studies on thermal, spectral, magnetic and biological properties of new ni(ii), cu(ii) and zn(ii)...
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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: [email protected]
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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