Studies on thermal, spectral, magnetic and biological propertiesof new Ni(II), Cu(II) and Zn(II) complexes with a bismacrocyclicligand bearing an aromatic linker

Cristina Bucur • Mihaela Badea • Mariana Carmen Chifiriuc • Coralia Bleotu •

Emilia Elena Iorgulescu • Irinel Adriana Badea • Maria Nicoleta Grecu •

Veronica Lazar • Oana-Irina Patriciu • Dana Marinescu • Rodica Olar

Received: 25 July 2013 / Accepted: 2 October 2013 / Published online: 23 October 2013

� Akademiai Kiado, Budapest, Hungary 2013

Abstract Novel complexes of M2LCl4�nH2O type (M:Ni,

n = 4; M:Cu, n = 3 and M:Zn, n = 0; L: ligand resulted

from 1,4-phenylenediamine, 3,6-diazaoctane-1,8-diamine and

formaldehyde one-pot condensation) were synthesized and

characterised by microanalytical, ESI–MS, IR, UV–Vis,1H NMR and EPR spectra, magnetic data at room tempera-

ture and molar conductivities as well. The electrochemical

behaviour of complexes was investigated by cyclic voltam-

metry. Simultaneous TG/DTA measurements were per-

formed in order to evidence the thermal behaviour of the

obtained complexes. Processes such as water elimination,

fragmentation and oxidative degradation of the organic ligand

as well as chloride elimination occurred during thermal

decomposition. The antimicrobial assays demonstrate that the

compounds exhibited good antibacterial activity, especially

against S. aureus and E. coli strains, the most active being the

copper(II) complex, which also exhibited the most prominent

anti-biofilm effect, suggesting its potential use for the

development of new antimicrobial agents. The biological

activity was correlated with log Pow values. All complexes

disrupt the membrane integrity of HCT 8 tumour cells.

Keywords Complex � Biofilm � Cytotoxicity �Decaazabismacrocycle � One-pot condensation �Thermal behaviour

Introduction

Polyazamacrocyclic ligands have the ability to generate

stable complexes with different metal ions thus having a

considerable potential for application in biochemistry

[1, 2], catalysis [3], structural effects elucidation [4], bio-

sensors design [5], and carbon dioxide capture [6] fields.

The research data on azabismacrocyclic ligands have

exponentially grown since the discovery of paraxylyl bi-

cyclam (AMD3100) as an anti-HIV cell entry inhibitor [7].

Known as Mozobil or Plerixafor, this compound was

approved for use as a stem cell mobilizing agent and hasElectronic supplementary material The online version of thisarticle (doi:10.1007/s10973-013-3460-1) contains supplementarymaterial, which is available to authorized users.

C. Bucur � M. Badea � D. Marinescu � R. Olar (&)

Department of Inorganic Chemistry, Faculty of Chemistry,

University of Bucharest, 90-92 Panduri Str.,

050663 Bucharest, Romania

e-mail: rodica_m_olar@yahoo.com

M. C. Chifiriuc � V. Lazar

Department of Microbiology, Faculty of Biology,

University of Bucharest, 1-3 Aleea Portocalelor Str.,

60101 Bucharest, Romania

C. Bleotu

Stefan S Nicolau Institute of Virology,

285 Mihai Bravu Ave., Bucharest, Romania

E. E. Iorgulescu � I. A. Badea

Department of Analytical Chemistry, Faculty of Chemistry,

University of Bucharest, 90-92 Panduri Str., 050663 Bucharest,

Romania

M. N. Grecu

National Institute of Materials Physics, POB MG-7,

077125 Magurele-Ilfov, Romania

O.-I. Patriciu

Chemical and Food Engineering Department, Faculty

of Engineering, ‘‘Vasile Alecsandri’’ University of Bacau,

157 Calea Marasesti, 600115 Bacau, Romania

123

J Therm Anal Calorim (2014) 115:2179–2189

DOI 10.1007/s10973-013-3460-1

recently entered in the clinical trial phase for the AIDS

treatment [8, 9].

This compound is interfering with the virus entrance in

the host cell, by blocking the chemokine coreceptor

CXCR4 and thereby preventing the virus attachment on the

cell surface [8]. Subsequent studies in the field have led to

the discovery of some other valuable complexes with

paraxylyl bicyclam and similar bismacrocycle ligands [10].

The coordination behaviour of these ligands was studied in

order to obtain a specific metallocyclam configuration able

to enhance the active sites affinity for CXCR4 [11, 12].

Very good results and improved in vivo activity were

obtained by the incorporation of Cu(II), Ni(II) or Zn(II) in

the xylyl bicyclam structure [4, 11–13].

The variation of the metal ion, azamacrocyclic ring

configuration and the macrocyclic linker has been shown to

have major effects on the molecular interactions involved

in the antiviral activity [14, 15]. Furthermore for dimetal-

lic(II) complexes with configurationally restricted bicycl-

ams the same correlation between antiviral activity and

binding to the coreceptor CXCR4 was observed [16, 17].

Data concerning synthesis of some azabismacrocyclic

complexes obtained by one-pot condensation reactions as

well as their antimicrobial potential for the eradication of

both planktonic and biofilm-embedded microbial strains

were recently reported [18–20].

In this paper, we report the synthesis, analytical, spectral,

magnetic, electrochemical and thermal characterisation of

new species of Ni(II), Cu(II) and Zn(II) complexes with

bismacrocyclic ligand 1,4-bis(N,N-1,3,6,9,12-pentaazacy-

clotridecane)-benzene resulted by one-pot condensation of

1,4-phenylenediamine, 3,6-diazaoctane-1,8-diamine(trien)

and formaldehyde, as well as the bioevaluation of their

antimicrobial and cytotoxic activities.

Experimental

Materials

The high-purity reagents were purchased from Sigma-

Aldrich (NiCl2�6H2O, CuCl2�2H2O, ZnCl2, Na2S�9H2O),

Merck (1,4-phenylenediamine, 3,6-diazaoctane-1,8-diamine

and methanol) and Loba (formaldehyde and triethylamine)

were used as received without further purification except for

1,4-phenylenediamine that was recrystallized from ethanol,

sublimed in vacuum and kept in dark conditions.

Instruments

Chemical analysis of carbon, nitrogen and hydrogen has

been performed using a Perkin Elmer PE 2400 analyser.

Metal content was determined with AAS on a Avanta

GBC spectrometer by using a stock standard solution

(Merck, 1 g mL-1), while the working solutions were

prepared by a suitable dilution of the sample obtained by

complex calcination at 450 �C and the successive treatment

of the residue with HCl and HNO3.

The molar conductance was determined for 10-3 M

solutions of complexes in dimethylsulphoxide (DMSO)

with a Multi parameter analyser CONSORT C861.

Mass spectra were recorded by electrospray ionization

tandem mass spectrometry (ESI–MS) technique operating

in the positive ion mode with the following procedure: a

sample was dissolved in acetonitrile:water (1:1) and after

30 s of ultrasounds bath the solution was injected directly

in mass spectrometer (Waters Micromass Quatro micro

API triple quadrupole mass spectrometer and controlled by

MassLynx software) with an electrospray interface. Nitro-

gen was used as desolvation gas at 6.66 dm3 min-1 and

350 �C. The capillary and cone voltage were 3.0 kV and

30 V, respectively, while the source temperature was

120 �C. The injection was performed with 0.2 mL min-1

flow.

IR spectra were recorded in KBr pellets with a Bruker

Tensor 37 spectrometer in the range 400–4,000 cm-1.

Electronic spectra by diffuse reflectance technique were

recorded in the range 200–1,500 nm on a Jasco V670

spectrophotometer by using spectralon as standard.

Magnetic measurements were done at room temperature

(RT), on a Lake Shore’s fully integrated vibrating sample

magnetometer (VSM) system 7404, calibrated with a Ni—

0.126 g sphere—SRM 772a; moreover, VSM was inter-

calibrated with an absolute calibrated Faraday balance or

calibrated with Hg[Co(NCS)4] as standard. The molar

magnetic susceptibilities were calculated and corrected for

the atomic diamagnetism.

EPR measurements were performed at RT in X

(9.2 GHz)- and Q (34 GHz)-band with Adani CMS 4800

and Bruker ELEXSYS 500 spectrometer that operate with

a field modulation of 100 kHz. The magnetic field cali-

bration was performed with a diphenylpicrylhydrazyl

standard marker with a narrow EPR line at g = 2.0036.1H NMR spectra were recorded on a Bruker Avance

spectrometer (working frequency 200 MHz) at 25 �C.

Chemical shifts were measured in parts per million from

internal standard tetramethylsilane.

Cyclic voltammograms were recorded by an electrochem-

ical system (potentiostat/galvanostat) Autolab PGSTAT 12.

Electrochemical studies were performed at RT under inert

atmosphere (Ar 99.9999 %) in DMSO containing tetrabutyl-

ammonium perchlorate (Bu4NClO4) 0.1 M as supporting

electrolyte. The reference electrode was Ag/AgCl/KCl sat.

The counter electrode was the platinum wire. The working

electrode was a platinum electrode with the effective area of

2180 C. Bucur et al.

123

electrode 7.065 mm2. The details concerning electrochemical

behaviour of compounds are presented as Online Resource 1.

The heating curves (TG and DTA) were recorded using a

Labsys 1200 SETARAM instrument, over the temperature

range of 20–900 �C with a heating rate of 10 �C min-1. The

measurements were carried out in synthetic air atmosphere

(flow rate 16.66 cm3 min-1) by using alumina crucible.

The X-ray powder diffraction patterns were collected on

a PANalytical X’Pert PRO diffractometer using Cu Ka

radiation (k = 1.5406 A) in the range of 2h from 5� to 80�.

The value of log Pow was determined by UV–Vis spec-

trophotometry. For this purpose an UV–Vis GBC type

Cintra 10e spectrophotometer working in the range

200–800 nm was used to record the absorption spectra of

complexes in both water and n-octanol. The wavelength of

maximum absorbance (kmax) in UV domain was determined

for each complex and further used for calibration curves

plotted in water. Aqueous solutions of studied compounds

having the concentration ranging between 0.025 and

0.05 mM were used in this study. Equal volumes of aqueous

solution of compounds and n-octanol were first mixed in an

extraction funnel, second shaken well and finally separated

and centrifuged. The complexes concentration in water was

determined by means of calibration curve.

Antimicrobial activity assays

The antimicrobial activity of the obtained complexes was

determined using American tissue cell culture (ATCC) ref-

erence strains (with ATCC code) as well as clinical isolates

from the collection of the Microbiology Department of the

Faculty of Biology, i.e. Gram-positive bacteria (B. subtilis

12488, B. subtilis 6633, E. faecalis ATCC 29212, S. aureus

ATCC 25923, MRSA 1684, S. aureus 13294 and S. epider-

mis 1736), Gram-negative bacteria (E. cloacae 61R, E. coli

ATCC 25922, E. coli 13147, E. coli 714, E. coli ESBL 1576,

K. pneumoniae 2968, K. pneumoniae 1771, P. aeruginosa

ATCC 1671 and P. aeruginosa 1397) and fungi (C. albicans

ATCC 249, C. albicans ATCC 128 and C. krusei 963).

Microbial suspensions corresponding to a 0.5 McFarland

density (1.5 9 108 CFU mL-1) obtained from 15 to 18 h

microbial cultures developed on solid media were used. The

antimicrobial activity was tested on Mueller–Hinton agar

medium, while a yeast peptone glucose medium was used in

case of fungal strains. The compounds (ligand and com-

plexes) were solubilised in DMSO and the starting stock

solution was of 1,000 lg mL-1 concentration. The qualita-

tive screening was performed by an adapted disc diffusion

method as previously described [21–23].

The quantitative assay of the antimicrobial activity was

performed by the liquid medium microdilution method, in

96 multi-well plates, in order to establish the minimal

inhibitory concentration (MIC). In this purpose, serial two-

fold dilutions of the compounds solutions (ranging between

1,000 and 1.95 lg mL-1) were performed in a 200 lL

volume of nutrient broth and each well was seeded with

50 lL microbial inoculum. Sterility control (wells con-

taining only culture medium) and culture controls (wells

containing culture medium seeded with the microbial

inoculum) were used. The influence of the DMSO solvent

was also quantified in a series of wells containing DMSO,

diluted accordingly with the dilution scheme used for the

complexes. The plates were incubated for 24 h at 37 �C,

and MIC values were considered as the lowest concentra-

tion of the tested compound that inhibited the visible

growth of the microbial overnight cultures.

The assessment of the complexes influence on the

microbial ability to colonize an inert substratum was per-

formed by the micro-titre method, following previously

described protocols [21]. The absorbance at 490 nm was

measured with an ELISA reader Apollo LB 911. All bio-

logical experiments were performed in triplicates.

Determination of cell cycle phases by flow cytometry

The HCT 8 cells were seeded in a 24-well cell culture in a

concentration of 105 per well. After 24 h the cells were

treated with solution of 100 lg mL-1 of compounds in

DMSO and after another 24 h all cells were collected,

washed twice with phosphate buffer saline (PBS) and fixed

with cold ethanol for at least 30 min at -20 �C. The cells

were further stained with propidium iodide (PI). The

acquisition was done using a Beckman-Coulter XL flow

cytometer and for analysis of disposition in different pha-

ses of cell cycle FlowJo software was used.

Flow cytometric determination of apoptosis by double

staining with annexin-V/PI method

The HCT 8 cells were incubated in a 24-well cell culture

cluster (105 per well), and 24 h later the cells were treated

with compounds at the final concentration 100 lg mL-l.

After 24 h all cells were collected and washed with PBS

twice. Cells were analysed for phosphatidylserine exposure

by an annexin-V FITC/PI double-staining method. Cells

were incubated with annexin-V FITC in dark condition at

RT for 10 min, then PI was added and cells were incubated

for another 10 min in the dark at RT. About 5,000 cells

were then acquired by Beckman-Coulter XL flowcytometer

and analysed using FlowJo software.

Synthesis and spectral data of complexes and ligand

To a suspension of trien (20 mmol), hydrated metal(II)

chloride (20 mmol), 2 mL triethylamine and 2 mL form-

aldehyde (37 %) in 150 mL methanol was added dropwise

Studies on Ni(II), Cu(II) and Zn(II) complexes 2181

123

to a solution of 1,4-phenylenediamine (10 mmol) in 50 mL

methanol. The reaction mixture was refluxed for 24 h until

a sparingly soluble compound, with various shades of

brown, was formed. The microcrystalline products were

filtered off, washed with methanol and air-dried.

[Ni2LCl2]Cl2�4H2O (1)

Analysis, found (%): Ni, 14.77; C, 33.29; H, 6.84; N, 17.28;

Ni2C22H52N10O4Cl4 requires (%): Ni, 15.05; C, 33.88; H, 6.72;

N, 17.96; ESI–MS in CH3CN:H2O (1:1) m/z: [Ni2C22

H44N10Cl3]?, 672.22; [Ni2C22H40N10Cl2]

?, 632.78; [Ni2C22

H38N10]?, 559.08; [NiC22H34N10]

?, 497.47; [NiC22H28N9]?,

477.02; [C22H50N10]?, 454.35; [C22H29N9]

?, 419.25;

[C20H39N8]?, 391.33; [C14H22N8]

?, 302.26; [C10H21N6]?,

225.25; [C10H21N4]?, 197.21.

[Cu2LCl4]�3H2O (2)

Analysis, found (%): Cu, 16.94; C, 34.37; H, 6.21; N,

18.25; Cu2C22H50N10O3Cl4 requires (%): Cu, 16.47; C,

34.25; H, 6.53; N, 18.15; ESI–MS in CH3CN:H2O (1:1)

m/z: [Cu2C22H48N10Cl3]?, 686.44; [Cu2C23H44N11Cl2]?,

672.32; [Cu2C23H48N11]?, 605.09; [C22H50N10]?, 454.37;

[C22H44N10]?, 448.80; [C20H39N8]?, 391.33; [C14H22N8]?,

302.40; [C10H21N4]?, 197.19.

[Zn2L]Cl4 (3)

Analysis, found (%): Zn, 17.95; C, 36.59; H, 6.19; N,

19.55; Zn2C22H44N10Cl4 requires (%): Zn, 18.14; C, 36.63;

H, 6.15; N, 19.42; 1H NMR (DMSO) d (ppm): 2.05 (s, 8H,

NH), 2.79 (m, 24H, (CH2)2), 3.33 (s, 8H, CH2), 7.31 (br,

4H, Ar–H); 13C NMR (DMSO) d (ppm): 36.04 (N–CH2–N),

39.10, 39.31, 39.52 ((CH2)2), 129.12, 130.06 (C6H4); ESI–

MS in CH3CN:H2O (1:1) m/z: [Zn2C23H48N10Cl2]?,

666.55; [Zn2C22H51N10Cl2]?, 657.16; [Zn2C22H48N10]?,

583.28; [Zn2C22H32N9]?, 553.32; [C22H43N10]?, 447.23;

[C22H29N9]?, 419.31; [C18H41N6]?, 341.26; [C10H21N4]?,

197.19.

The ligand (1,4-bis(N,N-1,3,6,9,12-

pentaazacyclotridecane)-benzene) synthesis

To a suspension of complex [Ni2LCl2]Cl2�4H2O in water

an excess of sodium sulphide nonahydrate was added and

the reaction mixture was magnetically stirred at 50 �C until

a black precipitate was formed. The suspension was filtered

off and the filtrate was treated with 2 N HCl until the

cessation of effervescence. After solvent evaporation, the

solid product was dissolved in methanol and purified by

thin liquid chromatography on silica gel 60 F-254 pre-

coated TLC plates (MeOH:CH2Cl2, 3:7). The ligand was

extracted then with DMSO from silica gel matrix. Analysis,

found (%): C, 59.57; H, 9.73; N, 31.31; C22H44N10 requires

(%): C, 58.90; H, 9.89; N, 31.22; 1H NMR (DMSO-d6) d(ppm): 2.25 (s, 8H, NH), 3.04 (t, 24H, (CH2)2), 3.88 (s, 8H,

CH2), 7.73 (d, 4H, Ar–H). ESI–MS in CH3CN:H2O (1:1) m/z:

[C22H50N10]?, 454.81; [C22H45N10]?, 449.58; [C22H38N8]?,

414.42; [C20H39N8]?, 391.10.

Results and discussions

Synthesis and characterisation of ligand and complexes

The one-pot reactions in formaldehyde excess of 2:2:1 molar

mixture of nickel(II), copper(II) or zinc(II) chloride, 3,6-dia-

zaoctane-1,8-diamine(trien) and 1,4-phenylenediamine in

alkaline medium produced the species M2LCl4�nH2O ((1) M:

Ni, n = 4; (2) M: Cu, n = 3 and (3) M: Zn, n = 0; L:

1,4-bis(N,N-1,3,6,9,12-pentaazacyclotridecane)-benzene)

(Scheme 1). The free ligand was synthesized by Ni(II)

complex treatment with sodium sulphide, followed by thin liquid

chromatography purification and DMSO recrystallization.

The chemical analyses are in accord with formulas pro-

posed for complexes and ligand (see ‘Experimental’ section).

The complex (2) behaves as non-electrolyte as their

molar conductance value (in DMSO) is 11 X-1 cm2 mol-1.

Instead for complexes (1) and (3) values of 73 and

238 X-1 cm2 mol-1 are an indicative of their behaviour as

1:2 and 1:4 electrolytes, respectively [24].

IR, 1H NMR and ESI–MS spectra

In comparison with 1,4-phenylenediamine and M(trien)Cl2(M: Ni, Cu, Zn) IR spectra the following comments can be

done for the ligand and complexes (Table 1):

(i) the absorptions in the range of 2,830–2,940 cm-1 can be

assigned to stretching vibration modes of the methylene

group [4] provided for bismacrocyclic ligand by both

3,6-diazaoctane-1,8-diamine and formaldehyde;

(ii) the strong band that appears around 1,515 cm-1

correspond to stretching vibration of C=C from

aromatic group [25] provided by 1,4-phenylenedi-

amine intermediate;

(iii) the broad absorption around 3,200 cm-1 arises from

the secondary amine stretching vibration [3, 22, 23].

This band is shifted by 51–66 cm-1 to lower

wavenumbers in comparison with the metal-free

bismacrocyclic ligand and indicates its coordination

through nitrogen atoms [26];

(iv) a broad band in the range 3,340–3,390 cm-1,

characteristic for the m(OH) stretching vibration,

can be noticed for the complexes (1) and (2) [27].

2182 C. Bucur et al.

123

The 1H NMR spectrum of ligand consists of two singlets

corresponding to NH and –NCH2N– protons, and one

triplet arising from –NCH2CH2N– chains provided by 3,6-

diazaoctane-1,8-diamine for bismacrocycle. The aromatic

protons are responsible for the doublet at 7.73 ppm

occurrences. The amino group coordination is further

supported by 1H NMR spectrum of complex (3) where the

resonance assigned to NH group is upfield-shifted relative

to the signal of the free ligand.

The formation of the ligand and complexes was studied

with ESI–MS spectra. A comparison between both molecular

and structural formulae of the studied compounds with the

m/z values of each MS spectra confirms the structures pre-

sented in Scheme 1. Thus, in the mass spectrum of ligand the

peak with m/z 449.58 was assigned to the molecular ion

[C22H44N10?H]?. For the complexes the molecular ions were

found as [Ni2C22H44N10Cl3]?, [Cu2C22H44N10Cl3?4H]? and

[Zn2C22H44N10Cl2?7H]?, respectively. Moreover, other

fragments with or without metal ion that can be related to the

ligand or complexes structure were identified such as

[C10H21N4]? (m/z: 197.19) observed in all complexes spectra

or [C20H39N8]? (m/z: 391.10) observed in the mass spectra of

ligand and complexes (1) and (2), respectively.

Electronic, EPR spectra and magnetic data

Electronic spectra correlated with magnetic data at RT

provided useful information concerning the oxidation state

of the metallic ion, stereochemistry and the ligand field

nature. Table 2 lists the electronic absorption bands toge-

ther with (vT)HT values of compounds at RT.

The intraligand p ? p* and n ? p* transitions appear

at 24,630 and 39,215 cm-1 in the ligand spectrum and are

different shifted in the complexes spectra as a result of

changes in the electronic density of the ligand upon

coordination.

The electronic spectrum of complex (1) exhibits bands

in visible and near-infrared regions with a pattern charac-

teristic for a square pyramidal stereochemistry of Ni(II)

with the high spin configuration [28]. The slightly higher

experimental (vT)HT value in comparison with calculated

one for dinuclear species of 2.00 cm3 K mol-1 further-

more sustains the proposed stereochemistry [29].

A broad band with tendency of splitting in three com-

ponents can be noticed in the visible region of the electronic

H H

H H

4H2O.Cl2NNi

NN

NNCl

Cl

NN

NN

NiN

H

H

H

H

2

M: Ni, Cu, Zn

+ 2MCl2 +

NH NH2

NH NH2

NEt3

4 CH2O +

ot C

H2N NH2

H

H

H

H

NCu

NN

NN

Cl

Cl Cl

Cl

NN

NN

CuN

H

H

H

H

3H2O.

H

H

H

H

NZn

NN

NN NN

NN

ZnN

H

H

H

H

Cl4

Scheme 1 Synthetic route to

prepare complexes

Table 1 IR absorption bands (cm-1) for 1,4-phenylenediamine

(1,4-FDA), bismacrocyclic ligand (L) and complexes (1)–(3)

1,4-

FDA

L (1) (2) (3) Assignments

– – 3,391vs 3,341vs – m(H2O)

3,303m – – – – mas(NH2)

– 3,293m 3,227s 3,242s 3,240s m(NH)

3,198s – – – – ms(NH2)

3,008w 3,031m 3,032m 3,036w 3,023w m(CHaromatic)

– 2,930m 2,931m 2,938m 2,925m mas(CH2)

– 2,843w 2,852m 2,831m 2,856m ms(CH2)

1,517vs 1,515m 1,518s 1,515s 1,518vs m(C=C)

1,448m 1,451vs 1,459m 1,456m 1,462m m(C=C) ? d(CH2)

– m(C=C)

– 1,304s 1,290m 1,291m 1,299m m(Caromatic–N)

– 1,141vs 1,172w 1,130m 1,176w m(Caliphatic–N)

– 903w 911w 923w 937w q(CH2)

867m 833m 831m 831m

vs very strong, s strong, m medium, w weak

Studies on Ni(II), Cu(II) and Zn(II) complexes 2183

123

spectrum of compound (2). The maximum at 18,180 and the

spectrum feature indicates an octahedral distorted stereo-

chemistry for this complex [28]. Considering that macro-

cycle unit is coordinated in equatorial positions around the

metal ion as observed for other saturated polyazamacrocy-

clic derivatives [30], the chloride anions in apical position

generate a tetragonal elongated distortion around the Cu(II)

ion. The additional band at 26,670 is tentatively assigned to

the ligand to metal charge transfer transition. The experi-

mental (vT)HT value close to calculated one indicate the

absence of any interaction between the metallic centres at

RT [29].

It was assumed that the Zn(II) complex also adopts an

octahedral stereochemistry having in view that a square

planar one is rarely found for this ion in compounds with

saturated azamacrocyclic ligands [30]. The additional band

in the spectrum of complex (3) can arise from a metal to

ligand charge transfer transition. As expected the Zn(II)

complex is diamagnetic.

EPR spectra of complex (2), recorded at RT in X- and

Q-band, are shown in Fig. 1. X-band spectrum is charac-

teristic for a Cu(II) complex with isolated centres and an

axial symmetry [31] with gll = 2.182 and g\ = 2.062, and

with hyperfine splitting All = 168 G while A\ is unsolved.

Q-band spectrum reveals the distribution of these param-

eters, the so-called ‘g-A-strain effect’, due to small varia-

tion in copper local site geometry, and hyperfine coupling.

Both spectra also show an additional line, well separated in

Q-band, due to the presence of a structural paramagnetic

defect with g = 2.005 and line width DH = 13.6 G.

Thermal behaviour of complexes

Thermal analysis techniques represent a useful tool for

determining the composition and thermal behaviour of

complexes [18–20, 32–35]. Furthermore, the understanding

the thermal behaviour of biological active species has

proved to be relevant in order to develop further applica-

tions [36, 37].

In order to obtain such information thermal behaviour of

complexes was investigated by simultaneous TG–DTA

analysis and the final residues were examined by powder

X-ray diffraction. The species isolated after the water

elimination were also isolated and characterised.

The simultaneous TG/DTA curves registered for com-

plex (1) are shown in Fig. 2 and indicate that the complex

undergoes three steps of thermal decomposition. The data

collected from these curves are summarised in Table 3.

The first step of thermal degradation of Ni(II) complex

consists in an endothermic elimination of water up to

166 �C, temperature range corresponding to crystallization

nature of these molecules [38]. The electronic spectrum of

residue isolated at 166 �C preserves all characteristics of

the parent compound, confirming that the water molecules

are uncoordinated. The second step is not a single one, but

an overlapping of four processes according to DTA curve

profile. The small exothermic peaks observed in the

Table 2 Absorption maxima (cm-1) from electronic spectra of

ligand and complexes (1)–(4), assignments and (vT)HT values

Compound Absorption

maxima/

cm-1

Assignments (vT)HT/

cm3 K mol-1

Found Calc.

L 45,450

36,360

30,770

p ? p*; n ? p* – –

[Ni2LCl2]Cl2�4H2O (1)

50,000

37,040

32,790

p ? p*; n ? p* 2.17 2.00

20,410 3B1 ? 3E

18,350 3B1 ? 3A2

14,925 3B1 ? 3E

13,245 3B1 ? 3A2

11,170 3B1 ? 3B2

7,140 3A2 ? 3E

[Cu2LCl4]�3H2O

(2)

41,670

37,040

p ? p*; n ? p* 0.78 0.84

26,670 LMCT

18,180 dxz, dyz ? dx2�y2

13,515 dxy ? dx2�y2

11,765 dz2 ? dx2�y2

[Zn2L]Cl4 (3) 50,000

38,460

33,330

p ? p*; n ? p* – –

22,470 MLCT

LMCT ligand to metal charge transfer, MLCT metal to ligand charge

transfer

250 275 300 325 350 375 400

Magnetic field/mT

gII

gII

gdefect

gdefect

1050 1125 1200 1275

X–bandν = 9.265767 GHz

Q–bandν = 34.15336 GHz

g T

g T

Fig. 1 Q- and X-band powder EPR spectra for complex (2)

2184 C. Bucur et al.

123

166–370 �C temperature range are probably due to both

endothermic and exothermic reactions that occur simulta-

neously such as cleavage and rearrangement of the bonds

as well as some moieties oxidative degradation. According

to the mass loss, about 32 % of the organic ligand is

eliminated through these processes up to 370 �C. In the

final step both the rest of organic part oxidative degradation

and the chloride anions elimination occur. As a result of

these reactions, several exothermic processes, the last one

is very strong, can be noticed on DTA curve. All these

transformations finally lead to nickel(II) oxide (found/calc.

overall mass loss: 80.8/80.8 %). The nature of final product

was confirmed by powder X-ray diffraction data (ASTM

78-0429). It is to be pointed that the thermal behaviour of

this compound exhibits the same pattern with that of the

similar compound with the bismacrocycle ligand derived

from 1,3-phenylenediamine [20].

The complex (2) loses the water molecules (Fig. 3) also

up to 166 �C as an indicative of their crystallisation nature

[38]. Again, the proof of the presence of uncoordinated

water is provided by the similarity of the electronic spec-

trum of residue at 166 �C to that of parent compound. The

ligand bonds cleavages together with some moieties oxi-

dation occur in the second step accompanied by two exo-

thermic processes visible on the DTA curve. About 23 %

of the organic ligand is lost during these processes. The

decomposition ends with one strong exothermic signal

resulted from oxidative processes. According with powder

X-ray diffraction profile the final product at 780 �C is CuO

(ASTM 5-661) (found/calc. overall mass loss: 79.3/79.4 %).

The decomposition starts at 168 �C for anhydrous

compound (3) and as a result two small exothermic pro-

cesses can be noticed on DTA curve up to 360 �C (Fig. 4).

The oxidative degradation of the remaining organic part

and chloride anion elimination proceed afterwards in sev-

eral overlapped exothermic processes. The residual mass at

716 �C corresponds to zinc(II) oxide (ASTM 036-1451)

stabilisation (found/calc. overall mass loss: 77.4/77.4 %).

Taking into account all the above data, complexes can

be formulated as [Ni2LCl2]Cl2�4H2O (1), [Cu2LCl4]�3H2O

(2) and [Zn2L]Cl4 (3), respectively (Scheme 1).

Antimicrobial activity

The antimicrobial activity of the complexes was deter-

mined on ATCC reference strains and clinical isolates

belonging to different bacterial and fungal species. The

complexes activity was assayed in comparison with that of

ligand and MtrienCl2 (M: Ni, Cu, Zn) species, used as

intermediates in the condensation process. It is to be

pointed out that the MtrienCl2 species did not exhibit a

significant activity, the diameter of the growth inhibition

zone being below 5 mm for all tested strains.

The tested compounds exhibited an improved antimi-

crobial activity against the planktonic microbial strains,

compared with the ligand (Table 4). In accordance with the

0

–20

–40

–60

–80

0 200 400 600 800 1000

Temperature/°C

120

100

80

60

40

20

0

–20

–40

Hea

t flo

w/μ

VE

xo

Mas

s va

riatio

n/%

Fig. 2 TG and DTA curves for [Ni2LCl2]Cl2�4H2O (1)

Table 3 Thermal behaviour data (in air atmosphere) for complexes

Compound Step Thermal effect Temperature

range/�C

Dmfound/% Dmcalc/%

[Ni2LCl2]Cl2�4H2O (1) 1. Endothermic 60–166 9.1 9.2

2. Exothermic 166–370 18.5 18.4

3. Exothermic 370–710 53.2 53.2

Residue NiO 19.2 19.2

[Cu2LCl4]�3H2O (2) 1. Endothermic 56–166 6.4 7.0

2. Exothermic 166–427 13.2 13.4

3. Exothermic 427–780 59.7 59.0

Residue CuO 20.7 20.6

[Zn2L]Cl4 (3) 1. Exothermic 168–360 7.3 7.5

2. Exothermic 360–716 70.1 69.9

Residue ZnO 22.6 22.6

Studies on Ni(II), Cu(II) and Zn(II) complexes 2185

123

MIC value, a good antimicrobial effect was considered for

MIC values lower than 250 lg mL-1 and a moderate

activity for MIC values between 250 and 500 lg mL-1.

The compound (1) exhibited a good activity on both Gram-

positive MRSA and the Gram-negative E. coli strains and

was moderately active on E. cloacae and P. aeruginosa

strains. Compound (2) was also very active on one S.

aureus and two E. coli strains and moderately active on C.

krusei strain. The compound (3) was the least active,

exhibiting good activity only on one E. coli strain and

moderate activity on P. aeruginosa and C. albicans strains.

It is to be pointed out the very good activity of compound

(1) against MRSA 1648 and of compound (3) on E. coli

ESBL 1576, strains with acquired resistance of epidemio-

logical importance to a beta-lactam antibiotic (i.e. methi-

cillin in case of MRSA and all beta-lactams, excepting

carbapenems in case of E. coli ESBL strain).

The most susceptible strains to the tested compounds were

E. coli followed by S. aureus, while B. subtilis, P. aeruginosa

and the fungal strains were less susceptible. Taken together

the results indicate that the complexes (1) and (2) proved to

be more active than complex (3) against planktonic strains.

Concerning the antibiofilm activity, the most efficient

compound against the adherent microbial cells was the

complex (2), which inhibited the biofilm formed by Gram-

negative, Gram-positive bacterial and fungal strains

(Table 5), followed by (3) and thereafter complex (2). The

ligand inhibited the biofilm formed by B. subtilis, while all

complexes were inactive.

The complexes activity may be related to the strength of

the metal-donor atom bonds, the cation size, receptor sites,

diffusion ability, lipophilicity and a combined effect of the

metal and the ligands for the inactivation of specific

0

–20

–40

–60

–80

0 200 400 600 800 1000–40

–20

0

20

40

60

80

100

120

Exo

Hea

t flo

w/μ

V

Temperature/°C

Mas

s va

riatio

n/%

Fig. 3 TG and DTA curves for [Cu2LCl4]�3H2O (2)

10

0

–10

–20

–30

–40

–50

–60

–70

–80

0 200 400 600 800 1000

–20

0

20

40

60

80

Temperature/°C

Hea

t flo

w/μ

VE

xo

Mas

s va

riatio

n/%

Fig. 4 TG and DTA curves for [Zn2LCl4] (3)

Table 4 The MIC (lg mL-1) values of the ligand and complexes

(1)–(3)

Strain L (1) (2) (3)

B. subtilis 12488 1,000 – – –

S. aureus ATCC 25923 – – – 500

S. aureus 13294 – – 15.62 –

MRSA 1648 – 15.62 – –

E. cloacae 61R – 250 – –

E. coli ATCC 25922 – 15.62 3.9 500

E. coli 13147 1,000 3.9 7.81 –

E. coli ESBL 1576 – – – 7.81

P. aeruginosa ATCC 1671 – 250 – 500

C. albicans ATCC 249 500 – – 250

C. krusei 963 – – 500 –

Table 5 The influence of the compounds on microbial biofilm for-

mation (the threshold concentration (lg mL-1) corresponding to the

inhibition domain)

Strain L (1) (2) (3)

B. subtilis 12488 Inhibition

250

– – –

E. faecalis

ATCC 29212

– – Inhibition

31.25

–

S. aureus ATCC

25923

– Inhibition

3.90

– Inhibition

3.90

MRSA 1684 – – – –

E. coli ATCC

25922

– – Inhibition

1.95

–

K. pneumoniae

2968

– – Inhibition

62.50

–

P. aeruginosa

ATCC 1671

– – – –

C. albicans

ATCC 249

– – – Inhibition

1.95

C. krusei 963 – – Inhibition

1.95

–

2186 C. Bucur et al.

123

microbial targets involved in microbial cells physiology or

infectivity (mediated by the capacity to adhere and colo-

nize a specific substratum). The target molecules may be

exposed at the level of microbial wall or may be located in

the cytosolic compartment. Thereby, the effectiveness

variation of the studied compounds against tested strains

may be assigned to the different permeabilities of the

cellular wall of the Gram-positive, Gram-negative or fun-

gal strains and/or to differences in the structure of the

intracellular targets [39].

In order to reach the intracellular targets, the compounds

must be either liposoluble in order to diffuse through the

lipid layer of the microbial wall or be hydrophilic, in order

to be actively internalized by porins. The compounds

lipophilicity were determined as results in order to obtain

information concerning the compounds ability to penetrate

the microorganisms’ lipid layers. The log P values obtained

for complexes (1), (2) and (3) of -0.37, -1.12 and -0.43,

respectively, indicate their hydrophilic character [40–42],

suggesting their inability to penetrate the microbial lipid

layers through passive diffusion.

In order to pass through porins, the molecules size must

be lower than the porin channel diameter, estimated for

E. coli porins to range from 1.0 to 2.0 nm [43]. Taking into

account that the complex (2) was the most active from this

series and that it exhibited the highest log P value, we

could hypothesize that: (i) either the compound is inter-

nalized through porins and is reaching its intracellular

target or (ii) the complex cations could establish electro-

static interactions with negatively charged functional

groups of the microbial wall, interfering with the complex

physiological roles of this structure, and thus with micro-

bial cell viability and pathogenicity. Furthermore, the fact

that from Irving Williams series, Cu(II) generates the most

stable compounds together with its stereochemical versa-

tility may also support this observation.

Cytotoxicity assay

The cytotoxicity evaluation of the ligand, and both com-

plexes with trien and azabismacrocyclic ligand was per-

formed using the human tumour cell line HCT 8 (human

ileocaecal adenocarcinoma). It is to be pointed that com-

plexes with trien did not exhibit any cytotoxicity. The cells

harvested by trypsin digestion after 24 h of treatment with

ligand and complexes labelled in suspension with annexin-

V FITC and PI were analysed to establish the percentage of

cells found in apoptosis and necrosis state. Only few cells

were positive for annexin-V FITC after treatment with

compound (1) (4.85 % early apoptosis and 18.10 % late

apoptosis) (Fig. 5). On the other hand, all compounds

induced an intensive cell staining by PI, indicating that

103

102

101

100

10–1

10–1 100101 102

103

Q30.167 %

Q20.479 %

Q19.86 %

Q489.5 %

Q464.2 %

Q112.9 %

Q218.1 %

Q34.85 %

Q20.804 %

Q30.125 %

Q468.7 %

Q130.4 %

Q141.1 %

Q20.108 %

Q30.066 %

Q458.7 %

FL1 LOG:: FITC10–1 100

101 102103

FL1 LOG:: FITC

10–1 100101 102

103

FL1 LOG:: FITC

10–1 100 101 102 103

FL1 LOG:: FITC

FL3

LO

G::

FL3

_PI

103

102

101

100

10–1

FL3

LO

G::

FL3

_PI

103

102

101

100

10–1

FL3

LO

G::

FL3

_PI

103

102

101

100

10–1

FL3

LO

G::

FL3

_PI

L 1

32

Fig. 5 Dot plots resulted from

the flow cytometry

quantification of HCT 8 cell

viability in the presence of

ligand and complexes assayed

by Annexin-FITC/PI Kit [cell

population from quadrants: (1)

top left Q1 necrotic cells

(positive for PI); (2) top right

Q2 late apoptosis (positive for

PI and FITC); (3) bottom right

Q3 early apoptosis (positive for

FITC); (4) bottom left Q4 viable

cells (negative for PI and

FITC)]

Studies on Ni(II), Cu(II) and Zn(II) complexes 2187

123

membrane integrity was severely damaged especially after

treatment with the compound (2) (41.10 %) and compound

(3) (30.40 %). Additionally, compound (3) increased the

percent of cells found in G2/M phase, an important

checkpoint that can block the entry into mitosis when DNA

is damaged. These results could indicate the potential anti-

tumour activity of this compound, but further investiga-

tions are needed in order to confirm this hypothesis.

Conclusions

Complexes of type M2LCl4�nH2O as well as the ligand 1,4-

bis(N,N-1,3,6,9,12-pentaazacyclotridecane)-benzene

(L) were synthesized and characterised.

The IR, NMR and ESI–MS data provide a solid support

for condensation process. Electronic spectrum of Ni(II)

complex is characteristic for the square pyramidal stereo-

chemistry, while that of Cu(II) complex displays the pattern

of octahedral surrounding. These data were furthermore

confirmed by magnetic behaviour at RT. EPR data for Cu(II)

complex support a tetragonal local symmetry.

Cyclic voltammetric studies indicated that each complex

presents a characteristic reduction peak situated at more

cathodic potential in comparison with DMSO solvated ion

indicating that the electrochemical process that occurs is more

difficult as result of the influence of the macrocyclic ligand.

The thermal analyses evidenced processes as water

elimination, fragmentation and oxidative degradation of

the organic ligand as well as chloride anion elimination.

The final product of decomposition was metal(II) oxide.

The results of the antimicrobial assays demonstrate that

the obtained complexes exhibited a good antibacterial

activity, especially against S. aureus and E. coli strains, the

most active compound being the Cu(II) complex, which also

exhibited the most prominent anti-biofilm effect, as well as

low cytotoxicity on HCT 8 cells, suggesting its potential use

for the development of new antimicrobial agents.

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