N,N-dimethylbiguanide complexes displaying low cytotoxicity as potential large spectrum antimicrobial agents

European Journal of Medicinal Chemistry 45 (2010) 3027e3034

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

N,N-dimethylbiguanide complexes displaying low cytotoxicity as potential large spectrum antimicrobial agents Rodica Olar a, *, Mihaela Badea a, Dana Marinescu a, Mariana-Carmen Chifiriuc b, Coralia Bleotu c, Maria Nicoleta Grecu d, Emilia-Elena Iorgulescu e, Veronica Lazar b a

Department of Inorganic Chemistry, University of Bucharest, 90-92 Panduri Street, Bucharest, Romania Department of Microbiology, Faculty of Biology, University of Bucharest, 1-3 Aleea Portocalilor Street, Bucharest, Romania Stefan S. Nicolau Institute of Virology, 285 Mihai Bravu Avenue, Bucharest, Romania d National Institute for Materials Physics, PO Box MG-7, Bucharest-Magurele, Romania e Department of Analytical Chemistry, University of Bucharest, 90-92 Panduri Street, Bucharest, Romania b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 September 2009 Received in revised form 28 January 2010 Accepted 23 March 2010 Available online 27 March 2010

The new complexes M(DMBG)2(ClO4)2 (M:Mn, Ni, Cu and Zn; DMBG: N,N-dimethylbiguanide) have been synthesized and characterized by IR, EPR, 1H NMR, 13C NMR as well as electronic spectroscopy data. Complex [Ni(DMBG)2](ClO4)2$2DMF (DMF: N,N-dimethylformamide) crystallizes in the monoclinic P2 (1)/c space group while [Cu(DMBG)2](ClO4)2 adopt monoclinic P21/c space group as X-ray single crystal data indicate. The redox behavior of complexes was investigated by cyclic voltammetry. The metal-free N, N-dimethylbiguanide and complexes exhibit specific anti-infective properties as demonstrated the low MIC values, a large antimicrobial spectrum and also inhibit the ability of Pseudomonas aeruginosa and Staphylococcus aureus strains to colonize the inert surfaces. The complexes exhibit also a low cytotoxicity levels on HeLa cells. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: Antimicrobial activity Bacterial biofilm Cytotoxicity Complex N,N-dimethylbiguanide

1. Introduction The involvement of some metal ions in regulation of physiological processes has stimulated the development of metal-based therapeutics. The pharmaceutical use of complexes arises from the fact that the positively charged metal centers are favored to bind to negatively charged biomolecules such as the amino acid residues as protein constituents, ATP and nucleic acids. Moreover, the ligands can interact with biomolecules through coordinative or hydrogen bonds as well as dipoledipol interactions [1]. Therefore, the use of the metal complexes as therapeutics has become increasingly important over the last years resulting in a variety of interesting drugs used in fields as cancer, arthritis, ulcer, diabetes, anemia and cardiovascular medicine [2]. Among these, the complexes demonstrating antimicrobial activity could be used as antibiotics or disinfectants. So far only silver compounds (nitrate and sulfadiazine) have been clinically used in order to prevent the opthalmia neonatorum development [3] or the bacterial infection in the severe burns treatment [4] but every year new complexes that present antimicrobial activity, being * Corresponding author. Tel: þ40214103178x118; fax: þ40214102279. E-mail address: [email protected] (R. Olar). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.03.033

active against bacteria, yeast and fungi are reported [5e29]. However, the developing of these species as drugs is not easy to realize because the side effects as toxicity and drug resistance could limit their clinical applications. The studies reveal that a significant antibacterial activity can be induced for complexes by using either a biocation or a metallic ion exhibiting inhibitory properties as well as a ligand that displays such activity. On the other hand, important characteristics that can be correlated with a good antimicrobial activities are as follows: (i) the presence of uncoordinated groups that permits the recognition by living organism and enhances the solubility (hydrophilic or hydrophobic) [29], (ii) the ability to form hydrogen bonding with a counter anion or solvent molecules [8], (iii) the dimension and nature of the substituted groups attached to the donor atoms that influence the ligand lipophilicity and membrane permeability, controlling thus the cell penetration [18], (iv) the small coordination number or the presence of some ligands easy to removed in interaction with biomolecules [6,9], (v) a stereochemistry that allows a favorable tridimensional interaction with biomolecules [29] and (vi) a high kinetic and thermodynamic stability in order to control the dissociation in the acidic medium from the stomach, the metabolization in sanguine flux and to induce a low substitution rate with the biomolecules [29,30].

3028

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

Recently, a good antimicrobial activity were evidenced for complexes with N,N-dimethylbiguanide (DMBG) [30,31]. Besides the chelating ability N,N-dimethylbiguanide is known for their large spectrum of biological activity behaving as antimicrobial [32], analgesic, antimalarial or glucose lowering agent [33] as well as antimetabolite for organisms that inhibit the folic acid metabolism [34]. The aim of this paper is to synthesize and characterize new complexes M(DMBG)2(ClO4)2 (M: Mn(II), Ni(II); Cu(II) and Zn(II)) with this ligand. The redox behavior of complexes has been investigated by cyclic voltammetry. The Ni(DMBG)2(ClO4)2 recrystallization from DMF gave Ni(DMBG)2(ClO4)2$2DMF for which the crystal structure was solved by single-crystal X-ray analysis. By using the same method, the stereochemistry as well as the coordination mode were established for Cu(DMBG)2(ClO4)2 also, but the crystals were very small and the crystal structure has no good quality. The new complexes were tested for their antimicrobial activity on reference and clinical bacterial and fungal strains recently isolated from catheter related infections. The cytotoxic activity of complexes was assayed on HeLa cells. 2. Results and discussion 2.1. Chemistry By using N,N-dimethylbiguanide (DMBG) as ligand and some biocations, complexes M(DMBG)2(ClO4)2 ((1) M:Mn; (2) M:Ni; (3) M:Cu and (4) M:Zn) have been characterized as mononuclear species by elemental analysis, IR, 1H NMR, 13C NMR, EPR and electronic spectra. The redox behavior of complexes has been investigated by cyclic voltammetry. The complexes with N,N-dimethylbiguanide (DMBG) were isolated by reaction of the appropriate metal perchlorate with the N,Ndimethylbiguanidium chloride in ethanol (Scheme 1). Complex (2) recrystallization from DMF gave crystals with composition (2)$2DMF while complex (3) recrystallization from water gave crystals with the same composition. The complexes are water, N,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO) fairly soluble. The elemental analyses indicate a 1:2 stoichiometry for all complexes (Table 1). 2.1.1. Infrared spectra The IR spectra of complexes reveal the characteristic bands of N, N-dimethylbiguanide coordinated through N2 and N4 atoms (Table 2) [30]. The new band around 1340 cm1 could be associated with the chelate ring formation for the biguanide derivatives [35]. IR spectrum of complex (2)$2DMF is similar to that of (2) except for additional band at 1677 cm1, assigned to vibration mode n (C]O) for tertiary amide group of DMF.

CH3 CH3 2 H3C

N

H N

NH

H3C NH2

N

H N

NH2

HN

NH M 2+ HN NH

+ M 2+/H3O+

NH H2N

N H

N

CH3

M: Mn, Ni, Cu, Zn Scheme 1. Synthesis of complexes.

CH3

Table 1 Elemental analysis for complexes. Complex

(1) (2) (3) (4)

Empirical formula

MnC8H22N10O8Cl2 NiC8H22N10O8Cl2 CuC8H22N10O8Cl2 ZnC8H22N10O8Cl2

%C

%H

%N

Found

Calcd.

Found

Calcd.

Found

Calcd.

18.48 18.54 18.43 18.35

18.76 18.62 18.45 18.38

4.32 4.34 4.35 4.28

4.33 4.30 4.26 4.24

27.49 27.18 26.92 26.88

27.34 27.14 26.89 26.80

For complexes (2) and (3) the two bands, one displaying broad feature centered around 1098 cm1 and another one having a sharp feature at 625 cm1 confirm the presence of perchlorate as uncoordinated. The presence of unidentate perchlorate generates a more complex feature for bands assigned to n3 and n1 stretching vibrations in the spectra of complexes (1) and (4) [36]. 2.1.2. NMR spectra The 1H NMR spectrum in DMSO-d6 for complex (4) exhibits a pattern assignable to the isolated methyl protons at 3.04 and, respectively, 4.82 ppm. Additional resonances arise from the amine and imine protons, respectively, at 6.51 and 7.15 ppm. The shift of the broad peak of ligand assigned to imine protons from 7.53 ppm to downfield (7.15 ppm) in complex spectrum indicates their coordination through N2 and N4 atoms. The appearance of two peaks at different values for the equivalent methyl protons attached at N1 of DMBG is in accord with the trans arrangement of the two coordinated DMBG molecules. The 13CNMR spectrum consists of peaks for the primary carbon atoms provided by methyl group at 37.9 ppm and for the quaternary carbon atoms from biguanide skeleton at 161.2 ppm. 2.1.3. Electronic spectral studies The solid state-d spectrum of (1) shows the characteristic bands of Mn(II) in pseudo-octahedral environment. The absorptions at 22730 and 15385 cm1 are assigned to the spin forbidden 6 A1 / 4A1, 4E(G) and 6A1 / 4T1 transitions [37]. The electronic spectra of (2) and (2)$DMF show a band centered at 20,830 and 20,700 cm1, respectively, assigned to the spin allowed transition 1 A1g / 1A2g in square-planar stereochemistry [37,38]. The band centered at 19,230 cm1 in the electronic spectrum of (3) was assigned to dxz;yz /dx2 y2 transition and indicates also a squareplanar surrounding [37,38]. 2.1.4. EPR studies The X-band spectrum of (1) is characteristic for a distorted octahedral symmetry. The resonance line is large (DB ¼ 59 mT) and the spectral parameters gef ¼ 1.99 and Aiso ¼ 9.2 mT indicate a high ionic degree of the bonds. The EPR spectrum registered at room temperature for complex (3) is characteristic for a mononuclear complex with an axial symmetry. The gk ¼ 2.178, gt ¼ 2.042, Ak ¼ 20.86 mT, and At  3.85 mT values are characteristic for a distorted square-planar geometry with a dx2 y2 ground state [39]. 2.1.5. Electrochemical measurements The cyclic voltammogram of complex (1) in DMF (þ0.8 to 0.6 V potential range) shows a well defined redox process corresponding to the Mn(II) / Mn(I) couple at Epc ¼ 0.0060 V and the associated anodic peak at Epa ¼ 0.1428 V for a scan rate 50 mV s1. The ratio of the peak current to the square root of the scan rate decreases as the scan rate increases. This is consistent with a complicated redox mechanism like ECE (electrochemical-chemical-electrochemical) type [40]. Depending of the scan rate, it can be show one or two cathodic peaks and only one anodic for complex (2). At a scan rate of 50 mV s1 the reduction peaks can be observed at Epc1 ¼ 0.1323 V

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

3029

Table 2 Characteristic IR absorption bands (cm1) for DMBG and complexes. Compound

nas (NH2)

n (NH)

ns (NH2)

n (C]N)

nchelate ring

n3 (ClO4)

n1 (ClO4)

n4 (ClO4)

DMBG (1)

3371s 3380s

3296 m 3301vs

3173s 3192vs

1635vs 1655s 1620vs

e 1335w

e 933 m

e 631 m 570 m

(2)

3459s

3375vs

3246vs

1341w

e

627 m

(2)$DMF

3455 m

3386s

3240 m

1340w

1080vs

e

628 m

(3)

3454s

3382vs

3250s

1325w

1085vs

e

627 m

(4)

3367vs

3332s

3195vs

1653vs 1620vs 1642vs 1630s 1673vs 1624vs 1650s 1628vs

e 1144s 1119s 1086s 1085vs

1340w

1143 m 1114s 1086s

932 m

628 m 570 m

and Epc2 ¼ 0.7769 V respectively, while the one corresponding to oxidation process appear at Epa ¼ 0.587 V. When the scan rate is higher then 50 mV/s, the cathodic peak at negative potential disappears and the ratio ipa/ipc z 1 indicates a reversible redox process for the couple Ni(II)/ Ni(0) without kinetic complications. Cyclic voltammograms recorded for complex (3) show at different scan rates one cathodic peak in the þ0.236 to þ0.3081 V potential range and one anodic peak at þ0.6611 to þ0.6276 V potential range, characteristic for the Cu(II)/Cu(I) couple. Depending on the scan rate, the cyclic voltammogram recorded on glassy carbon electrode (GC) displays a cathodic peak at ca. 0.7200 V for (4). As increasing the scan rate, the cathodic peak potential is shifted to negative values while the ratio ipc/v1/2 is decreasing. The plot of cathodic current as a function of the square root of the scan rate is not linear. These results indicate an electrochemicalechemical (EC) mechanism corresponding to Zn(II) / Zn (I) reduction, followed by a chemical reaction that can be a comproportionation reaction between complexes with Zn(I) or Zn(II). Based on all the above data, the following coordination was proposed for complexes (Fig. 1).

2.2. Biological activity 2.2.1. Antimicrobial activity The results of the antimicrobial properties screening demonstrated a specific antimicrobial activity, both concerning the antimicrobial spectrum and the MIC value. The MIC values widely ranged between 4 mg/mL and 1024 mg/mL, i.e. from very high bactericidal effect on Listeria monocytogenes (preserved at a very low MIC value of 4 mg/mL for compounds (3)) (Table 1) to a very low one on Staphylococcus aureus and Candida albicans (with corresponding high MIC values of 1024 mg/mL). The compounds exhibit a medium to high inhibitory activity against Pseudomonas aeruginosa, thus they could represent possible new antibacterial drugs against this multiresistant strains, which CH3 H3C

N

CH3

H N

NH2

H3C

N

HN NH O 3ClO M OClO 3 HN NH H2N

N H

N

HN

NH2 NH (ClO4)2

M HN

CH3

CH3

M: Mn, Zn

H N

H2N

NH N H

M: Ni, Cu

Fig. 1. The coordination proposed for complexes.

N

CH3

CH3

cause very severe and difficult, if not impossible to treat nosocomial infections. All tested compounds, excepting (4) proved also to be effective against Salmonella enteritidis, while the compound (3) showed medium antimicrobial activity directed more generally against Enterobacteriaceae (i.e. Escherichia coli and S. enteritidis). As concerning the intensity of the antimicrobial activity of the metal perchlorates comparatively with their complexes, our results showed that in the case of compound (2), the complex exhibit a superior antimicrobial activity comparatively with the perchlorate. The compound (2)$2DMF proved to be less active comparing with (2) on the tested strains. These results could be due to decreasing of the active compound (2) amount at the same concentration of samples. However, in all cases, the antimicrobial activity of the complexes and metal perchlorates was much higher than that of DMBG (Table 3). It is to be noticed that Mn(II), both as metal perchlorate as well as complex exhibit the best antimicrobial activity with a large spectrum and the lowest MIC values for all bacterial as well as the fungal tested strains. 2.2.2. Inhibitory effect of complexes on the ability of P. aeruginosa and S. aureus to colonize the inert substratum P. aeruginosa and S. aureus are considered at present two of the most fearful opportunistic pathogens, due to their frequent incidence in the etiology of the community-acquired and nosocomial infections and to their high rates of natural and acquired resistance to different antimicrobial agents, including the majority of the large scale used antibiotics. Their clinical impact is increased by the ability of these bacterial strains to colonize the inert materials used in medicine and to determine prosthetic devices associated chronic infections, very hard to treat and often with severe outcome [41e43]. In this study we have tested the potential inhibitory effect of the complexes on the ability of some S. aureus and P. aeruginosa to colonize the cellular substratum represented by HeLa cells and the inert one, quantified by the production of slime (hydrophilic bacterial exopolysaccharide implicated in the bacterial adherence to inert substrata) [41,42]. The tested compounds inhibit the slime production and the ability of the bacterial strains to colonize the inert substratum. This general inhibitory effect could be explained by the potential inhibitory effect of the divalent metals on the bacterial exopolysaccharide secretion. This was previously demonstrated for calcium using a mucoid P. aeruginosa strain [43]. These results are accounting for the potential use of these compounds in the prevention of the bacterial biofilm development on prosthetic devices, as well as for the design of new antiseptics and disinfectants with efficient protective action against bacterial colonization of tissue and inert surfaces [42].

3030

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

Table 3 The minimum inhibitory concentration (MIC, mg/mL) for dimethylbiguanide, complexes (1)O(4) and perchlorates. Compounds

E. coli

S. enteritidis

P. aeruginosa

B. subtilis

L. monocytogenes

S. aureus

C. albicans

DMBG (1) Mn(ClO4)2$6H2O (2) (2)$2DMF Ni(ClO4)2$6H2O (3) Cu(ClO4)2$6H2O (4) Zn(ClO4)2$6H2O

1024 512 256 256 1024 >1024 512 256 512 256

1024 256 128 256 1024 >1024 >1024 256 512 512

1024 128 512 256 1024 >1024 256 128 1024 512

512 32 32 256 512 >1024 128 256 128 32

1024 128 128 512 1024 >1024 4 4 128 512

512 512 256 1024 1024 >1024 512 512 512 512

512 128 128 1024 1024 512 512 256 512 128

Concerning the influence of the tested compounds on the ability of bacterial strains to colonize and invade the cellular substratum, the obtained results were different for the two bacterial strains. In case of S. aureus, all complexes, except for (4) completely abolished the ability of the tested strain to colonize the HeLa cells. By comparison, Mn(II) and Ni(II) salts slightly inhibited this virulence hallmark (Fig. 2). When the tested substance was added over the eukaryotic cell monolayer before adding the bacterial suspension, the compounds (1), (3) and (4), as well as the Zn(II) perchlorate inhibited also the ability of bacterial cell to adhere to the eukaryotic cells, demonstrating the interference of the tested compounds with the intracellular signaling mechanisms. P. aeruginosa proved to be more sensitive to the tested compounds, its adherence ability being abolished by the compounds (2), (3) and (4) and by all four perchlorates. Unlike the previous treatment of the eukaryotic cells with the tested compounds, only compounds (2) and (3) and the Cu(II) perchlorate inhibit the bacterial adherence to the cellular substratum (Fig. 3). The higher susceptibility of P. aeruginosa to the tested compounds in comparison with that of S. aureus indicates that the compounds are better internalized in the Gram-negative cells, probably due to the presence of external lipid layer correlated with the liposolubility of the tested substances. Having in view that b-actin is implicated in the intimate adherence and subsequent internalization of the bacterial cells, the expression of this cytoskeletal protein in the HeLa cells in the presence of the tested compounds was assessed. The results showed that the expression of b-actin assay is decreased in the presence of all tested compounds (Fig. 4). 2.2.3. Cytotoxicity assay The complexes cytotoxicity assessed by flow citometry revealed that all complexes exhibited very low cytotoxicity levels on HeLa cells, thus presenting a great advantage in the potential selection of

Fig. 2. Graphic representation of the influence of the tested compounds on the ability of Staphylococcus aureus strain to adhere and invade the HeLa cells (gray e treatment of prokaryotic cells with 500mM substance for 24 h; black e treatment of eukaryotic cells with 500 mM substance for 24 h).

these compounds for the in vivo use as antimicrobial agents (Table 4). It was also tested the influence of complexes, ligand and perchlorates on the eukaryotic cell cycle (Table 5). The analysis of HeLa cell cycle distribution after them exposure for 40 h to 100 mM metal perchlorate salts and complexes, show that Cu (II) salts/complex induced an increase of S and G2/M phases. The anti-tumor activity of copper (II) complexes has been already reported and might be due to the ability of these substances to intercalate into the stacked base pairs of DNA and to induce oxidative DNA damage involving the generation of reactive oxygen species [44,45]. However, the molecular mechanisms by which our specific complex combinations develop an anti-tumoral effect remain to be proved by in-depth studies. It is also to be noticed the slight increase of S phase in the presence of Mn(II) and Ni(II) salts or their complexes (Table 5). Metal perchlorates and complexes exhibited specific antiinfective properties as demonstrated by the low MIC values, with a large antimicrobial spectrum. The study on the complexes with a good antimicrobial activity has evidenced that the activity depends both by their liposolubility and ability to inhibit different enzymes into the cell. Thus, the hydrophobicity and charge of complexes should be balanced in order to allow enhanced membrane permeability of the different bacterial strains. It was observed that ligands, which form chelate ring, enhance the complex lipophilicity as result of electrons delocalization. Inside of the cell the most complexes disturb the physiological processes. This arises by blocking the active sites in the enzymes of microorganisms through metal or ligand coordination and/or by hydrogen bonds formation [46]. The N,N-dimethylbiguanide beside behaving as chelate agent has both supplementary coordination sites and ability to generate hydrogen bonds, as indicate the X-ray results for

Fig. 3. Graphic representation of the influence of the tested compounds on the ability of Pseudomonas aeruginosa strain to adhere and invade the HeLa cells (black e treatment of prokaryotic cells with 500 mM substance for 24 h, gray e treatment of eukaryotic cells with 500 mM substance for 24 h).

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

3031

Fig. 4. Histogram of the b-actin expression in HeLa cells (a), and after treatment of eukaryotic cells with (4) and Zn(II) perchlorate 100 mM for 24 h (b, c).

protonated derivatives as well as complexes. Thus, complexes (1) and (3) were the most effective agents, probably due the enzyme inhibition through both ligand and metal ions. 2.3. The X-ray crystal structures Three-dimensional structures of (2)$2DMF and (3) were determined by single-crystal X-ray diffraction. The crystal structure of (2)$2DMF consists of discrete [Ni (DMBG)2]2þ cations and perchlorate (Fig. 5). The Ni(II) is tetracoordinated with a square planar surrounding each metal ion being linked to two chelate DMBG. In the coordination polyhedra the two-methyl groups of the ligands are in a trans-configuration. The NieN bond lengths of 1856(3)e1876(3) Å fit well with those found for [Ni(DMBG)2]Cl(OH) [47] and are a little longer than those observed in [Ni(DMBG)2] [48]. It was observed that the interaction of Ni(II) with the DMBG is not so stronger than those observed for complexes with this ligand in deprotonated form. The hydrogen bonds are developed between coordinated DMBG molecules, perchlorate anions and N,N-dimethylformamide. The single crystal X-ray on the complex (3) reveals columns of [Cu(DMBG)2]2þ oriented in the same direction and generated through pep staking interaction. The neighbouring columns are held together by hydrogen bonds with perchlorate anions. The Cu (II) ions adopt a square planar stereochemistry and the methyl groups of the ligands are in a trans-configuration similar with complex (2)$2DMF. 3. Conclusion Complexes of type M(DMBG)2(ClO4)2 ((1) M:Mn, (2) M:Ni, (3) M: Cu, and (4) M:Zn; DMBG: N,N-dimethylbiguanide) were synthesized. The complexes were characterized by elemental analyses, spectral and magnetic studies as well as cyclic voltammetry. Based

on these studies, the complexes were formulated as mononuclear species with N,N-dimethylbiguanide coordinated through N2 and N4 atoms. The optical and EPR studies indicate a square planar stereochemistry around the Ni(II) and Cu(II) respectively an octahedral one for Mn(II). Complexes [Ni(DMBG)2](ClO4)2$DMF and [Cu(DMBG)2](ClO4)2 were characterized by single crystal X-ray diffraction. Metal-free N,N-dimethylbiguanide and their complexes exhibit specific anti-infective properties (low MIC values) and a large antimicrobial spectrum. The low cytotoxicity levels on HeLa cells are representing a great advantage for the in vivo use of the tested complexes as antimicrobial agents. The tested complexes exhibit also an inhibitory effect on the bacterial adherence and invasion of the cellular substratum. All complexes generally inhibit the bacterial ability to colonize the inert substratum, accounting for their potential use in the prevention of the bacterial biofilms development on prosthetic devices, as well as for the design of new antiseptics and disinfectants with protective action against bacterial colonization of tissue and inert surfaces. 4. Experimental protocols All complexes were synthesized from commercially available starting materials used without further purification. Metal salts (Merck) were of analytical grade. IR spectra were recorded in KBr pellets with an FTIR-Biorad 135 instrument in 400e4000 cm1 range. Electronic spectra of the solids (380e1200 nm) were obtained by diffuse reflectance technique, using MgO as standard, with a VSU-2P Zeiss Jena instrument. 1 H NMR spectra were recorded on a Bruker Avance DPX250 spectrometer (working frequency 250 MHz) at 25  C. Chemical shifts were measured in parts per million from internal standard TMS. 13 C NMR spectra were recorded on a Bruker Avance DPX250

Table 5 The results of the influence of the compounds on the eukaryotic cell cycle.

Table 4 Levels of cytotoxicity induced by the complexes on Hela cells. Complex

Viable cells

Apoptosis %

Secondary apoptosis

Necrosis %

Mn (ClO4)$6H2O Ni(ClO4)2$6H2O Cu(ClO4)2$6H2O Zn(ClO4)2$6H2O (1) (2) (3) (4)

98.72 98.10 97.72 98.00 98.03 97.52 96.43 98.11

0.21 0.22 0.28 0.28 0.06 0.24 0.49 0.10

0.80 1.42 1.72 1.50 1.57 1.92 2.73 1.57

0.27 0.26 0.28 0.22 0.34 0.32 0.35 0.26

Control Mn (ClO4)2$6H2O Ni(ClO4)2$6H2O Cu(ClO4)2$6H2O Zn(ClO4)2$6H2O (1) (2) (3) (4)

G0/G1

S

G2/M

68.85 67.97 61.18 54.12 71.12 70.29 60.17 55.42 69.27

25.48 26 32.41 41.23 23.99 24.75 33.41 32.91 22.78

5.74 5.41 4.38 13.79 6.66 5.57 5.49 12.34 8.74

3032

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

involved anisotropic displacement parameters for all nonhydrogen atoms. The drawings were creating with the Diamond program by Crystal Impact GbR [51]. The detailed structural data have been deposited with CCDC-626506 and CCDC-762618, respectively. A summary of crystal data, data collections and refinement for (2)$2DMF and (3) is given in Table 6. 4.2. Biological assays

Fig. 5. A view of the molecular structure of (2)$2DMF with the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii.

spectrometer (working frequency 62.9 MHz) at 25  C. The EPR measurements were performed on microcrystalline samples using a JEOL JES-ME upgrade spectrometer equipped with X- and K-band cavities (9.5 GHz and 24 GHz, respectively) and a Jeol system for low- and high-temperature experiments (80e550 K). Cyclic voltammograms were obtained by an electrochemical system (potentiostate/galvanostate) 273 A, EG&G Princeton Applied Research. Electrochemical studies were performed at room temperature in DMF containing NaClO4 0,1 M or tetrabutylamonium tetrafluoroborate (TBATFB) 0.05 M using a KO264 microcell. The reference electrode was Ag/AgCl (KCl 3 M) while the counter electrode was the platinum wire. The working electrode was a platinum (for complexes (1)e(3)) and glassy carbon (GC) (for complex (4)) solid milielectrode (the effective aria of electrode 3.14  102 cm2) according to compounds electronegativity. The solutions were thoroughly degassed with pure argon prior to determination. X-ray measurements were performed on a Brucker SMART APEX diffractometer with graphite-monochromated Mo-Ka radiation (l ¼ 0.71073 Å). Elemental analyses (C, H, N) were performed with a Perkin Elmer PE 2400 instrument. 4.1. Chemistry 4.1.1. Preparation of complexes To a solution of M(ClO4)2$6H2O (5 mmoles) in ethanol (25 mL) was slowly added DMBG$HCl (10 mmoles) and few drops of HClO4 (3 M), under continuous stirring and heating at 50  C. The reaction mixture was then stirred at 50  C for 2 h until a microcrystalline product was formed. The product was filtered off and washed several times with a small volume of cold ethanol, ethylic ether and air-dried. Crystals suitable for X-ray determination were obtained by DMF recrystallization of complex (2) and by water recrystallization for complex (3), respectively. 4.1.2. X-ray measurements For X-ray crystallographic studies, each single-crystal was mounted on a cryoloop. Data collection was performed at room temperature and processed using the program SMART [49] on a Brucker SMART APEX and STOE-IPDS II diffractometer, both using graphite-monochromated Mo-Ka radiation (l ¼ 0.71073 Å). Data collection was performed at room temperature. The structures were solved by direct methods with the program SHELXS [49] and refined by full-matrix least-squares techniques with SHELX-97 [50]. Hydrogen atoms were included in calculated idealized positions and constrained with the use of a riding model. The final models

4.2.1. Microbial strains The antimicrobial activity of the investigated compounds was tested against bacterial and fungal strains recently isolated from clinical specimens as well as reference strains belonging to the following genera and species: Gram positive (methicillin resistant Staphylococcus aureus, L. monocytogenes, Bacillus subtilis), Gramnegative (E. coli, S. enteritidis, P. aeruginosa) and C. albicans. The microbial strains were identified by aid of VITEK I automatic system. VITEK cards for identification and susceptibility testing (GNS-522) were inoculated and incubated according to the manufacturer’s recommendations. The results were interpreted by using software version AMS R09.1. In our experiments there were used bacterial suspensions of 1.5  108 CFU/mL or 0.5 McFarland density obtained from 15e18 h bacterial cultures developed on solid media. The antimicrobial activity was tested on MuellereHinton medium recommended for the bacterial strains and Yeast Peptone Glucose (YPG) medium for C. albicans. We used the solutions of the new compounds in DMSO or DMF with 2048 mg/mL concentration. 4.2.2. Qualitative screening of the antimicrobial properties of the tested compounds The qualitative screening was performed by an adapted disk diffusion method. Petri dishes with Mueller Hinton (for bacterial strains) /YPG (for yeasts) medium were seeded with bacterial inoculums as for the classical antibiotic susceptibility testing disk diffusion method [52e55]; 5 mm diameter paper filter disks were placed on the seeded medium, at 30 mm distance. Subsequently, the disks were impregnated with 5 mL tested compound solution (1024 mg/mL concentration). The plates were left at room Table 6 Summary of crystal data for [Ni(DMBG)2](ClO4)2$2CHON(CH3)2 ((2) $2DMF) and [Cu(DMBG)2](ClO4)2 (3) Compound

[Ni(DMBG)2](ClO4)2$2 CHON(CH3)2

[Cu(DMBG)2](ClO4)2

Color/shape Empirical formula Formula weight Temperature Crystal system Space group Unit cell dimensions

Orange/block C14H36Cl2N12NiO10 662.16 298(2) K Monoclinic P2(1)/c a ¼ 6.2466(6) Å a ¼ 90 b ¼ 17.4612(18) Å b ¼ 102.812(2) c ¼ 13.3791(14) Å g ¼ 90 1423.0(2) Å3 2 1.545 mg/m3 0.937 mm1 692 1.131 R1 ¼ 0.0551, wR2 ¼ 0.1385

Pink/needle C18H22Cl2CuN10O8 520.80 293(2) K Monoclinic P21/c a ¼ 5.0370(8) Å a ¼ 90 b ¼ 16.7286(19) Å b ¼ 93.550(14) c ¼ 11.378(2) Å g ¼ 90 956.9(3) Å3 2 1.807 mg/m3 1.483 mm1 534 1.104 R1 ¼ 0.1146, wR2 ¼ 0.2589

R1 ¼ 0.0640, wR2 ¼ 0.1446 0.475 and 0.365 e A-3

R1 ¼ 0.1282, wR2 ¼ 0.2680 2.579 and 1.211 e A-3

Volume Z Calculated density Absorption coefficient F(000) Goodness-of-fit on F2 Final R indices [I > 2s(I)] R indices (all data) Largest difference peak and hole

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

temperature for 20e30 min and then incubated at 37  C for 24 h. The positive results were read as the occurrence of an inhibition zone of microbial growth around the disk. 4.2.3. Quantitative assay of the antimicrobial activity It was performed by binary micro dilution method, in 96 multiwell plates, in order to establish the minimal inhibitory concentration (MIC) [52,53,55]. In this purpose, serial binary dilutions of the tested compounds (ranging between 1024 and 4 mg/mL) were performed in a 200 mL volume of nutrient broth and each well was seeded with 50 mL microbial inoculum. The plates were incubated for 24 h at 37  C, and MICs were read as the last concentration of the compound, which inhibited the microbial growth. 4.2.4. Study of the influence of the complexes on the P. aeruginosa and S. aureus ability to colonize the inert substratum by slime test The bacterial strains were cultivated on nutrient agar and incubated at 37  C for 24 h. The next day, isolated colonies were suspended in sterile distilled water in order to obtain microbial suspensions with a density corresponding to 0.5 MacFarland nephelometric standard and 50 mL of these suspensions were distributed in 96-multiwell plates with 200 mL nutrient broth/well. In order to assess the influence of the tested complexes on the bacterial adherence to the plate wells, the same experiment was repeated in the presence of subinhibitory concentrations of the tested compounds. After incubation, the wells were washed three times with sterile distilled water and stained with alcoholic 1% safranin solution for 30 min. After staining the wells were washed with distilled water and dried at room temperature. The positive result was registered as the occurrence of a red ring of different color intensities adhered to the plate well. 4.2.5. Cell cultures The cell cultures used in cytotoxicity assay were represented by HeLa (ECACC # 93021013) cells. The cells were grown in Dulbecco’s Modified Essential medium DMEM (Sigma) supplemented with 10% fetal calf serum (Sigma) at 37  C, 5% CO2, in a humid atmosphere. 4.2.6. Flow cytometry and annexin V assay HeLa cells were grown in DMEM supplemented with 10% fetal calf serum (FCS) and 24 h later 500 mM compounds were added. Cells from the supernatant and monolayer were harvested and 1 105 cells were stained with annexin V and propidium iodide using the Immunotech Annexin V-FITC Kit according to the manufacturer’s instructions (Beckman Coulter Company, France). Cells were analyzed by flow cytometry using a Coulter EPICS XL flow cytometer (Beckman Coulter). Green fluorescence (525 nm; FITC annexin V) and red fluorescence (613 nm; propidium iodide) were measured. 4.2.7. Cell cycle distribution Cell cycle distribution and percentage of HeLa cells treated for 40 h with 100 mM compounds, within the sub-G1 peak were estimated by PI staining. Cells (1 106/mL) were fixed in 70% cold ethanol, washed twice in PBS, then incubated 15 min, at 37  C, with RNAse A (10 mg/mL), and 1 h with propidium iodide (10 mg/mL). After staining of cells with propidium iodide the acquisition was done using Beckman Coulter XLM flow cytometer. Data were elaborated using Beckman Coulter XLM software and expressed as fractions of cells in the different cycle phases. Samples were run in triplicate, and each experiment was repeated three times. Diagrams of growing cultures display the characteristic x-axis distribution according to the DNA content, the first peak corresponding to the diploid (2c) peak, i.e. to cells in the G0/1 phase, and the second peak

3033

to cells with 4c DNA content, i.e. to cells in G2/M phase. Cells with an intermediate DNA content are in the S phase. When DNA is fragmented, as in apoptotic cells, the affinity with the intercalating PI dye is decreased and a so-called hypodiploid peak (or area) becomes apparent to the left of the G0/1 peak [56,57]. 4.2.8. Study of the influence of the complexes on the P. aeruginosa and S. aureus ability to colonize the cellular substrate represented by HeLa cells The experiments were performed in three variants: (1) bacterial cells added over the cellular monolayer as well; (2) bacterial cells treated with 500 mM of the tested compounds for 24 h; (3) bacterial cells added over the monolayer after the treatment of the eukaryotic cells with 500 mM substance for 24 h. 4.2.9. b-actin quantification After treatment of HeLa cells with 500 mM compounds for 24 h, 5  105 cells were collected, washed twice in PBS, 0.1% BSA, and then cells were incubated for 1 h with monoclonal antibody b-actin (Sigma-Aldrich, MO, USA). The labeled cells were analyzed using a Beckman Coulter EPICS XL flow cytometer. Ten thousand events were acquired and data were analyzed with WinMDI software. Positive cells were determined as percentages of gated cells. References [1] C. Xin Zhang, S.J. Lippard, New metal complexes as potential therapeutics. Cur. Opin. Chem. Biol. 7 (2003) 481e489. [2] I. Bertini, H.B. Grey, E.I. Stiefel, J.S. Valentine, Biological Inorganic Chemistry. Structure and Reactivity. University Science Books, Sausalito, California, 2007. [3] C.S.R. Credé, Die Verhütung der Augenentzündung der Neugeborenen. Arch. Gynakol. 70 (1881) 50e53. [4] C.L. Fox Jr., S.M. Modak, Mechanism of silver sulfadiazine action on burn wound infections. Antimicrob. Agents Chemother. 5 (1974) 582e588. [5] K. Nomiya, S. Takahashi, R. Noguchi, S. Nemoto, T. Takayama, M. Oda, Synthesis and characterization of water-soluble silver(I) complexes with L-histidine (H2his) and (S)-()-2-pyrrolidone-5-carboxylic Acid (H2pyrrld) showing a wide spectrum of effective antibacterial and antifungal activities. Crystal structures of chiral helical polymers [Ag(Hhis)] n and {[Ag(Hpyrrld)]2}n in the solid state. Inorg. Chem. 39 (2000) 3301e3311. [6] N.C. Kasuga, K. Sekino, C. Koumo, N. Shimada, M. Ishikawa, K. Nomiya, Synthesis, structural characterization and antimicrobial activities of 4- and 6-coordinate nickel(II) complexes with three tiosemicarbazones and semicarbazone ligands. J. Inorg. Biochem. 84 (2001) 55e65. [7] R. del Campo, J.J. Criado, E. Garcia, M.R. Hermosa, A. Jimenez-Sanchez, J. L. Manzano, E. Monte, E. Rodriguez-Fernandez, F. Sanz, Thiourea derivatives and their nickel(II) and platinum(II) complexes: antifungal activity. J. Inorg. Biochem. 89 (2002) 74e82. [8] N.C. Kasuga, K. Sekino, M. Ishikawa, A. Honda, M. Yokoyama, S. Nakano, N. Shimada, C. Koumo, K. Nomiya, Synthesis, structural characterization and antimicrobial activities of 12 zinc(II) complexes with four thiosemicarbazone and two semicarbazone ligands. J. Inorg. Biochem. 96 (2003) 298e310. [9] K. Nomiya, S. Yamamoto, R. Noguchi, H. Yokoyama, N.C. Kasuga, K. Ohyama, C. Kato, Ligand-exchangeability of 2-coordinate phosphinegold(I) complexes with AuSP and AuNP cores showing selective antimicrobial activities against gram-positive bacteria. Crystal structures of [Au(2-Hmpa)(PPh3)] and [Au(6Hmna)(PPh3)] (2-H2mpa ¼ 2-mercaptopropionic acid, 6-H2mna ¼ 6-mercaptonicotinic acid). J. Inorg. Biochem 95 (2003) 208e220. [10] A. Chaudhary, N. Bansal, A. Gajraj, R.V. Singh, Antifertility, antibacterial, antifungal and percent disease incidence aspects of macrocyclic complexes of manganese(II). J. Inorg. Biochem. 96 (2003) 393e400. [11] M. Alexiou, I. Tsivikas, C. Dendrinou-Samara, A.A. Pantazaki, P. Trikalitis, N. Lalioti, D.A. Kyriakidis, T.P. Kessissoglou, High nuclearity nickel compounds with tree, four or five metal atoms showing antibacterial activity. J. Inorg. Biochem. 93 (2003) 256e264. [12] Z.H. Chohan, C.T. Supuran, A. Scozzafava, Metaloantibiotics: synthesis and antibacterial activity of cobalt(II), copper(II), nickel(II) and zinc(II) complexes of kefzol. J. Enzym. Inhib. Med. Chem. 19 (2004) 79e84. [13] Z.H. Chohan, H. Pervez, A. Rauf, K.M. Khan, G.M. Maharvi, C.T. Supuran, Antibacterial and antifungal mono- and di-substituted symmetrical and unsymmetrical triazine-derived Schiff-bases and their transition metal complexes. J. Enzym. Inhib. Med. Chem. 19 (2004) 161e168. [14] K. Nomiya, A. Yoshizawa, K. Tsukagoshi, N.C. Kasuga, S. Hirakawa, Synthesis and structural characterization of silver(I), aluminium(III) and cobalt(II) complexes with 4-isopropyltropolone (hinokitiol) showing noteworthy biological activities. Action of silver(I)eoxygen bonding complexes on the antimicrobial activities. J. Watanabe, J. Inorg. Biochem 98 (2004) 46e60.

3034

R. Olar et al. / European Journal of Medicinal Chemistry 45 (2010) 3027e3034

[15] A.Th. Chaviara, P.J. Cox, K.H. Repana, R.M. Papi, K.T. Papazisis, D. Zambouli, A. H. Kortsaris, D.A. Kyriakidis, C.A. Bolos, Copper(II) Schiff base coordination compounds of dien with heterocyclic aldehydes and 2-amino-5-methyl-thiazole: synthesis, characterization, antiproliferative and antibacterial studies. Crystal structure of CudienOOCl2. J. Inorg. Biochem. 98 (2004) 1271e1283. [16] N.M. Rageh, Tautomeric structures, electronic spectra, acidebase properties of some 7-aryl-2,5-diamino-3(4-hydroxyphenyazo)pyrazolo[1,5-a]pyrimidine6-carbonitriles, and effect of their copper(II) complex solutions on some bacteria and fungi. Spectrochim. Acta A 60 (2004) 1917e1924. [17] G.G. Mohamed, Z.H. El-Wahab, Mixed ligand complexes of bis(phenylimine) Schiff base ligands incorporating pyridinium moiety. Synthesis, characterization and antibacterial activity. Spectrochim. Acta Mol. Biomol. Spectros. 61 (2005) 1059e1068. [18] M.S. Iqbal, I.H. Bukhari, M. Arif, Preparation, characterization and biological evaluation of copper(II) and zinc(II) complexes with Schiff bases derived from amoxicillin and cephalexin. Appl. Organomet. Chem. 19 (2005) 864e869. [19] Z.D. Matovi c, V.D. Mileti c, G. Samard zi c, G. Pelosi, S. Ianelli, S. Trifunovi c, Square-planar copper(II) complexes with tetradentate amido-carboxylate ligands. Crystal structure of Na2[Cu(obap)]2. Strain analysis and spectral assinments of complexes. Inorg. Chim. Acta 358 (2005) 3135e3144. [20] F. Arjmand, B. Mohani, S. Ahmad, Synthesis, antibacterial, antifungal activity and interaction of CT-DNA with a new benzimidazole derived Cu(II) complex. Eur. J. Med. Chem. 40 (2005) 1103e1110. [21] P. Drevensek, T. Zupan ci c, B. Pihlar, R. Jerala, U. Kolotsch, A. Plaper, I. Turel, Mixed-valence Cu(II)/Cu(I) complex of quinolone ciprofloxacin isolated by a hydrothermal reaction in the presence of L-histidine: comparison of biological activities of various coppereciprofloxacin compounds. J. Inorg. Biochem. 99 (2005) 432e442. [22] M.C. Rodriguez-Argüelles, E.C. Lopez-Silva, J. Sanmartin, P. Pelagatti, F. Zani, Copper complexes of imidazole-2-, pyrrole-2- and indol-3-carbaldehyde thiosemicarbazones: inhibitory activity against fungi and bacteria. J. Inorg. Biochem. 99 (2005) 2231e2239. [23] O. Ibopishak Singh, M. Damayanti, N. Rajen Singh, R.K. Hemakumar Singh, M. Mohapatra, R.M. Kadam, Synthesis, EPR and biological activities of bis(1-nbutylamidino-O-alkylurea)copper(II) chloride complexes: EPR evidence for binuclear complexes in frozen DMF solution. Polyhedron 24 (2005) 909e916. [24] K. Singh, M. Singh Barwa, P. Tyagi, Synthesis and characterization of cobalt(II), nickel(II), copper(II) and zinc(II) complexes with Schiff base derived from 4amino-3-mercapto-6-methyl-5-oxo-1,2,4-triazine. Eur. J. Med. Chem. 42 (2007) 394e402. [25] D.P. Singh, R. Kumar, J. Singh, Synthesis and spectroscopic studies of biologically active compounds derived from oxalyldihydrazide and benzil, and their Cr(III), Fe(III) and Mn(III) complexes. Eur. J. Med. Chem. 44 (2009) 1731e1736. [26] S. Belaid, A. Landreau, S. Djebbar, O. Benali-Baitich, G. Bouet, J.-P. Bouchara, Synthesis, characterization and antifungal activity of a series of manganese(II) and copper(II) complexes with ligands derived from reduced N,N0 -O-phenylenebis(salicylideneimine). J. Inorg. Biochem. 102 (2008) 63e69. [27] A. Kulkarni, S.A. Patil, P.S. Badami, Synthesis, characterization, DNA cleavage and in vitro antimicrobial studies of La(III), Th(IV) and VO(IV) complexes with Schiff bases of coumarin derivatives. Eur. J. Med. Chem. 44 (2009) 2904e2912. [28] S.P. Fricker, Metal based drugs: from serendipity to design. Dalton Trans. (2007) 4903e4917. [29] K.H. Thompson, C. Orvig, Metal complexes in medicinal chemistry: new vistas and challenges in drug design. Dalton Trans. (2006) 761e764. [30] R. Olar, M. Badea, E. Cristurean, V. Lazar, R. Cernat, C. Balotescu, Thermal behavior spectroscopic and biological characterization of cobalt(II), zinc(II), palladium(II) and platinum(II) complexes with N,N-dimethylbiguanide. J. Therm. Anal. Calorim. 80 (2005) 451e455. [31] R. Olar, M. Badea, D. Marinescu, V. Lazar, C. Balotescu, Thermal behavior of some N,N-dimethylbiguanide derivatives displaying antimicrobial activity. J. Therm. Anal. Calorim. 88 (2007) 323e327. [32] C.J. Bailey, R.C. Turner, Metformin. New Engl. J. Med. 334 (2003) 574e579.

[33] G. Siest, F. Roos, J.J. Gabou, Dosage du N-N-dimethyl biguanide (glucophage) par le diacetyle en milieu alcalin. Bull. Soc. Pharm. Nancy 58 (1963) 29e38. [34] P.V. Babykutty, C.P. Prabhakaran, R. Anantaraman, C.G.R. Nair, Electronic and infrared spectra of biguanide complexes of the 3d-transition metals. J. Inorg. Nucl. Chem. 36 (1974) 3685e3688. [35] B.J. Hathaway, Oxyanions. in: G. Wilkinson, R.D. Gillard, J.A. McCleverty (Eds.), Comprehensive Coordination Chemistry, first ed. Pergamon Press, Oxford, U.K, 1987. [36] A.B.P. Lever, Inorganic Electronic Spectroscopy. Elsevier, Amsterdam, London, New York, 1986. [37] L. Sacconi, F. Mani, A. Bencini Nickel, B.J. Hathaway, Copper. in: G. Wilkinson, R.D. Gillard, J.A. McCleverty (Eds.), Comprehensive Coordination Chemistry, first ed. Pergamon Press, Oxford, U.K, 1987. [38] J.R. Pilbrow, Transition Ion Electron Paramagnetic Resonance. Clarendon Press, Oxford, 1990. [39] P. Zanello, Inorganic Electrochemistry: Theory, Practice and Application. Royal Society of Chemistry, Cambridge, U.K, 2003. [40] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15 (2002) 167e193. [41] J.W. Costerton, P.S. Steward, E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections. Science 284 (1999) 1318e1322. [42] M.E. Davey, G.A. O’Toole, Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64 (2000) 847e867. [43] J. Tan, B. Wang, L. Zhu, DNA binding and oxidative DNA damage induced by a quercetin copper(II) complex: potential mechanism of its antitumor properties. J. Biol. Inorg. Chem. 14 (2009) 727e739. [44] E.K. Efthimiadou, M.E. Katsarou, A. Karaliota, G. Psomas, Copper(II) complexes with sparfloxacin and nitrogen-donor heterocyclic ligands: structureeactivity relationship. J. Inorg. Biochem. 102 (2008) 910e920. [45] A.Y. Louie, T.J. Meade, Metal complexes as enzyme inhibitors. Chem Rev. 99 (1999) 2711e2734. [46] P. Lemoine, M. Chiadmi, V. Bissery, A. Tomas, B. Viossat, Les Composés de la Metformine avec les Ions CoII, CuII and NiII. Acta Cryst. C52 (1996) 1430e1436. [47] P. Yurkanis Bruice, Organic Chemistry, third ed. Prentice Hall Inc., Pearson Education, 2001. [48] M. Zhu, L. Lu, P. Yang, X. Jin, Bis(1,1-dimethylbiguanido)nickel(II). Acta Cryst. E58 (2002) m272em274. [49] Bruker, SMART (Version 5.625), SAINTþ (Version 6.0), SHELXTL (Version 6.10) and SADABS (Version 2.03). Bruker AXS Inc, Madison, Wisconsin, USA, 2001. [50] G.M. Sheldrick, SHELXL-97. University of Gottingen, Germany, 1997. [51] DIAMOND e Visual Crystal Structure Information System, CRYSTAL IMPACT Postfach 1251, D-53002 Bonn, Germany, 2001. [52] V. Lazar, M.C. Balotescu, L. Moldovan, G. Vasilescu, L.-M. Petrache, D. Bulai, R. Cernat, Comparative evaluation of qualitative and quantitative methods used in the study of antibacterial and antifungal activity of hydroalcoholic vegetal extracts. Roum. Biotech. Lett. 10 (2005) 2225e2232. [53] M.-C. Balotescu, E. Oprea, L.-M. Petrache, C. Bleotu, V. Lazar, Antibacterial, antifungal and cytotoxic activity of Salvia officinalis essential oil and tinctures. Roum. Biotech. Lett. 10 (2005) 2471e2479. [54] National Committee for Clinical Laboratory Standards, Performance Standards for Antimicrobial Susceptibility Testing. NCCLS Approved Standard M100eS9. National Committee for Clinical Laboratory Standards, Wayne, PA, 1999. [55] National Committee for Clinical Laboratory Standards, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Approved Standard M7-A2. National Committee for Clinical Laboratory Standards, Villanova, PA, 1990. [56] K. Awtar, Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell. Biol. 66 (1975) 188e193. [57] R.G. Hawley, T.S. Hawley, Flow Cytometry Protocols, second ed. Humana Press, 2004.