Synthesis and characterization of Ni (II), Cu (II), Fe (II) and Fe3O4 nanoparticle complexes with tetraaza macrocyclic Schiff base ligand for antimicrobial activity and cytotoxic activity against cancer and normal cells

Article abstract of DOI:10.1002/aoc.4860

This work describes a simple synthesis of complexes of the type [M(C₃₂H₂₈N₄)Cl₂], where M?=?Ni (II), Cu (II) and Fe (II) and a novel complex of magnetite nanoparticle (Fe₃O₄NP) inside (INS)

Full text of DOI:10.1002/aoc.4860

Received: 11 October 2018
DOI: 10.1002/aoc.4860
Revised: 14 January 2019
Accepted: 20 January 2019
F U L L P A P E R
Synthesis and characterization of Ni (II), Cu (II), Fe (II) and
Fe3O4 nanoparticle complexes with tetraaza macrocyclic
Schiff base ligand for antimicrobial activity and cytotoxic
activity against cancer and normal cells
Laroussi Chaabane1,4 | Hassiba Chahdoura2 | Wassim Moslah3 | Mejdi Snoussi2 |
Emmanuel Beyou4 | Mohammed Lahcini5,6 | Najet Srairi‐Abid3 |
Mohamed Hassen V. Baouab1
1 Unité de Recherche Matériaux et Synthèse
This work describes a simple synthesis of complexes of the type [M(C32H28N4)
Cl2], where M = Ni (II), Cu (II) and Fe (II) and a novel complex of magnetite
nanoparticle (Fe3O4NP) inside (INS) tetraaza macrocyclic Schiff base ligand
(C32H28N4): [Fe3O4NP‐INS‐(C32H28N4)], which was prepared by using a novel
co‐precipitation method of coordinated ferric ion (Fe3+) in the complex
[Fe(C32H28N4)Cl2] under mild conditions. The synthesized compounds were
characterized and compared with a various physic‐chemical techniques like:
Fourier transform infrared (FT‐IR), ultraviolet–visible spectroscopic techniques
Organique (UR17ES31) Institut Préparatoire
aux Etudes d’Ingénieurs de Monastir,
Université de Monastir‐Tunisie, Bd. de
l’environnement, 5019 Monastir, Tunisie
2 Laboratoire de Recherche “Bioressourses,
Biologie Intégrative & Valorisation”,
Institut Supérieur de Biotechnologie de
Monastir, Avenue Tahar Hadded, BP 74,
5000 Monastir, Tunisia
3 Université de Tunis El Manar, Institut
Pasteur de Tunis, LR11IPT08 Venins et
biomolécules thérapeutiques, 1002 Tunis,
Tunisia
4 UMR CNRS5223, Ingénierie des
Matériaux Polymères, Université Lyon 1,
F‐69622 Villeurbanne, France
5 Laboratory of Organometallic and
Macromolecular Chemistry‐Composites
Materials, Faculty of Sciences and
Technologies, Cadi Ayyad University,
Avenue Abdelkrim Elkhattabi, B.P. 549,
40000 Marrakech, Morocco
6 Mohammed VI Polytechnic University,
Lot 660, Hay Moulay Rachid, 43150 Ben
Guerir, Morocco
1
(UV–Vis), 1‐dimensional (1D) H‐NMR, 13C‐NMR spectroscopic techniques,
mass spectra, Powder X‐ray diffraction (PXRD), Vibrating sample magnetome-
ter (VSM), Scanning electron microscopy (SEM), elemental analysis and molar
conductance measurements. Furthermore, the highest saturation magnetiza-
tion was 26.56 emu.g−1 obtained from [Fe3O4NP‐INS‐(C32H28N4)] (diameter
of Fe3O4NPs~20.87 nm) that prove easy separation by an external magnetic
field. In vitro screening of all the compounds against different species of bacte-
ria and fungi shows that [Fe3O4NP‐INS‐(C32H28N4)] is effective against the
tested strains as compared to the tetraaza macrocyclic ligand and selected com-
plexes. The cytotoxic activity of the all compounds was also examined in 3
human tumor cell lines as U87, MDA‐MB‐231 and LS‐174. The complex
[Fe3O4NP‐INS‐(C32H28N4)] shows moderate and strong cytotoxic activity
against brain cancer, colon cancer and breast cancer (U87, MDA‐MB‐231 and
LS‐174 respectively), without showing cytotoxicity towards peripheral blood
mononucleocyte (PBMC) cells.
Correspondence
Mohamed Hassen V. Baouab, Unité de
Recherche Matériaux et Synthèse Organique
(UR17ES31) Institut Préparatoire aux
Etudes d’Ingénieurs de Monastir, Université
de Monastir-Tunisia. Bd. de
KEYWORDS
antimicrobial/cytotoxic activity, complexes, magnetite nanoparticle, Tetraaza macrocyclic
l’environnement, 5019 Monastir, Tunisie.
Email: hbaouab@yahoo.fr
Funding information
France‐Tunisia‐Morocco
Appl Organometal Chem. 2019;e4860.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
1 of 19
https://doi.org/10.1002/aoc.4860

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CHAABANE ET AL.
to keep the stability of Fe3O4NPs. Many kinds of mate-
rials have been discovered in recent years use as the
coating including small organic molecules or surfac-
tants, biocompatible polymers and noble metals.[32,33]
Among these coating materials, biocompatible polymers
are very exceptional since the dense biocompatible poly-
mers may prevent Fe3O4NPs from chemical contact with
corrosive liquid media.[34] Recently, Rahimi, et al. dem-
onstrated that Fe3O4NPs coated with biocompatible
polymers can prevent agglomeration of nanoparticles,
minimize protein adsorption, leading to increased circu-
lation time in the blood stream and showing cytotoxicity
towards normal cells.[35] To exhibit the truly remarkable
magnetic properties and biological activities, tetraaza
macrocyclic molecules are novel surface complexes of
nano‐materials that have good chemical stability and
low toxicity.[4–6,34] In this paper, we report the coordina-
tion of tetraaza macrocyclic complexes of Ni (II), Cu (II)
and Fe (II) derived from the condensation of benzil (1,2‐
diphenylethane‐1,2‐dione) with ethylenediamine. We
have also proposed the novel complex of magnetite nano-
particle inside tetraaza macrocyclic Schiff base ligand,
which were produced by the novel co‐precipitation
method under mild conditions. All the complexes were
characterized by various physic‐chemical techniques like
FT‐IR, Uv–Vis, NMR, ESI, PXRD, VSM, SEM, conduc-
tance measurements and elemental analysis. Finally, the
antimicrobial and cytotoxic activities of the synthesized
compounds were evaluated and compared.
1 | INTRODUCTION
In recent years, the coordination chemistry of macrocy-
clic ligands has attracted the interest of bioinorganic
chemists, owing to their amazing unique chemical pro-
prieties and biological activities.[1–4] Macrocyclic Schiff
bases are important in macrocyclic chemistry because
they can selectively chelate certain metal ions depending
on the number, type and position of their donors, the
ionic radii of the metal centers, and the coordinating
property of counterions.[5–8] However, the chemistry of
transition metal complexes is very useful in fundamental
studies, such as biological, catalysis and magneto
chemistry.[9–12] A number of important macrocyclic metal
complexes show interesting biological activities including
antibacterial,[13] antifungal,[14] anticancer[15] and
antiproliferative effects.[16] Additionally, synthetic
tetraazamacrocycles (N4) have been considered typically
good models due to the presence of four nitrogen donor
sites restricted to stabilize uncommon oxidation states of
coordinated metal ions.[17,18]
By now, the synthesis of oxide nano‐materials is of
great importance in the development of modern nano-
technology due to their application within biomedi-
cine.[19] Among all the presently studied nano‐materials,
magnetite is one of the most popular ones and magnetite
nanoparticles (Fe3O4 NPs) have a lengthily material stud-
ied because of specific characteristics such as shape,
hydrophobicity, size distribution, surface chemistry,
and superparamagnetic allowing them to be optimized
for a specific function.[20] Moreover, Fe3O4NPs offer a
high potential of application in biomedical and bioengi-
neering fields, such as cancer thermotherapy, magnetic
resonance imaging (MRI), labeling and sorting of cells,
as well as separation of biomedical products.[21–23]
Among the practically used methods for the synthesis
of Fe3O4NPs, the following main ones should be men-
tioned: co‐precipitation,[24,25] solvothermal[26,27] and
Ionothermal.[28,29] The co‐precipitation method is the
most used technique to synthesize Fe3O4NPs because
of its easy operation, lower reaction temperature and
eco‐friendly procedure.[30] In this technique, the initial
solution including Fe2+ and Fe3+ salts is reduced with
alkaline solution like NaOH or NH3.H2O. Usually, the
reaction is intended with Fe2+/Fe3+ ratio equal to 1/2
that is sometimes partially varied in order to compensate
for oxidation probability.[21] However, the co‐
precipitation or precipitation method shows difficulty
to control the size distribution and prevent the aggrega-
tion of Fe3O4NPs due to the strong dipole–dipole mag-
netic attractions and interactions of the particles.[31]
Therefore, it is important to provide proper surface coat-
ing and developing some effective protection strategies
2 | EXPERIMENTAL
2.1 | Materials used
All of the solvents and chemicals were purchased com-
mercially available reagent grade and used without puri-
fication. Dimethylformamide (DMF), dimethylsulfoxide
(DMSO), ethanol, methanol, benzil, ethylenediamine,
nickel (II) chloride hexahydrate (NiCl2.6H2O), copper
(II) chloride dihydrate (CuCl2.2H2O), ferrous chloride
tetrahydrate (FeCl2.4H2O), ferric chloride hexahydrate
(FeCl3.6H2O) salts and ammonium hydroxide (NH4OH)
were procured from Sigma‐Aldrich, France.
2.2 | Characterizations
Elemental analysis of all compounds was carried out
using Perkin‐Elmer 240Q elemental analysis. The molar
conductance of ligand and its metal complexes was mea-
sured at 25 °C in DMF using a coronation digital (CON
6000) conductivity meter. The Infrared spectra (FT‐IR)
were recorded in the range of 4000–400 cm−1 using a

CHAABANE ET AL.
3 of 19
FT‐IR 460 spectrometer (Perkin‐Elmer, Washington, DC,
USA) in KBr disks; 1 mg of the powdered samples was
diluted with 10 mg KBr. The electronic spectra were
was low as soon as the metal (II) chlorides was added
with ligand (C32H28N4) (1). Hot stirring ethanolic solu-
tion (20 ml) of ethylenediamine (2 mmol) was slowly
added bivalent metal salts M (Cl2) (2 mmol) dissolved in
20 mL of ethanol. The reaction mixture was heated at
80 °C under nitrogen atmosphere for 30 min. Then,
benzil (2 mmol) was added in the refluxing mixture and
refluxing was continued for 8‐12 hr. The solid product
precipitate was filtered, washed with diethyl ether and
dried under vacuum over anhydrous P4O10; yield 76–93%.
recorded on
a
Perkin‐Elmer spectrophotometer. 1H
(300 MHz) and 13C (75 MHz) NMR spectra were run at
Bruker 300 spectrometer in DMSO‐d6. The chemical shifts
were reported in ppm and coupling constants were mea-
sured in Hz. The synthesised compounds were also charac-
terized by ESI Mass spectroscopy (model ApexII 7e)
Bruker. The X‐Ray Diffraction measurements of the pow-
der (PXRD) reflection data were obtained from a Rigaku
Ultima IV diffractometer. The data were collected with a
step size of 0.05°, scan rate of 1°/s, and 2θ angle scanned
from 2θ to 110°. The diffractometer uses a Cu Kα radiation
source (λ = 1.5406 Ǻ) operated at 40 kV and 44 mA. The
magnetic proprieties were measured using a super‐
conducting quantum design (MPMS,SQUIR‐VSM) at
25 °C and in a magnetic field ranging from ‐2 T to +2 T.
First, the field cooled (FC) and zero field cooled (ZFC) mea-
surements were performed by lowering the sample temper-
ature from 320 to 180 K at Zero field. Then, the material
prepared was heated from 180 to 320 K at 100 Oe and again
cooled from 320 to 180 at 100 Oe. The surface morphologies
of the samples were characterized by the scanning electron
microscope (SEM, FEI Quanta 250 FEG, Eindhoven,
Netherlands), at a typical accelerating voltage of 15 Kv.
• [Ni(C32H28N4)Cl2] (2)
Yield (1.55 g, 93%). m.p. 210–212 °C; FT‐IR (cm−1, KBr):
ⱱ (CH2), 3229; ⱱ(C=N), 1570; ⱱ(C=C), 1453; ⱱ (Ni‐N),
465; 1H NMR (DMSO‐d6, 300 MHz): δ 3.53 (S, 8H, ‐
CH2), 7.18–7.28 (m, 20H, Ar‐H); 13C NMR: (DMSO‐d6,
75 MHz): 45.20, 127.99, 129.42, 137.62, 159.37; Anal.
Calcd. for C32H28Cl2N4Ni (598.19): C, 64.24; H, 4.68; N,
9.36; Ni, 9.82%. Found: C, 64.20; H, 4.71; N, 9.28; Ni,
9.72%. MS (ESI) calcd. For [Ni(C32H28N4)Cl2] [M]+:
596.10; found: 597.05 [M + H+]+ .
• [Cu(C32H28N4)Cl2] (3)
Yield (1.32 g, 76%). m.p. 253–255 °C; FT‐IR (cm−1, KBr):
ⱱ (CH2), 3235; ⱱ(C=N), 1563; ⱱ(C=C), 1447; ⱱ (Cu‐N),
462; 1H NMR (DMSO‐d6, 300 MHz): δ 3.58 (S, 8H, ‐
CH2), 7.24–7.34 (m, 20H, Ar‐H); 13C NMR: (DMSO‐d6,
75 MHz): 45.25, 128.00, 129.44, 137.60 and 159.40; Anal.
Calcd. for C32H28Cl2N4Cu (603.04): C, 63.73; H, 4.68; N,
9.29; Cu, 10.54%. Found: C, 63.69; H, 4.71; N, 9.25; Cu,
10.56%. MS (ESI) calcd. For [Cu(C32H28N4)Cl2] [M]+:
601.09; found: 601.11 [M + H+]+ .
2.3 | Chemical synthesis
2.3.1 | Synthesis of
2,3,8,9‐tetraphenyl‐1,4,7,10‐
tetraazacyclododeca‐1,3,7,9‐tetraene:
Ligand (C32H28N4) (1)
Benzil (1 g, 4.76 mmol) was added to a solution of
ethylenediamine (2.86 g, 4.76 mmol) in ethanol (10 m).
The reaction mixture was refluxed for 3 hr. The suspension
was cooled to room temperature in ice cold water. After fil-
tration, the white product was washed with diethyl ether
and dried in a vacuum. Yield (1.82 g, 82%). m.p. 154–
156 °C; FT‐IR (cm−1, KBr): ⱱ (CH2), 3100; ⱱ(C=N), 1600;
• [Fe(C32H28N4)Cl2] (4)
Yield (1.47 g, 87%). m.p. 234–236 °C; FT‐IR (cm−1, KBr):
ⱱ (CH2), 3276; ⱱ(C=N), 1559; ⱱ(C=C), 1441; ⱱ (Fe‐N),
470; 1H NMR (DMSO‐d6, 300 MHz): δ 3.64 (S, 8H, ‐
CH2), 7.22–7.33 (m, 20H, Ar‐H); 13C NMR: (DMSO‐d6,
75 MHz): 45.30, 128.02, 129.46, 137.61 and 159.43; Anal.
Calcd. for C32H28Cl2N4Fe (595.34): C, 64.56; H, 4.74; N,
9.41; Fe, 9.38%. Found: C, 64.43; H, 4.82; N, 9.49; Fe,
9.41%. MS (ESI) calcd. For [Fe(C32H28N4)Cl2] [M]+:
594.11; found: 594.14 [M + H+]+
1
ⱱ(C=C), 1494; H NMR (DMSO‐d6, 300 MHz): δ 3.50 (S,
8H, ‐CH2), 7.16–7.24 (m, 20H, Ar‐H). 13C NMR: (DMSO‐
d6, 75 MHz): 45.20, 127.99, 129.42, 137.62 and 159.35. Anal.
Calcd. for C32H28N4 (468.23): C, 82.02; H, 5.83; N, 11.76%.
Found: C, 82.05; H, 5.98; N, 11.95%. MS (ESI) calcd. For
(C32H28N4) [M]+: 468.23; found: 468.41 [M + H+]+ .
2.3.2 | Synthesis of the Ni (II), cu (II) and
Fe (II) complexes
2.3.3 | Synthesis of [Fe3O4NP‐INS‐
(C32H28N4)] (5)
Ni (II), Cu (II) and Fe (II) complexes were prepared by
condensation method because the yield of all complexes
[Fe3O4NP‐INS‐(C32H28N4)] (5) was prepared based on
the novel chemical co‐precipitation method in the

4 of 19
CHAABANE ET AL.
presence of aqueous ferric ion (Fe3+) and [Fe(C32H28N4)
Cl2] (4). First, 1.5 g [Fe(C32H28N4)Cl2] (4) was dissolved
in 200 ml of 2:1 ethanol/water with severely stirring at
70 °C. Then, 3 g of FeCl3.6H2O was added to the solution
and stirred vigorously at 70 °C under nitrogen atmo-
sphere for 30 min. After, 20 ml NH4OH (8 M) was poured
drop wise into solution until pH raised to about 10 and
stirring continued for 1 hr. When ammonium was being
added to the solution, it was led to changing of solution
color from orange to dark brown and then became black
denoting the formation of Fe3O4NPs inside the tetraaza
macrocyclic Schiff base ligands, as schematically repre-
sented in Figure 1. After that, the complexes of magnetite
nanoparticles were collected by centrifugation and
washed with ethanol and deionized water to remove
chloride ions and excess the coating agent. Finally, the
black product was dried under vaccum at 60 °C for over-
night to remove the excess water.
2.4 | Biological assays
2.4.1 | Antimicrobial activity
Bacterial and fungal strain
All the complexes were screened for their in vitro antimi-
crobial activities against some bacterial strains, viz.
Staphylococcus
aureus
(ATCC
25923),
Listeria
monocytogenes (ATCC 19115), Vibrio parahaemolyticus
(CECT 511) and Vibrio vulnificus (CECT 529) (gram‐pos-
itive bacteria), Vibrio parahaemolyticus (ATCC 17802),
Staphylococcus epidermidis (CECT 231), Pseudomonas
aeruginosa (ATCC 27853) and Salmonella typhimurium
(ATCC 1408) (gram‐negative bacteria), and some fungal
strains, viz. Candida albicans, Candida tropicalis, Can-
dida glabrata, and Candida parapsilosis.
Disc‐diffusion assay
The tested microbial was carried out by agar disc‐
diffusion method, according to the protocol described by
Vuddhakul et al.[36] First, the test tubes containing
9 mL of Mueller‐Hinton broth (for bacteria) and
Sabouraud Chloramphenicol broth (for Candida) with
10 μl of the microorganisms were inoculated at 37 °C
for 24 hr. The optical density was adjusted to 107 to 108
UFC/mL (0.1 at OD600 for bacteria and 0.4 at OD540 for
• [Fe3O4NP‐INS‐(C32H28N4)] (5)
Yield (1.95 g, 88%). m.p. > 300 °C; FT‐IR (cm−1, KBr):
ⱱ (CH2), 2949; ⱱ(C=N), 1547; ⱱ(C=C), 1436; ⱱ (Fe‐O),
550; ⱱ (Fe‐N), 425; VSM (emu.g−1, T) HC(T), 0.162; MS
(emu/g), 26.56; MR (emu/g), 18.07; SR, 0.68; PXRD (2θ,
Cu Kα): 30.15°, 35.52°, 43.19°, 53.61°, 57.29° and 62.65°.
FIGURE 1 Schematic representation of the [Fe3O4NP‐INS‐(C32H28N4)] (5), which was prepared by using a novel co‐precipitation method
of coordinated ferric ion in the complex [Fe(C32H28N4)Cl2] (4) under mild conditions

CHAABANE ET AL.
5 of 19
fungal strains). The inoculums of the microorganisms
were streaked onto SB agar plates using a sterile cotton
mop as recommended by the CA‐SFM EUCAST 2017.
Sterile filter discs (diameter 6 mm, Biolife Italy) were
placed at the surface of the appropriate agar mediums
and 10 μl of the product dissolved in 10%
dimethylsulfoxide (DMSO) was dropped onto each disc.
Ampicillin (10 mg/ml; 10 μl/disc) and Amphotericin B
(10 mg/ml; 10 μl/disc) were used as reference antibiotics.
The antibacterial activities were evaluated by measuring
an inhibition zone formed around the disc. All tests were
performed in triplicate.
Modified Medium (DMEM) (Gibco™, Sigma) eagle
enriched with 10% fetal bovine serum (FBS), antibiotic
mixture (100 μg/ml) and l‐glutamine (100 μg/ml) and
placed in CO2 incubator with 5% of CO2 at 37 °C. The
three cell lines U87, MDA‐MB‐231 and LS‐174 were
seeded in 96 well plate (104 cells/well) and treated with
different concentrations (10, 30, 50 and 100 μg/ml) of
each compounds for 24 hrs. MTT (1 mg/ml) was added
to cells and incubated for 3 hr at 37 °C. The formed
formazan
crystals
were
then
dissolved
in
dimethysulfoxide (DMSO) and the absorption was mea-
sured at 560 nm with a microplate reader (MULTISKAN,
Labsystems). Cell viability was expressed as a percentage
of the viable cell number of the mock‐treated cells. Addi-
tionally, peripheral blood mononuclear cells (PBMC)
were isolated from healthy human volunteer (Mr. Nader
KAYET) by Ficoll‐Paque (Hospital Fattouma Bourguiba,
Laboratories; 2154BM, Monastir, Tunisia) density gradi-
ent centrifugation as per standard procedure. 12 PBMC
(2 × 105 cells/well) have been cultured in complete
RPMI‐1640 media as usual and incubated with ligand
(C32H28N4) (1), [Fe3O4NP‐INS‐(C32H28N4)] (5) and metal
complexes of Schiff base ligand (Ni (II), Cu (II) and Fe
(II)) (100 μg/ml) (2–4) for 48 hrs using trypan blue exclu-
sion assay. This methods yields approximately more than
95% feasible PBMC.
Micro‐well determination of MIC and MBC/MFC
The determination of the minimal inhibition concentra-
tions (MIC) and the minimal bactericidal/fungicidal con-
centrations (MBC/MFC) were made following the
technique of dilution in medium liquid, as described by
Snoussi et al..[37] For this, the microbial inoculums were
prepared from 12 hr broth cultures and spectrophotomet-
rically were also adjusted to 107 UFC/ml. First, the differ-
ent amounts of the compounds were dissolved in 10%
dimethylsulfoxide (DMSO) and diluted with culture broth
to the concentration of 0.5 mg/ml, and then serial twofold
dilutions were performed by addition of culture broth to
obtain concentrations ranging from 0.5 to 0.003 mg/ml
in 5 ml sterile glass tubes containing nutrient broth. A
95 μl of nutrient broth was distributed in a 96‐well, as
well as a sterility control. The test microbial cultures were
dotted in a predefined pattern by aseptically transferring
5 μl of each microbial suspension onto each wells. The
first well containing the nutrient broth Mueller Hinton
or Sabouraud Chloramphenicol without product and
5 μl of the inoculum was exploited as a negative control.
The microplates were incubated at 37 °C for approxi-
mately 24 hr. For the reading, the minimum inhibitory
concentration (MIC) was defined as the smallest concen-
tration of the compounds to inhibit the growth of the
microorganisms. MBC and MFC values were determined
as the highest dilution of the sample. Therefore we
observe a clear fluid with no development of turbidity
or visible growth, respectively. These studies were per-
formed in triplicate.
3 | RESULTS AND DISCUSSION
3.1 | Chemistry
Ligand (C32H28N4) and mononuclear transition metal
complexes of Ni (II), Cu (II) and Fe (II) were synthesized
by the condensation of benzil and ethylenediamine (2:2).
The complexes were formulated as: MLCl2, where
M = Ni (II), Cu (II) and Fe (II); L = Ligand (C32H28N4).
[Fe3O4NP‐INS‐(C32H28N4)] were successfully prepared by
using a co‐precipitation of coordinated ferric ion (Fe3+
)
inside the complex [Fe(C32H28N4)Cl2] (Figure 1). The syn-
thesized compounds are colored solids, insoluble in non-
polar organic solvents, and completely soluble in polar
solvents with a strong donor strength, as dimethysulfoxide
(DMSO) or/and dimethylformamide (DMF).
2.4.2 | Cytotoxic activity against cancer
3.1.1 | Molar conductance
and normal cells
The molar conductivities of 10−3 mol dm−3 solution of
the metal (II) complexes of Schiff base ligand in
dimethyformamide (DMF) showed values in the range
2.3–6.5 Ω−1 cm2 mol−1. The conductance data, as shown
in Table 1, have demonstrated the non‐electrolytic nature
of these complexes. Furthermore, the elemental analysis
U87 human glioblastoma cancer cells, MDA‐MB‐321
human breast cancer cells and LS‐174 T human colorectal
adenocarcinoma cells were generously provided by dealt
with Dr. Khadija Essafi‐Benkhadir (Pasteur Institue of
Tunsia). This cell lines have been cultured in Dulbecco's

6 of 19
CHAABANE ET AL.
TABLE 1 Analytical data of the tetraaza macrocyclic ligand and its metal complexes of Ni(II), Cu(II) and Co(II)
Elemental analysis (Calcd.) (%)
Compounds
Mol. Wt.
C
H
N
M
M. C (Ω−1 cm2 mol−1
)
μeff (B.M)
C32H28N4 (1)
468.23
598.19
82.05 (82.02) 5.98 (5.83)
64.20 (64.24) 4.71 (4.68)
63.69 (63.73) 4.71 (4.68)
64.43 (64.56) 4.82 (4.74)
11.95 (11.76)
9.28 (9.36)
9.25 (9.29)
9.49 (9.41)
‐
‐
‐
[Ni(C32H28N4)Cl2](2)
9.72 (9.82)
6.5
3.3
2.1
4.7
[Cu(C32H28N4)Cl2](3) 603.04
[Fe(C32H28N4)Cl2](4) 595.34
10.56 (10.54) 2.3
9.41 (9.38) 3.8
results of the synthesized metal complexes also conform
with calculated values, showing that these complexes
have 1:1 non‐electrolytes and may be formulated as:
[M(L)Cl2].[38,39]
(1) exhibited no bands corresponding to a free carbonyl
group of benzil or free primary diamine of
a
ethylenediamine. The appearance of novel bands near
1547–1600 cm−1 demonstrated the condensation of the
primary amino groups with carbonyl groups and the for-
mation of a tetraaza macrocyclic Schiff base ligand, as
these bands may be attributed to the ⱱ(C=N) vibrations.
Compared to (a), displacement to lower frequency of the
ⱱ(C=N) vibrations in (b), (c), (d) and (e) complexes
may be explained by the delocalization of metal electron
density (t2g) to the ᴨ‐system of the ligand (C32H28N4) (1),
indicating that the coordination has taken place through
nitrogen of (‐C=N‐) groups Figure 3.[40–43] Various bands
appearing at 425‐470 cm−1 indicate ⱱ(M‐N) vibrations.
Two of these bands confirms the coordination of the
azomethine group of the ligand with the metal ions.
Coupled with the monodentate coordination of the anions
as was discussed above, an octahedral configuration may
be proposed for these complexes, with the ligand forming
a square plane and the anions residing in axial positions.
The bands present in the range 2949–3276 cm−1 may be
attributed to the ⱱ(‐CH2) stretching vibrations of the
benzil and ethylenediamine moieties. The various absorp-
tion bands in the region 1436–1494 cm−1 could be attrib-
uted to the ⱱ(C=C) aromatic stretching vibrations of the
benzene ring. Compared to the neat Fe3O4NPs (Figure 2
f), the [Fe3O4NP‐INS‐(C32H28N4)] (5) (Figure 2e)
displayed similar characteristic peaks. The strong peak
observed around at 550 cm−1 could be attributed to the tet-
rahedral and octahedral strectching vibration of the Fe‐O
bond of Fe3O4NPs.[25]
3.1.2 | Electronic spectral studies and
magnetic measurements
The electronic spectral measurements were used for con-
veying the stereochemistry of metal ions in the complexes
based on the number of d–d transition peak and positions.
The Nujol mull electronic absorption spectral data of the
complexes and effective magnetic moment values (B.M.)
are reported (Table 2). The electronic spectra of the com-
plexes show one bond in the range of 32573–33557 cm−1
,
2
2
which can be attributed to Eg Teg transitions. An inter-
pretation of these bands indicates that the complexes have
octahedral geometries and might possess D4h symmetry.
However, the metal (II) complexes of Schiff base ligand
show magnetic moments in the range of 2.1 to 4.7 B.M.
at room temperature (Table 1). The values are going
high‐spin configuration and indicating the presence of
an octahedral environment around the metal ions.
3.1.3 | FT‐IR spectra
The FT‐IR spectra of all complexes were performed on a
FT‐IR 460 spectrometer (Perkin‐Elmer, Washington, DC,
USA) in the range of 4000–400 cm−1 and represented in
Figure 2. The infra‐red spectra of the ligand (C32H28N4)
TABLE 2 Spectral absorption bands of the Ni (II), Cu (II) and Fe
(II) complexes
3.1.4 | 1H NMR and 13C NMR
Band position
The 1H‐NMR data for metal (II) complexes of ligand
(C32H28N4) (1) show a singlet at 3.50–3.64 ppm which
may be ascribed to the methylene protons (‐CH2; 8H).
However, the multiples in the region 7.16–7.34 ppm
may be attributed to aromatic ring protons. The 13C‐
NMR data displays peaks at 159.35–159.43 ppm, indicat-
ing that the azomethine moieties were in a different envi-
ronment (Figure 4). The 13C NMR data are in agreement
Compound
(cm−1
)
Assignment Geometry
2
[Ni(C32H28N4)
Cl2](2)
33557 cm−1
(298 nm)
32573 cm−1
(307 nm)
2Eg → T2g
Octahedral
Octahedral
Octahedral
2
[Cu(C32H28N4)
Cl2](3)
2Eg → T2g
2
[Fe(C32H28N4)
Cl2](4)
33444 cm−1
(299 nm)
2Eg → T2g

CHAABANE ET AL.
7 of 19
FIGURE 2 FT‐IR spectra of ligand (C32H28N4) (a), [Ni(C32H28N4)Cl2] (b), [Cu(C32H28N4)Cl2] (c), [Fe(C32H28N4)Cl2] (d), [Fe3O4 NP‐INS‐
(C32H28N4)] (e) and Fe3O4NPs(f)
3.1.5 | Mass spectra
The mass spectra of ligand and its Ni (II), Cu (II) and Fe
(II) complexes were recorded and their stoichiometric
compositions were compared. The mass spectrum of mac-
rocyclic ligand L = C32H28N4 shows a well‐defined molec-
ular ion peak at m/z = 468.41 amu which coincides with
the formula weight of the Schiff base. The mass spectra of
all the synthesized macrocyclic complexes displayed
molecular
ion
597.05
peaks
amu
(100%)
[Ni(C32H28N4)Cl2]
as
follows:
(2),
m/z
m/z
=
=
601.11 amu [Cu(C32H28N4)Cl2] (3) and
m/z = 594.14 amu [Fe(C32H28N4)Cl2] (4). This informa-
tion is in good conformity with the proposed molecular
formulae and structures of the macromolecular metal
complexes. There are various other peaks in the spectra
which are due to the thermal cleavage of the complexes
assigned to various fragments of the dissociated molecule.
FIGURE 3 Structurally significant Infrared IR bands (C=N) (in
cm−1) for ligand (C32H28N4) (1),[Ni(C32H28N4)Cl2] (2),
[Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2] (4) and [Fe3O4 MNP‐
INS‐(C32H28N4)] (5)
3.1.6 | X‐ray diffraction
with the IR result in which different peaks for C=N group
are recorded. Additionally, the NMR spectrum of the
complex: [Fe3O4NP‐INS‐(C32H28N4)] shows broad signals
due to the presence of ferromagnetic of Fe3O4NPs.
The PXRD patterns of the bare Fe3O4NPs, ligand
(C32H28N4) (1) and the [Fe3O4 NP‐INS‐8(C32H28N4)] (5)
are shown in Figure 5. The PXRD spectrum of ligand
(C32H28N4) (1) is relatively featureless except for a peak

8 of 19
CHAABANE ET AL.
FIGURE 4 13C NMR (25 °C, DMSO‐d6) of ligand (C32H28N4) (1), [Ni(C32H28N4)Cl2] (2), [Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2] (4)
at low 2θ values. Six main peaks in the green trace at 30.2°,
35.5°, 43.1°, 53.5°, 57.3° and 62.7°, were observed and are
marked by their indices (220), (311), (400), (422), (511) and
(440) planes. Positions and relative intensities of all the
peaks are in accordance with the face‐centered cubic
(fcc) system of Fe3O4NPs, which agrees with previously
synthesized pure magnetite nanoparticles.[24] Patterns of
Fe3O4NPs and [Fe3O4NP‐INS‐(C32H28N4)] (5) were
included for comparison. The narrow shape peaks of
materials indicate that the [Fe3O4 NP‐INS‐(C32H28N4)]
(5) has relatively high crystallinity and without the
appearance of the impurities phase of goethite α‐FeO
(OH) and hematite (Fe2O3) corresponding to the diffrac-
tion peaks of (110), (104) at 2θ positions of 21.2° and
33.1°. Furthermore, the PXRD analysis result for
[Fe3O4NP‐INS‐(C32H28N4)] (5) has shown characteristic
peaks of Fe3O4NPs corresponding to the crystal planes of
(220), (311), (400), (422), (511) and (400), revealing com-
plexion inside tetraaza macrocyclic Schiff base ligand.
The average crystal size in the diameter of the crystals
can be estimated through full‐Width at half maximum
(FWHM) of the strangest reflection of the peak, using
the Scherrer approximation,[44–46] which assumes the
small crystallite size to be the cause of line broadening
(Equation 1)
Where, D is the crystallite mean size, λ is the wavelength
of Cu Kα radiation (λ = 0.154 nm), k is the grain shape
factor taken as a unity contemplating that the particles
are spherical in shape and have a value of 0.89 (0.9) is
used, β is the broadening of diffraction line measured at
half maximum in intensity, in the 2θ scale and θ is the
Bragg's angle. The calculated crystal size of pure
Fe3O4NPs and [Fe3O4NP‐INS‐(C32H28N4)] (5) was
100.65 and 20.87 nm respectively. Owing to the advan-
tages of magnetite nanoparticles complex inside tetraaza
macrocyclic Schiff base ligands will decrease repulsive
forces to balance the magnetic and the Vander
Waals attractive forces acting on the Fe3O4NPs which
can be attributed to inhibition of crystal growth by a
complexion agent. For comparison, Taleb et al. reported
a size (22 nm) of Fe3O4NPs present on surface of
microfibrillated cellulose linked by oxalyl and maleic
acid.[47]
3.1.7 | Magnetic properties measurement
The magnetic proprieties of [Fe3O4NP‐INS‐(C32H28N4)]
(5), was studied by measuring the magnetic‐hysteresis
(MH) at room temperature with vibrating sample magne-
tometer. The magnetic hysteresis loop analysis proved the
ferromagnetic character of the [Fe3O4NP‐INS‐
(C32H28N4)] (5) (Figure 6), the saturated magnetization
k λ
D ¼
(1)
β Cos θ

CHAABANE ET AL.
9 of 19
systems with uniaxial anisotropy, suggesting that the
response of the spin magnetic moment of particles is led
by the competition between interparticle and
intraparticle interactions.
FC and ZFC measurements were carried out on the
[Fe3O4NP‐INS‐(C32H28N4)] (5) for a more detailed analy-
sis of its magnetic properties (Figure 7). In the case of the
FC measurement, the magnetization direction of each
particle is sheltered whereas the net magnetization for
ZFC is zero and all the magnetic spins are slanted ran-
domly. In the case of nanoparticles the ZFC typically
decreased and FC increased in parallel with the decrease
in temperature. Heating the sample caused the ZFC mag-
netization to increase and to approach a blocking temper-
ature, that is, the temperature to which the domains are
oriented. In the case of a homogenous particle size distri-
bution the change should be abrupt.[49] The maximum
value in the ZFC curve for the mixture occurred above
320 K (Figure 7). We attribute the high blocking temper-
ature to a large particle size and the magnetic interaction
among particles. The ZFC curve of the [Fe3O4NP‐INS‐
(C32H28N4)] (5) decreased approaching zero when the
temperature decreased. Unlike the ZFC, the FC curve of
the [Fe3O4NP‐INS‐(C32H28N4)] (5) decreases slightly with
increasing temperature. The FC and ZFC curves of the
neat magnetic particles followed a similar trend.
3.1.8 | Morphology analysis
FIGURE 5 PXRD of ligand (C32H28N4) (1), [Fe3O4 NP‐INS‐
(C32H28N4)] (5) and Fe3O4NPs
The morphology of Fe3O4NPs and [Fe3O4NP‐INS‐
(C32H28N4)] (5) were compared by the scanning electron
microscopy (SEM) technique to have knowledge of the
nanoparticles spreading on the support (Figure 8).
Figure 8a shows that the magnetite nanoparticles were
presumably aggregated which could be attributed to the
strong dipole–dipole magnetic attractions and interac-
tions between the particles. Figure 8b shows that the
Fe3O4NPs were closely encapsulated in the tetraaza mac-
rocyclic ligands and some neighboring particles were
aggregated together with these surfaces. Furthermore, it
is worthy to note that the magnetite nanoparticles are
spherical with diameters ranging from 20 nm to 130 nm
homogeneously dispersed in the [Fe3O4NP‐INS‐
(C32H28N4)] (5). It has already been known that the mag-
netic properties of the Fe3O4NPs were greatly influenced
by their dimension, when the MS generally decreases
with reducing of crystallite size.[50]
(MS) was 26.56 emu g−1, which represents 27% of pure
Fe3O4NPs (98.15 emu g−1 [48]
suggesting that the loading
)
of tetraaza macrocyclic ligand (1) obviously decreased the
magnetic response to an applied magnetic field. Within
the external magnetic field, [Fe3O4NP‐INS‐(C32H28N4)]
(5) moved slowly, and finally aggregated completely at
10 sec. Once aligned in an applied magnetic field, the
magnetic domains of our ferromagnetic materials will
have magnetic memory which cannot return to the initial
state without the expenditure of the endowed energy. To
ensure this, hysteresis loop was monitored for the resul-
tant magnetic hybrid nanopowder materials in the
applied field ranging from −2 T to 2 T. The remnant mag-
netization (MR), coercivity (HC), and squareness
(Sr = MR/MS) were calculated (Figure 6) and listed in
Table 3. Hence the remnent magnetization was found to
be 18.07emug−1 whereas the coercive field (HC) attained
0.162 T. From these data it is possible to indicate that
remanence ratio of the nanocomposite sample is smaller
than the one reported for isolated magnetic particles
3.1.9 | A possible reaction mechanism
The advantage and novelty of using the complex
[Fe(C32H28N4)Cl2] (4) as the ferrous source which, it is

10 of 19
CHAABANE ET AL.
FIGURE 6 The magnetization curves measured at 320 K for the [Fe3O4NP‐INS‐(C32H28N4)] (5)
TABLE 3 Magnetic property of the [Fe3O4NP‐INS‐(C32H28N4)] (5)
Coercivity (M = 0)
HC (T)
Magnetization (H = 0.01 T)
MS (emu/g)
Retentivity MR (emu/g)
Samples
Tb (K)
Sr
[Fe3O4NP‐
320
0.162
26.56
18.07
0.68
INS‐(C32H28N4)] (5)
aHC = Coercivity.
bMS = Saturation magnetization.
cMR = Remnant magnetization.
dSr = Squareness.
important to provide proper surface complexes and devel-
oping some effective protection strategies to keep the sta-
bility of Fe3O4NPs and decrease their an aggregation
phenomena. This new strategy reported for [Fe3O4NP‐
INS‐(C32H28N4)] (5).
The new co‐precipitaion method reported for
[Fe3O4NP‐INS‐(C32H28N4)]
synthesis
using
[Fe(C32H28N4)Cl2] and ferric salts in alkaline conditions
are given in Equations (2)–(7).
NH4OH→NH3þ þ OH−
(2)
2þ
½FeðC32H28N4Þꢀ
Â
Ã
þ 2OH−→ FeðC32H28N4ÞðOHÞ2
(3)
(4)
ðsÞ
FIGURE 7 The ZFC/FC curves of [Fe3O4NP‐INS‐(C32H28N4)] (5)
(Applied field = 9.10−3 T and temperature range = 180‐320 k)
Fe3þ þ 3OH−→ Fe ðOHÞ3 ðsÞ

CHAABANE ET AL.
11 of 19
Giving an overall reaction:
Fe ðOHÞ3 ðsÞ→ Fe ðOOHÞðsÞ þ H2O
(5)
(6)
2þ
2Fe3þ þ ½FeðC32H28N4Þꢀ þ 8OH−
(7)
Â
Ã
FeðC32H28N4ÞðOHÞ2
Â
Ã
ðsÞ
¼ 2FeðOHÞ3 FeðC32H28N4ÞðOHÞ2
→½Fe3O4NP−INS−ðC32H28N4Þꢀ ðsÞþ 8H2O
þ 2FeðOOHÞðsÞ→½Fe3O4NP−INS−ðC32H28N4ÞꢀðsÞ
ðSÞ
þ 2H2O
FIGURE 8 SEM images of (a) Fe3O4NPs and (b) [Fe3O4NP‐INS‐(C32H28N4)] (5)

12 of 19
CHAABANE ET AL.
FIGURE 9 Template coordination of magnetite nanoparticle inside Schiff base ligand after co‐precipitation between ferric ion (Fe3+) and
[Fe(C32H28N4)]2+
These reactions are very fast. First, the [Fe(C32H28N4)
(OH)2](s) and Ferric hydroxides are precipitated. Second,
the ferric hydroxide decomposes to FeOOH, due to the
low ethanol/water activity of resulting NH4Cl solution
in a slower reaction (Equation 5). The solid state reaction
between FeOOH and [Fe(C32H28N4)(OH)2](s) takes
place and produces magnetite (Equation 6). Additionally,
ferrimagnetic iron oxide having cubic inverse spinel
structure with oxygen anions forming a face cubic
closed packing and iron (cations) located at the intersti-
tial tetrahedral sites or/and octahedral sites.[51] In the
[Fe3O4NP‐INS‐(C32H28N4)] (5), Fe3O4NPs fabricated
through this approach show a relatively high crystallin-
ity. Indeed, the lower MS value 26.56 emug−1 compared
to the one reported for bulk iron oxide (90–100 emug
−1) is due to the prevention of aggregation phenom-
ena.[26] From the results described above, we also suggest
an explanation reaction in Figure 9 for the [Fe3O4NP‐
INS‐(C32H28N4)] (5).
it is estimated that the chelation of the Ferric ions (Fe3+
)
on the [Fe(C32H28N4)]2+ can also prevent nucleation
and inhibit the increase of the crystal nuclei, leading to
small nanoparticles of magnetite (diameter of
Fe3O4NPs~20.87 nm) (Equation 7). Indeed, it is a

CHAABANE ET AL.
13 of 19
FIGURE 10 (a) Antibacterial activity of ligand (C32H28N4) (1), [Ni(C32H28N4)Cl2] (2), [Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2] (4) and
[Fe3O4NP‐INS‐(C32H28N4)] (5) determined by disc diffusion method (10 mg/disk), Ampicillin (10 mg/ml) expressed as mean SD (n = 3).
(b) Antifungal activity of ligand (C32H28N4) (1), [Ni(C32H28N4)Cl2] (2), [Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2] (4) and [Fe3O4NP‐INS‐
(C32H28N4)] (5) determined by disc diffusion method (10 mg/disk), Amphotericin B (10 mg/mL) expressed as mean SD (n = 3)
or the absence of an inhibition zone diameter (Figure 10
a and b and Table 4). The MIC and MBC/MFC values
as shown in Table 5 and Table 6. Indeed, metal com-
plexes of Schiff base ligand (Ni (II), Cu (II) and Fe (II))
showed a good antibacterial activity against Pseudomonas
aeruginosa and Staphylococcus aureus. Ni (II), Cu (II) and
Fe (II) complexes were specialized in inhibiting Gram‐
negative bacteria strain (P. aeruginosa), with 20.67–
3.2 | Biological activities
3.2.1 | Antibacterial and antifungal
activities
When tested on different bacteria strains, the compounds
were found to have remarkable activity. The effects were
qualitatively and quantitatively assessed by the presence

14 of 19
CHAABANE ET AL.
TABLE 4 Antibacterial and antifungal activity [Zone of inhibition (mm)] of 1, 2, 3, 4 and 5 Compounds
1 2 3
IZa
4 5 Drug
IZa
Microorganisms
IZa
IZa
IZa
IZa
Bacterial strains
Ampicillin
Staphylococcus aureus ATCC 25923
Listeria monocytogenes ATCC 19115
Vibrio parahaemolyticus CECT 511
Vibrio vulnificus CECT 529
Vibrio parahaemolyticus ATCC 17802
Staphylococcus epidermidis CECT 231
Pseudomonas aeruginosa ATCC 27853
Salmonella typhimurium ATCC 1408
Yeast strains
22.0 0.58
20.0
0
0
0
0
0
0
19.67 0.58
14.67 0.58
21.0 0.58
35.0 1.0
20.0 1.0
6.0
15.0
6.0
0
0
0
0
0
6.0
6.0
6.0
6.0
6.0
6.0
17
0
0
0
0
0
6.0
6
0
0
21.33 0.58
21.67 0.58
23.33 0.58
17.0 1.74
23.33 0.58
11.67 0.58
12.33 0.58
6.0
21.67 0.58
6.0
24.0
6.0
6.0
6.0
0
0
6.0
0
6.0
21.33 0.58
22.33 0.58
33.33 0.58
21.33 0.58
6.0
20.67 0.58
21.33 0.58
23.33 0.58
21.00 1.0
6.0 0.
6.00
0
0
25.0
0
16 1.0
Amphotericin B
Candida glabrata
24.33 0.58
22 1.0
24.0
0
23
24.33 0.58
24
23 1.0
0
21.67 0.58
24.33 0.58
25.33 0.58
24.33 1.0
25.00 0.58
25.33 0.58
25
24.33 0.58
10.33 1.15
11.33 0.57
11.0 1.0
Candida albicans
24 1.0
24.33 0.58
24.33 0.55
Candida parapsilosis
21.67 0.5
25.33 0.58
0
0
Candida tropicalis
6.0
0
1 = Ligand (C32H28N4), 2 = [Ni(C32H28N4)Cl2], 3 = [Cu(C32H28N4)Cl2], 4 = [Fe(C32H28N4)Cl2] and 5 = [Fe3O4NP‐INS‐(C32H28N4)]
aIZ: inhibition zone (mm) around discs impregnated with 1, 2, 3, 4 and 5 compounds (10 mg/disk), Ampicillin (10 mg/ml) or Amphotericin B (10 mg/ml)
expressed as mean SD (n = 3).
TABLE 5 Antibacterial and antifungal activity (MIC in mg/ml) of 1, 2, 3, 4 and 5 compounds
1 2 3
4 5 Drug
MICa
Microorganisms
MICa
MICa
MICa
MICa
MICa
Bacterial strains
Ampicillin
0.166
Staphylococcus aureus ATCC 25923
Listeria monocytogenes ATCC 19115
Vibrio parahaemolyticus CECT 511
Vibrio vulnificus CECT 529
Vibrio parahaemolyticus ATCC 17802
Staphylococcus epidermidis CECT 231
Pseudomonas aeruginosa ATCC 27853
Salmonella typhimurium ATCC 1408
Yeast strains
0.250
0.250
0.007
0.250
0.250
0.250
0.062
0.250
0.250
0.062
0.250
0.250
0.250
0.003
0.007
0.166
0.250
0.250
0.166
0.250
0.007
0.250
0.007
0.007
0.062
0.062
0.125
0.125
0.062
0.031
0.007
0.003
0.078
0.023
0.078
0.023
0.011
0.023
0.011
0.023
0.250
0.250
0.250
0.250
0.125
0.007
0.250
Amphotericin B
Candida glabrata
0.062
0.125
0.166
0.166
0.062
0.062
0.166
0.166
0.031
0.062
0.125
0.166
0.031
0.125
0.031
0.166
0.031
0.024
0.390
0.097
0.200
Candida albicans
0.062
0.031
0.031
Candida parapsilosis
Candida tropicalis
1 = Ligand (C32H28N4), 2 = [Ni(C32H28N4)Cl2], 3 = [Cu(C32H28N4)Cl2], 4 = [Fe(C32H28N4)Cl2] and 5 = [Fe3O4NP‐INS‐(C32H28N4)]
aMIC: minimal inhibitory concentration (mg/ml).
21.33 mm of inhibition zone and 0.007–0.062 mg/mL,
0.015–0.031 mg/mL as MIC and MBC, respectively. The
importance of this unique property of the investigated
Schiff base complexes lies in the fact that, it could be
applied in the treatment of infections caused by any of
these particular strain.[52] However, Ni (II), Cu (II) and

CHAABANE ET AL.
15 of 19
TABLE 6 Antibacterial and antifungal activity (MBC and MFC in mg/ml) of 1, 2, 3, 4 and 5 compounds
1 2 3
4 5 Drug
Microorganisms
MBCa/MFCb MBCa/MFCb MBCa/MFCb MBCa/MFCb MBCa/MFCb MBCa/MFCb
Bacterial strains
Ampicillin
Staphylococcus aureus ATCC 25923
Listeria monocytogenes ATCC 19115
Vibrio parahaemolyticus CECT 511
Vibrio vulnificus CECT 529
Vibrio parahaemolyticus ATCC 17802
Staphylococcus epidermidis CECT 231
Pseudomonas aeruginosa ATCC 27853
Salmonella typhimurium ATCC 1408
Yeast strains
>0.500
0.500
0.166
>0.500
0.500
0.062
0.015
0.500
>0.500
>0.500
0.250
>0.500
0.500
0.166
>0.500
0.031
0.500
0.031
0.062
0.250
>0.500
0.500
0.166
0.166
0.250
0.250
0.125
0.062
0.031
0.031
0.625
0.093
0.625
0.093
3.00
>0.500
0.500
0.500
0.500
0.062
0.250
3.00
0.031
0.031
12.0
0.500
>0.500
0.093
Amphotericin B
Candida glabrata
0.166
0.250
0.500
0.500
0.166
0.166
0.500
0.500
0.166
0.125
0.250
0.500
0.125
0.250
0.250
0.500
0.166
0.781
6.250
0.195
0.390
Candida albicans
0.250
0.166
0.166
Candida parapsilosis
Candida tropicalis
1 = Ligand (C32H28N4), 2 = [Ni(C32H28N4)Cl2], 3 = [Cu(C32H28N4)Cl2], 4 = [Fe(C32H28N4)Cl2] and 5 = [Fe3O4NP‐INS‐(C32H28N4)]
aMBC: minimal bactericidal concentration (mg/ml)
bMFC: minimal fungicidal concentration (mg/ml).
Fe (II) complexes could be applied fairly in the treatment
of some common diseases caused by S. aureus. The data
obtained showed that the magnetite complex shows great
antimicrobial activity than that of the metal complexes
and free ligand (C32H28N4) (1). It is found that
[Fe3O4NP‐INS‐(C32H28N4)] has higher antibacterial activ-
ity against both Gram‐positive (S. aureus, L.
monocytogenes, V. parahaemolyticus and V. vulnificus)
and Gram‐negative (S. epidermidis, P. aeruginosa and S.
typhimurium) bacterial strains than the free ligand
(C32H28N4) (1) and metal complexes for both concentra-
tions. In this case, S. typhimurium was noted to be the
most sensitive, with 33.33 mm of inhibition zone and
0.031 mg/mL, 0.062 mg/mL as MIC and MBC, respec-
tively. It is known that the membrane of Gram‐negative
bacteria is surrounded by an outer membrane containing
lipopolysaccharides. This would suggest that the chela-
tion could facilitate the ability of a magnetite nanoparti-
cles complex and metal complexes to cross a cell
membrane of the Gram‐negative bacteria and can be
explained by Tweedy's chelation theory.[53] Furthermore,
Chelation/complexation increases the delocalization of π‐
electrons over the whole chelate ring and enhances the
penetration of the metal complexes into lipid membranes
or blocking of the magnetite nanoparticles complex bind-
ing the sites in the enzymes of microorganisms.[54] These
complexes also disturb the respiration process of the cell
and thus block the synthesis of proteins, which restricts
further growth of the organism. Moreover, coordination
reduces the polarity of the metal ion mainly because of
the partial sharing of its positive charge with the donor
groups within the chelate ring system created during
the coordination.[55–57] This process, in turn, increases
the lipophilic nature of the central metal atom, which
supports its permeation more efficiently through the lipid
layer of microorganisms, thus destroying them more
aggressively.[58] On the other hand, our results showed
that all compounds have displayed good antifungal activ-
ity against Candida glabrata, Candida albicans, Candida
parapsilosis and Candida tropicalis, with 21.67–
25.33 mm of the inhibition zone and 0.031–0.166 mg/
mL, 0.166–0.500 mg/ml as MIC and MFC, respectively
(Tables 4,5 and 6 and Figure 10b). Additionally, the
results of anti‐fungal screening, indicate that [Fe3O4NP‐
INS‐(C32H28N4)] show more activity than the Ni (II), Cu
(II) and Fe (II) metal complexes. These results may be
due to a higher stability constant of the Fe3O4NPs com-
plex than the studied metal complexes.
3.2.2 | Cytotoxicity activity
The (C32H28N4) ligand (1), [Fe3O4NP‐INS‐(C32H28N4)]
(5) and metal complexes of Schiff base ligand (Ni (II),
Cu (II) and Fe (II)) were studied at different concentra-
tions (10, 30, 50 and 100 μg/mL), on the viability of three

16 of 19
CHAABANE ET AL.
human tumoral cell lines: U87 (Brain), MDA‐MB‐231
(Breast) and LS‐174 (Colon) for 24 hr. The results showed
that the effects of all the molecules are dose dependant on
the viability of both U87 and MDA‐MB231 cell lines. For
LS‐174 although all the molecules have dose‐dependent
effect, the complex (3) reaches the saturation (12.32% of
cell viability) at 10 μg/ml concentration showing that
LS‐174 cell line are the most sensitive to this complex.
The compound [Fe3O4NP‐INS‐(C32H28N4)] (5) showed
the highest effect on cell viability of all the studied cell
lines with IC50 values of 8 μg/ml, 4.3 μg/ml and
18 μg/ml on U87, MDA‐MB231 and LS‐147 cells, respec-
tively. This can be explained by the oxidation stress which
is an important mechanism of cytotoxic actions of
Fe3O4NPs. In addition, the ability of Fe3O4NPs to gener-
ate reactive oxygen species (ROS) served to evaluate their
toxic and cytotoxic effects. This may be due to their
unique physicochemical properties and direct or indirect
effects of [Fe3O4NP‐INS‐(C32H28N4)] (5) on different
organelles, as they enter cells via a “Trojan‐horse” mech-
anism. After their intracellular accumulation, Fe3O4NPs
are stored in lysosomal vacuoles. Mitochondria are the
main sources of cellular ROS reported that [Fe3O4NP‐
INS‐(C32H28N4)] (5) exhibited cytotoxicity by inducing
ROS production.[59]
The complexes (2) and (4) (IC50 values of 143 μg/ml,
182 μg/ml and 111.2 μg/ml, 50 μg/ml, 51,6 μg/ml and
31,6 μg/ml against U87, MDA‐MB231 and LS‐174 cells,
respectively) didn't show important activity especially
when compared to the ligand (1) (Figure 11(a‐c) and
Table 7). On another hand, the ligand (C32H28N4) (1)
showed less cytotoxicity effect than complexes (3) which
present the highest cytotoxicity activity in all the studied
cancer cell lines under identical conditions, may be show-
ing a higher Cu (II) ions exchange between complex (3)
and cancer cell membranes. This would suggest that the
chelation could facilitate the ability of Cu (II) to cross
cancer cell membranes and can be explained by Tweedy's
chelation theory.[60,61] From the data it was also observed
that the activity depends upon the type of metal ion and
its stability in the following order of the metal ion: Cu
(II) > Fe (II) > Ni (II). As a consequence, ligand
(C32H28N4) (1) acts as a ship for transport and specific
deliberation of metal ions for a cross into cancer cell
membranes or to generate reactive oxygen species (ROS)
served to evaluate their toxic and cytotoxic effects from
Fe3O4NPs.
FIGURE 11 Effect of ligand (C32H28N4) (1), [Ni(C32H28N4)Cl2]
(2), [Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2] (4) and [Fe3O4NP‐
INS‐(C32H28N4)] (5) on the viability of human tumor cells. U87 (a),
MDA‐MB231 (b) and LS174 (c) cells were treated with different
concentrations of each complex (10, 30, 50 and 100 μg/ml) for 24 h.
Cell viability was analyzed by MTT assay. Results represent the
mean S.E.M of three independent (N = 3) experiments (*
p < 0.05, ** p < 0.01 and *** p < 0.005 with respect to CN)
To examine the cytotoxic effect of the ligand
(C32H28N4) (1), [Fe3O4NP‐INS‐(C32H28N4)] (5) and metal
complexes of Schiff base ligand (Ni (II), Cu (II) and Fe
(II)) against normal PBMC cells, trypan blue dye exclu-
sion assay was performed. PBMC cells have been treated
with highest dose (100 μg/mL) of the synthesized
compounds for 48 hours. The complexes (2), (3) and (4)
(45%, 52% and 85% of cell viability of PBMC, respectively)

CHAABANE ET AL.
17 of 19
TABLE 7 Effect of 1, 2, 3, 4 and 5 on Viability of U87, MDA‐MB‐
231 and LS‐174 cell lines
show important cytotoxic effect on PBMC cells especially
when compared to the ligand (C32H28N4) (1) (Figure 12).
The higher cytotoxic effect of the metal complexes in the
normal PBMC may be owing to the effect of metal ions on
the normal cell membrane compared to the parent ligand
under experimental conditions. Metal chelates stand non-
polar and polar properties together; this makes them suit-
able for permeation to the cells.[62,63] Also, no significant
cytotoxic effect was observed in the normal PBMC at the
highest concentration (100% μg/mL) of [Fe3O4NP‐INS‐
(C32H28N4)] (5) that significantly affected the cancer
cells, suggesting that the effect of the [Fe3O4NP‐INS‐
(C32H28N4)] (5) was selective for cancer cells (Figure 11
). Finally, these results confirm that [Fe3O4NP‐INS‐
(C32H28N4)] (5) possess good anticancer activity without
showing cytotoxicity towards normal PBMC cells com-
pared with metal (II) complexes.
Tissue
Brain
Breast
Colon
MDA‐MB‐
LS‐
Cell line
U87 231
174
Viability (%)
DMSO 0.10%
(Control)
Conc.
(μM)
100 100
100
Code
1
10
30
50
100
71.03 85.59
68.58 72.03
54.86 49.40
43.45 40.70
63.86
25.79
22.24
16.22
2
3
4
5
10
30
50
100
87.54 69.26
85.86 68.18
83.65 64.23
62.33 63.86
81.40
79.64
59.71
60.44
10
30
50
100
77.34 81.22
62.86 29.58
55.96 19.27
15.65 17.80
12.31
11.28
11.31
11.04
4 | CONCLUSION
In this study a tetraaza (N4) macrocyclic ligand and its
corresponding transition metal complexes (Cu (II), Ni
(II) and Fe (II)) were synthesized. Additionally, Fe3O4NP
was fabricated successfully inside the tetraaza macrocy-
clic Schiff base ligand through a co‐precipitation method.
All the synthesized compounds were characterized by
physic‐chemical techniques like: FT‐IR, UV–Vis, NMR,
ESI, PXRD, SEM, VSM, conductance measurements and
elemental analysis. Finally, the biological activities of
the synthesized compounds were investigated against
some microbial strains and four cell lines. The results
showed that [Fe3O4NP‐INS‐(C32H28N4)] and the metal
Cu (II) complexes possess good antimicrobial activity.
Also the results showed that [Fe3O4NP‐INS‐(C32H28N4)]
possess good anticancer activity without showing cytotox-
icity towards normal cells compared with metal (II)
complexes.
10
30
50
100
75.02 87.68
66.30 68.22
49.37 56.01
41.59 33.68
66.87
49.62
46.86
11.96
10
30
50
100
60.40 38.31
47.50 21.43
7.93 19.34
5.61 9.25
90
12.86
11.63
4.50
1 = Ligand (C32H28N4), 2 = [Ni(C32H28N4)Cl2], 3 = [Cu(C32H28N4)Cl2],
4 = [Fe(C32H28N4)Cl2] and 5 = [Fe3O4NP‐INS‐(C32H28N4)]
ACKNOWLEDGEMENTS
The authors greatly acknowledge financial support
between cooperation France‐Tunisia‐Morocco for funding
the work through the research group N° 17/TM10. We
would to thank Dr. Khadija Essafi‐Benkhadir (Pasteur
Institue of Tunsia) for generously providing human
cancer cells. The authors also would like to thank
Pr. BENONISTER David (Oxford University, UK) for
English language correction.
FIGURE 12 Cytotoxic effect of ligand (C32H28N4) (1),
[Ni(C32H28N4)Cl2] (2), [Cu(C32H28N4)Cl2] (3), [Fe(C32H28N4)Cl2]
(4) and [Fe3O4NP‐INS‐(C32H28N4)] (5) on normal PBMC cells. Data
represented as mean S.E.M of three independent experiments.
*P < 0.01 and **P < 0.005 versus control group
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.

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