Synthesis, spectral, antitumor, antioxidant and antimicrobial studies on Cu(II), Ni(II) and Co(II) complexes of 4-[(1H-Benzoimidazol-2-ylimino)-methyl]-benzene-1,3-diol

Article abstract of DOI:10.1016/j.saa.2015.03.020

A new Schiff base of 2-aminobenzimidazole with 2,4-dihydroybezaldehyde (H₃L), and its Cu(II), Ni(II) and Co(II) complexes have been synthesized and characterized by elemental analyses, molar conductance, thermal analysis (TGA), inductive couple

Full text of DOI:10.1016/j.saa.2015.03.020

Accepted Manuscript
Synthesis, spectral, antitumor, antioxidant and antimicrobial studies on Cu(II),
Ni(II) and Co(II) complexes of 4-[(1H-Benzoimidazol-2-ylimino)-methyl]-ben-
zene-1,3-diol
Nadia El-wakiel, Mai El-keiy, Mohamed Gaber
PII:
S1386-1425(15)00316-9
http://dx.doi.org/10.1016/j.saa.2015.03.020
SAA 13434
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
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Revised Date:
Accepted Date:
28 October 2014
8 February 2015
1 March 2015
Please cite this article as: N. El-wakiel, M. El-keiy, M. Gaber, Synthesis, spectral, antitumor, antioxidant and
antimicrobial studies on Cu(II), Ni(II) and Co(II) complexes of 4-[(1H-Benzoimidazol-2-ylimino)-methyl]-
benzene-1,3-diol, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://
dx.doi.org/10.1016/j.saa.2015.03.020
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Synthesis, spectral, antitumor , antioxidant and antimicrobial studies on
Cu(II) , Ni(II) and Co(II) complexes of 4-[(1H-Benzoimidazol-2-
ylimino)-methyl]-benzene-1,3-diol
Nadia El-wakiel1*, Mai El-keiy2, Mohamed Gaber1
1Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt
2 Division of Biochemistry, Chemistry Department, Faculty of Science, Tanta
University, Tanta, Egypt
Abstract
A new Schiff base of 2-aminobenzimidazole with 2,4-dihydroybezaldehyde (H3L),
and its Cu(II), Ni(II) and Co(II) complexes have been synthesized and characterized
by elemental analyses, molar conductance, thermal analysis (TGA), inductive poupled
plasma (ICP), magnetic moment measurements, IR, EI-mass, UV-Vis. and ESR
spectral studies. On the basis of spectral studies and analytical data, it is evident that
the Schiff base acts as dibasic tridentate ligand coordinating via deprotonated OH, NH
and azomethine nitrogen atom. The results showed that Co(II) and Ni(II) complexes
have tetrahedral structure while Cu(II) complexes has octahedral geometry. The
kinetic and thermodynamic parameters of the thermal decomposition stages have been
evaluated. The studied complexes were tested for their in vitro antimicrobial activities
against some bacterial strains. The anticancer activity of the ligand and its metal
complexes is evaluated against human liver carcinoma (HEPG2) cell. These
compounds exhibited a moderate and weak activity against the tested HEPG2 cell
lines with IC50 of 9.08, 18.2 and 19.7 µg/ml for ligand, Cu(II) and Ni(II) complexes,
respectively. In vitro antioxidant activity of the newly synthesized compounds has
also been evaluated.
Keywords
Benzimidazole. Schiff bases complexes, Antimicrobial, Antitumor, Antioxidant
* Corresponding author: E-mail: drnadia6@yahoo.com
Tel: (+2 040 3410037) Fax: (+2040 3350804).

1. Introduction
Transition metal complexes are of interest in the development of metal based
anticancer agents [1-3]. The clinical success of platinum-based drugs as anticancer
agents is severely affected by the serious side effects, toxicity and acquired drug
resistance [4-6]. These drawbacks drive inorganic chemists to develop innovative
strategies for the preparation of more effective, less toxic, target specific and
preferably non- covalently bound anticancer drugs.
Imidazoles are common of large number of natural products and
pharmacologically active molecules. As biologically active compounds,
benzimidazoles and their derivatives exhibited many biological applications such as
antitumor [7], anticancer [8], antimicrobial [9], antifungal [10], and anti-inflammatory
activities [11]. Imidazole, benzimidazole and their derivatives are part of an important
class of N-heterocycles which can be excellent organic ligands to generate various
complexes upon ligation [12- 21]. Schiff bases are used extensively as ligands in
coordination chemistry. In view of their interesting structural properties and wide
ranging uses, studies of the Schiff base complexes have attracted the attention of
many investigators [22-27]. Metal complexes of Schiff base compounds are a class of
important compounds in medicinal and pharmaceutical field. They have wide
applications in some biological aspects [26, 28-33]
The present work aims to synthesize a new Schiff base ligand namely, 4-[(1H-
Benzoimidazol-2-ylimino)-methyl]-benzene-1,3-diol and its complexes with Co(II),
Ni(II) and Cu(II) ions. The complexes have been characterized by several tools of
analyses such as elemental analyses, magnetic moment measurements, , ICP , molar
conductance, ESR, IR, UV–Vis spectra. In vitro antioxidant and antimicrobial activity
of the free ligand and its metal complexes have been assessed. Also, the antitumor
activity of the synthesized compounds has been studied.

2. Experimental
2.1. Materials
All compounds used in the present study were of pure grade available from BDH,
Aldrich or Sigma. The solvents used for the spectral studies were spectroscopic grade
from Aldrich.
2.2. Synthesis of Schiff-base ligand (H3L)
The Schiff base ligand (Fig. 1), was prepared by the condensation of 1 mmol of 2-
aminobenzimidazole with 1 mmol of 2,4-dihydroybezaldehyde in methanol. A 1:1
mixture of them was refluxed for 24 h. The reaction mixture was then poured onto ice
water. The product which separated immediately was filtered off, washed several
times with cold water and finally dried in a vacuum desiccator over anhydrous
calcium chloride.
2.3. Synthesis of complexes
To a solution of ligand (0.253g, 1 mmol) in 30 ml ethanol, an ethanolic (20 ml)
solution of the hydrated metal(II) chloride(0.170 g CuCl2.2H2O, 0.237g NiCl2.6H2O
and 0.237g CoCl2.6H2O, 1 mmol) was added slowly with constant stirring. The
reaction mixture was heated to reflux for 4 hr. The colored precipitate appeared after
adding 3 drops of triethylamine as buffering agent. The formed solid product was
removed by filtration, washed several times with ethanol and dried over anhydrous
CaCl2. The purity of the formed compounds was monitored by TLC.
2.4. Instrumentation
The elemental microanalyses of the solid compounds were performed at the micro
analytical center, Cairo University using Perkin-Elmer 2400 CHN Elemental
analyzer. Metal content was estimated using inductive coupled plasma (ICP) Perkin
Elmer/Optima 7000 DV at central laboratory, Tanta University. Molar conductivities
in DMF (10-3 M) at room temperature (25°C) were measured using conductance
bridge of the type 523 conductivity bridge. The Infrared spectra were recorded using a
JASCO FT/IR-4100 spectrophotometer within the range 4000-400 cm-1 as KBr discs.
Standard electron impact mass spectra (EI) were recorded using a Finnigan MAT

8222 Spectrometer at 70 eV at micro analytical unit of Cairo University. The
electronic absorption spectra were recorded using a Shimadzu UV-Vis 240
spectrophotometer. The room temperature magnetic susceptibility of the solid samples
was measured using magnetic susceptibility balance (Johnson Mtthey) 436 Devon
Park Drive employing the Gouy's method. The thermogravimetric analysis (TGA) of
°
the solid samples was performed within the temperature range 25-800 C using the
°
Shimadzu TG-50 thermogravimetric analyzer with heating rate (10 C/min.) under
nitrogen atmosphere. The X-band electron spin resonance (ESR) spectra of powder
samples were recorded using Joel JES-FE2XG spectrometer model equipped with an
E101 micro wave bridge at room temperature. The magnetic field was calibrated with
diphenylpicrylhydrazyl (DPPH).
2.5. Antibacterial Studies
Antimicrobial activity of the tested samples was determined using a modified
Kirby-Bauer disc diffusion method [34] at the micro-analytical unit of Cairo
University. Briefly, 100 µl of the test bacteria was grown in 10 ml of fresh media until
they reached 106 cells/ml for bacteria [35]. 100 µl of microbial suspension was spread
onto agar plates corresponding to the broth in which they were maintained. Isolated
colonies of each organism that might be playing a pathogenic role should be selected
from primary agar plates and tested for susceptibility by disc diffusion method [36].
From the available many media, NCCLS recommends Mueller-Hinton agar since it
results in good batch-to-batch reproducibility. Disc diffusion method for filamentous
fungi was tested by using approved standard method (M38-A) developed by the
NCCLS [37] for evaluating the susceptibilities of filamentous fungi to antifungal
agents. Disc diffusion method for yeasts developed by using approved standard
method (M44-P) by the NCCLS [38]. Plates inoculated with Gram positive bacteria as
Staphylococcus aureus NCTC 6356 and Gram negative bacteria as Escherichia coli
NRRL-B-3704. They were incubated at 35-37 °C for 24-48 hours and, then the
diameters of the inhibition zones were measured in millimeters [39]. Standard discs of
tetracycline (antibacterial agent) and amphotericin B (antifungal agent) served as
positive controls for antimicrobial activity but filter discs impregnated with 10 µl of
solvent (distilled water, DMSO) were used as a negative control. The agar used is
Meuller-Hinton agar that is rigorously tested for composition and pH. Further the
depth of the agar in the plate is a factor to be considered in the disc diffusion method.

This method is well documented and standard zones of inhibition have been
determined for susceptible and resistant values. Blank paper disks (Schleicher &
Schuell, Spain) with a diameter of 8.0 mm were impregnated 10 µl of tested
concentration of the stock solutions. When a filter paper disc impregnated with a
tested chemical is placed on agar the chemical will diffuse from the disc into the agar.
This diffusion will place the chemical in the agar only around the disc. The solubility
of the chemical and its molecular size will determine the size of the area of chemical
infiltration around the disc. If an organism is placed on the agar it will not grow in the
area around the disc if it is susceptible to the chemical. The area of no growth around
the disc is known as a "Zone of inhibition" or "Clear zone". For the disc diffusion, the
zone diameters were measured with slipping calipers of the National Committee for
Clinical Laboratory Standards [32, 33]. Agar-based methods such as Etest and disk
diffusion can be good alternatives because they are simpler and faster than broth-
based methods [40, 41].
2.6. Antioxidant studies
The antioxidant activity of ligand and its complexes as well as the standard
ascorbic acid was assessed on the basis of the radical scavenging effect of the stable
DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical. The radical scavenging effects
of antioxidants on DPPH are due to their hydrogen donating ability which causes an
absorbance drop at 517 nm. In the DPPH radical scavenging, antioxidants react with
the DPPH radical, which is a stable free radical and exists naturally in deep violet
color, to turn into a yellow colored (diphenyl--picryl hydrazine). The degree of
discoloration indicates the radical scavenging potential of the antioxidant [42]. 1µg
of the studied samples and ascorbic acid (reference compound) was dissolved in 1 ml
of DMSO. 250 µl of each solution were added to 1 ml DPPH/DMSO solution (6
mg/50 ml) and the total volume was adjusted to 3 ml with DMSO. After overtaxing,
the mixture was incubated for 30 min at room temperature. Absorbance was
measured at 517 nm. Absorbance of blank sample containing the same amount of
DMSO and DPPH solution was prepared and measured as well. The experiment was
carried out in triplicate. The scavenging potential was compared with a solvent
control (0% radical scavenging) and ascorbic acid.
2.7. Measurement of Potential cytotoxicity

The human cancer cell line used for in vitro screening experiment is liver
˚
Carcinoma (HEPG2) cell lines. It was obtained frozen in liquid nitrogen (-180 C)
from the American Type Culture Collection. The tumor cell lines were maintained in
the National Cancer Institute, Cairo, Egypt, by serial sub-culturing. Potential
cytotoxicity of the compounds was tested using Skehan et al. method [43].
Cells were plated in 96-multiwell plate (104 cells/well) for 24 hrs before treatment
with the compound to allow attachment of cell to the wall of the plate. Different
concentration of the compounds under test (0, 5, l0, 12, 25 and 50 µg/ml) were
added to the cell monolayer. Triplicate wells were prepared for each individual dose.
Monolayer cells were incubated with the compounds for 48 hrs at 37 °C and in
atmosphere of 5% CO2. After 48 hrs, Cells were fixed, washed and stained with
Sulfo-Rhodamine-B stain. Excess stain was washed with acetic acid and attached
stain was recovered with Tris EDTA buffer. Color intensity was measured in an
ELISA reader. The relation between surviving fraction and drug cone is plotted to
get the survival curve of each tumor cell line after the specified compound. An Elisa
reader (TECAN SUNRISE), Potential cytotoxicity of the compounds was measured
in (Pharmacology Unit, Cancer Biology Department, National Cancer Institute,
Cairo University). Doxorubicin was used as standard cytotoxins
3. Results and Discussion
All the metal chelates are colored and stable towards air and moisture. The
analytical results of the complexes are consistent with the proposed molecular
formulae and confirm the formation of 1:1 and 2:1 (M:L) complexes (Table 1). The
observed molar conductance values of all complexes in 10–3 molar DMF solution
reveals their non-electrolytic nature [44]
3.1. 1H-NMR Spectru of the Ligand
1H-NMR spectrum of the Schiff base H3L was recorded in d6-DMSO. The 1H-NMR
spectrum of the ligand exhibited a multiple at 6.39-7.64 ppm which assigned to
aromatic protons. The azomethine proton (-CH=N-) appeared as a singlet at 8.13ppm.
The ligand spectrum showed a peak at 9.90 ppm, belonging to the phenolic OH
proton. The most downfield signals observed as a singlet is due to NH proton of
imidizole moiety and appeared at 12.52 ppm [45].

3.2. EI-mass spectra
The constitutions and purities of the prepared ligand ( H3L) and its metal complexes
are confirmed using ESI mass spectrometry. The mass spectra of H3L, [CuHL(H2O)3],
[Ni2HLCl2.EtOH] and [CoHL.EtOH] showed peaks at m/z 253, 368.25, 485.20, and
355.20, respectively, corresponding to the molecular weight of the parent ion [H3L+]
and [ML+].The results of both elemental analyses and MS of the prepared complexes
are in satisfactory agreement with each other's which confirm the proposed molecular
formula (Table 1).
3.3. Infrared spectral studies
The IR spectra of the Schiff base and its metal complexes along with their tentative
assignments are reported in Table (2) and shown in Fig(2).
The IR spectrum of the free ligand showed two bands at 3443 and 3299 cm-1 which
assigned to phenolic OH and NH groups, respectively. All complexes showed a broad
absorption band within the range 3443-3384 cm–1 due to ν(OH) of water or ethanol
molecules attached to metal(II) ion. The disappearance ν (N-H) band of imidazole ring
for all complexes indicated the participation of the deprotonated N-H in chelation.
The band appearing at 1604 cm-1 is due to υC=N of azomethine group of the free
ligand. This band was shifted to lower frequency (13-27 cm-1) on complex formation.
This shift is due to coordination of azomethine nitrogen to the central metal ion. The
band δOH appeared at(1327 cm-1 for the free ligand H3L) was shifted to 1311 , 1308
and 1309 cm-1 for complexes 1, 2 and 3, respectively. The above observations were
supported by the appearance of a new band at 569-555 and 461-498 cm-1, which were
tentatively assigned to the ν(M–O) and ν(M–N) modes, respectively [46].
3.4. Thermogravimetric analysis of the complexes
The thermal behavior of the solid metal complexes was studied using TG
technique. The stages of decomposition, temperature range, weight loss percentages
as well as the decomposition products are given in Table 3. The thermal
decomposition of complexes 1 and 3 takes place in two steps. The first decomposition
step appears within the temperature range 29-177 and 29-120°C with mass loss of
14.63 and 12.35% which corresponded to elimination of coordinated water and
ethanol molecules for complexes 1 and 3,respectively. The second step within the

temperature range 177-512 and 120-519 °C with mass loss of 19.80 and 16.21%
represents the degradation of the organic ligand leaving CuO + C and CoO as the
final product. The thermal decomposition of complex 2 takes place in three steps. The
first decomposition step appears within the temperature range 30-162 °C with mass
loss of 9.45 % corresponding to the elimination of coordinated ethanol molecule. The
second step appears within the temperature range 162-328 °C with mass loss of 14.89
% due to the loss of chloride ions The third step appears within the temperature range
328-460°C with mass loss of 23.72% due to the degradation of the organic ligand
leaving NiO as the final product.
The thermodynamic parameters of the different decomposition processes of the
complexes namely, enthalpy (∆H*), entropy (∆S*) and free energy of the
decomposition (∆G*) as well as the activation energy (E*), order (n) and pre-
exponential factor (A), were evaluated graphically by using Coats–Redfern [47].
The calculated values of n, E*, A, ∆S*, ∆H* and ∆G* for the decomposition steps are
given in Table (4).
From the results listed in Table (4), the following features can be deduced:
Kinetics of the thermal decomposition stages of all complexes under study obeys, in
most cases, the first order kinetics. The values of ∆G* increased for the subsequently
decomposition stages due to increasing the values of (T∆S*) from one stage to
another due to the structural rigidity of the remaining complex after the expulsion of
one and more ligand as competed with the precedent complex, which require more
energy. The positive values of ∆G* suggested that all the decomposition steps are
non-spontaneous process. The positive ∆H* values indicate that the decomposition
processes are endothermic. The negative values of the activation entropies ∆S*
indicate a more ordered activated complex than the reactants and/or the reactions are
slower than normal [48].
3.5. Electronic absorption and magnetic moment measurements
The electronic absorption spectra of the complexes 1-3 were studied in Nujol mull.
The spectrum of Cu(II) complex 1 displayed a band at 22026 cm-1 assigned to
2Eg→2T2g transition assuming octahedral geometry around the central Cu(II) ion. The
electronic absorption spectrum of Ni(II) complex showed two bands at 16286 and

3
3
22522 cm-1 assignable to T1(F) →3A2 , T1(F)→3T1(p), respectively,assuming the
tetrahedral geometry around the Ni(II) ion. The electronic absorption spectrum of
Co(II) complex displayed two bands at 15527 and 21929 cm-1 which is assigned
4
to4A2→4T1(F)
and A2→4T1(P)
transition, respectively, due to tetrahedral
arrangement.
The ligand field parameter B ( interelectronic repulsion of the d electrons in
complex), β (The Nephelauxetic effect) and 10Dq are calculated according to the
equation reported for the tetrahedral Co(II) complex [49]
µeff = 3.87(1- 4λ/10Dq) , λ = -178Cm-1
B = [4(ν3-15Dq) 2 – 10Dq2] / [60((ν3-15Dq) – 180Dq]
ν1 = 10Dq
β = B(complex) / B(free ion)
where B (free ion) for Co(II) is 963cm−1. The 10Dq, B and β values are 7060 cm-1,
695.66 cm-1 and 0.62, respectively. These results show that the interelectronic
repulsion of the d-electrons in a complex is less than in the free ion. The value of B in
a complex is 72% of the free-ion value. The β value is related directly to covalence.
The reduction of B by complex formation is caused by delocalization of the d-electron
cloud on the ligand, which is in turn caused by the formation of covalent bond. The
data show the Co(II) complex has covalent character [50].
The magnetic moment data of the solid complexes at room temperature showed
that all complexes are paramagnetic. The magnetic moment value of Cu(II) complex
(1.90 BM) is higher than the theoretical spin only value of Cu(II) complex refers to
spin – orbital coupling. The magnetic moment value of Ni(II) complex (2.65 BM) is
lower than spin only value of Ni(II) complex may be due dimmerization and
antiferromagnetic interaction[51]. The magnetic moment value of Co(II) complex
(4.26 BM) is higher than the theoretical spin only value of Co(II) complex referring
to orbital contribution for Co(II) complex[51]. These values indicate octahedral
structure for complex 1 and tetrahedral structure for complexes 2 and 3.
3.6. ESR spectra

The X-band ESR spectra of the powdered Cu(II) Schiff base complex was
recorded at room temperature (25oC). The profile of the ESR spectrum of Cu(II)
complex is consistent with octahedral geometry around Cu(II) center which agree
with the electronic spectral data. The ESR spectrum of Cu(II) complex exhibited two
g-values. The calculated values of gII and g┴ of Cu(II) complex showed the order gII >
g┴ > 2.0023 which is consistent with d
ground state [52]. The g-value of Cu(II)
x2–y2
complex has a positive contribution from the value of free electron (2.0023) due to the
measurable covalent character in the bonding between the Schiff base ligand and
Cu(II) ion. The value gII <2.3 indicated that the Cu(II)-Schiff base complex has the
covalent character [53, 54]
The geometric parameter G, which measure the exchange interaction between the
metal center in a polycrystalline compounds is calculated by using the expression:
G= (gII -2.0023)/ (g┴-2.0023)
The calculated G value was found greater than 4 (Table 5) which indicates no
interaction between the adjacent Cu(II) centers [54,55]
It is usual to determine the covalent bonding parameters for the Cu(II) ion in
various ligand field environments. Molecular orbital coefficient, α2 (a measure of the
covalency in the in-plane σ-bonding between a copper 3d orbital and the ligand
orbitals), β2 (covalent in-plane π –bonding) and γ2 (out-of-plane π –bonding) were
calculated by using the following equations[56-58].
α2 = - (A║/0.036) + (g║ - 2.0023) + 3/7(g┴ - 2.0023) + 0.04
β2 = (g║ - 2.0023) E / 8λα2
γ2 = (g┴ - 2.0023) E / 2λα2
The α2 value = 0.5, it indicates a complete covalent bonding, while the value of α2
= 1.0 suggests complete ionic bonding. The observed value of α2 (0.59) indicates that
the Cu(II) complex has covalent character. For the Cu(II) complex, the high value of
β2 compared to α2 indicated that the in-plane π –bonding is less covalent than the in-
plane σ-bonding
The ESR parameters are used to evaluate the orbital reduction factors K by using
2
the expression [59] K = (K║ +2 K┴ )/ 3, where K║ and K┴ are the parallel and
2
perpendicular components of the orbital reduction factor. The low value of K than

unity was indicative of its covalent nature which in agreement with the conclusion
obtained from the value of gII. Hathaway [60] has pointed out that for pure σ-
bonding, K ≈ K = 0.77 and for in-plane π-bonding K < K , while for out-of plane π-
⊥
⊥
||
||
bonding K > K . For the studied Cu(II) complex, the observed order K (0.51) >
⊥
||
||
K (0.31) implies a greater contribution from out-of-plane π-bonding than for in-plane
⊥
π-bonding in metal–ligand π-bonding[60].
Based on the analytical, spectral data and magnetic moment data, the complexes
were formulated in Fig 3.
3.7. Antibacterial Results
The synthesized ligand and its complexes were screened for their antibacterial
activity against E. coli (Gram-negative bacteria) and S. aureus (Gram-positive
bacteria). The standard drugs, tetracycline and amphotericin B, were also tested for
their antibacterial activities at the same concentration and conditions of the test
compounds (Table 6 ).
From the value for gram's positive bacteria (S. aureus ) one can arrange the
complexes as Cu(II) > Co(II) > Ni(II) > Ligand. On the other hand, the inhibition
values against gram's negative bacteria (E. coli) can arranged the compound as Co(II)
> Ligand > Cu(II) > Ni(II). The antimicrobial activity of the ligand and its metal
complexes could not reach the effectiveness of the standard antimicrobial drugs such
as tetracycline or amphotericin B.
3.8. Antioxidant results
Hydroxyl radical is highly reactive oxygen centered radical formed from the
reactions of various hydroperoxides with transition metal ions. Among all the free
radicals, hydroxyl radical is by far the most potent and therefore the most dangerous
oxygen metabolite and hence the elimination of this radical is one of the major aims
of antioxidant administration[61]. Hydroxyl radical is known to be capable of
abstracting hydrogen atoms from membrane lipids and brings about peroxide reaction
of lipids. The antioxidant activity of free ligand , complexes and the standard

ascorbic acid were assessed on the basis of the radical scavenging effect of the stable
DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical. The results were shown in Fig.
S1.
By comparing the antioxidant activity of the ligand (IC50 value is 77.85 µg/ml)
with that of the metal complexes (IC50 values of Cu(II), Ni(II) and Co(II) complexes
are 83.73, 99.86 and 57.77 µg/ml, respectively) it become clear that: Co(II) complex
.
possesses higher scavenging activity towards OH than the parent ligand. It exhibited
excellent scavenging activity against the DPPH radical. The values were found to be
close to the values of standard ascorbic acid (IC50 value is 67.44 µg/ml). Cu(II) and
.
Ni(II) complexes possesses higher scavenging activity towards OH than the free
ligand. For Co(II) complex, the observed lower IC50 value in antioxidant assays
demonstrated that Co(II) complex has the potential as drugs to eliminate the radicals.
The antioxidant activity was decreased in the order Co(II) > free ligand >Cu(II) >
Ni(II).
3.9. Antitumor results
The anticancer activity of the free ligand and its Cu(II) and Ni(II) complexes was
determined in vitro against human cancer cell line liver Carcinoma (HEPG2). The
values of IC50, compared with the standard drug doxorubicin are collected in table 7.
Fig.S2 represents the cytotoxicity of ligand , complexes and doxorubicin (Dox) drug
against the liver Carcinoma (HEPG2) cell line, using different concentrations of
compounds or Dox. Untreated cells were used as a control. From these figures it is
clear that Ni(II)complex inhibited the growth of the tested cells than Cu(II) complex.
This indicated that the type of ion may be the reason for the different anticancer
activity [62].
The SB ligand and its Ni (II) and Cu (II) complexes showed an inhibition of cell
viability and gave the IC50 values of 9.03, 18.20 and 19.7 µg/ml against HEPG2,
respectively, compared with IC50 value of ( 4.73 µg/ml) for the standard cytotoxin
drug doxorubicin. According to Shier [63] the compounds exhibiting IC50 activity
within the range of 10-25 µg/ml are considered weak anticancer drugs, while those of
IC50 activity between 5 and 10 µg/ml are moderate and compounds of activity below
5.00 µg/ml are considered strong agents. Accordingly, the studied complexes showed

a weak antitumor activity, but the free ligand showed a moderate activity against the
HEPG2.
4. Conclusion
A new Schiff base of 2-aminobenzimidazole with 2,4-dihydroybezaldehyde
(H3L), and its Cu(II), Ni(II) and Co(II) complexes were synthesized and
characterized by elemental analysis, spectral studies ( mass spectra, IR, ESR, UV-
Vis) and magnetic data. The electronic and ESR spectral studies suggested
tetrahedral geometry for Ni(II) and Co(II) complexes. The Cu(II) complex have
octahedral geometry. The kinetic and thermodynamic parameters of the thermal
decomposition stages have been evaluated using Coats-Redfern methed. The
antimicrobial activity of the ligand and its metal complexes could not reach the
effectiveness of the standard antimicrobial druges such as
tetracycline or
amphotericin B. The antioxidant activity complex exhibited excellent scavenging
activity against the DPPH radical. The values were found to be close to the value of
standard ascorbic acid. The antioxidant activity decreases in the order Co(II) > free
ligand >Cu(II) > Ni(II). In vitro antitumor activity of these complexes showed a
weak antitumor activity, but the free ligand showed a moderate activity against the
HEPG2.
Acknowledgement
The authors would like to thank Tanta University (Grant No: Tu 02-13-02 ) for the
financial support of this project. We would like to extend our grateful thanks to
Prof. Dr Tark Mostafa for his assistance in evaluating the antioxidant activities.

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Table 1. Physical characteristics, analytical and molar conductance data of the
complexes.
% Elemental analysis (found)
No
Lig
1
Color
(Λm)a
Yield
%
Mol. wt
compound
C
H
N
M
--
16.60
(16.15)
253
H3L
Dark
66.40
4.38
85
82
(253)*
C14H11N3O2
yellow
(66.31)
(4.80)
11.39
368.50
[CuHL(H2O)3]
C14H15CuN3O5
Brown
0.19
45.59
4.07
17.23
(368.25)*
(11.09)
(45.04)
(4.02)
(17.31)
2
Reddish
brown
0.90
8.65
[Ni2HLCl2.EtOH]
C16H15Ni2Cl2N3O3
39.57
3.11
24.47
485.20
80
82
(8.27)
(40.09)
(3.03)
(24.17) (485.20)*
3
[CoHL.EtOH]
C16H15CoN3O3
Olive
0.50
53.94
4.20
11.82
16.49
355.90
(53.38)
(4.08)
(11.34)
(16.40) (355.20)*
All the synthesized complexes decompose without melting above 300 °C,
aΛm = Molar conductance (Ω−1 cm2 mol−1).
*molecular weight from mass spectrum

Table 2. IR Spectral data (cm-1) of the ligand and its Cu(II) , Ni(II) and Co(II)
complexes
νM- N
Compounds
νOH
νNH
νC=N
Schiff
1604
νOH.
νM- O
H3L
[CuHL(H2O)3]
3443
3443
3299
--
1327 ------
-----
461
491
498
1577
1591
1584
1311
1308
1309
555
564
569
[Ni2HLCl2.EtOH] 3409
[CoHL.EtOH] 3384
--
--

Table 3. Thermogravimetric analysis data (TGA) of the metal complexes
Mass loss %
Temp. range
No
Assignment
(°C)
Found
Calc.
Loss of coordinated water molecules
Loss of coordinated chloride ions
10.98
23.18
77.55
11.42
22.53
77.94
29-200
200-370
370-727
1
Dissociation of the organic ligand with formation of
CuO + C as final products
Loss of coordinated ethanol molecule
Loss of coordinated chloride ions
87-167
167-267
267-690
18.61
13.80
87.86
18.00
13.87
88.47
2
3
Dissociation of the organic ligand with formation of
CoO as final product
Loss of coordinated ethanol molecule
12.35
83.09
29-120
12.95
83.46
Dissociation of the organic ligand with formation of
CoO as final product
120-519

Table 4. The activation parameters (∆ H*, ∆S*, ∆G*) for decomposition of Cu(II),
Ni(II) and Co(II) complexes
Complex
n
ste
p
r
E*
∆ H*
A
-∆S*
∆ G*
Cu(II)
0
1
1st
2nd
0.9529
0.9100
6.8
3.58
4.15E+8
319327
0.090
0.152
38.60
119.4
complex
53.45
49.62
Ni(II)
0
1
1
1st
2nd
3rd
0.9953
0.9906
0.9923
16.39
59.86
92.7
13.52
56.99
89.8
3329959
14054
506
0.111
0.175
0.203
51.55
117.3
159.6
complex
Co(II)
1
1
1st
2nd
0.9952
0.9833
44.3
82.2
41.44
75.34
1797.5
3942
0.192
0.193
107.6
234.9
complex
E*, ∆H* , ∆G* (KJ.mol-1), ∆S* (J.K-1.mol-1), A: the pre-exponential factor(s-1)
Table 5. ESR spectral data of Cu(II) complex at 25oC
Compound
G
Kǁ
K┴
K
α2
β2
γ2
gǁ
g┴
gav
[CuHL(H2O)3] 6.20
0.51
0.31
0.15
0.59 0.86
0.52 2.267 2.04
2.21

Table 6. Antimicrobial activities of the investigated ligand and its complexes.
Compounds
Inhibition zone diameter(mm/ mg compound)
E. coli
St.aur
H3L
12
11
10
17
33
22
9
[CuHL(H2O)3]
[Ni2HLCl2.EtOH]
[CoHL.EtOH]
Tetracycline
Ampicilin
12
10
11
30
18
Table 7. Antitumor data of the investigated ligand and its complexes.
compound
IC50 ug/ml
stander
4.73
ligand
9.08
Cu complex
18.2
Ni complex
19.7

N
H
C
N
N
H
OH
HO
Fig. 1 The structure of Schiff base (H3L).

Co(II) complex
250
200
150
100
50
Ni(II) complex
Cu(II) complex
SB ligand
4000
3500
3000
2500
2000
1500
1000
500
wavenumber cm-1
Fig.2 IR spectra of the ligand and its Cu(II) , Ni(II) and Co(II) complexes.

N
H
C
OH2
Cu
N
O
N
OH2
OH2
HO
Cu(II) complex
N
H
C
N
N
Cl
Ni
Ni
OHEt
Cl
O
HO
Ni(II) complex
N
H
C
N
N
Co
OHEt
O
HO
Co(II) complex
Fig. 3 The structures of the metal complexes

Ni(II) complex
100
80
60
40
20
0
Cu(II) complex
H3L ligand
Ascorbic acid
Co(II) complex
1
2
3
compound
4
5
Fig.S1 Trends in the inhibition of hydroxyl radical by the H3Lligand and its
complexes.

100
80
60
40
20
00
00
untreated
Cu(II) complex
Ni(II) complex
SB ligand
5.0
12.5
25
50
Dox.
Fig. S2 In vitro cytotoxicity of H3L ligand ,complexes and doxorubicin (Dox) drug
against human liver Carcinoma cell line (HEPG2)

Graphical Abstract
100
80
60
40
20
00
00
untreated
5.0
Cu(II) complex
Ni(II) complex
12.5
25
SB ligand
50
Dox.
In vitro cytotoxicity of H3L ligand ,complexes and doxorubicin (Dox) drug against
human liver Carcinoma cell line (HEPG2)