) Complexes of 1 , 2 , 4-Triazole on Murine Peritoneal Macrophages

Os complexos polinucleares [{PdCl 2 (μ-Htrz)} n ] (1) e [{PdBr 2 (μ-Htrz)} n ] (2) (Htrz = 1,2,4-triazol) foram sintetizados neste trabalho. O composto 1 foi preparado a partir da substituição da acetonitrila do precursor [PdCl 2 (MeCN) 2 ] pelo 1,2,4-triazol. A adição posterior de brometo de potássio ao meio reacional resultou na formação do complexo 2. Os complexos inéditos foram isolados, purificados e caracterizados por análise elementar, espectroscopias vibracional no infravermelho e eletrônica no UV-visível e curvas de análise termogravimétrica (TG). Os resultados experimentais sugerem que, em ambos os casos, a coordenação do 1,2,4-Htrz ocorra via átomos N(2) e N(4), atuando como pontes entre centros de paládio. O poliedro de coordenação quadrado-planar ao redor do paládio(II) é determinado pelos dois átomos de N do heterociclo e por dois ligantes cloro (1) ou bromo (2), em uma provável configuração trans. As curvas TG indicaram que a natureza do ligante aniônico não afeta significativamente a estabilidade térmica de 1 e 2. Os produtos finais de decomposição térmica foram identificados como paládio metálico pela técnica de difratometria de raios X de pó. Testes preliminares envolvendo a avaliação dos efeitos dos compostos 1, 2 e Htrz na produção de H 2 O 2 e NO em culturas de macrófagos peritoneais de camundongos BALB/c foram realizados in vitro.


Introduction
One of the first cells to respond to invasion by pathogens and tumor cells are macrophages, which play a pivotal role in the development of specific and non specific immune response.When appropriately activated, macrophages are capable of killing pathogens and tumor cells by a number of processes that can involve the phagocytosis of foreign particles and the production and release of numerous soluble cytotoxic molecules such as cytokines (e.g.][3] Hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) are reactive oxygen and nitrogen intermediates essential in cell signaling and are effector molecules for the microbicidal and cytotoxic response of macrophages after stimulation. 4,5itric oxide and reactive oxygen species affect virtually every step of the development of inflammation.Large amounts of NO, generated primarily by inducible nitric oxide synthase (iNOS) can be toxic and pro-inflammatory.Similarly, O 2 − produced by NADPH oxidases may lead to toxic effects, when produced at high levels during oxidative burst. 6Despite the fact that nitric oxide is not a strong oxidant, NO reacts at a nearly diffusion limited rate with superoxide to yield peroxynitrite ( − OONO), a strong oxidant.Activated inflammatory cells and agoniststimulated endothelial cells generate peroxynitrite which is able to oxidize several biological molecules to nitrate or protein tyrosine residues and other phenolics. 7,8When iNOS is synthesized, it releases higher NO quantities than constitutive nitric oxide synthase (cNOS) and the production of NO continues until L-arginine or the cofactors are depleted or when cellular death occurs. 9Prolonged exposure to a large amount of NO, as in activation of iNOS, inhibits the activity of several enzymes, such as aconitase, cytochrome c oxidase and ribonucleotide reductase.Thus, NO may become cytotoxic or cytostatic. 10Of considerable therapeutic value are the agents that modulate the activity of NO.In particular, those that reduce the formation of NO may be beneficial in pathophysiological conditions where excessive production of NO is a contributory factor.These include diseases such as septic shock, neurodegenerative disorders, and inflammation. 11It is worth to point out that agents with strong anti-inflammatory and anti-oxidative activities may display anti-tumor properties since tumor promotion is closely linked to inflammation and oxidative stress. 12ome authors have shown that murine macrophages treated with cisplatin become activated and acquire enhanced capacity to lyse tumor cells in vitro, produce increased amount of interleukin-1 (IL-1) and TNF, reactive oxygen metabolites (ROI), reactive nitrogen intermediates (RNI), lysozyme and arginase. 13The fact that cisplatin can be used not only as an antitumor drug but also as a potent non-specific immunopotentiating agent has prompted us to initiate a research program in order to synthesize new metal-based compounds with the ability to enhance the host defense responses.Our previous results have indicated that the release of H 2 O 2 levels in cultures of activated mice peritoneal macrophages was increased in the presence of compounds of the type [PdX(dmba)(dppp)] (X = Cl, N 3 , NCO, NCS; dmba = N,N-dimethylbenzylamine; dppp = 1,3-bis(diphenylphosphine)propane). 14etal-based compounds of 1,2,4-triazole have received attention due to their potential uses as antimicrobial and antitumoral drugs. 15,161,2,4-Triazoles are heterocycles which possess three nitrogen atoms in a five-membered ring.As ligands, 1,2,4-triazoles are extremely versatile and display the ability to act as monodentate, exobidentate N(1),N (2)  and exobidentate N(2),N(4) when neutral or monodentate, exobidentate N(1),N(2), exobidentate N(1),N(4) and N(1),N (2),N(4)-tridentate when deprotonated. 17n pursuing our interest on chemical and biological aspects of palladium(II) complexes, 14,18,19 we describe in this work the synthesis, spectroscopic characterization and thermal investigation of the two new 1,2,4-triazolylbridged polynuclear complexes [{PdCl 2 (µ-Htrz)} n ] (1) and [{PdBr 2 (µ-Htrz)} n ] (2).We also investigated the immunomodulatory effects of the compounds 1,2,4-triazole (Htrz), 1, 2, and cisplatin by the release of NO and H 2 O 2 in the culture supernatants of treated murine peritoneal cells.

Materials
The materials employed in the syntheses were all commercially available and were used without purification.All solvents were dried and stored over molecular sieves prior to use.Cisplatin aqueous solution (d = 1 g mL -1 , F. H. Faulding & Co Limited -Australia) was generously supplied by Centro Oncológico da Região de Araraquara (C.O.R.A.).

Instrumental
Elemental analyses of carbon, nitrogen and hydrogen were performed on a microanalyser Perkin-Elmer model 240 at Central Analítica -Instituto de Química -USP-SP.Infrared spectra (IR) were recorded as KBr pellets on a Nicolet FTIR-Impact 400 spectrophotometer in the spectral range of 4000-400 cm -1 .Diffuse reflectance spectra in UV-visible range were registered on a Cary 5000 spectrophotometer using MgO as reference.Thermal analyses (TG) were carried out using a Mettler Toledo TG-50, under flow of dry synthetic air (100 mL min -1 ), temperature up to 900 o C and heating rate of 20 o C min -1 , in α-alumina sample holders.X-ray powder patterns of the residues were obtained with a Siemens D-5000 X-ray diffractometer using CuK α radiation (λ= 1.541 Å) and setting of 40 kV and 20 mA.The peaks were identified using ICDD bases for Pd o (ASTM 05-0681) and PdO (ASTM 06-0515). 21

Animals
Female 6-8 weeks old BALB/c mice weighing 18 to 25 g were purchased from Universidade Estadual de Campinas (UNICAMP) central animal facilities CEMIB (Centro Multidisciplinar para Investigação Biológica), SP, Brazil.They were maintained in polycarbonate boxes at 23 ± 2 ºC, 56 ± 2% humidity, at 12 h light/dark cycle, kept under specific-pathogen-free conditions (positive-pressure cabinet) and provided sterilized food and water ad libitum in accordance with the protocols of the Universidade Estadual Paulista (UNESP).All the experiments were carried out following the Federal Government legislation on animal care.

Peritoneal macrophage
Thioglycollate-elicited peritoneal exsudate cells (PEC) were harvested from female BALB/c mice using 5.0 mL of sterile PBS, pH 7.4.The cells were washed twice by centrifugation at 200 rpm for 5 min at 4 °C and re-suspended in appropriate medium for each test.

Statistical analysis
Results are representative of three independent experiments and they are presented as Means ± Standard Deviation from quadruplicate observations.Data were analyzed statistically by the ANOVA by variance test and after by Tukey-Kramer post-test, using significance level p < 0.05, in the Graph Instat Pad software.
The coordination polymers 1 and 2 were obtained as air stable solids.The elemental analyses and thermogravimetric data, together with IR spectroscopy results, are consistent with the proposed formulae for the synthesized compounds.The polymeric nature of the obtained compounds was inferred by their low solubility, high thermal stability and amorphous nature as evidenced by powder X-ray diffraction analysis (data not shown).Although 1,2,4-triazole is a simple molecule, very few X-ray structures of its derivatives were reported.[28]

Spectroscopic studies
The IR spectrum of the free Htrz exhibits two bands attributed to the ring torsion mode (τ 1 and τ 2 ) at 684 and 651 cm -1 , respectively, since 1,2,4-triazole is found predominately, in the solid state, as the 1H-tautomer of C s symmetry.The coordination mode of 1H-1,2,4-triazole ligand can be inferred on basis of the number of ring torsion bands at 700-600 cm -1 range. 29Two ring torsion absorptions are expected when Htrz possesses C s local symmetry (monodentate and exobidentate N(2),N(4) modes) resembling the IR spectrum of the solid free base.On the other hand, the exobidentate N(1),N (2) and N(1),N (2),N(4)-tridentate coordination modes (C 2v ) are characterized by the disappearance or decrease in intensity of the band associated with the first ring torsion vibration (τ 1 ).Therefore, the presence of the two bands at 656 (τ 2 ) and 629 cm -1 (τ 1 ) for 1 and 2 strongly supports the local C s point group for 1H-1,2,4-triazole in these compounds.
In the diffuse reflectance electronic spectra of 1 and 2, the shift of the π X → d Pd (X = Cl, Br) LMCT (ligand-to-metal charge transfer) band from 258 (1) to 262 nm ( 2) is a strong evidence of the displacement of chloro by bromo ligands.Intense absorptions observed at 340-345 nm are attributed to MLCT (metal-to-ligand charge transfer) excitations of the type d Pd → π* Htrz .The d-d absorptions were not observed and are probably obscured by the intense charge transfer bands. 31he analytical and spectroscopic data obtained for compounds 1 and 2 suggest that both exhibit 1D polymeric structures made by linear arrays of palladium(II).The coordination environment around palladium is expected to be square planar with the sites occupied by two halide groups {Cl -(1), Br -(2)}, and two N atoms, one from each of the two N(2),N(4)-bridging Htrz ligands, in a trans configuration (Figure 2).
The assumption of the trans configuration of the products was based on the structure of the precursor.According to Belluco, complexes of the type cis-[PdX 2 (RCN) 2 ] exhibit two IR strong bands in the ν CN region, whereas the trans analogues are characterized by only one ν CN band. 32Taking into account that the precursor used in our work just showed one intense ν CN band (2330 cm -1 ) in the IR spectrum, we infer the trans configuration for this compound.Such assumption is also in accordance with the literature. 33,34

Thermal analysis
The TG curves for the compounds 1 and 2 are shown in Figure 3.
The TG curves of these compounds showed a similar thermal degradation pattern in which the ligands are initially released in one or two stages, together with uptake of O 2 , leading to a mixture of Pd 0 and PdO.The slight mass increase up to ca. 800 °C is ascribed to the oxidation of the remaining Pd 0 to PdO.Finally, the decomposition of PdO to Pd 0 is completed at ca. 840 °C.The final weight percent residues of 45.36 and 30.56 % found for complexes 1 and 2, respectively, agree well with their respective calculated content of Pd {43.19 % (1) and 31.74 % (2)}.

Cell viability of macrophages in the presence of the compounds Htrz, 1, and 2
Concentration-response cytotoxicity curves for 24 h exposure of macrophages to compounds Htrz, 1 and 2 were performed and the data were compared with the reference standard cisplatin (Table 1).The IC 25 and IC 50 results indicate that Htrz is significantly cytotoxic to macrophages.However, coordination of Htrz to palladium(II) in 1 dramatically reduces its cytotoxic activity.This fact can be associated to the modification of the electronic density around the Htrz molecule modifying the cellular targets of the new compound.It can be also observed from Table 1    that the cytotoxicity was enhanced with the substitution of chloro (1) by bromo ligands (2), whose mechanism of interaction between the compounds and macrophages remains unknown.The reference standard cisplatin was found to be more cytotoxic than both palladium(II) compounds.

Effect of Htrz, 1, and 2 on H 2 O 2 production by peritoneal macrophages
In order to determine the effect of Htrz, 1, and 2 on H 2 O 2 production by macrophages, these cells were treated with IC 25 and IC 50 concentrations of the tested compounds for 1 h.The results depicted in Figure 4 summarize the release of H 2 O 2 expressed as nmol mL -1 after 1 h of incubation with Htrz, 1, and 2.
Compounds 1 and 2 were able to stimulate H 2 O 2 production by macrophages in a dose-dependent manner, releasing levels ca.10-17 times higher than Htrz and cisplatin, at IC 50 concentration.In addition, complexes 1 and 2 also induced the production of higher H 2 O 2 levels when compared with the positive control PMA, a powerful immunostimulator.
Reactive oxygen species (ROS), including hydrogen peroxide, are strong oxidants which are toxic to cells and cause cellular and tissue damage at high concentrations. 35owever, a sublethal concentration of hydrogen peroxide acts as potentially important signaling molecules in both intra and intercellular reactions in a number of different cell types. 36The high production of H 2 O 2 by macrophages stimulated by 1 and 2 suggest that palladium(II) compounds would display a positive effect when used at lower concentrations than those tested in this work.
Measurement of H 2 O 2 inhibition was also carried out in this study (data not shown).However, no inhibitory effect was detected for compounds 1 and 2 since they stimulated the production of high H 2 O 2 levels by macrophages.

Effect of Htrz, 1, and 2 on NO production by peritoneal macrophages
The results illustrated in Figure 5 show the release of NO expressed as µmol mL -1 of supernatant after 24 h of incubation with Htrz, 1, 2, and cisplatin.
When macrophages were treated with IC 50 concentrations of 1 and 2 for 24 h, the release of NO was reduced by about 3-4 times compared to that obtained at IC 25 .This significant reduction in NO production is related to the cytotoxic effects of the compounds in the experimental conditions used.Compounds 1 and 2 also stimulated the production of NO levels of ca.3-4 times higher than Htrz and cisplatin, at IC 25 concentration.Nevertheless, palladium(II) compounds induced the production of lower NO concentrations when compared with the positive control LPS.Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, is an important inflammatory mediator through innate host defense and well known for its high capacity to prime mouse macrophages to generate nitric oxide (NO) via NF-κB. 37,38Stimulation of murine macrophages by  LPS results in the expression of an inducible NO synthase (iNOS), which catalyzes the synthesis of large amounts of NO from L-arginine and molecular oxygen. 39ince that the reduction of NO production is related to the cytotoxicity of the tested compounds, current assays using lower concentrations are underway in our laboratories.

Conclusions
The synthesis, characterization and immunological evaluation of two new triazolyl palladium(II) derivatives have been described in this work.The results of this study suggest that palladium(II) complexes display interesting immunomodulating activity on murine macrophages, despite the fact that the mechanisms of this action currently remain uncharacterized.It was evident that the substitution of chloro by bromo from the coordination sphere of the metal in the [{PdX 2 (µ-Htrz)} n ] compounds results in the substantial increase of cytotoxicity towards macrophage cultures.Our preliminary in vitro findings also showed that macrophages respond to complexes 1 and 2 by enhancing H 2 O 2 levels and releasing lower NO amounts than the positive control.
Further studies are in progress in our laboratories to elucidate the mechanism involved on the production of reactive toxic intermediates by palladium(II) compounds as well the cytokines production by the palladium(II) complex stimulated macrophages.

Figure 4 .
Figure 4. Effects of Htrz, 1, 2 and cisplatin at IC 25 (a) and IC 50 (b) concentrations on H 2 O 2 production by mice peritoneal macrophages in vitro.Cells incubated with 0.2 µmol L -1 of PMA alone were used as a positive control.Each bar represents the mean ± S.D. of four animals.Representative results of one experiment repeated four times are given.*p < 0.001

Table 1 .
Cytotoxicity of Htrz, 1, 2, and cisplatin in BALB/c peritoneal macrophages in vitro a not obtained since the molar weights of polymers 1 and 2 are unknown.