Nickel(II) complexes of N′-(2-thienylcarbonyl)thiocarbamates O-alkyl-esters: Structural and spectroscopic characterization and evaluation of their microbiological activities

Journal of Molecular Structure 990 (2011) 86–94

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Nickel(II) complexes of N0 -(2-thienylcarbonyl)thiocarbamates O-alkyl-esters: Structural and spectroscopic characterization and evaluation of their microbiological activities Lígia R. Gomes a,b,⇑, John Nicolson Low c, Marisa A.A. Rocha d, Luís M.N.B.F. Santos d, Bernd Schröder e, Paula Brandão e, Carla Matos a, José Neves a,f a

CIAGEB – Faculdade de Ciências de Saúde, Escola Superior de Saúde da UFP, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal REQUIMTE – Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, P-4169-007 Porto, Portugal c Department of Chemistry, University of Aberdeen, Meston Walk, Old Aberdeen AB24 3UE, Scotland, United Kingdom d Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, P-4169-007 Porto, Portugal e CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal f IBB-Instituto para a Biotecnologia e a Bioengenharia, Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, P-4710-057 Braga, Portugal b

a r t i c l e

i n f o

Article history: Received 29 November 2010 Received in revised form 9 January 2011 Accepted 12 January 2011 Available online 22 January 2011 Keywords: Thiocarbamates O-alkyl-esters Ni(II) complexes Rx Spectroscopic characterization Antimicrobial activity

a b s t r a c t In the present work a set of five Ni(II) complexes with general formula [Ni(L)2] and with HL = N-thenoylthiocarbamic-O-n-alkylylesters (n = 2–6) has been prepared and characterized in solution by UV–vis and NMR spectroscopies. Three of them were also characterized in the solid state by X-ray diffractometry. The energy rotation of the thiophene ring of ligand was evaluated theoretically. Liposomes of complexes were prepared in order to evaluate their ability to interact with the membrane. Furthermore, their biological activities were evaluated in a set of bacteria (gram+ and gram) and yeasts. The X-ray structure determination confirms that bidentate ligand forms a tetra co-ordinated complex with an S2O2 co-ordination sphere around the nickel(II) ion in a cis configuration. The metal centre is coordinated in a square planar fashion. NMR spectra taken in solution show a diamagnetic signal compatible with a square-planar geometry around the metal centre. The values obtained for the liposome/water partition coefficients (Kp) show that [Ni(ttete)2] and [Ni(ttpre)2] have a similar membrane partition ability, whilst the [Ni(ttbue)2] derivative presents a significantly higher Kp, describing a stronger interaction within the membrane. For all the compounds, [Ni(ttpre)2] has a higher efficacy against Gram negative bacteria and yeasts nevertheless, the anti-yeast and anti-bacterial activity values of all tested compounds are lower than ones of the reference compounds. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction There are several metal complexes of derivatives of dialkyldithiocarbamic acids, of alkylene-bis-dithiocarbamic acids and of thiourea which have become known and widely used as fungicides in plant protection. The antifungal activities of new dialkylthiourea derivatives and their metal complexes have been exploited in the last decade. A synopsis of the use of metal complexes of thiourea, triazole and thiadiazine compounds as antifungal agents has been recently made [1]. In parallel with the search for antimicrobial activity, the interest in platinum(II) and palladium(II) complexes ⇑ Corresponding author at: CIAGEB – Faculdade de Ciências de Saúde, Escola Superior de Saúde da UFP, Universidade Fernando Pessoa, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal. Tel.: +351 225 074 630; fax: +351 225 074 637. E-mail address: [email protected] (L.R. Gomes). 0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2011.01.021

of dithiocarbamates has risen in the last two decades, since they appear to exhibit high anticancer activity associated with reduced nephrotoxicity, as compared to cisplatin and its analogues. Palladium(II) and nickel(II) derivatives containing a sulphur chelating ligand appear to be able to bind strongly to the metal centre, thus preventing interactions with sulphur-containing enzymes, reactions that are believed to be responsible for the nephrotoxicity induced by platinum(II)-based drugs [2,3]. The structural and chemical properties of mono-thiocarbamates complexes were reviewed some decades ago [4,5]. Thioureas and thiocarbamates and their metal complexes have also been of considerable interest in the chemical field because of the wide variation in their modes of binding and stereochemistry [6–8]. In the present work a set of five Ni(II) complexes with general formula [Ni(L)2] and with HL = N-thenoylthiocarbamic-O-n-alkylylesters (n = 2–6) as schematically shown in Fig. 1, has been

L.R. Gomes et al. / Journal of Molecular Structure 990 (2011) 86–94

O

R

O

R

S

S Ni

N

87

O

N O

S

S

[Ni(ttete)2]: Bis[O-etylester-N'-(2-thienylcarbonyl)thiocarbamato] nickel(II)

R=-CH2CH3

[Ni(ttpre)2]: Bis[O- propylester-N'-(2-thienylcarbonyl)thiocarbamato] nickel(II)

R=-CH2CH2CH3:

[Ni(ttbue)2]: Bis[O- butylester -N'-(2-thienylcarbonyl)thiocarbamato] nickel(II)

R=-CH2CH2CH2CH3

[Ni(ttpte)2]: Bis[O- pentylester -N'-(2-thienylcarbonyl)thiocarbamato] nickel(II)

R=-CH2CH2CH2CH2CH3

[Ni(tthxe)2]: Bis[O- hexylester -N'-(2-thienylcarbonyl)thiocarbamato] nickel(II)

R=-CH2CH2CH2CH2CH2CH3

Fig. 1. General formula of the compounds studied. [Ni(ttete)2]: Bis[O-etylester-N0 -(2-thienylcarbonyl)thiocarbamato] nickel(II), R = ACH2CH3; [Ni(ttpre)2]: Bis[O-propylesterN0 -(2-thienylcarbonyl)thiocarbamato] nickel(II), R = ACH2 CH2CH3; [Ni(ttbue)2]: Bis[O-butylester-N0 -(2-thienylcarbonyl)thiocarbamato] nickel(II), R = ACH2CH2CH2CH3; [Ni(ttpte)2]: Bis[O-pentylester-N0 -(2-thienylcarbonyl)thiocarbamato] nickel(II), R = ACH2CH2CH2CH2CH3; [Ni(tthxe)2]: Bis[O-hexylester-N0 -(2-thienylcarbonyl)thiocarbamato] nickel(II), R = ACH2CH2CH2CH2CH2CH3.

prepared and characterized in solution by UV–vis and NMR spectroscopies and three of them were characterized in the solid state by X-ray diffractometry. Liposomes of complexes were prepared in order to evaluate their ability to interact with the membrane. Furthermore, their biological activities were evaluated in a set of bacteria (gram+ and gram) and yeasts. 2. Experimental 2.1. Instruments and measurements NMR spectra were recorded on a Bruker ARX-300 using CDCl3 as solvent and TMS as internal standard. UV–vis spectra were recorded using a diode array spectrophotometer, an Agilent 8453 UV–visible spectroscopy system with temperature control. For evaluation of the partition coefficient in liposomes the spectra of all suspension was collected in a double-beam spectrophotometer (Perkin Elmer, Lambda 25), equipped with UV WinLab V2.85 software for data acquisition, with buffer as reference sample. 2.2. Synthesis of ligands and complexes All reagents and solvents were reagent grade. 2.2.1. Ligands The N-thenoylthiocarbamic-O-n-alkylesters studied in this paper were prepared according to the described procedure [9]. Essentially they were prepared by the addition of thenoylisothiocyanate (dissolved in toluene) to the corresponding alcohol (two molar excess), at room temperature under stirring and slowly heated up to 333 K for 1 h. After 24 h, the reaction crude was dissolved in methanol and water. Continuous washing leads to pure, yellow crystals. 2.2.2. Complexes The preparation of the corresponding complexes has been also described elsewhere [10] and was carried out as follows: 10 mmol N-thenoyl-thiocarbamic-O-alkylester were dissolved in ethanol. To this solution 5 mmol Ni(CH3COO)24H2O dissolved in ethanol were added slowly under stirring. The precipitate was filtered off and dissolved in dichloromethane. Ethanol was added slowly without stirring, the mixture was kept aside. The complexes formed were filtered off, dried and recrystallized from acetone,

resulting in reddish-dark brown crystals, with Tfus = 444.7 K (Ni(ttete)2), 422.7 K (Ni(ttpre)2), 431.8 K (Ni(ttbue)2), 441.5 K (Ni(ttpte)2), 408.4 K (Ni(tthxe)2), respectively. The purity of the complexes was verified by elemental analysis and 1H NMR;

2.2.3. Elemental analysis The mass fraction w of C, H, N and S were as follows: for [Ni(ttete)2], (C16H16N2O4S4)2Ni, found 102 w(C) = 39.7, 102 w(H) = 3.2, 102 w(O) = 14.0, 102 w(N) = 5.9, 102 w(S) = 25.8, calculated 102 w(C) = 39.44, 102 w(H) = 3.31, 102 w(O) = 13.13, 102 w(N) = 5.75, 102 w(S) = 26.3; for [Ni(ttpre)2], (C18H20N2O4S4)2Ni, found 102 w(C) = 41.8, 102 w(H) = 4.0, 102 w(O) = 14.7, 102 w(N) = 5.3, 102 w(S) = 25.8, calculated 102 w(C) = 41.95, 102 w(H) = 3.91, 102 w(O) = 12.42, 102 w(N) = 5.44, 102 w(S) = 24.89; for [Ni(ttbue)2], (C20H24N2O4S4)2Ni, found 102 w(C) = 44.5, 102 w(H) = 4.3, 102 w(O) = 13.0, 102 w(N) = 5.1, 102 w(S) = 24.3, calculated 102 w(C) = 44.21, 102 w(H) = 4.45, 102 w(O) = 11.78, 102 w(N) = 5.16, 102 w(S) = 23.61; for [Ni(ttpte)2], (C22H28N2O4S4)2Ni, found 102 w(C) = 46.0, 102 w(H) = 4.9, 102 w(O) = 13.5, 102 w(N) = 4.8, 102 w(S) = 22.9, calculated 102 w(C) = 46.24, 102 w(H) = 4.94, 102 w(O) = 11.20, 102 w(N) = 4.90, 102 w(S) = 22.45; for [Ni(tthxe)2], (C24H32N2O4S4)2Ni, found 102 w(C) = 48.1, 102 w(H) = 4.8, 102 w(O) = 13.7, 102 w(N) = 4.6, 102 w(S) = 21.4, calculated 102 w(C) = 48.08, 102 w(H) = 5.38, 102 w(O) = 10.68, 102 w(N) = 4.67, 102 w(S) = 21.40.

2.2.4. 1H NMR (CDCl3, 300 MHz) For [Ni(ttete)2]: 7.91–7.89 (d, 2H, Arom), 7.66–7.64 (d, 2H, Arom), 7.18–7.15 (t, 2H, Arom), 4.58–4.50 (qua, 4H, CH2), 1.45– 1.40 (t, 6H, CH3). For [Ni(ttpre)2]: 7.91–7.89 (d, 2H, Arom), 7.66–7.64 (d, 2H, Arom), 7.18–7.15 (t, 2H, Arom), 4.46–4.41 (qua, 4H, CH2), 1.86– 1.79 (m, 4H, CH2); 1.07–1.02 (t, 6H, CH3). For [Ni(ttbue)2]: 7.91–7.89 (d, 2H, Arom), 7.66–7.64 (d, 2H, Arom), 7.18–7.15 (t, 2H, Arom), 4.51–4.46 (qua, 4H, CH2), 1.80– 1.60 (m, 4H, CH2); 1.45–1.40 (m, 4H, CH2) 0.99–0.94 (t, 6H, CH3); for [Ni(ttpte)2]: 7.91–7.89 (d, 2H, Arom), 7.66–7.64 (d, 2H, Arom), 7.18–7.15 (t, 2H, Arom), 4.50–4.45 (qua, 4H, CH2), 1.84–1.75 (m, 4H, CH2); 1.60–1.58(m, 4H, CH2) 1.45–1.40 (m, 4H, CH2) 0.99– 0.94 (t, 6H, CH3); for [Ni(tthxe)2]: 7.91–7.89 (d, 2H, Arom), 7.66– 7.64 (d, 2H, Arom), 7.18–7.15 (t, 2H, Arom), 4.50–4.45 (qua, 4H,

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CH2), 1.84–1.74 (m, 4H, CH2); 1.61–1.57 (m, 4H, CH2); 1.44–1.31 (br, 6H, CH2) 0.97–0.92 (t, 6H, CH3). 2.3. X-ray crystallography X-ray quality single crystals for compounds were obtained by slow evaporation of a methanolic/dichloromethane (1:1) solution of the complex [Ni(ttete)2] and a dichloromethane solutions of [Ni(ttpte)2] and [Ni(tthxe)2]. The intensity data were collected by a Bruker-Nonius CCD diffractometer. Data collection, cell refinement and data reduction were made with the software package of the diffractometer: COLLECT [11] for data collection; SMART and SAINT [11] for cell refinement and for data reduction. Absorption correction was performed with SADABS [12]. The structure was solved and refined using the software: OSCAIL [13] and SHELXL97 [14]. H atoms were treated as riding atoms with CAH(aromatic), 0.95 Å, CAH(CH2), 0.99 Å, with Uiso(H) = 1.2Ueq(C), CAH(methyl), 0.98 Å, with Uiso(H) = 1.5Ueq(C). The thiophene groups in [Ni(ttpte)2] and in [Ni(tthxe)2] were found to be disordered in both ligands by rotation through 180° about the C1AC11 bond. Details of the treatment of the disorder for each compound can be found in the respective deposited .cif files. Molecular graphics were produced by ORTEPIII [15] and PLATON [16]. The complete set of structural parameters in CIF format is available as an Electronic Supplementary Publication from the Cambridge Crystallographic Data Centre (CCDC 717799, 780484 and 795858, [Ni(ttete)2], [Ni(tthxe)2] and [Ni(ttpte)2] respectively. Crystal data, data acquisition conditions and refinement parameters for the compounds are listed in Table 1. 2.4. Theoretical calculations One-dimensional potential energy scans have been performed, using Turbomole V6.2 [17]. Relaxed scans around the dihedral

angle formed by the thiophene ring and the acyl carbon have been accomplished at the RI-DFT BP level using the def-TZVP basis set [18]. Two different starting geometries of the complex have been considered, one with the thiophene sulphur atoms both pointing outwards, another with both sulphur atoms both pointing inwards, while the thiophene rings are in plane with the coordination center. A similar scan at the same conditions has been made for the most stable conformer of the free ligand. 2.5. Liposome preparation Egg phosphatydilcholine (EPC) and Hepes were purchased from Sigma–Aldrich, and used as supplied. Solutions were prepared with 1  102 mol dm3 Hepes buffer, being the ionic strength adjusted to 0.1 mol dm3 with NaCl. Double-deionized water (conductivity less than 0.1 lS cm1) was used for all solutions. Liposomes were prepared by evaporation to dryness of a lipid solution in chloroform with a stream of nitrogen. The lipid film, was sufficient to yield a final lipid concentration of around 1  103 mol dm3, was then left under vacuum overnight to remove all traces of the organic solvent. After the addition of Hepes buffer, multilamellar vesicles (MLVs) were formed by vortexing the mixture. The EPC concentration in all vesicle suspensions was determined by phosphate analysis using the Fiske and Subbarow phosphomolybdate method [19]. 2.6. Evaluation of the liposome/water partition coefficient (Kp) In the derivative spectrophotometry studies, two series of buffered suspensions containing increasing concentrations of EPC (in the range 0–250 lM) were prepared: (1) one contained a fixed concentration of the compound under analysis and (2) the corresponding reference solutions were prepared identically but without compound. All suspensions were then vortexed and incubated in

Table 1 Crystal data, data acquisition conditions and refinement parameters for Ni (II) complexes.

Empirical formula Formula weight (g mol1) Temperature (K) Wavelength (Mo Ka) (Å) Crystal system, space group Unit cell dimensions a (Å) b (Å) c (Å) b (°) Volume (Å3) Z, calc. density, q (Mg m3) Absorpt. Coeff., l (mm1) Max. (Tmax) and Min. transmission (Tmin) Crystal size (mm) F(0 0 0) h range for data collection (°) Index ranges

Reflection collected Independent reflections Reflections with I > 2r(I); completeness to h = 27.5° Absorption correction Refinement method GooF Data/parameters/constrains Final R indices (I > 2r(I)) R indices (all data) Maximum and minimum difference peaks (e Å3) CCDC

[Ni(Httete)2]

[Ni(Httpte)2]

[Ni(Htthxe)2]

C16H16N2NiO4S4 Mr = 487.26 T = 150 (2) 0.71073 Monoclinic, P21/c

C22H28N2NiO4S4 Mr = 571.41 T = 150 (2) 0.71073 Monoclinic, C2/c

C24H32N2NiO4S4 Mr = 599.47 T = 150 (2) 0.71073 Monoclinic, C2/c

10.732(12) 11.158(2) 16.111(14) 92.701(4) 1927.1 (9) 4, 1.679 1.47 0.944, 0.758 0.20  0.20  0.04 1000.0 1.9–27.5 13 6 h 6 13 14 6 k 6 14 20 6 l 6 20 67 034/4 432/4 004 Rint = 0.031 100% Multi-scan Full-matrix least squares on F2 S = 1.17 4432/246/0 R = 0.039, wR = 0.094 R = 0.044, wR = 0.096 Dqmax = 0.73 Dqmin = 0.44 717799

13.8399(12) 10.2489(8) 19.5922(18) 114.438(6) 2530.1 (4) 4, 1.500 1.13 0.896, 0.946 0.10  0.10  0.05 1192.0 1.9–27.6 18 6 h 6 18 13 6 k 6 13 25 6 l 6 25 18,225/2923/3050 Rint = 0.198 99.4% Multi-scan Full-matrix least squares on F2 S = 0.99 2923/146/15 R = 0.061, wR = 0.136 R = 0.156, wR = 0.292 Dqmax = 0.93 Dqmin = 0.69 795858

23.1365(17) 10.3362(6) 15.8017(12) 130.944(3) 2454.4 (3) 4, 1.395 1.00 0.825, 0.971 0.20  0.16  0.03 1256.0 1.9–28.3 30 6 h 6 29 13 6 k 6 13 20 6 l 6 21 27,295/3534/3050 Rint = 0.030 99.8% Multi-scan Full-matrix least squares on F2 S = 1.04 3532/178/15 R = 0.026, wR = 0.061 R = 0.034, wR = 0.066 Dqmax = 0.37 Dqmin = 0.24 780484

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the dark for two hours at room temperature to allow them to reach equilibrium. The second derivative spectra were calculated after subtracting the blank data and spectra smoothing, by the use of Origin 6.1, using 12 points in the derivative calculus. The partition coefficients were determined by performing a non-linear fitting of the proposed mathematical model [20,21] to the experimental data using the same computer program. At least two independent experiments were conducted for each compound under analysis. The theoretical background and mathematical formalism of the methodology has been described elsewhere [20,21] and some additional information is given in supporting information.

properly labelled and incubated at 37 °C for 24 h for bacteria and at 30 °C for 48 h (fungi). At the end of 24 h incubation growth (turbidity in broth) was observed and compared with the controls. The lowest concentration of the compounds that prevented visible growth was considered to be minimal inhibitor concentrations (MICs). Solvent, media and positive growth controls were also run simultaneously.

2.7. Antimicrobial activity

The nickel(II) complex [Ni(ttete)2] crystallizes in the monoclinic space group P21/c while [Ni(ttpte)2] and [Ni(tthxe)2] crystallise in the monoclinic space group C2/c The asymmetric unit for [Ni(ttete)2] is shown in Fig. 2. In the remaining complexes, the asymmetric unit corresponds to half of the complex being the two parts related by a mirror plane perpendicular to the coordination plane that passes through the nickel atom. ORTEP diagrams for [Ni(ttpte)2] and [Ni(tthxe)2] are presented in Figs. 3 and 4. The Xray structure determination confirms that the complexes show the expected molecular structure. In all of them, the bidentate ligand forms a tetra co-ordinated complex with an S2O2 co-ordination sphere around the nickel(II) ion in a cis configuration. The metal bonds to two ligands and the coordination is taking place via the amidic oxygen and the thiocarbamic sulphur atoms of each ligand. N0 -(aryl)thiocarbamatoalkylesters coordinate usually in an S,O-bidentate manner and have been found to exhibit S,S-cis stereo-chemical configuration, although several examples of coordination through the S atom and an S,N-donor-atom set have been reported [23,24]. In these three complexes, the metal center is four coordinated in a square planar fashion. For [Ni(ttete)2] the maximum deviations of the atoms from the best plane formed by the five central atoms are: 0.006 (1), 0.006 (1), 0.005 (1), 0.005 (1) and 0.001 (1) Å, for O1, O3, S1, S3 and Ni1, respectively. These deviations are lower than those presented by [Ni(ttpre)2] that shows a slight distortion from square-planar geometry [25]. [Ni(ttpte)2] and [Ni(tthxe)2] exhibit square-planar geometries for their coordination spheres as imposed by the crystallographic symmetries.

The ‘‘in vitro’’ anti-bacterial activity of the complexes was investigated in several representative Gram positive bacteria (Sarcina lutea, ATCC 9341, Staphylococcus aureus, ATCC 29213 S. aureus methicillin-resistant (clinical isolate) and Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 Klebsiella pneumonia ((clinical isolate)). Also antifungal activity was tested against yeasts (Candida albicans ATCC 10231, Candida krusei ATCC 6258. All microrganisms were obtained from the culture collection of Fernando Pessoa University. Stock solutions of [Ni(ttete)2] were prepared in dimethyl sulfoxide (DMSO) which had no effect on the microorganisms in the concentrations studied. Controls with DMSO were adequately done. All of the dilutions were done in the media to concentrations ranging from 208  103 g dm3 to 1.5  103 g dm3. A 1:10 dilution of 24 h culture of the test microorganism was made. Broth was used to adjust the diluted culture until the turbidity compared with McFarland standard number 0.5. The MIC values were determined according a modified method [22]. In a 96 well microplate columns of the microorganisms (50  106 dm3) were tested. For each one, 50 lL of broth, was dispensed into the microplate and 1:2 serial dilution of the stock solution was carried out starting from 100  106 dm3. Positive controls were made only with test microorganism and broth and negative controls were made with media sterility. The above procedure was duplicated. All inoculated microplates were

3. Results and discussion 3.1. Crystal structures for complexes

Fig. 2. ORTEP view of [Ni(ttete)2] showing the atom labelling. Ellipsoids represent 30% probability level.

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Fig. 3. ORTEP view of [Ni(ttpre)2] showing the atom labelling. Ellipsoids represent 30% probability level.

Fig. 4. ORTEP view of [Ni(tthee)2] showing the atom labelling. Ellipsoids represent 30% probability level.

The bond lengths between atoms of the co-ordination rings are within the range reported for similar complexes, both derived from thioureas [26–28] and from thiocarbamates. In particular, the values obtained for [Ni(ttete)2] are similar to those presented for the analogous compound [Ni(ttpre)2] [25]. Nevertheless, all complexes show some significant (at 3r level) differences in bond distances values between atoms of the thiocarbamic ester functional group when compared with the free ligands [29], Table 2. In the complexes the CAO (carbonyl) group and CAS from (of the thiocarbamic residue) bonds are longer (respectively about 0.03 and 0.08 Å) and the CAN bond (from thiocarbamic ester group) is about 0.08 Å shorter, with a value of 1.298(4) assuming a formal value of double bond. Thus in the complexes the ligands are in the thiol form rather than in the thione form. In [Ni(ttete)2] the Csp2 = Csp2 and the Csp2-S bond lengths of the thiophene ring are slightly longer than those found in the free ligands but within the expected range [30].

Table 2 Comparison of selected bond lengths for the free ligands and complexed ligands (Å). Bond lengths (Å)

tteteb

ttprea

ttbue

ttpte

tthxe

C21AO21 (H) (Ni) C22AS22 (H) (Ni)

– 1.250(3) – 1.707(3)

1.224(5) 1.258(4) 1.627(4) 1.715(3)

1.212(3) – 1.622(3) –

1.215(3) 1.267(5) 1.627(3) 1.705(5)

1.2218(14) 1.2608(16) 1.6368(13) 1.7190(14)

a For Httpre the X-ray parameters were taken as the average of the four molecules. b For Ni(ttete)2 the X-ray parameters were taken as the average of the two ligands.

The supramolecular structure of [Ni(ttete)2], Fig. 5, is dominated by a CAH  p interaction, 2.90 Å long (139.0°) between C34AH34 and the gravity centre of the thiophene ring formed by C11AS11AC12AC13AC1four atoms located at (x, 0.5  y, 0.5 + z) forming zig-zag two centrosymmetric related chains along the c-axis as depicted in Fig. 5.

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Fig. 5. Diagram showing the supramolecular structure for [Ni(ttete)2]. The CAH  p interaction is between C34AH34 at (x, y, z) with Cg of the thiophene ring at (x, 1/2  y, 1/2 + z) making a zig-zag chain running parallel to [1 1 0].

Supramolecular structures of [Ni(ttpte)2] and [Ni(tthxe)2] may be stabilised by similar CAH  p interactions, nevertheless, in those complexes the thiophene rings are disorder by a twofold rotation around the C1AC11 single bond, thus preventing the accurate determination of the geometric parameters and therefore the characterization of the potential CAH  p interactions. 3.2. Theoretical studies This twofold rotation is also present in [Ni(ttpre)2] and it is common for the thiophene ring. Nevertheless it is interesting to observe that in solid state [Ni(ttete)2] the sulphur atoms of the ring are turned on to the outside part (OUT) of the co-ordination sphere while in the remaining complexes these atoms can be found in the other direction (IN/OUT) with a ratio of 75/25 for [Ni(tthxe)2] and 82/18 for [Ni(ttpte)2] and 82/18 and 74/26 for the two ligands in [Ni(ttpre)2]. It is of interest to note that the major components

may be stabilized by a weak CAH  O intermolecular contact between CX4 of the thiophene ring and the ester OX1 atom at (x, y, z), Fig. 6. Furthermore, structural analysis of the ligands does not show disorder of the thiophene rings [29]. So a theoretical study in which a potential energy surface scan with the torsion angle around the C1AC11 single bond in [Ni(ttete)2], [Ni(ttpre)2] and Httpre was performed. The results are shown graphically in Fig. 7 and these results give rise to the conclusion that the potential barrier for rotation for Httpre free ligand (22 kJ mol1) is lower than that for the complex (38 kJ mol1 and 41 kJ mol1 for the outer and inner rotation respectively). Also the outer rotation barrier in [Ni(ttete)2] is similar to that found in the propyl analogue. For the complexes, the calculated rotation values for potential barriers are high enough to disable the conformational interconversion of the thiophene ring in solution, suggesting that these arrangements occur preferentially in the ligands during the formation of the complexes.

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state. When compared with those of the corresponding free ligands (HL) [29]. The Ni(II) complexes spectra shows the disappearance of the broad NAH signals at 9.06–9.00 ppm range compatible with the assumed anionic character of the ligand when coordinated. In the complexes the aromatic thiophene protons are split into three distinct peaks while in the ligands only two signals are recorded. In the ligands they were recorded as a multiplet at 7.66 ppm (2H) and as a triplet at 7.16 (1H) and in the complexes as two (duplets) at 7.65 ppm and 7.90 ppm and a triplet at 7.15 The observed separation of about 0.27 ppm between the two peaks for the aromatic protons indicates the change of the aromaticity of the aromatic ring upon coordination with the metallic centre. When the X-ray structures of the complexes are compared with those of the ligands it can be seen that there is an increase of about 0.03 Å on the CX1ACX2 bond length in the complex. The aliphatic protons are practically unaffected by complex formation.

3.4. UV–vis spectra

Fig. 6. Diagram showing the week intermolecular contact CAH  J between C14A/ C34A at (x, y, z) with O21 and O41 at (x, y, z), respectively, for [Ni(ttpre)2].

3.3. NMR spectra The 1H NMR spectra of Ni(II)L2 were recorded in CDCl3 and shows the diamagnetic character of the complexes which is compatible with the assumed square-planar geometry of the solid

Electronic spectra of [Ni(L2)] were recorded, at 298.15 K, in the interval 260–1100 nm in two solvents with different coordination numbers (dimethyl sulfoxide and dichloromethane). The typical spectra of [Ni(L2)] in DMSO is shown in Fig. 8. The spectra of the complexes are practically identical in both solvents, where one low intensity band at kmax  680 nm (e  15 L mol1 cm1) is observed, followed by a more intense band with its maximum at 430 nm (e  102 L mol1 cm1). At higher energies several high intensity bands (e > 104 L mol1 cm1) are also detected, which are typical of ligand based transitions as can be inferred by the spectra analysis of the ligands. For nickel(II) complexes with ligands containing oxygen donor atoms in which the ligand has a p donor capacity two bands at low energies corresponding to d–d transitions could be observed [31]. Nevertheless, for complexes with soft donors such as sulphur with delocalized p systems the energy values for the occupied orbitals can be very similar and only one d–d band is observed. Similar spectra are commonly observed for low-spin square-planar Ni(II) complexes with O2S2 co-ordination spheres [32,33] for which the low intensity band has been assigned to d–d transitions, and the high-energy bands to charge-transfer transitions. Assignment of the high-energy bands

R

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dihedral angle /° Fig. 7. Potential energy surface scan for thenoyl ring rotation on complexes and ligand.

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L.R. Gomes et al. / Journal of Molecular Structure 990 (2011) 86–94 0.0010 0.0008 0.0006 0.0004

Ad

0.0002 0.0000 -0.0002

260

275

290

305

λ (nm)

-0.0004 -0.0006 -0.0008 -0.0010

Fig. 9. The second derivative absorption spectra of [Ni(ttete)2] in the presence of increasing lipid concentrations from 0 to ca. 200 lM; the concentration of [Ni(ttete)2] is kept constant.

Fig. 8. Typical UV–Vis spectrum of [Ni(L)2] obtained in DMSO. Here the example is the complex with alkyl = propyl group.

is complicated due to the overlapping of interligand and chargetransfer transitions. 3.5. Liposome water partition coefficient Liposomes are a valuable cell model since they can mimic the structure and anisotropy present in biological membranes in a simple manner. With partition and transport across membranes being a fundamental step in all biological phenomena (drug absorption, action and toxicity), liposomes have been employed in an attempt to understand the interaction of xenobiotic with membranes [21]. Although traditionally a compound’s lipophilicity can be related to the octanol–water partition coefficient, some lack of correlation between this parameter and biological properties has been shown [34], revealing the need to employ more structural systems, such as liposomes, to determine the partition coefficient (Kp) [35]. The liposome/water partition coefficient (Kp) can be defined as the ratio between the compound concentration in the lipid phase and in the aqueous phase. Spectroscopic techniques, such as spectrophotometry and fluorimetry, are among the most usual procedures applied in the determination of this parameter, due to their sensitivity, straightforward procedures and possibility to analyse signals originating from both (lipid and aqueous) phases without the need to apply separation procedures, especially when allied to spectral derivation [36,37]. The second derivative absorption spectra of [Ni(ttete)2] in the presence of increasing lipid concentrations are shown in Fig. 9: a decrease in absorbance is observed when changing the lipid concentration from 0 to ca. 200 lM the concentration of [Ni(ttete)2] is kept constant. Together with the [Ni(ttete)2] spectra, blank derivative spectra are shown, to show the effect of the elimination of the liposome background signal. The spectra show isosbestic points, an indication that the system has two states: compound in polar bulk aqueous and in nonpolar EPC bilayer phases. The experimental second derivative spectrophotometric data, was fitted using a non-linear least-squares regression method, at wavelengths where the scattering is completely eliminated (see supporting information for details). At least two independent assays were made for each compound and the values of Kp obtained in Hepes buffer (pH = 7.4) were: 2692 ± 294, 2967 ± 427 and 6236 ± 466 (log Kp = 3.43 ± 0.05, 3.47 ± 0.07 and 3.79 ± 0.03) for [Ni(ttete)2], [Ni(ttpre)2] and [Ni(ttbue)2]. The analogues [Ni(ttpte)2] and [Ni(tthxe)2] had too low water solubility to enable the application of the methodology. Values for the liposome/water partition coefficients (Kp) show the [Ni(ttete)2] and [Ni(ttpre)2] have a similar membrane partition ability, whilst the [Ni(ttbue)2] derivative

presents a significantly higher Kp, describing a stronger interaction within the membrane. In fact, the butyl derivative presents very low water solubility, thus a preference to the lipid medium would be expected for this molecule, as well as for the homologues not analysed, [Ni(ttpte)2] and [Ni(tthxe)2]. In vivo, these analogues could be expected to present a higher affinity for the cell membrane, and so a probable higher absorption and distribution. Nevertheless, some notes of caution should be made at this point: (I) a higher Kp values is not always an indication of better in vivo bioavailability: in fact, an adequate hydrophilic/lypophilic balance (HLB) is better for good bioability behaviour; (II) the fact that a molecule is more lipophilic does not determine that it will have a greater interaction with lipid membranes. That can be true for analogues or homologues, but not for molecules with entirely different structures. In fact, [Ni(ttete)2] and [Ni(ttpre)2], although less water soluble than other molecules we have studied, present a similar Kp [20,21], which is related to the bulkiness of these molecules. Liposomes present a membrane structure similar to the cellular one, in which the lipophylic hydrocarbon region is sandwiched between two ordered polar headgroup regions. An adequate interaction with this membrane requires the compound’s ability to interpenetrate between the phospholipids headgroups and tails, so amphiphilic, often charged and often hydrophilic molecules, show that ability, and tend to have higher Kp values than bulk, non-charged and highly lipophilic molecules like the ones in this study. 3.6. Antimicrobial activity The in vitro antimicrobial activities against a number of Gram positive, Gram negative bacteria and yeasts were assayed with [Ni(ttbue)2] and [Ni(ttpre)2]. The remaining complexes presented very low solubility preventing the performance of assays. The results obtained are presented in Table 3. All compounds inhibited Table 3 MIC (106 g cm3) values for the in vitro antimicrobial activities of [Ni(ttete)2] and [Ni(ttpre)2] against a number of representative Gram positive, Gram negative bacteria and yeasts.

[Ni(ttpre)2] [Ni(ttbue)2] Ref. drugs Gentamycin Amikacin

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SA

SA (MRSA)

EC

KP

PA

CA

CK

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200 400

200 200

100 50

100 100

100 50

50 50

25 50

1

2

1 2

2

Sarcina lutea (SL), Staphylococcus aureus (SA), Staphylococcus aureus methicillinresistant (SAMRSA), Escherichia coli (EC), Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Candida albicans (CA), Candida krusei (CK).

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the growth of bacteria with MIC values ranging between 208 and 104 lg/cm3 and showed anti-yeast activity with MICs between 208 and 26 lg/cm3. According to the anti-bacterial studies, the efficacy against Gram negative bacteria is higher than against Gram positive bacteria. The lower anti-yeast efficacy of the compound may be due to the differences between the cell structures of bacteria and yeast. While the cell walls of fungi contain chitin, the cell walls of bacteria contain murein [38]. In addition, fungi contain ergosterol in their cell membranes instead of the cholesterol found in the cell membranes of animals [39,40]. The present findings further confirm the relative efficacy of the assayed compounds against Gram negative bacteria and yeasts as observed by similar compounds [41]. Unfortunately, the anti-yeast and anti-bacterial activity values of all tested compounds are lower than the reference compounds, thus these compounds cannot be proposed for further antimicrobial studies. Acknowledgments B. Schröder and M.A.A. Rocha acknowledge Fundação para a Ciência e a Tecnologia (FCT) and the European Social Fund (ESF) under the 3rd Community Support Framework (CSF) for the award of research grants with reference SFRH/BPD/38637/2007 and SRFH/BD/60513/2009, respectively. References [1] E. Rodríguez-Fernández, J.L. Manzano, J.J. Benito, R. Hermosa, E. Monte, J.J. Criado, J. Inorg. Biochem. 99 (2005) 1558. [2] G. Faraglia, D. Fregona, S. Sitran, L. Giovagnini, C. Marzano, F. Baccichetti, U. Casellato, R. Graziani, J. Inorg. Biochem. 83 (2001) 31. [3] R.T. Dorr, in: H.M. Pinedo, J.H. Schornagel (Eds.), Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy, vol. 2, Plenum Press, New York, 1996, pp. 131–154. [4] B.J. McCormick, R. Bereman, D. Baird, Coord. Chem. Rev. 54 (1984) 99. [5] K.S. Siddiqi, S. Khan, S.A. Nami, M.M. El-ajaily, Spectrochim. Acta A Mol. Biomol. Spectrosc. 67 (2007) 995. [6] A. Castiñeiras, R. Carballo, T. Pérez, Polyhedron 20 (2001) 441. [7] J.S. Casas, M.S. García-Tasende, J. Sordo, Coord. Chem. Rev. 209 (2000) 197. [8] O.P. Pandey, S.K. Sengupta, M.K. Mishra, C.M. Tripathi, Bioinorg. Chem. Appl. 1 (2003) 35. [9] M.A.V. Ribeiro da Silva, I.M.M. Monteiro, L.M.N.B.F. Santos, B. Schröder, J. Chem. Thermodynam. 39 (2007) 767.

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