Immunomodulatory effects of palladium(II) complexes of 1,2,4-triazole on murine peritoneal macrophages

ARTICLE

Immunomodulatory effects of palladium(II) complexes of 1,2,4-triazole on murine peritoneal macrophages

Amadeu M. Bego^I; Regina C. G. FremI,^*; Adelino V. G. Netto^I; Antonio E. Mauro^I; Sandra R. Ananias^II; Iracilda Z. Carlos^II; Michelle C. da Rocha^II

^IDepartamento de Química Geral e Inorgânica, Instituto de Química de Araraquara, Universidade Estadual Paulista, PO Box 355, 14801-970 Araraquara-SP, Brazil

^IIDepartamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas de Araraquara, Universidade Estadual Paulista, PO Box 502, 14801-902 Araraquara-SP, Brazil

ABSTRACT

The 1,2,4-triazolyl-bridged polynuclear complexes [{PdCl₂(µ-Htrz)}_n] (1) and [{PdBr₂(µ-Htrz)}_n] (2) have been obtained in this work. Compound 1 is prepared by the displacement of acetonitrile from [PdCl₂(MeCN)₂] by 1,2,4-triazole (Htrz). Further addition of potassium bromide to the reaction medium afforded complex 2. The new complexes have been isolated, purified and characterized by means of elemental analysis, IR and UV-visible electronic spectroscopies and thermogravimetric (TG) curves. The experimental data suggested that, in both cases, the coordination of 1,2,4-Htrz takes place through the N(2) and N(4) atoms, bridging the palladium centers. The square-planar coordination polyhedron of palladium(II) is determined by two nitrogen atoms from the triazole ligands, while the other two coordination positions are occupied by the chloro (1) or bromo (2) ligands. TG curves indicated that the nature of the anionic ligand does not affect significantly the thermal stability of 1 and 2. The final products of the thermal decompositions were identified as metallic palladium by X-ray powder diffractometry. Preliminary tests involving the evaluation of the effects of compounds 1, 2 and Htrz on H₂O₂ and NO production in cultures of peritoneal macrophages from BALB/c mice were carried out in vitro.

Keywords: palladium(II) complexes, 1,2,4-triazole, macrophages, nitric oxide, hydrogen peroxide

RESUMO

Os complexos polinucleares [{PdCl₂(µ-Htrz)}_n] (1) e [{PdBr₂(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 [PdCl₂(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 H₂O₂ 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. tumor necrosis factor, TNF-α), reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI).^1-3 Hydrogen peroxide (H₂O₂) 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.^4,5 Nitric 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, O₂^- produced by NADPH oxidases may lead to toxic effects, when produced at high levels during oxidative burst.⁶ Despite 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 agonist-stimulated endothelial cells generate peroxynitrite which is able to oxidize several biological molecules to nitrate or protein tyrosine residues and other phenolics.^7,8 When 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.⁹ Prolonged 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.¹⁰ Of 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.¹¹ It 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.¹²

Some 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.¹³ The 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 H₂O₂ levels in cultures of activated mice peritoneal macrophages was increased in the presence of compounds of the type [PdX(dmba)(dppp)] (X = Cl, N₃, NCO, NCS; dmba = N,N-dimethylbenzylamine; dppp = 1,3-bis(diphenylphosphine)propane).¹⁴

Metal-based compounds of 1,2,4-triazole have received attention due to their potential uses as antimicrobial and antitumoral drugs.^15,16 1,2,4-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.¹⁷

In 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-triazolyl-bridged polynuclear complexes [{PdCl₂(µ-Htrz)}_n] (1) and [{PdBr₂(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 H₂O₂ in the culture supernatants of treated murine peritoneal cells.

Experimental

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.).

Synthesis of [PdCl₂(MeCN)₂]

The precursor compound was prepared as described for [PdCl₂(PhCN)₂].²⁰ In an erlenmeyer containing 40 mL of boiling acetonitrile under intense magnetic stirring, palladium(II) chloride (2.00 g; 11.3 mmol) was slowly added. The reaction mixture was stirred at 80º C for about 30 min. The yellow suspension was filtered off and the obtained solid was washed with acetonitrile and dried under vacuum. Yield 75%. Anal. Calcd. for C₄H₆Cl₂N₂Pd (%): C, 18.52; N, 10.80; H, 2.34. Found: C, 18.28; N, 10.40; H, 2.49.

Synthesis of [{PdCl₂(µ-Htrz)}_n] (1)

Compound 1 was prepared by adding dropwise [PdCl₂(MeCN)₂] (300 mg; 1.16 mmol) suspended in 15 mL of H₂O to a solution of 1,2,4-triazole (88 mg; 1.27 mmol) in 2 mL of a mixture H₂O:HCl 1:1. After stirring for 30 min at 50 ºC, the yellow suspension was filtered off and the obtained solid was washed with methanol and dried under vacuum. (Yield 45%). mp > 350 ºC. Anal. Calcd. for C₂H₃Cl₂N₃Pd (%): C, 9.75; N, 17.05; H, 1.23. Found: C, 9.98; N, 17.36; H, 1.01. Selected IR frequencies (ν_max/cm^-1): 3171, νCH; 2991, νCH; 1535, ν_ring; 1418, ν_ring; 1317, dCH; 1080, δCH; 775, γNH; 656, γ_ring; 627, γ_ring.

Synthesis of [{PdBr₂(µ-Htrz)}_n] (2)

Compound 2 was prepared by adding dropwise [PdCl₂(MeCN)₂] (300 mg; 1.16 mmol) suspended in 15 mL of H₂O to a solution of 1,2,4-triazole (88 mg; 1.27 mmol) in 2 mL of a mixture H₂O:HCl 1:1. The resulting yellow suspension became yellow-orange after addition of KBr (344 mg; 2.89 mmol) dissolved in 15 mL of H₂O. After stirring for 30 min at 50 ºC, the suspension was filtered off and the obtained solid was washed with methanol and dried under vacuum. (Yield 50%). mp > 350 ºC. Anal. Calcd. for C₂H₃Br₂N₃Pd (%): C, 7.16; N, 12.53; H, 0.90. Found: C, 6.89; N, 12.36; H, 1.12. Selected IR frequencies (ν_max/cm^-1): 3169, νCH; 2991, νCH; 1533, ν_ring; 1417, ν_ring; 1317, δCH; 1082, dCH; 777, γNH; 656, γ_ring; 627, γ_ring.

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 900º C and heating rate of 20 ºC 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 Pdº (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 ºC and re-suspended in appropriate medium for each test.

Cytotoxicity evaluation

For the determination of the cytotoxic effect, the PEC were plated at a concentration of 5×10⁶ per well in RPMI 1640 (Gibco), supplemented with 10% fetal bovine serum (FBS) (Cult-Lab), 100 U mL^-1 of penicillin, 100 µg mL^-1 of streptomycin, 2 mmol L^-1 of L-glutamine, and 5×10^-2 mol L^-1 of 2-mercaptoethanol (Sigma); this mixture was named complete RPMI-1640 (RPMI 1640-C). Then, samples of 100 µL of peritoneal cells suspension (5 × 10⁶ cell mL^-1) in RMPI-1640-C medium were added to each well of a 96-well tissue culture plate with 100 µL of different concentrations of samples: 1,2,4-triazole (Htrz), [{PdCl₂(µ-Htrz)}_n] (1), [{PdBr₂(µ-Htrz)}_n] (2), and cisplatin (standard drug) containing 0.1% DMSO and RPMI 1640-C medium. Cells were incubated for 24 h at 37 ºC in a humidified atmosphere containing 7.5% of CO₂. After incubation, the medium was poured off, and macrophages were incubated with 100 µL of solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) (MTT) in each well. MTT assay was performed and the plates were incubated for 3 h at 37 ºC and 7.5% CO₂. The formazan formed was dissolved with 100 µL of isopropyl alcohol and the optical density was measured using a microplate reader (Multiskan, Labsystem) equipped with a 540 nm filter and 620 nm reference filter. The optical density of dissolved formazan in the control (untreated cells) was taken as 100% of viability.²²

H₂O₂ measurement

PEC at 2 × 10⁶ cells mL^-1 were suspended in a solution containing 140 µmol L^-1 of NaCl, 10 µmol L^-1 of potassium phosphate buffer, pH 7.0, 5.5 µmol L^-1 of dextrose, 0.56 µmol L^-1 of phenol red, and 0.01 µg mL^-1 of type II horseradish peroxidase (HRP). Next, 100 µL of this suspension was added to each of the wells of a 96-well flat-bottom tissue culture plate and exposed to IC₂₅ and IC₅₀ concentrations of samples: 1,2,4-triazole (Htrz), [{PdCl₂(µ-Htrz)}_n] (1), [{PdBr₂(µ-Htrz)}_n] (2), and cisplatin (standard drug) containing 0.1% DMSO (50 µL) and phorbol myristate acetate (PMA) 0.2 µmol L^-1 (50 µL). Cells incubated only with PMA were used as a positive control. The cells were incubated for 1 h at 37 ºC in a 7.5% CO₂ atmosphere. The reaction was stopped with 50 µL of 5 mol L^-1 of NaOH and the samples were read at 620 nm with a Multiskan Ascent ELISA reader (Labsystems) against a blank containing phenol red solution and 5 mol L^-1 of NaOH. The results were expressed as nmol of H₂O₂ mL^-1 cells, from a standard curve established in each test consisting of known molar concentrations of H₂O₂ in buffered phenol red.^23,24

NO measurements

The nitric oxide (NO) production was determined by measuring nitrite (NO₂^-), a stable degradation product of nitric oxide by assaying culture supernatants.²⁵ PEC at 5 × 10⁶ cells mL^-1 was incubated with IC₂₅ and IC₅₀ concentrations of the samples: 1,2,4-triazole (Htrz), [{PdCl₂(µ-Htrz)}_n] (1), [{PdBr₂(µ-Htrz)}_n] (2), and cisplatin (standard drug) containing 0.1% DMSO and RPMI 1640-C medium for 24 h at 37 ºC in a 7.5% CO₂ atmosphere. Cell-free supernatant (50 µL) was mixed with 50 µL of Griess reagent (sulfanilamide 0.1%, phosphoric acid 3%, naphthylethylenediamine 0.1%) and incubated at room temperature for 10 min. Cells incubated with lipopolysaccharide (LPS) (1 mg mL^-1) were used as a positive control. After incubation, the absorbance was determined by using a microplate reader (Multiskan, Labsystem) equipped with a 540 nm filter. Nitrite concentration was determined using dilutions of sodium nitrite in culture medium as standards.

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^TM software.

Results and Discussion

The precursor [PdCl₂(MeCN)₂] reacts with 1,2,4-triazole (Htrz) in water to afford [{PdCl₂(µ-Htrz)}_n] (1). The [{PdBr₂(µ-Htrz)}_n] (2) is readily synthesized by metathesis of the chloro groups in 1 with potassium bromide. A general scheme illustrating the strategy employed for the synthesis of the complexes 1 and 2 is illustrated in Scheme 1.

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. Most probably this is caused by the fact that this ligand almost always produces intractable solids with transition metal salts, which is a typical characteristic of almost all linear coordination polymers.^17,26-28

Spectroscopic studies

The IR spectrum of the free Htrz exhibits two bands attributed to the ring torsion mode (τ₁ and τ₂) 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.²⁹ Two 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 (τ₁). Therefore, the presence of the two bands at 656 (τ₂) and 629 cm^-1 (τ₁) for 1 and 2 strongly supports the local C_s point group for 1H-1,2,4-triazole in these compounds.

There is an important IR spectral difference between monodentate and exobidentate N(2),N(4) coordination mode of 1H-1,2,4-triazole.³⁰ The first ring torsion absorption (τ₁) occurs at ca. 640 cm^-1 for monodentate complexes, whereas for the bidentate mode such band always appears at lower wavenumbers. Thus, the τ₁ bands at 629 cm^-1 found in the IR spectra of 1 and 2 (Figure 1) clearly indicate the N(2),N(4) coordination mode of the 1H-1,2,4-triazole.

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.³¹

The 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-[PdX₂(RCN)₂] exhibit two IR strong bands in the ν_CN region, whereas the trans analogues are characterized by only one ν_CN band.³² Taking 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 O₂, leading to a mixture of Pdº and PdO. The slight mass increase up to ca. 800 ºC is ascribed to the oxidation of the remaining Pdº to PdO. Finally, the decomposition of PdO to Pdº is completed at ca. 840 ºC. 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 IC₂₅ and IC₅₀ 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 H₂O₂ production by peritoneal macrophages

In order to determine the effect of Htrz, 1, and 2 on H₂O₂ production by macrophages, these cells were treated with IC₂₅ and IC₅₀ concentrations of the tested compounds for 1 h. The results depicted in Figure 4 summarize the release of H₂O₂ expressed as nmol mL^-1 after 1 h of incubation with Htrz, 1, and 2.

Compounds 1 and 2 were able to stimulate H₂O₂ production by macrophages in a dose-dependent manner, releasing levels ca. 10-17 times higher than Htrz and cisplatin, at IC₅₀ concentration. In addition, complexes 1 and 2 also induced the production of higher H₂O₂ 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.³⁵ However, 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.³⁶ The high production of H₂O₂ 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 H₂O₂ 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 H₂O₂ 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 IC₅₀ concentrations of 1 and 2 for 24 h, the release of NO was reduced by about 3-4 times compared to that obtained at IC₂₅. 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 IC₂₅ 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.^37,38 Stimulation 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.³⁹

Since 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 [{PdX₂(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 H₂O₂ 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.

Acknowledgments

This work was sponsored by grants from FAPESP, CNPq and CAPES.

Received: February 21, 2008

Web Release Date: February 6, 2009

FAPESP helped in meeting the publication costs of this article.

• 1. Schmidt, H. H. H. W.; Walter, U.; Cell 1994, 78, 919.
• 2. Moreira, R. R. D.; Carlos, I. Z.; Vilegas, W.; Biol. Pharm. Bull. 2001, 24, 201.
• 3. Nathan, C.; Cell 1995, 82, 873.
• 4. Nathan, C. F.; Hibbs, J. B. J.; Curr. Opin. Immunol. 1991, 3, 65.
• 5. Forman, H. J.; Torres, M.; Am. J. Respir. Crit. Care Med. 2002, 166, S4.
• 6. Guzik, T. J.; Korbut, R.; Adamek-Guzik, T.; J. Physiol. Pharmacol. 2003, 54, 469.
• 7. Ischiropoulos, H.; Nelson, J.; Duran, D.; Al-Medhdi, A.; Free Radical Biol. Med. 1996, 20, 373.
• 8. Linares, E.; Giorgio, S.; Mortara, R. A.; Santos, C. X. C.; Yamada, A. T.; Augusto, O.; Free Radical Biol. Med. 2001, 30, 1234.
• 9. Dusting, G. J.; MacDonald, P. S.; Ann. Med. 1995, 27, 395.
• 10. Achike, F. I.; Kwan, C. Y.; Clin. Exp. Pharmacol. Physiol. 2003, 30, 605.
• 11. Hobbs, A. J.; Higgs, A.; Moncada, S.; Annu. Rev. Pharmacol. Toxicol. 1999, 39, 191.
• 12. Bhimani, R. S.; Troll, W.; Grunberger, D.; Frenkel, K.; Cancer Res. 1993, 53, 4528.
• 13. Shishodia, S.; Sodhi, A.; Shrivastava, A; Int. J. Immunopharmac. 1997, 19, 683.
• 14. De Almeida, E. T.; Mauro, A. E.; Santana, A. M.; Netto, A. V. G.; Carlos, I. Z.; Quim. Nova 2005, 28, 405.
• 15. Nomiya, K.; Noguchi, R.; Ohasawa, K.; Tsuda, K.; Oda, M.; J. Inorg. Biochem. 2000, 78, 363.
• 16. Reisner, E.; Arion, V. B.; Eichinger, A.; Kandler, N.; Giester, G.; Pombeiro, A. J. L.; Keppler, B. K.; Inorg. Chem. 2005, 44, 6704.
• 17. Haasnoot, J. G.; Coord. Chem. Rev. 2000, 200, 131.
• 18. Da Rocha, M. C.; Santana, A. M.; Ananias, S. R.; de Almeida, E. T.; Mauro, A. E.; Placeres, M. C. P.; Carlos, I. Z.; J. Braz. Chem. Soc. 2007, 18, 1473.
• 19. Caires, A. C. F.; De Almeida, E. T.; Mauro, A. E.; Hermely, J. P.; Valentini, S.; Quim. Nova 1999, 22, 329.
• 20. Heck, R. F.; Palladium Reagents in Organic Synthesis: Best Synthetic Methods, Academic Press: New York, 1985.
• ²¹
  Powder Diffraction File of the Joint Committee on Power Diffraction Standards, published by the International Center of Diffraction Data, Swarthmore, PA, USA, 19081, 1982.
• 22. Mossman, T.; J. Immunol. Methods 1983, 65, 55.
• 23. Pick, E.; Keisari, Y.; J. Immunol. Methods 1980, 38, 161.
• 24. Pick, E.; Mizel, D. J.; J. Immunol. Methods 1981, 46, 211.
• 25. Green, L. C. D.; Wagner, D. D. A.; Glogowski, J.; Skepper, P. L.; Wishnok, J. S.; Tannenbaum, S. R.; Ann. Biochem. 1982, 126, 131.
• 26. Janiak, C.; J. Chem. Soc., Dalton Trans. 2003, 2781.
• 27. Archer, R. D.; Coord. Chem. Rev. 1993, 128, 49.
• 28. Bailar Jr., J. C.; In Preparative Inorganic Reactions, Jolley, W. L., ed., Interscience: New York, 1964.
• 29. Haasnoot, J. G.; Vos, G.; Groeneveld, W. L.; Z. Naturforsch, B: Chem. Sci. 1977, 32, 533.
• 30. Haasnoot, J. G.; Vos, G.; Groeneveld, W. L.; Z. Naturforsch, B: Chem. Sci 1977, 32, 1421.
• 31. Lever, A. B. P.; Inorganic Electronic Spectroscopy, Elsevier: Amsterdam, 1968.
• 32. Belluco, U.; Crociani, B.; Boschi, T.; Inorg. Chem. 1970, 9, 2021.
• 33. Storhoff, B. N.; Lewis Jr., H. C.; Coord. Chem. Rev. 1977, 23, 1.
• 34. Walton, R. A.; Can. J. Chem. 1968, 46, 2347.
• 35. Schraufstatter, I. U.; Hyslop, P. A.; Hinshaw, D. B.; Spragg, R. G.; Sklar, L. A.; Cochrane, C. G.; Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 4908.
• 36. Chua, C. C.; Hamdy, R. C.; Chua, B. H.; Free Radical Biol. Med. 1998, 25, 891.
• 37. Barratt, G. M.; Raddassi, K.; Petitm, J. F.; Tenu, J. P.; Int. J. Immunopharmacol. 1991, 13, 159.
• 38. Forstermann, U.; Gath, I.; Schwarz, P.; Closs, E. I.; Kleinert, H.; Biochem. Pharmacol. 1995, 50, 1321.
• 39. Palmer, R. M.; Ashton, D. S.; Moncada, S.; Nature 1988, 333, 664.

Publication Dates

• Publication in this collection
  25 May 2009
• Date of issue
  2009

History

• Accepted
  06 Feb 2009
• Received
  21 Feb 2008