Synthesis, spectroscopic characterization, DFT studies and biological assays of a novel gold(I) complex with 2-mercaptothiazoline

Polyhedron 30 (2011) 2354–2359

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Synthesis, spectroscopic characterization, DFT studies and biological assays of a novel gold(I) complex with 2-mercaptothiazoline Camilla Abbehausen a,⇑, Juliana F. Castro a, Marcelle B.M. Spera a, Tassiele A. Heinrich b, Claudio M. Costa-Neto b, Wilton R. Lustri c, André L.B. Formiga a, Pedro P. Corbi a a b c

Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo – USP, 14049-900 Ribeirão Preto, SP, Brazil Department of Biological and Health Sciences – UNIARA, 14801-320 Araraquara, SP, Brazil

a r t i c l e

i n f o

Article history: Received 4 April 2011 Accepted 22 June 2011 Available online 28 June 2011 Keywords: Gold(I) 2-Mercaptothiazoline Infrared spectroscopy NMR spectroscopy Antibacterial activity Cytotoxic assays

a b s t r a c t A new gold(I) complex with 2-mercaptothiazoline (MTZ) with the coordination formula [AuCN(C3H5NS2)] was synthesized and characterized by chemical and spectroscopic measurements, DFT studies and biological assays. Infrared (IR) and 1H, 13C and 15N nuclear magnetic resonance (NMR) spectroscopic measurements indicate coordination of the ligand to gold(I) through the nitrogen atom. Studies based on DFT confirmed nitrogen coordination to gold(I) as a minimum of the potential energy surface with calculations of the hessians showing no imaginary frequencies. Thermal decomposition starts at temperatures near 160 °C, leading to the formation of Au0 as the final residue at 1000 °C. The gold(I) complex with 2mercaptothiazoline (Au–MTZ) is soluble in dimethyl sulfoxide (DMSO), and is insoluble in water, methanol, ethanol, acetonitrile and hexane. The antibacterial activities of the Au–MTZ complex were evaluated by an antibiogram assay using the disc diffusion method. The compound showed an effective antibacterial activity against Staphylococcus aureus (Gram-positive) and Escherichia coli and Pseudomonas aeruginosa (Gram-negative) bacterial cells. Biological analysis for evaluation of the cytotoxic effect of the Au–MTZ complex was performed using HeLa cells derived from human cervical adenocarcinoma. The complex presented a potent cytotoxic activity, inducing 85% of cell death at a concentration of 2.0 lmol L 1. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Inorganic compounds have been used for the diagnosis and treatment of different human diseases. Chrysotherapy is the medicinal term for treatments based on gold compounds. The uses of gold in medicine were first reported by the Arabic and Chinese physicians. In the middle ages, gold mixtures were applied for the treatment of a wide range of conditions and also as an elixir of youth. In the end of the 19th century, Koch first demonstrated

Abbreviations: MTZ, mercaptothiazoline; NMR, nuclear magnetic resonance; IR, infrared; Au–MTZ, gold(I) complex with mercaptothiazoline; MIC, minimal inhibitory concentration; TMS, tetramethylsilane; DMSO, dimethyl sulfoxide; LANL2DZ, Los Alamos National Laboratory, 2nd version on double zeta function; B3LYP, Becke 3-Lee Young Parr functional; ATCC, American type collection cell; MH, Mueller– Hinton agar; BHI, brain–heart infusion medium; CFU, colony forming unit; DMEM, Dulbecco’s Modified Eagle’s Medium; FCS, Fetal Calf Serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt; PBS, phosphate buffered saline. ⇑ Corresponding author. Tel.: +55 19 35213130; fax: +55 19 35213023. E-mail addresses: [email protected] (C. Abbehausen), [email protected] (P.P. Corbi). 0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2011.06.021

the bacteriostatic effects of gold cyanide, [Au(CN)2] , providing the primary scientific basis concerning the pharmacological activities of gold compounds [1,2]. Today, the application of gold compounds in medicine is mainly centered on the treatment of inflammatory processes, with emphasis on rheumatoid arthritis. Gold(I) compounds seem to alleviate the symptoms associated with this medical condition. The most used compound in the treatment of rheumatoid arthritis is triethylphosphine–gold(I) tetraacetatothioglucose, or auranofin. Solganol and myochrysine are other examples of gold(I) compounds used for the treatment of arthritis. According to the literature, the gold compounds act as pro-drugs, providing controlled gold-delivery at the sites of inflammation [3]. Studies on the mechanism of action of gold complexes showed inhibition of cyclooxigenases, selenium glutathione peroxidase and thioredoxine reductase. More recently, auranofin was also shown to possess antineoplasic activity against HeLa cells in vitro and in vivo. The antitumor activity of auranofin is considered responsible for the growth in syntheses and anticancer assays of several gold(I) and gold(III) complexes with different ligands [4–6]. Recently, Rubbiani et al. prepared new gold(I) complexes with Nheterocyclic carbene derivatives and investigated their antiprolif-

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erative activities against human tumor cells. The complexes showed a significant antiproliferative effect against the tested cells [7]. The design of gold compounds for antitumor activity is based not only on gold(I) complexes but also on gold(III), due to their structural similarities to the Pt(II) complexes. Recently, Shaik et al. reported the synthesis and characterization of a new stable cationic Au(III) complex containing a iminophosphorane ligand and its cytotoxic activities against HeLa and Jukart-T cells [8]. Gabbiani et al. reported the synthesis of structurally related oxobridged binuclear Au(III) complexes with bipyridine and bipyridine-derivative ligands. The complexes exhibited significant antiproliferative effects toward HeLa cells and an appreciable stability under physiological conditions [9]. Gold(I) compounds have also been evaluated as antimicrobial and antiparasitic agents. Gold complexes with S-donor ligands demonstrated inhibition of growth of Gram-positive and Gramnegative bacteria and of fungi [10]. Complexes with N-donor heterocyclic ligands and triphenylphosphine were also prepared and showed a pronounced capacity of inhibition of Staphylococcus aureus and Bacillus subtilis, both Gram-positive bacteria [11]. Recently, gold(I) analogues of a platinum complex with acridine, an S,N-donor ligand, exhibited selective activity against Mycobacterium tuberculosis, responsible for tuberculosis. The activity of such compounds stimulated the evaluation of other gold(I) complexes in the treatment of tuberculosis, with promising results [12]. More recently, gold(I) complexes with N-acetyl-L-cysteine [13] and with ibuprofen [14], a non-steroidal anti-inflammatory drug, were synthesized and characterized in our laboratories. Preliminary studies showed their antibacterial activities against Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (S. aureus) microorganisms. Mercaptothiazoline (C3H5NS2, MTZ) is a heterocyclic N,S-donor ligand, which has been described as a precursor for the synthesis of a variety of biologically active molecules [15]. The structure of MTZ is presented in Fig. 1. Synthesis and crystallographic studies of a new Cu(I) complex with MTZ, with the coordination formula [Cu(MTZ)2Cl], have been described [16]. The coordination of MTZ to the metal center is monodentated through sulfur atom in an unusual trigonal CuL3 arrangement. Raper et al. [17,18] also described the synthesis and crystallographic studies of a polymeric Cu(I) complex with the composition {[Cu(MTZ)]4(C6H5CH3)}n, while Dehand and Jordanov studied a novel Pd(II) complex with MTZ [19]. No biological studies were performed for such complexes. This paper reports the synthesis, characterization, and antibacterial and cytotoxic studies of a novel gold(I) complex with MTZ.

a)

H3a

H3b

C3

H2b

C2

c) S C1

SH1a

N

H2a

H3a S

H3b

C3

H2b

C2

N

H2a

Au

C1

SH1

C4

b)

N

H3a

H3b

C3

H2b

C2

S C1 S NH1b

H2a Fig. 1. Tautomeric structures of MTZ (a, b) and the Au–MTZ complex (c).

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2. Experimental 2.1. Materials and equipments 2-Mercaptothiazoline (98%) and potassium dicyanoaurate(I), K[Au(CN)2], were purchased from Sigma–Aldrich. Hydrochloric acid was purchased from Merck. Elemental analysis for carbon, hydrogen and nitrogen were performed using a Perkin–Elmer Model 2400 CHNS/O Analyzer. Electrospray ionization mass spectrometry (ESI-MS) measurements were carried out using a Waters Quattro Micro API. Samples were evaluated in the positive mode in an 1:1 acetonitrile:water solution with addition of 0.10% (v/v) formic acid. IR spectra were measured using a Bomem MB-Series Model B100 FT-IR spectrophotometer in the range 4000–400 cm 1 with resolution of 4 cm 1. Samples were prepared as KBr pellets. The 1H NMR spectra were recorded on a Bruker 250 MHz Avance II spectrometer operating at 250.1 MHz. 13C NMR spectra were recorded on a Varian INOVA 500 MHz (11.744T) operating at 125.7 MHz. The 1H and 13C NMR chemical shifts were referenced to tetramethylsilane (TMS). The 15N NMR data were recorded on a Bruker Avance III 400 MHz (9.395T) operating a 400.1 MHz. The 15N NMR spectra were indirectly obtained by the [1H–15N] Multiple Bond Coherence technique (HMBC). Samples were prepared in deuterated dimethyl sulfoxide solutions (DMSO-d6). Thermogravimetric and differential thermal analysis (TGA/DTA) were performed on a Simultaneous TGA/DTA SEIKO EXSTAR 6000 Thermoanalyzer, using the following conditions: synthetic air, flow rate of 50 cm3 min 1 and heating rate of 10 °C min 1, from 25 °C to 900 °C. The residue after thermal treatment at 900 °C was analyzed on a Shimadzu XRD-6000 diffractometer using Cu Ka radiation (k = 1.5406 Å), voltage of 40 kV and current of 30 mA, with a graphite monochromator and at room temperature. 2.2. Synthesis of the complex The gold(I) complex with 2-mercaptothiazoline (Au–MTZ) was synthesized by the reaction of 1.0 mL of an aqueous solution containing 3.5  10 4 mol of K[Au(CN)2] with 7.5 mL of a freshly prepared aqueous solution of MTZ hydrochloride, containing 3.75  10 4 mol of the ligand. The synthesis of the complex was carried out with stirring and at room temperature. After 1 h of constant stirring, the white solid obtained was collected by filtration, washed with cold water and dried in a desiccator over P4O10. The yield was 85%. Anal. Calc. for Au(CN)(C3H6NS2): C, 13.9; H, 1.77; N, 8.16. Found: C, 14.6; H, 2.20; N, 8.30%. ESI-MS (m/z): 434.9 [Au(C3H5NS2)2+H]+ (100%); 342.9 [Au(CN)(C3H5NS2)+H]+ (30%); 436.9 [Au(C3H5N34S2)2+H]+ (20%). The complex is soluble in dimethyl sulfoxide (DMSO). It is insoluble in water, ethanol, methanol, acetonitrile and hexane. No single crystals were obtained, even several attempts using mixtures of solvents, in order to provide a full X-ray characterization of the complex. 2.3. Molecular modeling Geometric optimizations were carried out using the GAMESS software [20] with a convergence criterion of 10 4 a.u. in a conjugate gradient algorithm. The LANL2DZ effective core potential was used for gold and the atomic 6-31G(d) basis set [21–25] for all other atoms. Density Functional Theory (DFT) calculations were carried out by using the B3LYP [26,27] gradient-corrected hybrid to solve the Kohn–Sham equations with a 10 5 a.u. convergence criterion for the density change. The coordination through the nitrogen atom was confirmed as a minimum of the potential energy surface with calculations of the hessians showing no imaginary frequencies.

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2.4. Antimicrobial assays Three referenced bacteria (E. coli – ATCC 25922, P. aeruginosa – ATCC 27853, and S. aureus – ATCC 25923) were used for the antibacterial experiments. The antibiogram assay was performed by the disc diffusion method [28,29]. The sensitivity of Au–MTZ complex was tested in Mueller–Hinton (MH) agar. The microorganisms were transferred to separate test tubes containing 5.0 mL of sterile brain heart infusion (BHI) medium and incubated for 18 h at 35– 37 °C. Sufficient inocula were added in new tubes until the turbidity equaled 0.5 McFarland (1.5  108 CFU mL 1). The bacterial inocula diluted with BHI (McFarland standard) were uniformly spread using sterile cotton swabs on sterile Petri dishes containing MH agar. Sterile filter paper discs (10 mm diameter) were aseptically impregnated with 400 lg of Au–MTZ according to the following procedure: 20.0 mg of Au–MTZ were suspended in 1000 lL of dimethylsulfoxide, homogenized in a vortex, and 20 lL of the solution were collected with a micropipette and transferred to the paper discs. Sterile discs impregnated with 800 lg of pure MTZ were used as a negative control. Discs impregnated with the Au–MTZ complex or with MTZ were dried and sterilized in a vertical laminar flow under UV radiation for 45 min before the experiment. All impregnated discs were placed on the surface of the solid agar. The plates were incubated for 18 h at 35–37 °C and examined thereafter. Clear zones of inhibition around the discs were measured and the complex sensitivity was assayed from the diameter of the inhibition zones (in millimeters). Experiments were performed in triplicate and the results were compared to the standard antibiotics gentamicine (GEN) and ceftriaxone (CRO).

2.5. Tumor cell culture and cytotoxic assays HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% of Fetal Calf Serum (FCS), using streptomycin and penicillin as antibiotics, in an atmosphere of 5% CO2 at 37 °C. All cell culture reagents were purchased from Invitrogen (Gaithersburg, MD), unless specified. Flasks and plates were purchased from Costar (Corning Inc., NY). Cisplatin, used as a positive control, was purchased from Acros and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) from Sigma. Cells were placed in a 48-well plate (4  104 cells/ well) 24 h prior to the beginning of the experiment. A stock solution of the Au–MTZ complex was prepared in dimethylsulfoxide (DMSO), which was directly diluted into the cells medium in order to achieve different concentrations. After dilution of the stock solution, DMSO was present in the cells medium at 1%. This amount of DMSO was assayed as the vehicle for the negative control. Fortyeight hours after addition of the complex, MTT salt was added (aiming for a final concentration of 0.50 mg mL 1) and the cells were incubated with this reagent for a period of 3 h [30,31]. After washing with PBS, isopropanol was added and cell viability was determined by absorbance measurements at 570 nm.

Coordination of the nitrogen atom of MTZ to Au(I) was evaluated by 15N NMR spectroscopy. The 15N NMR spectra are provided in Fig. 2. The 15N chemical shifts for Au–MTZ and free MTZ were indirectly obtained from the 2D spectra via the heteronuclear [1H–15N] multiple bond coherence technique (HMBC), as described for other metal complexes with N-donor ligands [32,33]. The assignment of the nitrogen resonance was performed by its correlation with protons H3a and H3b in Fig. 1. In the MTZ spectrum, the 15 N chemical shift was observed at 154.4 ppm while for the complex it was observed downfield at 169.7 ppm. The observed Dd of 15.3 ppm indicates coordination of MTZ to Au(I) through the N atom [34,35]. The 1H NMR spectra of MTZ and the Au–MTZ complex are shown in Fig. 3. It is important to note the presence of the two tautomeric forms (thione and thiol, Fig. 1a and b) of MTZ in solution. Thus, in order to make a consistent assignment of the hydrogen atom bonded either to sulfur or nitrogen atoms (H1a or H1b in Fig. 1a and b, respectively) the coexistence of the two tautomers needs to be considered [36,37]. In the spectrum of MTZ, the proton bonded to the N atom (Fig. 3) is observed as a singlet at 10.1 ppm. When the proton is bonded to the S atom the 1H NMR signal is observed at 3.30 ppm. These assignments are in agreement with other thiazolidine derivatives previously described in the literature [38]. Upon coordination, the proton bonded to the nitrogen is observed as a very low intensity peak at 11.9 ppm when compared to the 1H NMR signal of the proton bonded to the S atom, which becomes more intense. The observed data suggest the predominance of the thiol tautomer (N@C–SH, Fig. 1b) and support the proposition of nitrogen coordination of MTZ to gold(I). Also, significant differences in the chemical shifts of H2a,b are observed when the NMR data of the ligand and the complex are compared, which reinforces coordination through the N atom. The hydrogens H2a,b are found at 4.18 in the spectrum of the complex. The 1H NMR data are summarized in Table 1. The 13C NMR data are also presented in Table 1. When the ligand and the complex spectra are analyzed, only minor differences in chemical shifts are observed. As described in Table 1, a Dd (d

3. Results and discussion 3.1. NMR spectroscopic measurements Solution-state 1H, 13C{1H} and 15N NMR spectra of the Au–MTZ complex were analyzed by comparison with the NMR spectra of pure MTZ. The tautomeric structures of MTZ are presented in Fig. 1a and b. In addition, the proposed structure of the Au–MTZ complex is presented in Fig. 1c.

Fig. 2. [1H–15N] HMBC spectra of MTZ and Au–MTZ.

2357

(b)MTZ

ppm 12

11

Transmitance (a.u.)

3.30 (SH)

10.1 (NH)

3.85

11.7 (NH)

(a)Au-MTZ

3.49

4.18

3.75 (SH) 3.65

C. Abbehausen et al. / Polyhedron 30 (2011) 2354–2359

10

Au-MTZ MTZ

4

3

4000

3500

3000

2500

2000

1500

Wavenumber / cm

1

Fig. 3. H NMR of Au–MTZ (a) and MTZ (b).

1000

500

-1

Fig. 4. IR spectra of MTZ and Au–MTZ. Table 1 13 C and 1H NMR chemical shifts for MTZ and Au–MTZ. Compound

MTZ Au–MTZ

Chemical shifts (ppm) H2

H3

C1

C2

C3

CN

3.85 4.18

3.49 3.65

201.6 198.9

54.10 53.88

35.56 34.26

– 105.0

complex–d ligand) of 2.7 ppm was observed for carbon atom C1 while for C2 and C3 the Dd values are 0.22 ppm and 1.30 ppm. The 13C NMR spectra are shown in the supporting information.

3.4. Thermal analysis Thermogravimetric and Differential Thermal Analysis (TG/DTA) were carried out in order to confirm the complex composition and also to provide new data concerning the thermal stability of the compound. Ligand oxidation starts at 160 °C and occurs in three steps leading to the formation of Au0 at 1000 °C. Anal. Calc. for loss of [(C3H5NS2)(CN)]: 42.05. Found: 39.07%. Anal. Calc. for residue Au0: 57.05. Found: 60.90%. The TG/DTA curves are also shown in the supporting information.

3.2. Mass spectrometric data 3.5. Molecular modeling The mass spectrometric analysis confirmed the composition of the Au–MTZ complex as early proposed by considering the elemental and NMR data. Under acidic medium the molecular ion is observed at 342.9 m/z. This mass corresponds to the monoprotonated complex [Au(CN)(C3H5NS2)+H]+. Also, the cyanide group can be displaced after fragmentation by a second molecule of MTZ and the [Au(C3H5NS2)2+H]+ species is observed at 434.9 m/z. 3.3. Infrared spectroscopic measurements The IR spectrum of Au–MTZ was analyzed in comparison to that of free MTZ. Both spectra are shown in Fig. 4. A medium intensity band at 3135 cm 1, which can be attributed to the N–H stretching mode, is observed in the spectrum of pure MTZ. The absence of the N–H stretching mode in the spectrum of the complex confirms coordination through the nitrogen atom, as proposed by 1H and 15N NMR data. Weak absorptions in the region 3000–2850 cm 1 are assigned to asymmetric and symmetric stretching modes of CH2 groups. In the free ligand they are observed at 2931 cm 1 and 2848 cm 1 while in the complex they are found in the range 3000– 2870 cm 1. A sharp strong band is observed at 1517 cm 1 in the spectrum of the free ligand, being assigned to C–N stretching. After coordination, this band is observed at 1537 cm 1, which indicates the predominance of the (N@C–SH) tautomeric form when the complex is formed. A medium intensity band at 2146 cm 1, present only in the spectrum of the complex, is attributed to the C„N stretching of the cyanide group coordinated to the metal [39,40].

Theoretical investigations of the coordination of MTZ to Au(I) through sulfur and nitrogen atoms were performed in order to confirm the coordination structure of the complex. According to these studies, coordination through nitrogen was shown to be the more stable, being in agreement with the experimental data discussed previously. The calculated Au–N distance is 211 pm, Au–CN distance is 198 pm and the angle NC–Au–N is 179°. The simulated IR is also in agreement with experimental values. It is possible to see a sharp weak band in 2556 cm 1, being assigned to the SH stretching mode in the simulated spectrum. This band is absent in the experimental spectrum due to its very weak intensity. Bands at 2983 cm 1 and 2940 cm 1 are assigned to the asymmetrical and symmetrical CH2 stretching modes. The C„N stretching is observed at 2208 cm 1 being in accordance with experimental data for the cyanide coordinated to Au(I) in the complex. The C–N stretching mode of the MTZ ring is observed at 1568 cm 1 and C–S stretching at 912 cm 1. The simulated spectra are shown in the supporting information. 3.6. Antimicrobial assays Antibiogram assays revealed the antibacterial activity of the Au–MTZ complex against Gram-negative and Gram-positive microorganisms. The impregnated paper discs with Au–MTZ exhibited inhibition zones for E. coli, P. aeruginosa and S. aureus of 30.0 ± 0.1 mm, 32 ± 0.1 mm and 26.0 ± 0.1 mm, respectively. The inhibition zones indicate that these bacterial strains are sensitive to the Au–MTZ complex, being similar to the results ob-

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Table 2 Antibiotic sensitivity profiles for the bacterial strains. Compounds

Au–MTZ (400 lg) MTZ (800 lg) DMSO CRO GEN

Results E. coli Inhibition zone diameter (mm)

P. aeruginosa Inhibition zone diameter (mm)

S. aureus Inhibition zone diameter (mm)

30.0 (±0.1)

32.0 (±0.1)

26.0 (±0.1)

0.0

0.0

0.0

0.0 >34 24.0 (±0.1)

0.0 35.0 (±0.1) 25.0 (±0.1)

0.0 >40 26.0 (±0.1)

tained for the standard antibiotics ceftriaxone and gentamicine. The observed results are also comparable to the recently published data of gold(I) and silver(I) complex with N-acetyl-L-cysteine [13,41], which exhibits antibacterial activities against E. coli, S. aureus and P. aeruginosa microorganisms. Pure mercaptothiazoline did not exhibit antibacterial activity against the tested bacterial strains using the same experimental conditions. The antibiotic sensitivity profiles of the bacterial strains are listed in Table 2. 3.7. Tumor cells cytotoxic assays

% de viabilidade Cell viability,celular %

The Au–MTZ complex was assayed for its cytotoxic activity using concentrations varying from 2.0 lmol L 1 to 200 lmol L 1, with the vehicle (DMSO) as a negative control and cisplatin as a positive control. The results show a significant cytotoxic effect for the Au–MTZ complex against the human adenocarcinoma HeLa cells even at the lowest concentration assayed (2.0 lmol L 1), with 85% of cell death (Fig. 5). An increase in the concentration of the Au–MTZ complex did not result in significant increase of cell death. Pure MTZ did not exhibit cytotoxic activities against HeLa cells with the same experimental conditions (data not shown). The observed results are comparable to the in vitro activity of a series of triphenylphosphine–gold(I) sulfanylcarboxylate complexes against human HeLa-229 cervix carcinoma cells [42]. The experimental data for the Au–MTZ complex are also comparable to the activities of benzimidazol-2-ylidene–gold(I) complexes [7] and to a series of chloro–gold(I) phosphine complexes [43], which exhibit antitumor activities against MCF-7 (human breast adenocarcinoma) and HT29 (colon carcinoma) cells in the micromolar range.

Fig. 6. Optimized structure for the Au–MTZ complex.

4. Conclusion A new gold complex with 2-mercaptothiazoline (Au–MTZ) with a 1:1 M composition (metal:ligand) was obtained and structurally characterized. The 1H, 13C and 15N NMR data, as well as mass spectrometric and IR spectroscopic measurements, support coordination of the ligand to Au(I) via nitrogen. Based on the chemical, spectroscopic and DFT results a schematic structure for the Au(I)–MTZ complex is shown in Fig. 6. Biological studies revealed the effective antibacterial activity of the complex against Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus) microorganisms, while the antitumoral assays showed a significant cytotoxic effect of the Au–MTZ against HeLa cells, with inhibition of cell proliferation in the lmol L 1 range. Acknowledgments This study was supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil – Proc. 2006/ 55367-2) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil – Proc. 141617/2010-1). Authors are also grateful to Professor Carol H. Collins from Campinas State University – UNICAMP for the manuscript revision. Appendix A. Supplementary data

125

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.poly.2011.06.021.

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75 50 25

[8]

0 DMSO

2µ2M

2020 µM

200 200 µM

Fig. 5. Cytotoxic analyses of the Au–MTZ complex in different concentrations (lmol L 1) against HeLa cells. Data from three independent experiments were normalized to the maximum values obtained for the control group and are expressed as mean ± s.e.m.

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