Gold complexes with benzimidazole derivatives - Springer Link

2 downloads 0 Views 362KB Size Report
Jan 19, 2014 - Elaine Soares Coimbra • Carmen Verıssima Ferreira • Silvia Mika Shishido • ... gold complexes were tested against three Leishmania species ...
Biometals (2014) 27:183–194 DOI 10.1007/s10534-014-9703-1

Gold complexes with benzimidazole derivatives: synthesis, characterization and biological studies Vinicius Zamprogno Mota • Gustavo Senra Gonc¸alves de Carvalho • Adilson David da Silva • Luiz Antoˆnio Sodre´ Costa • Patrı´cia de Almeida Machado Elaine Soares Coimbra • Carmen Verı´ssima Ferreira • Silvia Mika Shishido • Alexandre Cuin



Received: 17 September 2013 / Accepted: 31 December 2013 / Published online: 19 January 2014 Ó Springer Science+Business Media New York 2014

Abstract Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of

Electronic supplementary material The online version of this article (doi:10.1007/s10534-014-9703-1) contains supplementary material, which is available to authorized users. V. Z. Mota  A. Cuin (&) LQBin, Laborato´rio de Quı´mica BioInorgaˆnica, Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-330, Brazil e-mail: [email protected]; [email protected] G. S. G. de Carvalho  A. D. da Silva Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 36036-330, Brazil

leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC50 values below 5.7 lM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells. Keywords Benzimidazole  Gold  Leishmania  Cancer  Cytotoxicity P. de Almeida Machado  E. S. Coimbra Departamento de Parasitologia, Microbiologia e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 33036-030, Brazil C. V. Ferreira  S. M. Shishido Laborato´rio de Bioensaios e Transduc¸a˜o de Sinal, Departamento de Bioquı´mica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP 13083-862, Brazil

L. A. S. Costa NEQC, Nu´cleo de Estudos em Quı´mica Computacional, Departamento de Quı´mica, Instituto de Cieˆncias Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG 33036-330, Brazil

123

184

Abbreviations NMR T D TG/DTA UV–Vis Lig1 Lig2

DMSO CDCl3 DFT TD Ab initio ECP SDD B3LYP

IEFPCM HOMO/LUMO MTT DMEM FBS Balb/c 3T3 SK-Mel 103 Panc-1 BHI LPS

Biometals (2014) 27:183–194

Nuclear magnetic resonance Triplet Duplet Thermogravimetric/differencial thermal analysis Ultraviolet–Visible spectroscopy 1-Benzyl-2-phenyl-1Hbenzimidazole 1-(4-Methoxybenzyl)-2-(4methoxyphenyl)-1Hbenzimidazole Dimethylsulfoxide Chloroform-d Density functional theory Time dependent method From Latin, means ‘‘from the beginning’’ Effective core potential Stuttgart/Dresden ECP Becke 3 parameter hybrid functional with non-local Lee– Yang–Parr expression Integral equation formalism on polarizable continuum model Highest occupied/lowest unoccupied molecular orbital 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide Dulbecco’s modified eagle’s medium Fetal bovine serum Mouse embryonic fibroblast Human melanoma cells Human pancreatic carcinoma Brain Heart Infusion Lipopolysaccharide

Introduction Gold compounds have been used in Medicine since 3500 B.C. for different purposes (Shaw 1999). In the last century the uses of gold-based drugs experienced an extraordinary increase with the development of new gold-based compounds such as auranofin, solganol and myochrisine for the treatment of rheumatoid

123

arthritis and these compounds have been used to alleviate the pain caused by this illness improving the life quality of the patients (Zatta 2009; Pereira et al. 2012; Costa et al. 2012). The successful application of gold(I) compounds in the treatment of rheumatoid arthritis led to the synthesis of new gold(I) and gold(III) compounds for the treatment of several diseases, including the preparation of potential anticancer-drugs. Particularly, the gold(III) complexes have shown to possess structural and electronic similarities with the anticancer Pt(II)-drugs, with emphasis on the formation of d8 square-planar compounds. Nevertheless, the mechanism of action of gold(III)-based drugs are quite different when compared to the mechanism of the Pt(II)-drugs (Cuin et al. 2011). It has been described that the cytotoxic effects of gold compounds are focused in a specific mitochondrial mechanism (Bindoli et al. 2009; Rubbiani et al. 2010). Some previous works have also provided evidences of the reactions of gold compounds with proteins (Hickey et al. 2008; Roberts et al. 1996; Christodoulou et al. 1994; Zou et al. 2000). Messori and co-works described a possible mode of action of antiproliferative gold-compounds based on systematic studies of various gold compounds with serum albumin (Gabbiani et al. 2012). Gold compounds have also been reported as antibacterial and antileishmanial agents (Navarro 2009). The uses of metals and their salts as antimicrobial or pathogenic agents declined sharply in the middle of the last century upon the introduction of penicillinderived antibiotics. On the other hand, with the actual increasing of microbial resistance to current antibiotics, there is a continuous demand for new classes of compounds that could be able to inhibit the growth of pathogenic microorganisms. In this way, benzimidazole derivatives such as mebendazole, chlormidazole and albendazole have been reported as antimicrobial and antiparasitic agents (Kazimierczuk et al. 2002; Navarrete-Vazquez et al. 2001). Leishmaniasis, for example, is a neglected tropical endemic disease (Navarro 2009), caused by parasitic protozoa of the genus Leishmania and it is still current over 98 countries spread on the five continents. The WHO reported endemic leishmaniasis transmission with an estimated 1.5–2 million new cases per year (WHO 2013). Chemotherapy is the effective way to treat all forms of this disease, but the current therapy exhibit several limitations, including toxicity, the very

Biometals (2014) 27:183–194

185

Synthesis Syntheses of ligands

Fig. 1 Proposed structures of benzimidazole derivatives— Lig1 and Lig2

expensive cost and the resistance to the used drugs, which has compelled the search for new antileishmanial agents (Croft and Olliaro 2011). Here we describe the synthesis, structural characterization, density functional theory (DFT)-based simulation and antileishmanial and anticancer studies of the organic compounds named Lig1 and Lig2 (see Fig. 1) and their new gold-based complexes named VZM006, VZM043, VZM012 and VZM045, which corresponds to [Au(Lig1)Cl3], [Au(Lig1)Cl], [Au(Lig2)Cl3] and [Au(Lig2)Cl], respectively.

Experimental Materials and measurements The starting compounds o-phenylenediamine (99.5 %), benzaldehyde (99.0 %), p-anisaldehyde (98 %), gold(I) chloride (98 %) and potassium tetrachloroaurate(III) (98 %) were purchased from Sigma-Aldrich Laboratory Co. All chemical reagents were used without further purification. Elemental analyses of C, H and N were performed on a CHNS-O EA 1110 Analyzer, CE Instruments. Thermal analysis was performed on a DTG-60 Simultaneous DTA-TG apparatus, Shimadzu using the following conditions: synthetic air, flow rate of 50 cm3 min-1 and heating rate of 10 °C min-1, from 25 to 900 °C. FT-IR spectra were recorded on a Spectrum 2000 FT-IR Perkin Elmer spectrophotometer in the range 4,000–300 cm-1. The samples were prepared as KBr pellets. The UV–Vis spectra were recorded in dimethylsulfoxide (DMSO) solution using a Shimadzu UV–Vis spectrophotometer in the range 300–800 nm.

Lig1 (1-benzyl-2-phenyl-1H-benzimidazole) was obtained mixing o-phenylenediamine (1.00 g, 9.25 mmol) and benzaldehyde (2.00 mL, 18.5 mmol) in 20 mL of ethanol. The mixture was stirred at room temperature for 6 h to give a yellow solution. The solution was left to stand overnight, giving rise to light yellow crystals. Yield, 70 %; Elemental analysis calcd (%) for C20H16N2: C, 84.4; H, 5.67; N, 9.85; Found (%) C, 84.3; H, 5.68; N, 10.1. Lig2 {1-(4-methoxybenzyl)-2-(4-methoxyphenyl)1H-benzimidazole} was isolated following the same procedure as Lig1 but p-anisaldehyde (2.25 mL, 18.5 mmol) was used instead benzaldehyde. The product was obtained as powder. Yield, 68 %; Elemental analysis calcd (%) for C22H20N2O2: C, 76.7; H, 5.89; N, 8.13; Found (%) C, 77.4; H, 5.92; N, 9.01. Syntheses of gold complexes VZM006 Potassium tetrachloroaurate(III)—K[AuCl4] (0.189 g, 0.50 mmol)—was dissolved in 6 mL of methanol and Lig1 (0.142 g, 0.50 mmol) was dissolved in 16 mL of methanol. The Lig1 solution was slowly added under vigorous stirring to the Au(III) solution. The final solution was maintained under stirring and at room temperature. After 40 min, a yellow solid was formed. The final solution was filtered and the solid was washed three times with *5 mL of cold methanol. The solid was collected and dried in a desiccator under P4O10. Yield 33 %. Elemental analysis calcd (%) for [Au(C20H16N2) Cl3]: C 40.9; H 2.71; N 4.79; Au 33.5. Found (%) C 40.4; H 2.83; N 4.71 and Au 33.1 (from TG curve). VZM012 This gold(III) compound was obtained following the same procedure as VZM006. However, Lig2 (0.172 g, 0.50 mmol) was used instead of Lig1. Yield 22 %. Elemental analysis calcd (%) for [Au(C22H20N2O2)Cl3]: C 40.8; H 3.12; N 4.32; Au 30.1. Found (%) C 40.3; H 3.34; N 4.34 and Au 29.5 (from TG curve). VZM043 Gold(I) chloride (0.116 g 0.5 mmol) was dissolved in 10 mL of ethyl ether. The Lig1 (0.142 g, 0.50 mmol) was also dissolved in 25 mL of ethyl

123

186

ether. The ligand solution was added to the Au(I) solution under stirring and at room temperature. After 1 h a pale yellow solid was formed. Yield, 25 %. Elemental analysis calcd (%) for [Au(C20H16N2)Cl]: C 46.5; H 3.12 N 5.42; Au 38.1. Found (%) C 46.9; H 3.16; N 5.51 and Au 37.5 (from TG curve). VZM045 Following the VMZ043 procedure the VZM045 was obtained. However, Lig2 (0.172 g, 0.50 mmol) was uses instead of Lig1. Yield, 25 %. Elemental analysis calcd (%) for [Au(C22H20N2O2)Cl]: C 45.8; H 3.49; N 4.90; Au 34.1. Found (%) C 46.5; H 3.02; N 5.20 and Au 34.0 (from TG curve). For all complexes the filtrated solutions were left to stand for several days, but no suitable crystals were obtained for X ray analysis. DFT simulation The AB initio calculations on each free ligand and the gold complexes were performed by a full unconstrained geometry optimization using typical convergence criteria and by vibrational harmonic frequencies calculations using the DFT in gas phase. The popular hybrid functional B3LYP was chosen (Becke 1993; Lee, Yang and Parr 1988; Miehlich et al. 1989) since it is well established as the most common DFT functional used for transition metal complexes providing good results when compared to the experimental data. The 6-31G basis set (Ditchfield, Hehre and Pople 1971; Hehre, Ditchfield and Pople 1972) used here includes one d polarization functions for C, H, Cl, N and O atoms as well as one diffuse function. The Stuttgart ECP (SDD) (Andrae et al. 1990) improved with one set of f polarization functions (af = 1.1386) for the gold atom was considered in all complexes calculations. This basis set scheme is shown in Supplementary Material #1 and it has been applied with success in a recent paper of dos (Dos Santos, Paschoal and Burda, 2012) Thermal contributions were calculated at 298.15 K and 1 atm. The time-dependent method (TD) (Stratmann et al. 1998) used to calculate the excited state energies provided a good assessment for the UV–Vis spectroscopic data, since the ligands are interesting conjugated systems. Only the main transitions with the highest oscillator strengths were evaluated. The polarizable continuum model using the integral

123

Biometals (2014) 27:183–194

equation formalism (IEFPCM) (Cance`s et al. 1997) was performed within TD calculations in order to simulate the presence of the solvent, DMSO. The solvent cavity was constructed using the UFF radii (Rappe´ et al. 1992). Also, for TD-DFT calculations the basis set for all atoms, except gold, was increased to 6-311??G(2d,2p). All calculations were executed using the Gaussian 09 program package (Mennucci and Tomasi 1997; Frisch et al. 2009). Biological assays Anti-leishmania activity assay Reagents Fetal bovine serum (FBS) was purchased from Cultilab (Campinas, Sa˜o Paulo, Brazil). Brain heart infusion (BHI) medium was purchased from Hime´dia (Mumbai, India). Hemin, folic acid, RPMI 1640 medium, Thioglycolate medium, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), lipopolysaccharide, N-(1-Naphthyl)ethylenediamine dihydrochloride and DMSO were purchased from SigmaAldrich (St. Louis, MO, USA). Phosphoric acid, sulfanilamide and L-glutamine were purchased from Vetec (Duque de Caxias, Rio de Janeiro, Brazil). Miltefosine was purchased from Cayman Chemical Company, Michigan, USA. Parasites and culture cell. Three species of Leishmania were used in this study: L. amazonensis (IFLA/Br/67/ PH8), L. braziliensis (MHOM/Br/75/M2903) and L. major (MRHO/SU/59/P). Promastigotes of L. amazonensis were cultured in Warren’s medium (BHI plus hemin and folic acid) and promastigotes of L. braziliensis and L. major were maintained in BHI medium plus L-glutamine and male urine sterile, both supplemented with 10 % FBS at 25 °C (Coimbra et al. 2013). In vitro antileishmanial activity Promastigote forms Anti-promastigote activity was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method based on tetrazolium salt reduction by mitochondrial dehydrogenases (Mossman 1983). Briefly, log phase promastigote forms were seeded in 96-well tissue culture plates. The parasites were exposed to the compounds, previously dissolved in DMSO, for 72 h at 25 °C and their viabilities were

Biometals (2014) 27:183–194

evaluated using the MTT assay. Controls with DMSO and without drugs were performed. All the tests were performed in duplicate. Miltefosine was used as the reference substance. The IC50 values were obtained from at least three independent experiments, and they were carried out at 5 % significance level (P \ 0.05, CI 95 %), calculated using a non-linear regression curve, by using GraFit Version 5 software (Erithacus Software Ltd., Horley, U.K). Amastigote forms Macrophages were obtained from Balb/c mice previously inoculated with 3 % thioglycolate medium (Machado et al. 2012; Coimbra et al. 2013). Briefly, peritoneal macrophages were plated on coverslips (13-mm diameter) previously arranged in a 24-well plate in RPMI 1640 medium supplemented with 10 % inactivated FBS and allowed to adhere for 24 h at 37 °C in 5 % CO2. Adherent macrophages were infected with L. amazonensis (IFLA/Br/67/PH8), L. braziliensis (MHOM/Br/75/M2903) or L. major (MRHO/SU/59/P) promastigotes in the stationary growth phase using a ratio of 1:10 at 33 °C for 3 h. Noninternalized promastigotes were eliminated and solutions of tested compounds were added. Then, the cells were maintained at 33 °C in 5 % CO2 for 72 h, fixed, and stained with Giemsa for parasite counting (optical microscopy, 1,000 9 magnification). Miltefosine was used as the reference substance. The IC50 values were obtained from two independent experiments. In parallel with the anti-amastigote Leishmania assay, the cytotoxicity of the compounds against murine macrophages was also performed. Mouse peritoneal macrophages were plated in 96-well culture plates, in RPMI 1640 medium supplemented with 10 % FBS. Adherent macrophages were incubated with the compounds, previously dissolved in DMSO, for 72 h at 37 °C. The viability of the macrophages was determined with the MTT assay, as described previously and was confirmed by comparing the morphology of the control group via light microscopy. Controls with DMSO and without drugs were also performed. All the tests were performed in duplicate. The IC50 values were obtained from at least two independent experiments. Cell culture and cytotoxicity assay against tumor cells Cisplatin, DMSO and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma, and ethanol from Synth.

187

Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum and antibiotics were purchased from Invitrogen (Gaithersburg, MD). Flasks and plates were purchased from TPP (Techno Plastic Products, St. Louis, USA). Balb/c 3T3 (mouse embryonic fibroblast, non-tumorigenic) cells were purchased from the National Institute of Health-Baltimore, USA. Human melanoma cells SK-Mel 103 were kindly provided by Dr. Silvya Stuchi (Sa˜o Paulo University-USP, Sa˜o Paulo, Brazil) and Panc-1 (human pancreatic carcinoma) cells were purchased from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil). Cells were routinely grown in DMEM supplemented with 10 % FBS and antibiotics (100 U/mL penicillin, 10 lg/mL streptomycin) in a humidified incubator with 5 % carbon dioxide at 37 °C. The concentrations of the compounds that led to reduction of 50 % on cell viability were used as parameter for cytotoxicity. Cisplatin was used as a positive control in the concentrations of 7.5 lM for Skmel 103 cells, 15.0 lM for Panc-1 cells and 25.0 lM for Balb/c 3T3 cells. DMSO 0.01 % v/v was used as negative control. Cells (1x105 cell/mL) were plated in 96 well plates and 24 h later the cells were exposed to the complex suspensions containing 1–300 lM. After 48 h the solutions were removed and 100 lL of MTT solution (0.50 mg/mL in FBS free culture medium) were added to each well. After incubation for 2 h at 37 °C, the MTT solutions were removed and the formazan crystals were solubilized in 100 lL of ethanol. The plate was shaken for 5 min on a plate shaker and the absorbance was measured at 570 nm in a microplate reader (Synergy HT, BioTek) (Mossman 1983).

Results and discussion Thermal behavior The thermal behavior of the four complexes was studied by thermogravimetric—TG and differential— DTA simultaneous analyses in the range 25–900 °C. VZM006 is stable up to 250 °C and then, the compound begins to lose mass in two consecutives steps. The first step finished near to 335 °C. Above 700 °C no mass loss is observed. The two steps mass losses were 65.5 %. The final residue was 33.1 %. Four exothermic peaks centered at 252, 348, 564 and 601 °C corresponding to the oxidation of the organic

123

188

Biometals (2014) 27:183–194

moiety were observed. The endothermic peak at 232 °C was assigned toVZM006 melting point. The TG curve for VZM043 shows two steps due to decomposition of the organic moiety. The first step occurs in the range 250–342 °C. The second step starts at 445 °C and upon 550 °C, only metallic gold was observed. The sum of mass losses was 62.5 %. DTA curve shows exothermic events centered at 282 and 443 °C. The melting point was observed as an endothermic peak at 218 °C. The melting point of VZM012 was observed in the DTA curve at 177 °C as an endothermic peak. Exothermic peaks centered at 240 and 578 °C were also observed, being attributed to the oxidative decomposition of the organic moiety of the complex. The mass loss starts at 233 and ends above 650 °C. The expected total mass loss is 69.6 % and the experimental value for the residue was found to be 29.5 %. The residue matches with the formation of metallic gold. Two consecutive mass loss steps were observed on VZM045 TG curve. The mass loss starts at 236 °C and upon 550 °C. Only one exothermic peak centered on 448 °C appears in the DTA curve. The total mass loss calculated was 65.9 %, while the experimental one was 66 %. The residue was of 34 %, being attributed to metallic gold. Structural comparison and IR spectroscopic analysis Since there are no crystallographic data available for the gold compounds, a comparison between the Table 1 Calculated bond lengths and angles for the DFT-optimized structures

123

experimental and calculated spectroscopic data of the free ligands and their gold complexes was performed in order to confirm the coordination sites of the ligands to the metal centers and also to propose the structures of the complexes. In this sense, Table 1 shows the calculated bond lengths and angles for all six optimized structures. Both gold(III) complexes exhibit similar length values for the Au–Cl bonds. Therefore, it is easy to identify that the Au–Cl(1) is smaller (2.339 for VZM006 and 2.341 for VZM012) than the other two Au–Cl bonds in these two ˚ . Also, the complexes, which are around 2.36 A calculated angles around the coordination sphere of Au(III) ions show a square planar geometry since the angles Cl–Au-Cl are near to 90° and N–Au-Cl (trans) C 179.5o (see Table 1). The optimized structures are shown in Fig. 2. The NMR studies were performed for all compounds, but these data were not presented here since the ligands were soluble only in organic solvents and gold(I) complexes were poorly soluble only in DMSO. The 1H and 13C NMR spectra for gold(III) complexes showed poor definition and they are also not presented here. The calculated 1H and 13 C -NMR spectra in DMSO phase using the polarizable continuum model for the compounds are included in the Supplementary Material #2. In order to compare the calculated harmonic vibrational modes the frequency calculations were performed as described in the methodology section. Lig1 shows the most intense band at 1,425.5 cm-1 in the theoretical infrared spectrum which can be assigned as a conjugated mode which includes an

˚ Bond length/A

Lig1

VZM006

VZM012

Lig2

VZM043

Au–Cl(1)



2.339

2.341



2.333

2.334

Au–Cl(2)



2.363

2.363







Au–Cl(3)



2.364

2.363







Au–N(1)



2.069

2.069



2.074

2.073

N(1)-C(1)

1.312

1.337

1.337

1.313

1.334

1.334

C(1)-N(2)

1.394

1.362

1.362

1.393

1.367

1.367

C(1)-C(2)

1.502

1.499

1.499

1.502

1.498

1.499

Angles/° Cl(1)-Au–N(1)



179.7

179.5



179.4

179.4

Cl(1)-Au–Cl(2)



90.5

90.6







Cl(1)-Au–Cl(3)



90.5

90.4







Au–N(1)-C(1)



126.8

126.8



127.5

127.5

N(1)-C(1)-N(2)

112.9

109.7

109.7

112.9

110.7

110.8

VZM045

Biometals (2014) 27:183–194

189

Fig. 2 Optimized structures of Gold complexes. VMZ006 (a), VMZ012 (b), VMZ043 (c) and VZM045 (d). The Nitrogen atoms are colored as blue, oxygen atoms are in red, carbon and hydrogen atoms are in gray and light gray respectively, chloride ions in green and gold I or III ions are colored as yellow. (Color figure online)

angular deformation (d) of -CH2- hydrogen atoms and the C–N stretching of pyrazole ring. Another important band lies on 1,567.8 cm-1 (1,600 cm-1 in experimental spectrum), which is assigned to the C=N stretching mode. Both bands can be seen in VZM006 and VZM043 complexes, although with some expected displacements. The first cited band is now in 1,468.7 cm-1 for VZM006 and 1,463.5 cm-1 for VZM043. The second one is 1,531.5 and 1,534.4 cm-1 for VZM006 and VZM043, respectively. These wavenumbers show a reasonable agreement with the experimental data, even for non-scaled harmonic frequencies. The N–C bonds are not so affected by the coordination of the nitrogen atom of the pyrazole ring to the gold ions, with differences comparable to those of Au–Cl bond lengths. Still, this

very slight difference may be of high significance especially when the experimental and calculated data show the coordination via the N-pyrazole. When a comparison is made with the other complexes, which includes two methoxy groups in paraposition in phenylene rings, the results are not exactly the same. In Lig2, it is possible to see new and close intense bands at 1,298.6 and 1,300 cm-1 which are conjugated to an mH3C-O–C(phenyl) asymmetrical mode. The C=N stretching mode lies in 1,564 cm-1 which is quite close to the same band in the free ligand Lig1. For the complexes VZM012 and VZM045 the comparisons with the free ligand (Lig2) is almost immediate. The bands cited above lying around 1,300 cm-1 are now displaced to 1,280 and 1,286 cm-1 (VZM012) and to 1,280 and 1,284 cm-1 (VZM045). Also, it is possible to

123

190

observe the C=N mode at 1,529 cm-1 for VZM012 and 1,532.8 cm-1 for VZM045. Nevertheless, it is important to state that some of these frequencies are resultant from conjugated modes where the highlighted bands appear as the intense ones. In this sense, the FT-IR spectra of the gold complexes with Lig1 and Lig2 are very similar to the spectra of the respectively free Ligand. On the other hand, in the spectra of the complexes the absorptions due to Au–Cl bond are observed at 364 (325), 362 (323), 335 (311) and 351 (310) cm-1 for VZM006, VZM012, VZM043 and VZM045, respectively. The values in parenthesis are from the DFT data. UV-Vis spectroscopic measurements A single band for Lig1, Lig2 and the gold(III) complexes—VZM006 and VZM012—is observed in the UV–vis spectra of the compounds. The maxima of the bands were centered at 305, 296, 359 and 302 nm, respectively. The bathochromic effect is more intense from Lig1 to its parent gold complex VZM006, in which the band is shifted by 44 nm, suggesting the metal–ligand coordination. From Lig2 to VZM012 the same band is displaced by 6 nm. The agreement between experimental and calculated UV–Vis data Fig. 3 Theoretical Electronic Absorption Transitions of Lig1 and VZM006

123

Biometals (2014) 27:183–194

related to bathochromic effect reinforces the geometries of compounds proposed in Fig. 2. The main electronic transitions calculated by TD-DFT are related to the frontier orbitals (HOMO, HOMO-1, LUMO and LUMO?1); the transitions with highest oscillator strengths were evaluated. The main transition observed in the calculated spectrum considering the DMSO dielectric constant on a PCM calculation appears only when more than 20 states (program default) are considered. In this case, for the gold(III) complex VZM006 this excitations lies at 377 nm and indicate an electronic transition to the LUMO orbital. In Fig. 3 the frontier molecular orbitals of Lig1 and VZM006 are shown for comparison. It is possible to see that the gold d orbital and chlorine p orbitals play an important role in the generation of LUMO, meaning that electronic density is clearly dislocated from the ligands to molecular orbitals of gold moiety. Biological activity Leishmania The compounds were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis: L. braziliensis, L. amazonensis and L. major

Biometals (2014) 27:183–194

191

Table 2 Effect of the compounds in promastigotes of Leishmania species Compounds

Promastigotes IC50 (lM)a L. amazonensis

L. braziliensis

L. major

Lig1

[100.00

[100.00

[100.00

Lig2

[100.00

[100.00

[100.00

K[AuCl4]

22.77 ± 2.91

20.07 ± 0.48

[100.00

VZM006

15.66 ± 0.86

9.90 ± 0.98

48.17 ± 0.26

VZM012

5.18 ± 0.56

1.29 ± 0.22

4.36 ± 0.31

VZM043

42.19 ± 0.38

18.40 ± 0.46

54.01 ± 1.13

Miltefosine

21.00 ± 2.92

28.07 ± 0.47

20.00 ± 0.52

a

Values of inhibitory concentration of 50 % of the growth parasites (IC50). IC50 values are the average of three independent experiments, respectively ± standard errors of the mean. Miltefosine was used as reference drug

(Coimbra et al. 2013; Den Boer et al. 2011). Antileishmanial activities of the compounds were determined in vitro against the insect promastigote and the intramacrophage amastigote forms, found in the mammalian host and responsible for the human disease. For antipromastigote assay, free ligand and gold complexes showed a good activity against Leishmania species with IC50 ranging from 1.29 to 54.01 lM (Table 2). The compounds Lig1 and Lig2 did not show leishmanicidal activity even at the maximum concentration tested (100 lM). The compound VZM012 showed the best activity against all Leishmania species and exhibited a strong leishmanicidal activity with IC50 value of 1.29 lM against L. braziliensis which is 21.75 times higher than the reference drug Miltefosine, against the same Leishmania species. For antiamastigote assay, only the

active compounds in promastigote forms Leishmania were tested in intracellular amastigotes. As can be seen in Table 2, the gold complexes also exhibited antiamastigote activity with IC50 ranging from 3.16 to 25.95 lM (Table 2). In general, the compounds were more active in intracellular amastigotes than extracellular parasite forms and the gold complexes showed a higher selectivity to the parasite cells than that of the free ligand. Clinical manifestations of leishmaniasis are associated to numerous Leishmania species and human immune response, and this information has import implications for clinical treatment. So, compounds with activity on several Leishmania species are very interesting in chemotherapy research against leishmaniasis and the gold complexes exhibited activity against all Leishmania species assayed. Leishmanicidal activity of metal complexes has been related by several authors (Carmo et al. 2011; Ilari et al. 2012). Investigation of the mechanism of action of the goldcontaining drugs indicated that these compounds interfere with parasitic enzymes including biosynthesis of polyamines, NADH fumarate reductase and cysteine proteases (Carmo et al. 2011; Ilari et al. 2012; Vieites et al. 2009). Macrophages are the main host cells for Leishmania parasites. Hence, murine macrophages were also used for the toxicity assays in order to verify the specificity of the anti-parasitic activity and nonspecific cytotoxic activity against mammalian cells. As observed in Table 3, the gold complexes did not show significant nonspecific cytotoxicity on macrophages at doses that are relevant for the anti-Leishmania activity. So, regarding the selectivity of the compounds, it

Table 3 Effect of the compounds in intracellular amastigotes of Leishmania species, murine macrophages and selectivity index Compounds

Amastigotes IC50 (lM)a (95 % CI)b

Macrophages CC50 (lM)c

L. amazonensis

L. braziliensis

L. major

SId L. amazonensis

L. braziliensis

L. major







K[AuCl4]

[25.00

[25.00

[25.00

59.68 ± 4.92

VZM006

25.95 (20.51–32.84)

6.84 (5.24–8,94)

15.43 (11.60–20.53)

84.75 ± 13.83

3.27

12.39

5.49

VZM012

16.25 (12.12–21.79)

4.06 (3.27–5.03)

5.69 (4.56–7.08)

60.28 ± 3.6

3.71

14.85

10.59

VZM043

5.77 (4.39–7.58)

3.16 (2.04–4.90)

14.90 (12.59–17.64)







Miltefosine

4.15 (2.90–5.96)

2.45 (2.21–2.94)

7.56 (6.35–8.98)

31.80

53.87

17.46

a

[150.00 131.99 ± 3.95

Results from two independent experiments are shown as IC50 values in lM

b

CI: Confidence Interval

c

Values of concentration that kills 50 % of the macrophages (CC50)

d

SI: selectivity index (CC50 of macrophages/IC50 of amastigotes). Miltefosine was used as reference drug

123

192 Table 4 Values of IC50 for free ligands, gold salt and gold complexes against tumor cells

IC50: values in lM; Balb/c 3T3 normal cells; Panc-1: pancreas Tumor cells; SKMel 103 Melanoma cells

Biometals (2014) 27:183–194

Compound

Cells Balb/c 3T3

SK-Mel 103

Lig1

74.44 ± 3.83

46.60 ± 3.27

22.20 ± 7.10

Lig2

78.06 ± 1.81

65.05 ± 3.99

58.37 ± 2.47

VZM006 VZM012

66.55 ± 2.42 64.41 ± 4.12

42.88 ± 3.05 47.12 ± 2.25

28.49 ± 0.98 33.45 ± 5.60

VZM043

71.77 ± 0.60

49.8 ± 6.28

46.32 ± 3.50

VZM045

78.02 ± 7.88

58.44 ± 13.76

56.50 ± 3.89

K[AuCl4]

223.57 ± 8.19

162.98 ± 17.09

125.33 ± 17.17

is important mention that, when the compounds were assayed against intracellular amastigotes, they were much more destructive to the parasitic form than to the host cells. In fact, VZM043 was about 50 times more selective for the parasite than for macrophages. Furthermore, this assay provides preliminary information about the toxicity against mammalian cells and the results pointed in this work are very encouraging, once the in vitro cytotoxicity assays can be used to predict human toxicity. Antitumor assays The cytotoxicity of Lig1 and Lig2 and their gold complexes was evaluated by the MTT reduction assay against Balb/c 3T3, SK-Mel 103 and Panc-1 cell lines. The results are presented in Table 4. Due to the poor solubility of complexes in water the experiments were conducted using DMSO as solvent. Even low soluble, the complexes VZM0012 and VZM045 were taken up by cells lending to more cytotoxic effect than free Lig2. The amounts of the VMZ012 complex that led to inhibition of 50 % on cell viability were 33.45 ± 5.6 lM for SK-Mel 103 while free Lig2 showed activity by 58.37 ± 2.47 and against Balb/c 3T3 cell, the VZM012 and Lig2 showed 64.41 ± 4.12 and 78.06 ± 1.81 lM, respectively. The K[AuCl4] used in the syntheses of VZM006 and VZM012 complexes has shown low cytotoxic effect against the tested cells, given concentration values higher than 100 lM depending on the cell line. No significant differences were observed for series of Lig1 and its gold(I and III) complexes. Considering the results obtained, the VZM012 complex was shown to enhance cytotoxic activity, although the complex was inferior to cisplatin in the same experimental

123

Panc-1

conditions. In the case of cisplatin, the IC50 was found 7.5 lM for SK-Mel 103.

Conclusions Gold(I) and gold(III) complexes with benzimidazole derivatives were synthesized and their respectively structures were proposed. Elemental and thermal analyses show a 1:1 metal/ligand proportion, leading to the compositions [Au(C20H16N2)Cl3], [Au(C22H20N2O2)Cl3], [Au(C20H16N2)Cl] and [Au(C22H20N2O2)Cl] for VZM006, VZM012, VZM043 and VZM045, respectively. The experimental and calculated IR data indicate coordination of benzimidazoles to gold(I or III) ions through the nitrogen atom of the imidazole moiety. Biological studies revealed that the complexes are effective against several promastigotes and amastigotes Leishmania species, with the IC50 values of VZM012 below than 5.7 lmol for three Leishmania species. Gold complexes have also shown to be cytotoxic against SK-Mel 103 and Panc-1 tumor cell lines.

Supplementary material Basis set scheme applied to all calculations involving gold complexes as inserted in the input for Gaussian 09 is presented in Supplementary Material #1. The calculated 1H and 13C -NMR spectra in DMSO phase using the polarizable continuum model for the compounds are presented in the Supplementary Material #2. Acknowledgments The authors are thankful to Brazilian agencies FAPEMIG (processes CEX APQ 00256/11, 02967/10,

Biometals (2014) 27:183–194 CBB APQ 1982-10 and CBB PPM 00302-13), CNPQ (474834/ 2012-3, 240094/2012-3 fellowship for Alexandre Cuin and 305160/2011-7 fellowship for Elaine Coimbra and CAPES (fellowship to Patrı´cia de Almeida Machado) for financial support.

References Andrae D, Haeussermann U, Dolg M, Stoll H, Preus H (1990) Energy-adjusted ab initio pseudo potentials for the second and third row transition elements. Theor Chem Acc 77:123–141 Becke AD (1993) Density-functional thermochemistry. 3.The role of exact exchange. J Chem Phys 98:5648–5652 Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A, Messori L (2009) Thioredoxin reductase: a target for gold compounds acting as potential anticancer drugs. Coord Chem Rev 253:1692–1707 Cance`s MT, Mennucci B, Tomasi J (1997) A new integral equation formalism for the polarizable continuum model: theoretical background and applications to isotropic and anisotropic dielectrics. J Chem Phys 107:3032–3041 Carmo AM, Silva FM, Machado PA, Fontes AP, Pavan FR, Leite CQF, Leite SR, Coimbra ES, da Silva AD (2011) Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and antitubercular agents. Biomed Pharmacother 65:204–209 Christodoulou J, Sadler PJ, Tucker S (1994) Gold-induced structural switch of cys34 in albumin: comparison of auranofin with aurothiomalate. Met-Based Drugs 1:527 Coimbra ES, Antinarelli LMR, Da Silva AD, Bispo MLF, Kaiser CR, De Souza MVN (2013) 7-Chloro-4-quinolinyl Hydrazones: a Promising and Potent Class of Antileishmanial Compounds. Chem Biol Drug Des 81:658–665 Costa GM, Corbi PP, Abbehausen C, Formiga ALB, Lustri WR, Cuin A (2012) Silver(I) and gold(I) complexes with penicillamine: synthesis, spectroscopic characterization and biological studies. Polyhedron 34:210–214 Croft SL, Olliaro P (2011) Leishmaniasis chemotherapy-challenges and opportunities. Clin Microbiol Infect 1:1478–1483 Cuin A, Massabni AC, Pereira GA, Leite CQF, Pavan FR, SestiCosta R, Heinrich TA, Costa-Neto CM (2011) 6-Mercaptopurine complexes with silver and gold ions: anti-tuberculosis and anti-cancer activities. Biomed Pharmac 65:334–338 Den Boer M, Argaw D, Jannin J, Alvar J (2011) Leishmaniasis impact and treatment access. J Clin Microbiol Infect 17:1471–1477 Ditchfield R, Hehre WJ, Pople JA (1971) Self-consistent molecular-orbital methods. IX. An extended Gaussiantype basis for molecular-orbital studies of organic molecules. J Chem Phys 54:724–728 Dos Santos HF, Paschoal D, Burda JV (2012) Exploring the potential energy surface for the interaction of sterically hindered trichloro(diethylenetriamine)gold(iii) complexes with water. J Phys Chem A 116(45):11015–11024 Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B,

193 Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery Jr JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, ¨ , Foresman JB, Ortiz JV, Dapprich S, Daniels AD, Farkas O Cioslowski J, Fox DJ, Gaussian 09, Revision A.02, Gaussian Inc., version A1, Wallingford CT, 2009 Gabbiani C, Massai L, Scaletti F, Michelucci E, Maiore L, Cinellu MA, Messori L (2012) Protein metalation by metal-based drugs: reactions of cytotoxic gold compounds with cytochrome c and lysozyme. J Biol Inorg Chem 17:1293–1302 Hehre WJ, Ditchfield R, Pople JA (1972) Self-consistent molecular orbital methods. XII. Further extensions of Gaussian-type basis sets for use in molecular orbital studies of organic molecules. J Chem Phys 56:2257–2261 Hickey JL, Ruhayel RA, Barnard PJ, Baker MV, Berners-Price SJ, Filipovska A (2008) Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J Am Chem Soc 130:12570–12571 Ilari A, Baiocco P, Messori L, Fiorillo A, Boffi A, Gramiccia M, Di Muccio T, Colotti G (2012) A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition. Amino Acids 42:803–811 Kazimierczuk Z, Upcroft JA, Upcroft P, Gorska A, Starosciak B, Laudy A (2002) Synthesis, antiprotozoal and antibacterial activity of nitro- and halogeno-substituted benzimidazole derivatives. Acta Biochim Polon 49:185–195 Lee C, Yang W, Parr RG (1988) Development of the collesalvetti correlation-energy formula into a functional of the electron-density. Phys Rev B 37:785–789 Machado PA, Hila´rio FF, Carvalho LO, Silveira ML, Alves RB, Freitas RP, Coimbra ES (2012) Effect of 3-alkylpyridine marine alkaloid analogues in leishmania species related to American cutaneous leishmaniasis. Chem Biol Drug Des 80:745–751 Mennucci B, Tomasi J (1997) Continuum solvation models: a new approach to the problem of solute’s charge distribution and cavity boundaries. J Chem Phys 106:5151–5158 Miehlich B, Savin A, Stoll H, Preuss H (1989) Results obtained with the correlation-energy density functionals of Becke and Lee, Yang and Parr. Chem Phys Lett 157:200–206 Mossman T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63 Navarrete-Vazquez G, Cedillo R, Hernandez-Campos A, Yepez L, Hernandez-Luis F, Valdez J, Morales R, Cortes R, Hernandez M, Castillo R (2001) Synthesis and antiparasitic activity of 2-(trifluoromethyl)-benzimidazole derivatives. Bioorg Med Chem Lett 11:187–190

123

194 Navarro M (2009) Gold complexes as potential anti-parasitic agents. Coord Chem Reviews 253:1619–1626 Pereira GA, Massabni AC, Castellano EE, Sodre´-Costa LA, Leite CQF, Pavan FR, Cuin A (2012) A broad study of two new promising antimycobacterial drugs: Ag (I) and Au (I) complexes with 2-(2-thienyl)benzothiazole. Polyhedron 38:291–296 Rappe´ AK, Casewit CJ, Colwell KS, Goddard WA III, Skiff WM (1992) UFF, a full periodic table force field for molecular mechanics and molecular dynamics simulations. J Am Chem Soc 114:10024–10035 Roberts JR, Xiao J, Schliesman B, Parsons DJ, Shaw CF III (1996) Kinetics and mechanism of the reaction between serum albumin and auranofin (and its isopropyl analogue) in vitro. Inorg Chem 35:424–433 Rubbiani R, Kitanovic I, Alborzinia H, Can S, Kitanovic A, Onambele LA, Stefanopoulou M, Geldmacher Y, Sheldrick WS, Wolber G, Prokop A, Wolfl S, Ott I (2010) Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties. J Med Chem 53:8608–8618 Shaw CF III (1999) Gold-based therapeutic agents. Chem Rev 99:2589–2600

123

Biometals (2014) 27:183–194 Stratmann RE, Scuseria GE, Frisch MJ (1998) An efficient implementation of time-dependent density-functional theory for the calculation of excitation energies of large molecules. J Chem Phys 109:8218–8244 Vieites M, Smircich P, Buggeri L, Marcha E, Gomez-Barrio A, Navarro M, Gara TB, Gambino D (2009) Synthesis and characterization of a pyridine-2-thiol N-oxide gold(I) complex with potent antiproliferative effect against Trypanosoma cruzi and Leishmania sp insight into its mechanism of action. J Inorg Biochem 103:1300–1306 WHO (World Health Organization). Leishmaniasis. Available at http://www.who.int/leishmaniasis/en/. Accessed 03 May 2013 Zatta P (2009) Special Issue: Bioinorganic and Biomedical Chemistry of Gold Preface. Coord Chem Rev 253:1597– 1598 Zou J, Taylor P, Dornan J, Robinson SP, Walkinshaw MD, Sadler PJ (2000) First crystal structure of a medicinally relevant gold protein complex: Unexpected binding of [Au(PEt3)](?) to histidine. Angew Chem Int Ed 39:2931