Some new nano-sized Fe(II), Cd(II) and Zn(II) Schiff base complexes as precursor for metal oxides: Sonochemical synthesis, characterization, DNA interaction, in vitro antimicrobial and anticancer activities

Article abstract of DOI:10.1016/j.bioorg.2016.10.009

The complexes of Fe(II), Cd(II) and Zn(II) with Schiff base derived from 2-amino-3-hydroxypyridine and 3-methoxysalicylaldehyde have been prepared. Melting points, decomposition temperatures, Elemental analyses, TGA, conductance measurements, infrared (IR

Full text of DOI:10.1016/j.bioorg.2016.10.009

Bioorganic Chemistry 69 (2016) 140–152
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Some new nano-sized Fe(II), Cd(II) and Zn(II) Schiff base complexes as
precursor for metal oxides: Sonochemical synthesis, characterization,
DNA interaction, in vitro antimicrobial and anticancer activities
⇑
Laila H. Abdel-Rahman, Ahmed M. Abu-Dief , Rafat M. El-Khatib, Shimaa Mahdy Abdel-Fatah
Chemistry Department, Faculty of Science, Sohag University, 82534 Sohag, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2016
Revised 6 September 2016
Accepted 30 October 2016
Available online 1 November 2016
The complexes of Fe(II), Cd(II) and Zn(II) with Schiff base derived from 2-amino-3-hydroxypyridine and
3-methoxysalicylaldehyde have been prepared. Melting points, decomposition temperatures, Elemental
analyses, TGA, conductance measurements, infrared (IR) and UV–Visible spectrophotometric studies
were utilized in characterizing the compounds. The UV–Visible spectrophotometric analysis revealed
1:1 (metal-ligand) stoichiometry for the three complexes. In addition to, the prepared complexes have
been used as precursors for preparing their corresponding metal oxides nanoparticles via thermal decom-
position. The structures of the nano-sized complexes and their metal oxides were characterized by X-ray
powder diffraction and transmittance electron microscopy. Moreover, the prepared Schiff base ligand, its
complexes and their corresponding nano-sized metal oxides have been screened in vitro for their antibac-
terial activity against three bacteria, gram-positive (Microccus luteus) and gram-negative (Escherichia coli,
Serratia marcescence) and three strains of fungus. The metal chelates were shown to possess more antimi-
crobial activity than the free Schiff-base chelate and their nano-sized metal oxides have the highest activ-
ity. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption
spectra, viscosity mensuration and gel electrophoresis. The DNA binding constants reveal that all these
complexes interact with DNA through intercalative binding mode. Furthermore, the cytotoxic activity
of the prepared Schiff base complexes on human colon carcinoma cells, (HCT-116 cell line) and hepatic
cellular carcinoma cells, (HepG-2) showed potent cytotoxicity effect against growth of carcinoma cells
compared to the clinically used Vinblastine standard.
Keywords:
DNA interaction
Antimicrobial activity
Nanoparticles
Metal oxides
Cytotoxic activity
Ó 2016 Elsevier Inc. All rights reserved.
1
. Introduction
Transition metal complexes derived from the Schiff base ligands
and animals. They are extensively utilized in the formulation of
healthcare products [4]. It has long been identified as a serious
co-factor in biological compounds, either as a compositional
template in protein folding or as a Lewis acid catalyst which can
readily adopt the coordination numbers 4, 5, or 6 [5,6]. There is
substantial importance in the coordination chemistry of cadmium
complexes as a result of the toxic environmental effect of
cadmium. The mobilization and immobilization of cadmium in
the climate, in organisms, and in some technical systems (such
as in ligand exchange chromatography) have been presented to
depend manifestly on the complexation of the metal center by
chelating nitrogen donor ligands [7]. The size and shape of the
nanomaterials are key factors for shaping their characteristics
such as, electrical, optical, magnetic, antimicrobial and catalytic
potency. Metal and metal oxide nanoparticles have found wide
diversity of uses, including heterogeneous catalysts, environmen-
tal remediation, electronic, chemical sensing devices, medicinal
fields, separations, thin films, inks, disinfection, and antimicrobial
with biological potency have been widely studied. Schiff bases
appear to be an important intermediate in a number of enzymatic
reactions including interaction of an enzyme with an amino or a
carbonyl group of the substrate. One of the most important kinds
of catalytic mechanism is the biochemical process which involves
the condensation of a primary amine in an enzyme that of a lysine
residue, with a carbonyl group of the substrate to form Schiff base
[
1]. Transition metal complexes with different oxidation states
have a strong role in bioinorganic chemistry and may offer the
basis of models for active sites of biological systems [2,3]. On the
other hand, ZnO NPs are of particular interest because they can
be prepared easily cheap and secure matter for human beings
⇑
Corresponding author.
E-mail address: ahmed_benzoic@yahoo.com (A.M. Abu-Dief).
http://dx.doi.org/10.1016/j.bioorg.2016.10.009
045-2068/Ó 2016 Elsevier Inc. All rights reserved.
0

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
141
activity [8]. The different applications of metal and metal oxide
OH
nanoparticles altered with morphology and size [9]. The detection
of metal ions of biological importance has attracted much
OH
2
+
N
attention. Zn ion fluorescent probes or sensors have achieved
2
+
OCH3
CH
special interest. Zn is an essential trace element and the second
2+
most abundant metal ion in humans (after Fe ) [10]. Thus, the
present aim of the work is to synthesize a Schiff base derived from
N
2
-amino-3-hydroxypyridine and 3-methoxysalicylaldehyde and to
prepare its transition metal complexes, characterize them and
inspect their antibacterial, antifungal and anticancer activities.
We also studied their interaction with DNA. Nano-sized particles
of that complexes and their corresponding metal oxides nanoparti-
cles also were prepared. We also investigate the antimicrobial
activity of the prepared metal oxides nanoparticles.
2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-
pyridin-3-ol
2
. Experimental
All the starting matters of chemicals used in this investigation
Such as 3-methoxysalicylaldehyde (v), 2-amino-3-hydroxypyridine
ahp), the metal salts (FeSO . (NH SO O, Zn(NO ꢀ6H O,
ꢀ6H
Cd(NO O), Calf thymus DNA (CT-DNA), bromophenol blue
ꢀ4H
dye, ethidium bromide and Tris[hydroxymethyl]-aminomethane
Tris) were acquired from Sigma–Aldrich Chemie (Germany).
(
4
)
4 2
4
2
3
)
2
2
3
)
2
2
(
Spectroscopic degree ethanol, dimethylformamide (DMF) and HCl
products were used.
HO
H N
O
O
2
2.1. Synthesis of Schiff base ligand
N
OH
v
ahp
+
An equimolar mixture of 2-amino-3-hydroxypyridine (0.110 g,
mmol) and 3-methoxysalicylaldehyde (0.152 g, 1 mmol) dis-
1
solved in 10 ml ethyl alcohol and mixture was refluxed for 1 h.
The reaction mixture was filtered, rinsed with water and recrys-
tallised from ethanol. The Schiff base was dried under reduced
pressure in a desiccator. The purity of prepared compounds was
checked by TLC utilizing silica gel G (yield: 88%) as shown in (cf.
Scheme 1):
1H NMR (d, ppm), in DMSO: (6.85–8.00 (m, 6H-Ar), 9.44 (s, 1H,
CH = N), 10.28 (s, 1H, OH), 3.34 (s, 3H, OCH
3
)).
C NMR (d, ppm), in DMSO: 166 (CH = N), 155, 150, 149, 144,
43, 125, 124, 123, 122, 120, 118 (11CH-Ar), 56 (OCH ).
1
3
Scheme 1. Synthesis of Schiff base ligand (ahpv), where ahp = 2-amino-3-hydrox-
ypyridine and v = 3-methoxysalicylaldehyde (o-vanillin).
1
3
2
.2. Preparation of complexes with nano-structures by sonochemical
2.4. Physical measurements
route
Melting point for Schiff base and decomposition points for com-
1
0 ml of a 0.1 M (1 mmol) solution of metal salt [(FeSO
ꢀ6H O, 0.392 g), (Zn(NO ꢀ6H O, 0.297 g), (Cd(NO
.308 g)] in Ethanol was positioned in a high-density ultrasonic
probe, elaborating at 24 kHz with a maximum power output of
00 W. Into this solution, 10 ml of a 0.1 M solution of the ligand
ahpv) (0.260 g, 1 mmol) was added drop wise. The acquired pre-
4
.(NH
4 2
) -
2
O,
plexes were carried out on a melting point apparatus, Gallenkamp,
UK. IR spectra of the metal complexes in KBr powder in the range
SO
0
4
2
3
)
2
2
3
)
2
ꢀ4H
ꢁ1
of 4000–400 cm were recorded making use of Shimadzu FTIR
model 8101 spectrophotometer. Molar conductivity measure-
ments were made by using JENWAY conductivity meter model
4
(
4
320 at 298 K using DMF as solvent. UV–visible spectra in DMF
cipitate was filtered off, rinsed with ethanol and then dried in air
were registered utilizing 10 mm matched quartz cells on PG spec-
as shown in (cf. Scheme 2):
1
13
1H NMR (d, ppm), in DMSO: (ahpvCd: 6.45–7.40 (m, 6H-Ar),
trophotometer model T + 80. H NMR and C NMR spectra were
carried out in DMSO on BRUKER model 400 MHz using TMS as
9
6
3
.20 (s, 1H, CH = N), 3.79 (s, 3H, OCH ); ahpvZn: 6.42–7.51 (m,
H-Ar), 9.26 (s, 1H, CH = N), 3.77 (s, 3H, OCH )).
3
6
an internal standard (d ppm) and DMSO-d as the solvent. The
Schiff base and their complexes were conquered to elemental anal-
yses which was made at the analytic unit of the central lab of Cairo
University by Elemental analyzer Perkin-Elmer model 240c. The
magnetic measures were performed on Gouy’s balance and the dia-
magnetic correction was made by Pascal’s constants. Thermo
2
.3. Synthesis of metal oxide nano particles
Nano-sized metal oxides were prepared by calcination of
0.05 g) of complexes with nano-structures in air at 500 °C with
(
gravimetric test was made under N
2
conditions with a warming
ꢁ1
ꢁ1
heating rate 10 °C min . The structures of the nano-sized oxides
or metals were identified by transmmitance electron microscopy
and X-ray powder diffraction.
rate 10 °C min on Shimaduz corporation 60H analyzer. The val-
ꢁ3
ues of absorbance of 1 ꢂ 10 M of each complex were measured
at various PH levels. The pH levels were checked by using a series

1
42
L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
OH
HO
N
OCH3
CH
N
2
-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-pyridin-3-ol
Sonochemical
meth o° d
Fe(NH )(SO ) .6H O
4
4 2
2
Zn(NO ) .6H O
Cd(NO ) .4H O
3 2
2
3
2
2
N
N
CH
N
CH
N
O
O
O
O
Zn
M
OCH3
.4H2O
OCH3
.H O
2
OH2
OH2
OH2
OH2
Scheme 2. The suggested structures of ahpvFe, ahpvCd, ahpvZn complexes: for ahpvFe and ahpvCd complexes: n = 1 and ahpvZn complex: n = 4.
of Britton universal buffers [11]. pH measurements were made
using HANNA 211 pH meter at 298 ± 1 K. TEM images were
recorded using transmittance electron microscope (TEM-2100),
Faculty of Science, Alex. University. An ultrasonic generator (Dr.
Hielscher UP400 S ultrasonic processor) elaporated with an
time, the test solution transpired and the growth of the
inoculated microorganisms was affected. Antimicrobial activity
was indicated by the presence of obvious zone of inhibition
around the wells. The zone of inhibition was evaluated in
mm [12]. The preparation of metal oxides followed this route:
First of all, Petri plates were made by (20 ml) of sterile Muller
Hinton Agar for bacteria and (20 ml) of potato Dextrose Agar
in case of yeast [13]. The 24 h prepared test cultures of
inoculums were swabbed on the top of the sclerotic media
and left to dry for 10 min. Previously prepared metal oxides
nanoparticles impregnated nipping at the different concentra-
tions of 10 and 20 (mg/ml) for bacteria and fungi were placed
aseptically on sensitivity plates with suitable controls. The
loaded discs were put on the surface of the medium and
left for 30 min at room temperature for compound diffusion.
Ofloxacin was used as positive control for bacteria and
Fluconazol was utilized as positive control for fungi. After that
all the plates were incubated for 24 h at 37 °C for bacteria and
‘
‘H22” sonotrode with diameter 22 mm, working at 24 kHz with
a maximum force output of 400 W, was utilized for the ultrasonic
irradiation. The chemical composition of the synthesized nanos-
tructures was also analyzed using energy dispersive analysis of
X-ray (EDAX) unit. X-ray powder diffraction (XRD) gauges were
performed using a Philips diffractometer made by X’pert with
monochromatized Cu K
a radiation. Particle size distribution of
the prepared imine complexes and their corresponding metal oxi-
des was evaluated using image J Launcher, broken-symmetry soft-
ware, version (1.4.3.6.7).
2.5. Antimicrobial activity
2
8–35 °C for fungi, respectively. The sensibility was registered
The in vitro biological screening activities of the investigated
compounds were examined against the gram negative bacteria
Escherichia coli and Serratia marcescence) and gram positive
by measuring the clear zone of growth inhibition of agar
surface around the discs in millimeter.
(
bacteria (Micrococcus luteus) by the well diffusion route using
agar nutrient as the medium. While antifungal effect was
carried out using potato Dextrose Agar as medium against
2.6. DNA binding experiments
(
Getrichm candidum, Aspergillus flavus and Fusarium oxysporum).
All the experiments including the interaction of the complexes
with DNA were performed in Tris–HCl buffer (55 mM, pH 7.4).
CT-DNA was purified by centrifugal dialysis before utilization. A
solution of calf thymus DNA in the buffer presented a ratio of UV
absorbance at 260 and 280 nm of about >1.87, indicating that the
DNA was sufficiently free from protein contamination [14]. The
concentration of DNA was estimated by monitoring the UV
The stock solutions were made ready by dissolving the com-
pounds in DMF. In a typical process, a well was made with
the help of borer on the nutrient medium plate which was
formerly inoculated with microorganisms. The well was made
full with the various concentrations of test solution using a
micropipette and incubated at 37 °C for 24 h (bacteria) and
ꢁ1
2
4
8 h (fungus). Ofloxacin and Fluconazol were used as standard
absorbance at 260 nm using
e260 = 6600 mol cm . The stock
against the bacteria and fungi respectively. During incubation
solution was saved at 5 °C and used within only two days.

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
143
2
.6.1. Absorption spectral studies
Absorption spectral studies were carried out in (55 mM Tris-HCl
cytotoxic potency of the ligand and its complexes was carried
out against Colon carcinoma cells, (HCT-116 cell line) and hepatic
cellular carcinoma cells, (HepG-2). The evaluation process was car-
ried out in vitro using the Sulfo-Rhodamine-B-stain (SRB) [19].
Cells were placed in 96-multiwell plate (10 cells/well) for 24 h
before processing with the complexes to allow attachment of cell
to the wall of the plate. Various concentrations of the compounds
buffer, pH 7.4) buffer at room temperature to investigate the bind-
ing affinity between CT - DNA and complex. The concentration of
CT - DNA was checked from the absorption intensity at 260 nm
4
ꢁ1
2
with a
e
value of 6600 mol cm . Absorption titration experiments
were performed by altering the concentration of the CT - DNA (0–
3
ꢁ3
0
lM) keeping the complex concentration (10 M) as constant.
under check in DMSO (0, 1, 2.5, 5 and 10
cell monolayer. Monolayer cells were incubated with the com-
pounds for 48 h at 37 °C and in atmosphere of 5% CO . After
lM) were added to the
The absorbance (A) was registered after each addition of CT -
DNA. The stock solution was stored at 5 °C and used within only
two days. In order to remove the absorbance of the CT-DNA an
equal amount of the same was added to both the compound solu-
tion and the reference solution. The intrinsic binding constant, Kb
for the complexes was determined from the spectral experiments
data using the following equation [15]:
2
48 h, cells were fixed, rinsed, and stained with Sulfo-Rhodamine-
B-stain. Excess stain was rinsed with acetic acid and attached stain
was treated with Tris EDTA buffer. Color intensity was rated in an
ELISA reader. IC50 was evaluated and potency was calculated with
regard to percentage of alteration of (vistabline standard) [20,21].
½
DNAꢃ
½DNAꢃ
1
¼
þ
ð1Þ
3
. Results and discussion
ð
e
a
ꢁ
e
f
Þ
ð
e
b
ꢁ
e
f
Þ
½K
b
ðe
b
ꢁ
e
f
Þꢃ
Here,
e
a
,
f
e , and
e
b
are apparent, free and fully bound complex
3.1. Physicochemical properties
extinction coefficients respectively, where; [DNA] is the concentra-
tion of DNA in base pairs. In particular, was evaluated from the
calibration curve of the isolated metal complex; following the Beer’s
law. was rated as the ratio between the measured absorbance
and the complex concentration, Aobs/[complex]. The data were
suited to the above equation with a slope equal to 1/(
and y-intercept equal to 1/[K )] and K was rated from the
e
f
All the compounds are tinted, solid and stable at room temper-
ature. The Analytical and physical data of ligand and complexes are
recorded in (Table 1). The metal complexes exhibit 1:1 (metal-
ligand) stoichiometry.
e
a
e
b
ꢁ
f
e )
b
(
e
b
ꢁ
e
f
b
1
13
3
.2. H NMR and C NMR spectra of ligand and their diamagnetic
ratio of the slope to the intercept. The standard Gibbs free energy
for DNA binding was evaluated from the following relation [16]:
complexes
1
–
The H NMR spectra of the L
1
H ligand gives the signal at 6.85–
D
Gb ¼ ꢁRT ln K
b
ð2Þ
8
.00 (m) d for aromatic proton and 9.44 (s) d for azomethine pro-
ton. The peak at 10.28 (s) d is due to AOH group, disappeared upon
addition of D O. The peak at 3.34 (s) d is due to AOCH group. The
C NMR are offering the signals at various values of d as follows: at
2
.6.2. Viscosity experiments for interaction of the prepared complexes
with DNA
Viscosity measurements were made using an Oswald micro vis-
2
3
1
3
d 169 ppm (CH = N) due to azomethine and at d 122–156 ppm
(11CH-Ar) for aromatic carbon atoms [22]. Also at d 56 ppm
cometer, kept at constant temperature at 25 ± 1 °C in thermostat.
The fluidity times were recorded for various concentrations of
1
(OCH ) due to carbon atom of methoxy group [23]. The H NMR
3
the complex (10–60
stant (50 M). Blending of the solution was made by bubbling
the N gas through the viscometer. The average value of the three
l
M), keeping the concentration of DNA con-
(DMSO-d , ppm) of ahpvCd and ahpvZn complexes shows singlet
signal at 9.20 and 9.26 for CH = N protons respectively, multiple
6
l
2
signals at 6.45–7.40 and 6.42–7.51 for six and six aromatic protons
1
measures was utilized to rate the viscosity of the samples. The buf-
respectively, The H NMR (DMSO-d , ppm) of ahpvCd and ahpvZn
6
fer flow time in seconds was registered as t°. The relative viscosi-
shows singlet signal at 3.79 and 3.77 for three OCH protons. It is
3
ties for DNA in the presence (
complex were calculated by using the relation
Where, t is the notified flow time in seconds and the values of
the relative viscosity ( °) were plotted against 1/R (R = [DNA]/
g
) and disappearance (
g
°) of the
shown that the signal for OH protons is disappeared. This indicates
g
= (t ꢁ t°)/t°.
happening of chelation of ligands with metal ions and deprotonat-
ing of AOH phenolic of pyridine and benzene ring. Also, the shift in
signals for CH = N protons confirm chelation and deprotonating of
phenolic AOH [23].
g/g
[
Complex]) [17].
2
.6.3. Agarose gel electrophoresis
The DNA binding experiment was conducted utilizing CT DNA
3.3. Infrared spectra
by gel electrophoresis with the corresponding metal complex.
The reaction mixture was incubated before electrophoresis exper-
The IR spectra of the complexes were compared with those of
the free ligands in order to determine the involvement of the coor-
dination positions in the chelation. Characteristic peaks in the
spectra of the ligand and complexes were considered and com-
pared. IR spectrum of the ahpv ligand exhibited the most charac-
iment at 37 °C for 45 min as follows: CT DNA 25
complex. The samples (mixed with bromophenol blue dye at a
:1 ratio) were electrophoresed for 30 min at 60 V on 1% agarose
gel using TBE buffer, pH = 8.1. After electrophoresis, the gel was
stained utilizing
g/cm3 ethidium bromide (EB) and pho-
tographed under UV light using Lumix Digital camera [18].
lM, 60 lM each
1
ꢁ
1
ꢁ1
teristic bands at 1613 cm
(CAO) [24]. The formation of the Schiff base was noted from the
absence of C@O and NH peaks in the ligand. The band at
613 cm due to the azomethine group of the Schiff base was
m(C@N, azomethine) and 1307 cm
2
l
m
2
ꢁ1
1
ꢁ1
2.7. Anticancer activity
shifted to lower frequencies (1593–1594 cm ) after complexa-
tion, indicating the bonding of nitrogen of the azomethine group
to the metal ions. The phenolic CAO stretching vibration that
appeared at 1307 cm in Schiff base shifted towards lower fre-
quencies in the complexes. This proposes deprotonation of the
phenolic OH group after its chelation with the metal ion.
The anticancer activity was made at the National Cancer Insti-
ꢁ1
tute, Cancer Biology Department, Pharmacology Department, Cairo
University. The absorbance or optical density (O.D.) of each well
was evaluated spectrophotometrically at 564 (nm) with an ‘‘ELIZA”
ꢁ
1
micro plate reader (Meter tech.
R
960, ‘‘USA”). Estimation of the
The appearance of broad bands at around 3455–3468 cm in the

1
44
L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
Table 1
The analytical and physical data of ligand and its metal complexes.
m (O 1 cm2 mol
ꢁ
ꢁ1
)
leff B.M.
Compound
Colour
(M. p) and Dec. p °C
M.wt
Elemental analysis found (calculated)
Conductance
K
N
H
C
ahpv
Orange
Brown
Yellow
Orange
250
295
>300
>300
260
10.76 (10.64)
7.57 (7.61)
6.56 (6.60)
7.04 (6.91)
4.61 (4.71)
4.86 (4.75)
4.22 (4.33)
5.03 (4.95)
60.00 (60.15)
42.18 (42.29)
36.58 (36.43)
39.25 (39.38)
–
33.7
8.5
–
4.95
dia
ahpvFe
ahpvCd
ahpvZn
369.8
426.4
397.38
13.15
dia
spectra of complexes may be due to water molecules [25–28]. A
3.7. Kinetic aspects
ꢁ1
band of medium intensity at 967–977 cm (OH rocking) suggests
the presence of coordinated water in all three complexes. In the
low frequency part, the band of weak intensity observed for the
The thermodynamic parameters of the degradation processes of
the complexes were calculated using the Coasts-Red fern equation
[36],
ꢁ1
complexes in the region 516–580 cm is attributed to MAO and
ꢁ1
in the region 417–517 cm to MAN as shown in Table S1.
ꢀlogðw ꢁ wÞÞꢁ
ꢀ
ꢂ
ꢃꢁ
ꢄ
1
AR
/E
2RT
E
E
log
¼ log
1 ꢁ
ꢁ
ð3Þ
2
ꢄ
ꢄ
T
2:303RT
3.4. Electronic spectra
where W
1
is the mass loss at the completion of the decay reaction.
W is the mass perishing up to temperature T, R is the gas constant
The nature of the ligand field around the metal ion was derived
⁄
and / is the heating rate. Since 1-2RT/E ꢅ1, the plot of the left hand
from the electronic spectra. The electronic absorption spectra of
ligands and their complexes were on record at the wavelength
range 800–200 nm and at 298 K. The ligand exhibits absorption
⁄
of equation against 1/T would give a straight line. E was then esti-
mated to form the slope and the Arrhenius constant, A, was
acquired from the intercept. The other kinetic parameters; the
bands in UV–Vis region around 355 nm which is specified to
⁄
⁄
entropy of activation (S ), enthalpy of energizing (H ) and the free
⁄
n ?
p
transition originating from the azo methane function of
⁄
energy variance of activation (G ) were calculated using the follow-
the Schiff base ligand [29]. The absorption bands of complexes at
max = 390–449 nm is assigned to charge transfer with in Schiff
base ligands [30]. Furthermore, A long and a broad band lying in
ing equations:
k
Ah
kT
Sꢄ ¼ 2:303R log
ð4Þ
ꢁ1
2)
the region 531 nm (emax = 110 mol cm . This band could be
mainly attributed to the d ? d transition in the structure of ahvpFe
complex [31] as shown in (Fig. S1). It is was shown that no d-d
transition in ahvpCd and ahvpZn complexes because it contains a
full d subshell.
ꢄ
ꢄ
H ¼ E ꢁ RT
ð5Þ
ð6Þ
ꢄ
ꢄ
ꢄ
G ¼ H ꢁ TS
where (k) and (h) are Boltzmann’s and Plank’s invariables, respec-
tively. The kinetic parameters are listed in (Table S3). It is shown
3
.5. Magnetic moment measurements
⁄
that G values increase due to increasing temperature. The positive
⁄
values of H dissect that degradation processes are endothermic. In
The paramagnetic compounds will be attracted while the dia-
⁄
most thermal steps, S values are negative submitting a decomposi-
magnetic compounds repelled in a magnetic field. Therefore, para-
magnetic substances will have positive susceptibilities. Thus, the
magnetic susceptibility measures determine geometry of the com-
plexes. Magnetic susceptibility measurements showed that Fe(II)
complexes has paramagnetic character with octahedral geometry
tion via abnormal pathway at those steps and the degradation pro-
cesses are unfavorable. The negative activation entropy value slices
that the activated complexes were more ordered than the reactant
and that the reactions were slow. The more ordered nature was
attributed to the polarization of bonds in the activated state, which
might take place through charge transfer electronic transitions.
[
32], but Cd(II) and Zn(II) complexes has diamagnetic character
with octahedral with tetrahedral geometry, respectively [33].
⁄
⁄
Finally, positive values were found for H and G respectively sym-
bolizing endothermic character for all thermal steps [37].
3.6. Thermal analysis
3.8. Spectrophotometric determination of the stoichiometry of the
Thermo gravimetric test was made under N
2
with a heating rate
prepared complexes
ꢁ1
of 10 °C min . The ahpvFe, ahpvCd and ahpvZn complexes have
weight losses of 4.74, 3.35 and 18.12%, respectively, which are
due to abstraction of water molecules of hydration. The weight
losses of 23.08, 31.50 and 25.00% corresponding to the abstraction
of the remaining thermally degradable part of the complex at tem-
perature range 169.5–510.1 °C with respect to ahpvFe complex.
The weight casualties of 23.57, 17.39, 6.61 and 21.65% correspond-
ing to the elimination of the residual thermally degradable part of
the complex at temperature range 142.4–528.3 °C with respect to
ahpvCd complex. The weight losses of 34.63 and 30.85% corre-
sponding to the elimination of the remaining thermally degradable
part of the complex at temperature range 188.6–526.1 °C with
respect to ahpvZn complex as shown in (Table S2). The weight
losses of 37.75 and 15.71% [34]. The final product explained from
a horizontal curve has been obtained suggesting formation of
metal product [35].
Stoichiometry of complexes is investigated using the two meth-
ods, continuous-variations method, and mole-ratio method. The
methods used and the experimental results showed, the stoi-
chiometry of the prepared complexes is 1:1. The detours of the
continuous variation method (cf. Fig. 1) showed maximum absor-
bance at mole fraction Xligand = 0.43 indicating the formation of
complexes with metal ion to ligand ratio 1:1. Moreover, the data
resulted from applying the molar ratio method confirm these
results (cf. Fig. S2) [38].
3.9. Determination of the apparent forming constants of the
synthesized complexes
f
The formation constants (K ) of the prepared complexes formed
in solution were obtained from the spectrophotometric measures

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
145
0
0
0
0
0
.8
.6
.4
.2
.0
0.8
ahpvFe+0.5
ahpvCd
ahpvZn+0.6
ahpvFe
ahpvCd
ahpvZn
0.6
0.4
0.2
0.0
0
.0
0.3
0.6
0.9
0
2
4
6
8
10
12
14
16
[
ahpv]/[ahpv]+[M]
pH
Fig. 1. Continuous variation plots for the prepared complexes in aqueous-alcoholic
ꢁ
3
mixtures at [M] = [ahpv] = 1 ꢂ 10 M and 298 K.
Fig. 2. Dissociation curves of the prepared complexes in aqueous alcohol mixtures
at [complex] = 1 ꢂ 10ꢁ M and 298 K.
3
by applying the continuous variation method [39] (Table 2). The
obtained K
plexes. The accounts of K
in the following order: ahpvCd > ahpvZn > ahpvFe complex. More-
over, the accounts of the stability constant (pK) and Gibbs free
f
accounts indicate the high stability of the studied com-
for the investigated complexes increase
zinc and oxygen elements, which presented in Fig. 3c. The compo-
sition of zinc and oxygen is 41.62% and 58.38%, respectively. No
other peak related with any impurity has been detected in the
EDS, which confirms that the grown NPs are composed only with
zinc and oxygen.
f
–
energy (
D
G ) of the complexes are evaluated. The negative results
of Gibbs free energy mean that the reaction is spontaneous and
favorable. The pH-profile presented in (cf. Fig. 2) displayed dissoci-
ation curves and a great stability pH range (4–10) of the prepared
complexes. This means that the preparation of the complex widely
stabilizes the Schiff base. Consequently, the suitable pH range for
the various applications of the complexes is from pH = 4 to
pH = 10. The results of elemental analysis, molar conductance,
magnetic susceptibility, infrared and electronic spectra help to
know the structure of the complexes [40].
3
.11. Particle size of the prepared complexes and metal oxides
The Fe(II), Cd(II) and Zn(II) oxides nanoparticles were
synthesized at 500 °C using Schiff base complexes as precursors
and their properties studied with the assistance of a transmission
electron microscope (TEM) and X-ray diffraction. Based on
TEM images and the calculated histogram (cf. Figs. 4(a)–(f) and
5
(a)–(f)), it is clear that the prepared complexes have particle size
of 12 nm, 23 nm and 36 nm for Fe(II), Cd(II) and Zn(II) complexes
respectively. It is observed that the complexes have a particle size
of 8 nm, 17 nm and 4 nm for nano-sized Fe O , CdO and ZnO
2 3
oxides respectively. These conclusions showed that the prepared
compounds have a high surface area and this can lead to many
important catalytic and potential properties [41]. Fig. 6 demon-
3
.10. Energy-dispersive X-ray spectroscopy (EDX) pattern of the
prepared metal oxides
2 3
The electron dispersive spectroscopy (EDS) analysis of Fe O
indicates the presence of Fe and O composition in the pure grown
iron oxide NPs (Fe ). It is clearly displayed that grown synthesize
strates the XRD patterns of the synthesized Fe
nanoparticles. The X-ray diffraction data were logged by using
Cu K radiation (1.5406 Å). The intensity data was gathered over
2 3
O , CdO and ZnO
2 3
O
materials contained only iron and oxygen elements, which pre-
sented in Fig. 3a. The composition of iron and oxygen is 63.31%
and 36.69%, respectively. No other peak related with any impurity
has been detected in the EDS, which confirms that the grown NPs
are composed only with iron and oxygen. The electron dispersive
spectroscopy (EDS) analysis of CdO indicates the presence of Cd
and O composition in the pure grown cadmium oxide NPs (CdO).
It is clearly displayed that grown synthesize materials contained
only cadmium and oxygen elements, which presented in Fig. 3b.
The composition of cadmium and oxygen is 27.74% and 72.26%,
respectively. No other peak related with any impurity has been
detected in the EDS, which confirms that the grown NPs are com-
posed only with cadmium and oxygen. The electron dispersive
spectroscopy (EDS) analysis of ZnO indicates the presence of Zn
and O composition in the pure grown zinc oxide NPs (ZnO). It is
clearly displayed that grown synthesize materials contained only
a
a 2h range of 5–80°. The medium grain size of the samples was
estimated with the help of the Scherrer equation, profiting the
diffraction intensity peak. X-ray diffraction studies confirmed that
2 3
the synthesized materials were Fe O , CdO and ZnO, that all the
diffraction peaks agreed with the reported standard data; no
characteristic peaks were denounced other than oxide, MO. The
mean grain size (D) of the particles was estimated from the
XRD line broadening mensuration using the Scherrer Equation
[
41,42]:
D ¼ 0:89k=b cos h
ð7Þ
where k is the wavelength (Cu K
a), b is the entire breadth at the
2 3
half-maximum (FWHM) of the Fe O , CdO and ZnO line and h is
the diffraction angle. An estimated line broadening of the diffraction
Table 2
The formation constant (K
–
f
), stability constant (pK) and Gibbs free energy (
D
G
) values of the synthesized complexes in aqueous-ethanol at 298 K.
–
ꢁ1
Complex
Type of complex
K
f
pK
DG (kJ mol )
9
1
9
ahpvFe
ahpvCd
ahpvZn
1:1
1:1
1:1
4.80 ꢂ 10
3.41 ꢂ 10
4.81 ꢂ 10
9.68
11.53
9.68
ꢁ55.20
ꢁ65.76
ꢁ55.20
1

1
46
L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
(a)
(
b)
(c)
2 3
Fig. 3. (a)EDX pattern of Fe O nanoparticles. (b) EDX pattern of CdO nanoparticles. (c) EDX pattern of ZnO nanoparticles.
peaks is a caption that the synthesized materials are in the
nanometer range. The reaction temperature highly affects the parti-
cle morphology of as-prepared Fe O , CdO and ZnO powders. The
2 3
finding of nanoparticle size mensuration of samples by XRD and
TEM elucidate that the size of the Fe
was about 4–17 nm.
2 3
O , CdO and ZnO nanoparticles

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
147
8
6
4
2
0
0
0
0
0
4
8
12 16 20 24 28
Diameter (nm)
(b)
(a)
8
0
0
0
0
0
6
4
2
4
8
12 16 20 24 28 32 36
Diameter (nm)
(d)
(c)
8
0
6
4
2
0
0
0
0
2
0
30
40
50
60
70
Diameter (nm)
(e)
(f)
Fig. 4. (a) TEM image of the prepared ahpvFe complex. (b) Calculated histogram for particle size distribution of ahpvFe complex. (c) TEM image of the prepared ahpvCd
complex. (d) Calculated histogram for particle size distribution of ahpvCd complex. (e) TEM image of the prepared ahpvZn complex. (f) Calculated histogram for particle size
distribution of ahpvZn complex.
3
.12. Antimicrobial activity
had moderate activity as matched with the standard, but all the
complexes were more active than free ligand. The nano sized metal
oxides have the highest activity. The higher inhibition zone of the
transition metal complexes than those of the ligand can be shown
based on the Overtone notion and the chelation theory. Upon
chelation, the polarity of the metal ion is decreased to a great
extent due to the overlap of the ligand orbital and the fractional
participating of the positive charge of the metal ion with
donor groups. Furthermore, it raises the delocalization of the
The in vitro antimicrobial actions of the Schiff base ligand, its
complexes and their metal oxides against three selected bacteria
Escherichia coli, Microccus luteus and Serratia marcescence) and
(
three fungi (Aspergillus flavus, Getrichm candidum and Fusarium
oxysporum), were determined. Any chemotherapeutic agent inhi-
bits the growth of microbes by microstatic mechanisms. All of
the compounds displayed good biological activity with the
micro-organism. On contrasting the biological wares of the Schiff
base ligand, its complexes and their metal oxides with those of a
standard bactericide and fungicide, it was clear that the complexes
p-electrons over the whole chelating ring and increases the
breakthrough of the complexes into lipid membranes and
the blocking of the metal attachment locations in the enzymes of

1
48
L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
100
80
60
40
20
0
0
5
10
15
20
Diameter (nm)
(b)
(a)
80
70
60
50
40
30
20
10
0
4
8
12 16 20 24 28 32
Diameter (nm)
(d)
(c)
80
70
60
50
40
30
20
10
0
1
0
15
20
25
30
35
Diameter (nm)
(
e)
(f)
Fig. 5. (a) TEM image of the prepared Fe
2 3 2 3
O . (b) Calculated histogram for particle size distribution of Fe O . (c) TEM image of the prepared CdO. (d) Calculated histogram for
particle size distribution of CdO. (e) TEM image of the prepared ZnO. (f) Calculated histogram for particle size distribution of ZnO.
micro-organisms [43–46]. The conclusions of the investigations
account for the antipathogenic manner of the compounds and this
efficacy is positively changed on complexation. Data are listed in
Tables (S4, S5) and Figs. 7 and 8. It is shown that antimicrobial
potency of metal oxides is better than that of their Schiff base
complexes. This could be simply demonstrated as smaller particles
normally have a larger surface to volume ratio which offers a more
efficient mean for antibacterial activities [47]. The minimum
inhibitory concentration (MIC) was estimated by serial dilution
route and reported in (cf. Table 3). Among complexes, ahpvFe
The ahpvFe complex (2 mg/ml) was found to be lowest MIC
against Escherichia coli. In the case of Microccus luteus, the ahpvCd
complex (2 mg/ml) showed the lowest MIC. The ahpvFe complex
(4 mg/ml) was found to be lowest MIC against Fusarium oxysporum.
In the case of Getrichm candidum, ahpvFe and ahpvCd
complexes (2 mg/ml) showed the lowest MIC. The ahpvZn complex
(3 mg/ml) was found to be highly effective as they exhibit the
lowest MIC against Aspergillus flavus. The antimicrobial studies
suggested that all the complexes showed significantly enhanced
antimicrobial activity against microbial strains in comparison to
the free ligands. Previous studies elsewhere suggested that
(
3 mg/ml) showed the lowest MIC against Serratia marcescence.

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
149
1
200
of this study. It was suspected that factors such as solubility, con-
ductivity, dipole moment, and cell permeability mechanism influ-
enced by the existence of metal ion might be the possible reason
for the increase in activity. The activities of the prepared com-
plexes were confirmed by calculating the potency index (cf.
Table 4) according to the following relation [48]:
8
4
00
00
0
2
1
400
44
800
1
200
inhibition zone of complex ðmmÞ
6
00
0
Activity index ðAÞ ¼
ꢂ 100
ð8Þ
inhibition zone of standard drug ðmmÞ
1
000
2
0
30
50
60
70
80
8
6
4
2
00
00
00
00
0
3
3
.13. DNA binding potency
2
0
40
60
80
.13.1. Electronic spectra of interaction with DNA
Titration with electronic absorption spectroscopy is an active
2θº
Fig. 6. XRD patterns of Fe
2
O
3
, CdO and ZnO nanoparticles.
route to check the binding mode of DNA with metal complexes.
The spectra were registered as a function of the addition of the buf-
fer solutions of pretreated CT-DNA to the buffer solutions of the
tested complexes. If the interaction mode is intercalation, the orbi-
tal of the intercalated ligand can couple with the orbital of the base
30
⁄
pairs, lowering the
p
–p
transition energy and lead to bathochro-
2
0
mism. If the conjunction orbital is partially filled by electrons, it
results in reducing the transition probabilities and lead to hypo-
chromism [49]. The extent of the hypochromism or hyper-
chromism in the metal-to-ligand charge transfer (MLCT) band is
commonly consistent with the force of intercalation interaction
1
0
0
10(mg/ml)
0(mg/ml)
2
[
50]. The electronic absorption spectra of ahpvFe complex in
the absence and presence of various concentrations of buffered
CT-DNA are given in Fig. S3. Addition of increasing amounts of
CT-DNA resulted in a reduction of absorbance for a complex. The
spectral parameters for the DNA interaction with the tested
complexes are shown in Table 5. The investigated complexes could
bind to DNA via an intercalative mode with the sequence:
ahpvFe > ahpvZn > ahpvCd complex.
Fig. 7. Zone of inhibition against Escherichia coli (bacteria) by the prepared
complexes and their metal metal oxides.
35
1
0(mg/ml)
0(mg/ml)
30
2
3.13.2. Viscosity measurements
25
For explaining the interaction nature between the tested com-
plexes and DNA, viscosity measures were carried out. Hydrody-
namic methods such as viscosity measures which are sensitive to
length increment or reduce of DNA are regarded as the most effec-
tive routes of studying the binding mode of compounds to DNA in
the absence of crystallographic structural data and NMR. For fur-
ther explaining of the binding mode, viscosity measurements were
made. Under appropriate conditions, a traditional intercalative
mode such as intercalation of drugs like ethidium bromide leads
to a significant increment in the viscosity of DNA solution due to
an increment in the segregation of base pairs at the intercalation
site and hence an increment in the overall DNA length. On other
hand, drug molecules attachment exclusively to the DNA grooves
lead to less pronounced in DNA solution viscosity [51] a partial
intercalation of compound may bend the DNA helix, resulting in
the reduction of its effective length and, concomitantly, its viscos-
ity [52]. The relative viscosity of DNA solution enhances signifi-
cantly as the amount of the compound raises, as shown in
(Fig. 9). This may be due to the admission of the aromatic ring in
2
0
1
5
1
0
5
0
Fig. 8. Zone of inhibition against Fusarium oxysporum (fungi) by the prepared
complexes and their metal oxides.
chelation tended to make the ligands act as more powerful and
potent bactereostatic agents [48], thus inhibited the growth of
microbe more than the parent ligands did and it is similar with that
Table 3
Results of minimum inhibition concentration (MIC) of the prepared Schiff base ligand and its complexes against different strains of bacteria and fungi.
Compounds
Minimum inhibition concentration (MIC)
Serratia Marcescence
Escherichia coli
Micrococcus Luteus
Fusarium oxysporum
Getrichm candidum
Aspergillus flavus
ahpv
8
3
4
6
6
2
4
7
4
5
2
3
8
4
7
7
13
2
2
8
5
4
3
ahpvFe
ahpvCd
ahpvZn
5

1
50
L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
Table 4
Results of activity index % of the prepared Schiff base ligand and its complexes against different strains of bacteria and fungi.
Compounds
Activity index %
Serratia Marcescence
Escherichia coli
Micrococcus Luteus
Fusarium oxysporum
Getrichm candidum
Aspergillus flavus
ahpv
39.3
78.6
71.4
64.3
40.9
72.7
59.1
63.6
38.5
87.2
71.8
79.5
37.5
66.6
54.1
58.3
38.4
87.1
71.1
79.4
35.4
67.7
74.1
77.4
ahpvFe
ahpvCd
ahpvZn
Table 5
Spectral parameters for DNA interaction with the synthesized complexes.
Chromism (%)a
Type of chromism
Binding constant
DG (kJ mol )
–
ꢁ1
Complex
k
max Free (nm)
k
max Bound (nm)
D
n (nm)
ꢂ 10 mol dm3
6
ꢁ1
K
b
[
[
[
Fe(ahpv)(H
Cd(ahpv)(H
Zn(ahpv)(H
2
O)
O)
O)
3
]ꢀH
]ꢀH
]ꢀ4H
2
O
531
14
393
15
449
93
526
419
390
314
466
353
5
5
3
1
17
40
36.3
32
19.6
21.4
89.6
22.6
Hyper
Hypo
Hypo
Hypo
Hypo
Hypo
0.01 ± 0.02
0.32 ± 0.02
0.19 ± 0.02
ꢁ22.80
ꢁ31.40
ꢁ30.09
4
2
3
2
O
3
2
3
2
O
2
a
Chromism (%) = [(Abs free ꢁ Absbound)/Absfree].
Schiff base into the DNA base pairs resulting in a crook in the DNA
helix, hence, increase in the segregation of the base pairs at the
intercalation place and increment in DNA molecular length. More-
over, the sequence of the observed increment in the values of vis-
cosity was renovated with the binding affinity to DNA i.e. ahpvFe
complex shows the highest binding affinity to DNA and the highest
viscosity.
3.13.3. Gel electrophoresis
Agarose gel electrophoresis is used for the DNA binding studies.
The Schiff base Fe(II), Cd(II) and Zn(II) complexes were studied for
their DNA binding activity by agarose gel electrophoresis method
(Fig. 10). The gel after electrophoresis clearly showed that the
intensity of all the treated DNA samples has partially detracted,
possibly because of the interaction with DNA. The partial binding
of DNA was observed in Fe(II), Cd(II) and Zn(II) complexes of the
Schiff base. The difference was clarified in the bands of the com-
plexes compared to that of the control DNA. This explains that
the control DNA alone does not show any visible cleavage whereas
the complexes show cleavage [53]. However, the nature of reactive
intermediates involved in the DNA binding by the complexes is not
obvious [54]. These results show that the metal ions play an impor-
tant role in the interaction with isolated DNA. As the compound
Fig. 10. DNA binding study Calf-thymus (CT)-DNA with Fe(II), Cd(II) and Zn(II)
complexes. Lane 1: Fe(II); Lane 2: Zn(II); Lane 3: Cd(II); Lane 4: Control DNA.
1
1
1
1
1
1
.5
.4
.3
.2
.1
.0
ahpvFe
ahpvCd
ahpvZn
EB
was observed to bind with DNA, it can be concluded that the com-
pound reduces the growth of the pathogenic organism by interac-
tion with genome. The studies reveal that partial binding of DNA
was observed by Fe(II), Cd(II) and Zn(II) complexes. The experi-
mental results indicated that the investigated complexes could
bind to DNA via intercalative mode.
3.13.4. Anticancer activity
The cytotoxic potency of Schiff base complexes (2), (3) and (4)
was evaluated against colon carcinoma cells, (HCT-116 cell line)
and hepatic cellular carcinoma cells, (HepG-2) within 0–10
l
M
0
.0
0.1
0.2
0.3
0.4
0.5
concentration range. The IC50 values were evaluated for each
compound and results are offered in Fig. 11 and Table S6. As
shown, most complexes displayed manifestly cytotoxic potencies
(which are greater than that of ligand) compared to vinblastine
[
Complex]/ [DNA]
Fig. 9. The effect of increasing the amount of the synthesized complexes on the
relative viscosities of DNA at [DNA] = 50 M, [complex] = 10–60 M and 298 K.
l
l

L.H. Abdel-Rahman et al. / Bioorganic Chemistry 69 (2016) 140–152
151
1
6
2
8
4
0
tive mode. Furthermore, nano-sized metal oxides were prepared
from their complexes and their antimicrobial activity was evalu-
ated. Very good cytotoxic activity of the selected complexes was
found with colon carcinoma cells, (HCT-116 cell line) and hepatic
cellular carcinoma cells, (HepG-2).
HCT-116
HepG-2
1
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.10.
0
09.
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[
[
HCT-116 cancer cells and (IC50 = 1.45–6.75
l l
5
(
4
4
l
l
l ll
[
l
l
1
l
l
[
(
6
l l
l
l
complexes are more potent than the free ligand. This indicated
beneficent of the antitumor potency upon coordination. The refine-
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[
[
[
(
[
1
[
[
[
4
. Conclusion
[
[
Some New nano-sized Fe(II), Cd(II) and Zn(II) Schiff base com-
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different analytical tools. The obtained results showed the exis-
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