Synthesis and characterization of Schiff base octaazamacrocyclic complexes and their biological studies

Article abstract of DOI:10.1016/j.jphotobiol.2014.10.004

A condensation reaction between 1,2-diphenylethane-1,2-dione dihydrazone (DPEDDH) and dimethyl or diethyloxalate in methanol resulted in a novel Schiff base octaazamacrocyclic ligand, (L): (6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-octaazacyclohexadecane-1,3,9,11-tetraene). Subsequently metal complexes of the type [MLX₂] and [CuL]X₂; (M = Mn(II), Co(II), Ni(II) and Zn(II); X = Cl or NO₃) were synthesized by the reaction of the free macrocyclic ligand (L) with the corresponding metal salts in 1:1 molar ratio. These complexes were characterized on the basis of analytical data, molar conductivity and magnetic susceptibility measurements, ESI-mass, IR, NMR (¹H and ¹³C), EPR and electronic spectral studies. The thermal stability of the complexes was also studied by TGA and DTA analyses. These studies show that all the complexes have octahedral arrangement around the metal ions except copper complexes which are square planar. The ligand and its complexes were screened for their antibacterial activity in vitro against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and were also studied for their anticancer activity against the human cancer cells lines: HeLa (Human cervical carcinoma), MCF7 (Human breast adenocarcinoma) and Hep3B (Human Hepatocellular carcinoma). The recorded IC₅₀ values for the tested compounds show moderate to good cytotoxicity against these cancer cell lines. The copper complex, [CuL]Cl₂, showed excellent antimicrobial activity against tested microorganisms which is almost equivalent to the standard drug ciprofloxacin.

Full text of DOI:10.1016/j.jphotobiol.2014.10.004

Accepted Manuscript
Synthesis and characterization of Schiff base octaazamacrocyclic complexes
and their biological studies
Hina Zafar, Abdul Kareem, Asif Sherwani, Owais Mohammad, Mohammad
Azam Ansari, Haris M. Khan, Tahir Ali Khan
PII:
S1011-1344(14)00301-7
DOI:
http://dx.doi.org/10.1016/j.jphotobiol.2014.10.004
JPB 9853
Reference:
To appear in:
Journal of Photochemistry and Photobiology B: Bi-
ology
Received Date:
Revised Date:
Accepted Date:
29 May 2014
9 October 2014
12 October 2014
Please cite this article as: H. Zafar, A. Kareem, A. Sherwani, O. Mohammad, M.A. Ansari, H.M. Khan, T.A. Khan,
Synthesis and characterization of Schiff base octaazamacrocyclic complexes and their biological studies, Journal
of Photochemistry and Photobiology B: Biology (2014), doi: http://dx.doi.org/10.1016/j.jphotobiol.2014.10.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and characterization of Schiff base octaazamacrocyclic
complexes and their biological studies
a
Hina Zafar, Abdul Kareem, Asif Sherwani , Owais Mohammad , Mohammad Azam
a
b
Ansari , Haris M Khan and Tahir Ali Khan*
b
Division of Inorganic Chemistry, Department of Chemistry, Aligarh Muslim University,
Aligarh -202002, India
a
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh- 202002, India
b
Nanotechnology and Drug Resistant Research lab, Department of Microbiology, Jawaharlal
Nehru Medical College, Aligarh Muslim University, Aligarh -202002, India
ABSTRACT
A condensation reaction between 1, 2-diphenylethane-1,2-dione dihydrazone (DPEDDH) and
dimethyl or diethyloxalate in methanol resulted in a novel Schiff base octaazamacrocyclic
ligand,
octaazacyclohexadecane-1,3,9,11-tetraene). Subsequently metal complexes of the type
MLX ] and [CuL]X ; (M = Mn(II), Co(II), Ni(II) and Zn(II) ; X = Cl or NO ) were
(L):
(6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-
[
2
2
3
synthesized by the reaction of the free macrocyclic ligand (L) with the corresponding metal
salts in 1:1 molar ratio. These complexes were characterized on the basis of analytical,
1
13
conductivity and magnetic susceptibility measurements, ESI-mass, IR, NMR ( H and C),
EPR and electronic spectral studies. The thermal stability of the complexes was also studied
by TGA and DTA analyses. These studies show that all the complexes have octahedral
arrangement around the metal ions except copper complexes which are square planar. The
ligand and its complexes were screened for their antibacterial activity in vitro against Gram-
positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and were
also studied for their anticancer activity against the human cancer cells lines: HeLa (Human
cervical carcinoma), MCF7 (Human breast adenocarcinoma) and Hep3B (Human
Hepatocellular carcinoma). The recorded IC50 values for the tested compounds show
2
moderate to good cytotoxicity against these cancer cell lines. The copper complex, [CuL]Cl ,
showed excellent antimicrobial activity against tested microorganisms which is almost
equivalent to the standard drug ciprofloxacin.
1

KEYWORDS: Octaazamacrocyclic complexes, Spectral studies, Thermal analyses,
Antibacterial activity and Anticancer activity.
*Senior Author Correspondence: Tahir Ali Khan, E-mail: takhan213@gmail.com
1
. Introduction
The biomedical inorganic chemistry has been a fascinating research area due to broad
applications of inorganic pharmaceuticals in various clinical therapy and diagnostics [1, 2].
Although platinum-based chemotherapeutic compounds have remarkable success with high
efficiency against human testicular, ovarian, bladder, head and neck carcinomas, several side
effects such as limited water solubility and the dose-dependent toxicities, mainly
nephrotoxicity, cytotoxicity and emetogenesis are the major drawbacks associated with
platinum based drugs [3, 4]. Thus in view of research for non platinum based drugs a large
number of studies have recently been carried out [3, 5-7]. Specially Cu(II) ion is known to
involve in many biological process having its role in increasing antitumor efficacy of organic
molecules[8-10]. Thus the copper complexes are the theme of Scientists due to their potential
use as antimicrobial, antiviral, anti-inflammatory and antitumor agents, enzyme inhibitors or
chemical nucleases [8, 9]. Now a large number of copper(II) chelate complexes that exhibit
cytotoxic activity through cell apoptosis or enzyme inhibition have been reviewed [8, 10].
The nitrogen and sulfur containing organic compounds and their metal complexes display a
wide range of biological activity as antitumor, antibacterial, antifungal and antiviral agents
[
11, 12]. Macrocyclic Schiff base ligands have received special attention because of their
mixed hard-soft donor character and versatile coordination behaviour [13] for their
enantioselective catalytic reactions [14], biological activities, i.e., toxicity against bacterial
growth, anticancerous and other biochemical properties [15]. The synthesis of
azamacrocyclic compounds received considerable attention during the last decade because of
their relationship to biomimetic and catalytic systems and their applications as chelating
agents in biology and medicine [16, 17]. The presence of N=C-C=N structural unit in Schiff
bases is responsible for the formation of a strong chelate ring resulting in possible electron
delocalization associated with extended conjugation [13]. The polyamide macrocycles
deserve special attention as they have been used for the construction of the corresponding
polyazamacrocyclic complexes [18-20]. We have a long term interest in synthesis and
characterization of macrocyclic complexes of a wide variety [19-23] and recently also studies
2

their biological properties [21-24]. Recently the antimicrobial and anticancer activities of
other materials have also been reported from our laboratories [25-27]. Herein, we report a
new series of novel Schiff base octaazamacrocyclic ligand (L):(6,7,14,15-tetraoxa-2,3,10,11-
tetraphenyl-1,4,5,8,9,12,13,16-octaazacyclohexadecane-1,3,9,11-tetraene) and its transition
metal complexes of the type, [MLX
Cl or NO ), which were also studied their biological properties: antimicrobial and
anticancer activities.
2
] and [CuL]X2, (M = Mn(II), Co(II), Ni(II) and Zn(II); X
=
3
2
. Experimental Section
2
.1. Materials and Methods
The chemicals benzil, hydrazinehydrate (Merck), dimethyl and diethyloxalate (both Fluka)
were used as received. Metal salts (all Merck) were commercially available pure samples.
Benzildihydrazone (DPEDDH) was prepared by the reaction of benzil and hydrazinehydrate
in methanol in 1:2 molar ratio. Ficoll-Histopaque, Fetal calf serum, RPMI1640 and
antibiotic-antimycotic solutions were procured from Sigma Aldrich, USA. Hep3B (Human
hepatocellular carcinoma), MCF7 (Human breast adenocarcinoma) and HeLa (Human
cervical carcinoma) cell lines were procured from Cell Repository–National Centre for Cell
Science Pune, (India).
2
.2. Synthesis of Macrocyclic Ligand,
(L):
6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-octaazacyclohexadecane-
1
,3,9,11-tetraene)
3
A methanolic solution (25 cm ) of 1,2-diphenylethane-1,2-dione dihydrazone (DPEDDH)
3
0.02mol, 4.76 gm) was placed in a round bottom flask, there a methanolic solution (25 cm )
(
of dimethyl (2.36 gm, 0.02 mol) or diethyloxalate (2.92 gm , 0.02 mol) was slowly added
with constant stirring for 8 h. The solution was then refluxed and stirred for 6 h. The reaction
mixture was then kept for evaporation resulting in a light yellow solid product, which was
filtered, washed several times with methanol and dried in vacuum.
2
.3. Synthesis of Macrocyclic Complexes,
Synthesis of dichloro/nitrato (6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-
octaazacyclohexadecane-1,3,9,11-tetraene) metal(II), [MLX ]; (M = Mn(II), Co(II), Ni(II)
2
3

and Zn(II) ; X = Cl or NO
3
and (6,7,14,15-tetraoxa-2,3,10,11- tetraphenyl-
1
,4,5,8,9,12,13,16-octaazacyclohexadecane – 1,3,9,11-tetraene) copper(II) chloride and
2 3
nitrate [CuL]X (X = Cl or NO )
An equimolar amount of the methanolic solution of the macrocyclic ligand, (L) and
corresponding metal salt (0.01mol) in 1:1 ratio were reacted in the round bottom flask. The
reaction mixture was stirred with heating for 12h leading to the isolation of solid product
which was filtered, washed several times with methanol and dried in vacuo.
3
. Measurements
The elemental analyses for carbon, hydrogen and nitrogen were obtained from the Central
1
13
Drug Research Institute Lucknow, India. The H and C-NMR spectra were recorded on a
JEOL GSX 300 MHz FX-1000 spectrometer using DMSO-d as a solvent and
tetramethylsilane (Me Si) as an internal standard. The estimation of halogen was done
gravimetrically [28] and the metals were estimated by titrating with standard EDTA solution
29]. The electronic spectra of the compounds in DMSO were recorded on a Pye-Unicam
6
4
[
8
800 spectrophotometer at room temperature. The EPR spectra were recorded on a JEOL JES
0
RE2X EPR spectrometer. Magnetic susceptibility were carried out at 25 C using a Faraday
balance. Electro spray ionization mass spectra (ESI-MS) of the complexes were recorded on a
Q-Tof Micromass spectrometer from Guru Nanak Dev University (Amritsar, India).
-1
The IR spectra were recorded in the region 4000–400 cm by using FT-IR Perkin Elmer
−
1
(
(
2400) spectrometer and in the region 700-30 cm by using FT-IR/FIR Perkin Elmer
Frontier) spectrometer. The TGA and DTA were performed on a Schimadzu Thermal
Analyze under nitrogen atmosphere using alumina powder as reference. The electronic
spectra of the compounds were recorded on a Pye-Unicam 8800 spectrophotometer at room
temperature. The antibacterial activity of the synthesized compounds was completed by the
disc diffusion method and broth dilution methods [25, 30].
3
.1. Cell viability assay (MTT)
The fresh Blood (10–15 mL) provided by Blood bank of Jawaharlal Nehru Medical College,
A.M.U, Aligarh (India), was diluted with the same volume of phosphate buffered saline
(
PBS). The diluted blood sample was carefully layered on Ficoll-Histopaque. The mixture
0
was centrifuged under at 900×g for 10 min at 20–22 C. The undisturbed lymphocyte layer
was carefully transferred out. The lymphocyte was washed and pelleted down with three
4

volumes of PBS for twice and resuspended RPMI-1640 media with 10% antibiotic and
antimycotic solution v/v fetal calf serum (FCS) [26,31]. The anticancer activity in vitro was
measured using the 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (MTT)
assay. The HeLa (Human cervical carcinoma), MCF7 (Human breast adenocarcinoma) and
Hep3B (Human hepatocellular carcinoma) cells were maintained in RPMI 1640 culture
medium supplemented with 10% heat-inactivated fetal calf serum. The cells were plated at a
3
density of 5 × 10 cells per well in a 96-well plate, and cultured for 24 h at 37°C. The cells
were subsequently exposed to drugs. The plates were incubated for 48 h, and cell
proliferation was measured by adding 20 µL of MTT dye (5 mg/mL in phosphate buffered
saline) per well. The plates were incubated for a further 4 h at 37°C in a humidified chamber
2
containing 5% CO . Formazan crystals formed due to reduction of dye by viable cells in each
well were dissolved in 150 µL dimethyl sulfoxide, and absorbance was read at 570 nm. The
absorption values were expressed as the cell present viability, according to the control group
as 100%.
4
. Results and Discussion
A [2+2] condensation reaction between 1, 2- diphenylethane - 1, 2-dione (DPEDDH) and
dimethyl or diethyloxalate (1:1 molar ratio) in methanol resulted a novel Schiff base
octaazamacrocyclic ligand, (L): (6,7,14,15-tetraoxa-2,3,10,11-tetraphenyl-1,4,5,8,9,12,13,16-
octaazacyclohexadecane-1,3,9,11-tetraene) this ligand (L) upon subsequent treatment with
appropriate metal salts in a 1:1 molar ratio in methanol yielded macrocyclic complexes of the
type, [MLX
2 2 3
] and [CuL]X (M = Mn(II), Co(II), Ni(II), and Zn(II) : X = Cl or NO )
(
Scheme 1). The ligand and its complexes are stable in at room temperature in atmosphere
and are soluble in dimethyleformaldehyde and dimethyl sulfoxide. The formation of ligand
framework and its complexes was further ascertained on the basis of results of elemental
analyses, molecular ion peak in ESI-mass spectra (Table 1), characteristic bands in the
1
13
FT-IR (Table 2) and resonance signals in the H and C NMR spectra. The overall geometry
of the complexes was inferred from the observed values of magnetic moments and electronic
spectra and the position of bands in the EPR for Cu(II) complexes (Table 3).
The molar conductivities in DMSO indicate [32] that copper(II) complexes are electrolytic
while others are non-electrolytic in nature.
5

4
.1. ESI-Mass Spectra
Mass spectra of tetraiminetetraamide ligand and their complexes showed m/z peaks at 583.70
65.20, 717.10, 759.80, 712.20, 770.1, 720.1, 775.90, 715.20, 769.50 and 725.70 that
7
correspond to
C
32
H
24
N
8
O
4
,
C
32
H
24
N
10MnO10
,
C
32
H
24
N
8
O
4
MnCl
2
,
C
32
H
24
N
10CoO10
,
C
32
32
H
24
24
N
N
8
8
O
O
4
4
CoCl
2
, C32
H
24
N
10NiO10, C32
H
24
N
8
O
4
NiCl
2
, C32
H
24
N
10CuO10, C32
H
24
N
8
O
4
CuCl
2
,
C
H
ZnCl
2
and C32
H
24
N
8
ZnO10 moieties, respectively. The proposed molecular
formulae of synthesized complexes were confirmed by comparing their molecular formula
weights with respective m/z values (Table 1) which are in good agreement for
aforementioned complexes shown in (Figure 1).
4
.2. IR Spectra
The most significant feature of IR spectra (Table 2) of macrocyclic ligand and its complexes
is the appearance of four characteristic bands in 1670-1715, 1510-1550, 1225-1255 and
-
1
6
35-670 cm regions which may be assigned to amide I [υ(C=O), amide II υ(C-N)
+
δ(N-H)], amide III δ(N-H), and amide IV wagging [ф(C=O)], vibration, respectively [19].
The amide I υ(C=O) is observed in the region expected for the metal free amide group [33]
ruling out the possibility of coordination through amide oxygen. Although the IR spectrum of
(
DPEDDH) there is appearance of two strong υ(N-H) bands at 3270 and 3220 but the IR
spectra of macrocyclic ligand and its complexes show only single sharp υ(N-H) band in the
-
1
3
230-3275 cm region, which is attributable to the uncoordinated amide nitrogen [33].
Reported macrocyclic complexes exhibit a strong intensity band in the range of
-
1
1
580–1595 cm assignable [34] to the coordinated υ(C=N). There is observed increase in the
υ(C=N) band intensity which may be due to the possible transformation of trans structure of
the (DPEDDH) into cis configuration upon complexation. The cis structure is stabilized by
the chelate ring formation, lowering the symmetry which consequently enhances the υ(C=N)
-
1
band intensity. The bands at 425–470 cm , originate from (M–N) vibration. The coordination
of nitrato and chloro groups have been confirmed due to the appearance of bands in the
-
1
2
20-240 and 270-290 cm regions which may be assigned to υ(M-O) and υ(M-Cl)
respectively. In addition, the IR spectra of nitrato complexes exhibit bands at 1280, 1040 and
-
1
8
50 cm which are consistent with the monodentate nature [19] of this group. However, free
-
nitrate absorption in Cu(II) complex appeared at 1385 cm which corresponds to an
1
uncoordinated nitrato group.
1
.3. H - NMR Spectra
4
6

1
The H NMR spectra of benzildihydrazone (DPEDDH) and the corresponding zinc
complexes have been recorded and compared (Figure 2). Benzildihydrazone shows a
multiplet at 5.12-6.03 ppm and a slightly broad signal at 3.93-4.71 ppm assignable [35] to the
phenyl ring (C
zinc complexes showed a band at 7.074-7.625 ppm, reasonably ascribed [36] to the amide
N-NH-C, 4 H) protons. A multiplet at 5.39- 6.54 ppm in both complexes is characteristic of
phenyl ring (C , 20 H) protons. Absence of characteristic peaks corresponding to the
6 5 2
H , 10 H) and amino (N-NH , 4 H) protons of the hydrazine moiety. Both the
(
6
H
5
protons of hydrazone and oxalate moieties, further confirm that the cyclization has indeed
taken place.
1
.4. C- NMR Spectra
3
4
1
3
The C-NMR spectrum of the mononuclear complexes gave signals characteristics of
different carbon atoms of macrocyclic framework that appeared at their appropriate positions
expected for proposed structure. However, the positions of resonance signals were found to
be slightly downfield shifted [37] in complexes as compared with the free Schiff base
macrocyclic ligand (Figure 3) confirming the coordination of the ligand to Zn(II) metal ion .
4.5. Magnetic Susceptibility Measurements
The magnetic moment values (Table 1) for Mn(II), Co(II) and Ni(II) complexes are in
8
support of octahedral geometry around metal ions. Nickel(II) with d configuration may have
two unpaired electrons in both high spin and low spin (weak or strong ligand field) octahedral
arrangements. However, it generally shows a tendency to form octahedral complexes with
week field ligands and square planar complexes with strong field ligands. The observed
magnetic moment values for Mn(II) and Co(II) complexes are consistent with the high spin
octahedral geometry which indicates that the ligand field is weak. Thus it is probable that
Ni(II) may assume an octahedral geometry rather than square planar. The magnetic moment
for the mononuclear Cu(II) complexes having no major interaction between two copper
moieties are reported in the range of 1.75 to 2.20 B.M. [38]. However, due to spin exchange
phenomena between two Cu(II) ions lower magnetic moments (1.2-1.6 BM) were observed in
the dimeric Cu(II) square planar complexes [39]. The observed magnetic moment values
(
1.87-1.92 B.M.) indicate that Cu(II) complexes are mononuclear.
4
.6. Electronic Spectra
7

The electronic spectra (Table 3) of manganese complexes gave two bands in the 22400-
-
1
-1
6
4
2
2550 cm and 18650-18850 cm regions which may be assigned to A1g→ T2g(F) and
6
4
A
1g→ T1g(P) transitions, respectively, corresponding to an octahedral environment around
the Mn(II) ion [20]. The cobalt complexes exhibit two bands appearing in the 21400-21700
-
1
4
4
and 15950-16200 cm regions which may reasonably correspond to T1g(F)→ T1g(P) and
4
4
T
1g(F)→ A2g(F) transitions, respectively, consistent with octahedral geometry [20] around
Co(II) ion. All the nickel complexes exhibit two electronic bands in 21200-24350 and 18200-
-
1
3
3
3
3
1
8750 cm regions which may be ascribed to A2g(F)→ T1g(P) and A2g(F)→ T1g(F)
transitions, respectively, suggesting [18] an octahedral geometry around the metal ion.
The energy level diagram for ligand field of distorted O or D4h symmetry predicts three
h
2
2
2
2
2
2
transitions: (I)
B
1g→ A1g (υ
1
), (II)
B
1g→ B2g (υ
2
)
and (III)
B
1g→ E
g
3
(υ ).
-1
The electronic spectra of copper complexes show a broad band maximum at 16050 cm
2
2
which may be assigned to B1g→ A1g transition. However, two weak shoulders appearing in
-
1
2
2
the regions 21500-21750 and 11750-12450 cm may be attributed to B1g→ E
g
and
2
2
B
1g→ B2g transitions, respectively, suggesting [40,41] a square-planar geometry around the
−
Cu(II) ion. The conductivity data (Table 1) for the copper complexes show that Cl and NO
−
3
groups are ionised and remain out of the coordination sphere. Further results of thermal
analysis (TGA/DTA, Section 4.8) of the copper complex, [CuL]Cl2 , shows the absence of
water/solvent molecules in/out of the coordination sphere. These studies rule out the
possibility of distorted octahedral geometry and are in strong support of square planar
structure for copper complexes. The EPR spectra further confirm square planar geometry for
the copper complexes.
4
.7. EPR Spectra
The EPR spectra of the powdered solid copper(II) macrocyclic complexes (Figure 4) were
recorded at room temperature and their g|| and g values have been calculated. Both the
⊥
complexes exhibit a similar single broad absorption band. The absence of hyperfine lines in
these complexes may be due to the strong dipolar and exchange interactions between the
copper(II) ions in the unit cell [42]. The g values are related by the expression G = (g|| -2) /
⊥ 2 3
(g -2) and these complexes, [CuL]X (X = Cl or NO ), gave axial symmetry parameter, G in
the 1.758 - 1.940 region (Table 3) indicating [42] very small exchange interaction among
copper(II) ions in these complexes, as the G values are less than 4. It is known that with
2
g the unpaired electron in tetragonal and square planar complexes lies in the dx –y
2
g
|| >
⊥
8

2
orbital giving B1g as the ground state [43]. The observed g|| and g
⊥
values (Table 3) appeared
in the region 2.1782-2.0925 and 2.087-2.0526, respectively. Thus, for these copper
complexes g|| >
⊥
g which support the fact that the ground state of Cu(II) is predominantly
2
2
1g having unpaired electron in the dx –y orbital. The unpaired electron will lie
2
B
2
predominantly in the dx –y orbital and two electrons in the dz orbital. Thus ligands
2
2
approaching along the Z – axis have to face very strong repulsive forces from the filled
2
dz orbital, consequently will not be successful to coordinate along the +Z and –Z directions
−
−
3
which is also supported by the conductometric study and thermal analysis (Cl , NO , water
or solvent molecules do not coordinate with the metal ions). The observed g values for both
the copper complexes lie in the range reported for square planar complexes [43-45], thus the
EPR spectral studies strongly support square planar structure for Cu(II) complexes.
Their square planar geometry has also been corroborated from electronic spectra.
4
.8. Thermal Analysis (TGA/DTA)
The thermal stability of the mononuclear octaazamacrocyclic complexes was investigated
using TGA (Figure 5). The thermogravimetric analyses (TGA) were carried out at a heating
0
0
rate of 20 C/min in a nitrogen atmosphere over a temperature range of 10–800 C.
The complexes have a different decomposition process. The relative high thermal stability
observed in all thermally investigated complexes may correspond to the absence of any
solvent / water molecules in/out of the coordination sphere. This is proposed referring to the
higher thermal stability observed for all investigated complexes in which the first
decomposition step is started at a relatively higher temperature. The thermograms of
0
TGA of mononuclear complexes exhibit decomposition between 100–550 C, which may be
due to the removal of the coordinated chloride or nitrate ions. The second decomposition
0
stage followed the previous one ended at 800 C, may be attributed to the removal of the
organic part of the compounds into their corresponding oxides. Further horizontal constant
curve may be due to the presence of metal oxides residue in the remaining part. The
differential thermal analysis (DTA) curves (Figure 5) of the mononuclear complexes show
0
endothermic peak in the temperature range 100–250 C assigned to loss of coordinated
chloride or nitrate ions. The sharp decomposition corresponding to the loss of organic moiety
in complexes can be seen in the DTA curves that contained one sharp exothermic peak falling
0
in the range of 250–450 C and indicate the formation of metal oxides [46, 47].
9

4
4
.9. Biological Activities
.9.1. Antibacterial Studies
The newly prepared complexes were screened for their antibacterial activity against
Staphylococcus aureus and Escherichia coli bacterial strains by disc diffusion method
7
25, 30]. Standard inoculums (1-2 X10 CFU/ml 0.5 McFarland standards) were spread onto
[
the surface of sterile agar plates. The discs measuring 6 mm in diameter were prepared using
Whatman No.1 filter paper and were sterilized by dry heat at 140°C for 1 h. The sterile discs
previously soaked in a known concentration of the test compounds were placed in the nutrient
agar medium. Ciprofloxacin (30 µg) was used as positive control, while the disk poured in
DMSO was used as negative control. The plates were inverted and incubated for 24 h at
3
7°C. The susceptibility was assessed on the basis of the diameter of the zone of inhibition
against Gram-positive and Gram-negative strains of bacteria. Inhibition zones were measured
and compared with the controls (Table 4 and Figure 6). Minimum inhibitory concentrations
(
MICs) were determined by the broth micro dilution method. The nutrient broth, which
contained logarithmic serially two fold diluted amount of test compound and controls were
5
inoculated with approximately 5x10 CFU/ml of actively dividing bacteria cells. The cultures
of the bacterial strains were incubated for 24 h at 37°C and the growth was monitored
visually and spectrophotometrically. The lowest concentration (highest dilution) required to
arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). To
obtain the minimum bacterial concentration (MBC), 0.1 ml volume was taken from each tube
and spread on agar plates displayed in Figure 7. The number of CFU was counted after 18-24
h of incubation at 35°C. MBC was defined as the lowest drug concentration at which 99.9%
of the inoculums were killed. The MIC and MBC are given in Table 4. The investigation of
antibacterial screening data revealed that all tested compounds showed moderate to good
2
antibacterial activity. Among the metal complexes studied the copper complex [CuL]Cl ,
showed highest antibacterial activity against S. aureus and E. coli nearly equivalent to
standard drug ciprofloxacin. Further as reported earlier [5, 8] antibacterial activity of free
ligand is found to be less than its metal complexes.
4
.9.2. Cytotoxic potential of macrocyclic complexes
The cytotoxic potential of the in house synthesized macrocyclic complexes against HeLa
Hep3B, and MCF7 cell lines was assessed by determining the number of viable cells
surviving after incubation with the macrocyclic complexes for the stipulated time period
1
0

using the MTT method as given in the experimental section. The cytotoxicity assay suggests
a variable cytotoxicity of inhouse synthesized complexes for HeLa, MCF7 as well as Hep3B
cell lines which can be attributed to the intrinsic anticancer property of these macrocyclic
complexes. Curves of dose-dependent effects of all the complexes on cell viability of
different human cancer cell lines (HeLa, MCF7, and Hep3B) are displayed in Figure 8.
Experiments revealed that there was substantial increase in cytotoxicity in the cell lines with
increasing exposure to drug concentration. The absorption values were expressed as the cell
viability in percent, according to the control group as 100%. Assays were performed in
triplicate on three independent experiments. The concentration required for 50% inhibition of
cell viability (IC50) was calculated using the software ‘‘Prism 3.0’’. For each of the tested
complexes IC50 was calculated and the results are summarized in Table 5. It is apparent from
the IC50 values that all the tested complexes show moderate to good cytotoxicity against
different human cancer cell lines. Further as aspected cytotoxicity of metal complexes is
comparatively more than the free ligand [7, 9]. It is remarkable that among these complexes
the copper complex, [CuL]Cl2, exhibits comparatively high cytotoxicity against all cancerous
cell lines with IC50 values of 12.7±2.5 µM against HeLa, 11.5±2.5 µM against MCF7 and
1
2.53±1.9 µM against Hep3B cell lies, respectively.
Further, it is observed that both antibacterial and anticancer activities of metal complexes are
comparatively higher than the metal free ligand under identical experimental conditions
which is also in accordance with the chelation theory [21, 48]. This enhancement in their
actvities may be due to the presence of azomethine bonds in the macrocyclic chelate ring.
As a result of coordination of the metal ion through nitrogen of the azomethine bonds of the
chelate ring the polarity of the metal atom is reduced due to partial sharing of its positive
charge with the ligand, as there arises the possiblility of π electron delocalization within the
chelate ring. Thus, in this process the lipophilic nature of the metal atom increases favouring
its permeation more efficient through the lipid layer of cell membranes.
It is also remarkable that copper complex shows higher antibacterial and anticancer activities
relative to other transition metals studied here [Mn(II), Co(II) and Ni(II)]. This behaviour of
copper is in agreement with the earlier studies [8, 10, 49]. Except the role of copper as an
essential trace element it exhibits considerable biochemical action forming complexes that
interact with bio molecules, mainly protein and nucleic acids. The role of copper is
significant as it can promote nucleic acid cleavage and therefore is used as metallodrug to
cause DNA damage [10, 50, 51]. The earlier reports reveal that copper(II) complexes exhibit
stronger DNA binding propensity [51-53] as compared to other transition metal complexes.
1
1

The later 3d transition metal ions, specially Cu(II), are classified as “borderline” between
hard’(a-class) and ‘soft’ (b-class) metals. Regarding their metal-DNA interactions they show
more affinity for both, the heterocyclic bases as well as phosphate group in contrast to ‘soft’
b-class) metals like Pt(II), therefore there appears enhancement in their binding strength with
DNA.
‘
(
5
. Conclusion
The newly synthesised free macrocyclic ligand (L): (6,7,14,15-tetraoxa-2,3,10,11-
tetraphenyl-1,4,5,8,9,12,13,16-octaazacyclohexadecane-1,3,9,11-tetraene) after reaction with
respective metal salts yielded transition metal macrocyclic complexes of the type [MLX
2
] and
[
CuL]X (M = Mn(II), Co(II), Ni(II) and Zn(II) ; X = Cl or NO ) having octahedral geometry
3
2
around metal ions except the copper complexes which are square planar.
After characterization further studied were carried out for their biological properties:
antibacterial and anticancer activities. The ligand and its complexes were screened in vitro
against Staphylococcus aureus and Escherichia coli bacteria and their cytotoxic potential was
studied against different human cancer cell lines: HeLa, MCF7 and Hep3B.
These studies reveal that metal complexes are comparatively more effective in their
antibacterial and anticancer activities than the metal free ligand. Although copper complex
shows moderate cytotoxicity against cancer cell lines but it shows excellent antimicrobial
activity against tested microorganisms which is almost equivalent to the standard drug
ciprofloxacin. These studies may explore further possibilities to provide new structural type
macrocyclic complexes which may be applied in the development of novel antimicrobial and
anticancer agents.
Acknowledgement
The authors thank the Chairman, Department of Chemistry, AMU, Aligarh (India) for
providing necessary research facilities.
References
[
1] L. Ronconi, P.J. Sadler, Using coordination chemistry to design new medicines, Coord.
Chem. Rev. 251 (2007) 1633–1648.
1
2

[
2] T. Storr, K.H. Thompson, C. Orvig, Design of targeting ligands in medicinal inorganic
chemistry, Chem. Soc. Rev. 35 (2006) 534–544.
3] M.J. Clarke, F. Zhu, D.R. Frasca, Non-Platinum Chemothera. Metallopharm. Chem. Rev.
9 (1999) 2511–2533.
4] F.J. Ramos-Lima, A.G. Quiroga, J.M. Perez, C. Navarro-Ranninger, Preparation,
characterization and cytotoxic activity of new compounds trans-[PtCl NH (3-
hydroxymethyl)-pyridine)] and trans-[PtCl NH (4-(hydroxymethyl)-pyridine)] Polyhedron.
2 (2003) 3379–3381.
5] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, The status of platinum anticancer drugs in
the clinic and in clinical trials, Dalton Trans. 39 (2010) 8113–8127.
6] A.G. Quiroga, F.J. Ramos-Lima, A. Alvarez-Valdés, M. Font-Bardía, A. Bergamo,
[
9
[
2
3
(
2
3
2
[
[
G.Sava, C. Navarro-Ranninger, Synthesis, characterization and tumor cell growth inhibition
of new transplatinum complexes with phosphane derivatives, Polyhedron 30 (2011) 1646–
1
650.
[
(
[
7] B.A. Chabner, T.G. Roberts, Jr. Chemotherapy and the war on cancer. Nat Rev Cancer. 5
2005) 65–72.
8] R. Katwal, H. Kaurt, B. Kishore Kapur, Applications of copper- Schiff’s base complexes:
A Review, Chem. Commun. 1 (2013)1-15.
9] A.M. Khedr, H.M. Marwani, Synthesis, Spectral, Thermal Analyses and Molecular
[
Modeling of Bioactive Cu(II)-complexes with 1,3,4-thiadiazole Schiff Base Derivatives.
Their Catalytic Effect on the Cathodic Reduction of Oxygen, Int. J. Electrochem.
Sci. 7 (2012) 10074 – 10093.
[
10] N. Pravin, N. Raman, Investigation of in vitro anticancer and DNA strap interactions in
live cells using carboplatin type Cu(II) and Zn(II) metalloinsertors, Eur. J. of Med. Chem.85
(
[
2014) 675-687.
11] F. Arjmand, B. Mohani, S. Ahmad, Synthesis, antibacterial, antifungal activity and
interaction of CT-DNA with a new benzimidazole derived Cu(II) complex, Eur. J. Med.
Chem. 40 (2005) 1103–1110.
[
12] M. Maji, M. Chatterjee, S. Ghosh, S.K. Chattopadhyay, B.M. Wu, T.C.W. Mak,
Chemistry of ruthenium(II) complexes of the tridentate NNS donor methyl 2-pyridyl ketone
-(4-tolyl)thiosemicarbazone. Isolation and structural characterisation of a novel
4
ruthenium(II) complex containing a co-ordinated imine of an α-N heterocyclic ketone, J.
Chem. Soc. Dalton Trans. (1999) 135–140.
1
3

[
13] S. Chandra, P. Pipil, EPR, IR, Mass and electronic spectroscopic studies on newly
synthesized 23,24-dioxo-1,6,9,12,17,20-hexaazacyclotracosa-1,5,12,16-tetraenemacrocyclic
ligand (L) and its transition metal complexes, J. Chem. and Pharm. Res. 5(5) (2013) 99-104.
[
14] J.S. Yoder, S. Cesario, V. Plotkin, X. Kelly, K. Shannon, M.S. Dworkin, Outbreak of
Enterotoxigenic Escherichia coli Infection with an Unusually Long Duration of Illness, Clin.
Infect. Dis. 42 (2006) 1513-1517.
[
15] G.D. Bajju, P. Sharma, A. Kapahi, M. Bhagat, S. Kundan, D. Gupta Oxovanadium(IV)
Complexes with Nitrogen Donors: Synthesis, Characterisation, and Biological Activities,
J. Inorg. Chem. (2013) 1-11.
[
16] D. Kumar, Sandhya, Synthesis, spectral and antimicrobial studies of tetraazamacrocyclic
complexes of bivalent transition metal ions of bioinorganic relevance, J. Chem. and Pharm.
Res. 6(6) (2014) 746-750.
[
17] S. Chandra, L.K. Gupta, S. Agrawal, Synthesis spectroscopic and biological approach in
the characterization of novel [N4] macrocyclic ligand and its transition metal complexes,
Trans. Met. Chem. 32 (2007) 558-563.
[
18] S. Chandra, M. Pundir, Spectroscopic characterization of chromium(III), manganese(II)
and nickel(II) complexes with a nitrogen donor tetradentate, 12-membered azamacrocyclic
ligand, Spectrochim. Acta Part A: Mol. and Biomo. Spect. 69 (2008) 1-7.
[
19] T.A. Khan, S.S. Hasan, A.K. Mohamed, K.S. Islam, M. Shakir, Metal complexes of 22-
membered mononuclear tetraimine-tetraamide macrocycles: Synthesis and characterization,
Synth. React. Inorg. Met.-Org.Chem.5 (2000) 815-828.
[
20] T.A. Khan, S. Naseem, Y. Azim, S. Parveen, M. Shakir, Metal-ion directed synthesis of
binuclear octaazamacrocyclic complexes of manganese(II), cobalt(II), nickel(II), copper(II)
and zinc(II) and their physicochemical studies, Trans. Met. Chem. 32(2007) 706-710.
[
21] T.A. Khan, S. Naseem, R. Hajra, M. Shakir, Synthesis, Physicochemical and
Antimicrobial Screening Studies of Complexes of Co(II), Ni(II), Cu(II), and Zn(II) with 18-
membered Schiff Base Octaazamacrocyclic Ligand, Synth. React. Inorg. Met.-Org.Chem.40
(
[
2010) 861–868.
22] H. Zafar, A. Kareem, A. Sherwani, M. Owais, T.A. Khan, Synthesis, characterization
and biological studies of homo and hetero-binuclear 13-membered pentaaza bis (macrocyclic)
complexes, J. Mol. Struct. (2014) PII: S0022-2860(14)00866-7 (In Press).
[
23] A. Kareem, H. Zafar, A. Sherwani, M. Owais, T.A. Khan, Synthesis, characterization
and in vitro anticancer activity of 18-membered octaazamacrocyclic complexes of Co(II),
Ni(II), Cd(II) and Sn(II) J. Mol. Struct.1075 (2014) 17–25.
1
4

[
24] T.A. Khan, H. Zafar, S.N. Khan, A.U. Khan, Metal-ion directed synthesis
of 18–membered tetrathia macrocyclic complexes of Co(II), Ni(II), Cu(II) and Zn(II), their
physico-chemical studies and DNA binding study of copper complex, Synth. React. Inorg.
Met.-Org.Chem. and Nano-Metal Chemistry (2014) (In press).
[
25] M.A. Ansari, M.K. Haris, A.K. Aijaz, M.K. Ahmad, Interaction of silver nanoparticles
with Escherichia coli and their cell envelope biomolecule, J. Basic Microbiol. 53 (2013)
–11.
26] N. Ahmed, N. Kumar Konduru, S. Ahmad, M. Owais, Synthesis of flavonoids based
1
[
novel tetrahydropyran conjugates (Prins products) and their antiproliferative activity against
human cancer cell lines, Eur. J. Med. Chem. 75 (2014) 233-246.
[
27] Shamsuzzaman, H. Khanam, A. Mashrai, A.Sherwani, M.Owais, N.Siddiqui, Synthesis
and anti-tumor evaluation of B-ring substituted Steroidal pyrazoline derivatives, Steroids.78
(
[
2013) 1263–1272.
28] Z. Khatoon, Kabir-Ud-Din, Potentiometric Investigations on the Cadmium(II) Amino
Acid-Imidazole systems (Amino Acid= Glycine, DL-Alanine or DL-Valine), Polyhedron (20)
(1990) 2437-2442.
29] A.I. Vogel, A Text Book of Quantitative Inorganic Analysis, 3 Ed, Longmans, 1961,
pp. 433-441.
30] M.A. Ansari, M.K. Haris, A.K. Aijaz, S. Asfia, Evaluation of antibacterial activity of
silver nanoparticles against MSSA and MRSA on isolates from skin infections, Biol. Med.
9
rd
[
[
(
[
2) 3 (2011)141–146.
31] A. Chauhan, S. Zubair, S.Tufail, A. Sherwani, M. Sajid, S.C. Raman, A. Azam, M.
Owais, Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of
liver cancer, Int. J. Nanomedicine. 6 (2011) 2305–2319.
[
32] W. Geary, The use of conductivity measurements in organic solvents for the
characterisation of coordination compounds, Coord. Chem. Rev. 7 (1971) 81-122.
33] M. Shakir, S.P. Varkey, A new synthetic route for the preparation of a new series of 14-
2-membered tetraoxomacrocyclic tetraamines and their transition metal complexes,
Polyhedron. 14 (1995) 1117-1127.
34] D.P. Singh, V. Grover, K. Kumar, K. Jain, Synthesis and characterization of divalent
[
2
[
metal complexes of the macrocyclic ligand derived from isatin and 1,2-diaminobenzene,
J. Serb. Chem. Soc. (2011)76(3) 385–393.
1
5

[
35] M. Shakir, A.K. Mohamed, S.P. Varkey, O.S.M. Nasman, Z.A. Siddiqi, Preparation and
Structural Characterization of 14-16-Membered Pendent Arm Macrocyclic Complexes of
Transition Metal Ions, Polyhedron 14(1995) 1277-1282.
[
36] P.R. Reddy, M. Radhica, P. Manuja, Synthesis and characterization of mixed ligand
complexes of Zn(II) and Co(II) with amino acids: Relevance to zinc binding sites in zinc
fingers, J. Chem. Sci., 1173 (2005) 239–246.
[
37] V. Sreenivas, G. Srikanth, M. Aruna, V.P. Kumar, P.M. Reddy, V. Ravinder,
Synthesis, Characterization and Antibacterial Activity and DNA cleavage Studies of tetra
dentate Schiff bases and their Zn(II) Complexes, Res. J. Chem. Sci. 4(6) (2014) 66-72.
[
(
[
38] F.A. Cotton and G. Wilkinson, “Advanced Inorganic Chemistry”, Wiley Eastern,
rd
3 edn.) New Delhi, 1972.
39] N.S. Biradar, B.R. Havinale, Dimeric Square Planar Complexes of Cu(II) with Aroyl
Hydrazones, Inorg. Chim. Acta 17 (1976) 157-160.
40] M. Shakir, Khan S. Islam, A.K. Mohamed, N. Jahan, Tetraamide macrocyclic complexes
of transition metals with ligands derived from hydrazine, Trans. Met. Chem. 22 (1997) 189-
92.
41] A.B.P. Lever, Crystal Field Spectra. Inorganic Electronic Spectroscopy, first ed.,
Elsevier; Amsterdam, 1968, pp. 249–360.
[
1
[
[
[
42] B.J. Hathaway, J.N. Bradley, R.D. Gillard, Essays in Chemistry Academic Press New
York, 1971.
43] B.J. Hathaway, D.E. Billing, The electronic properties and stereochemistry of mono-
nuclear complexes of the copper(II) ion, Coord. Chem. Rev. 5 (1970)143–207.
44] R. Kannappan, S. Tanase, Ilpo Mutikainen, Urho Turpeinen, Jan Reedijk, Square-planar
[
copper(II) halide complexes of tridentate ligands with π – π stacking interactions and
alternating short and long Cu…Cu distances, Inorg. Chim. Acta 358 (2005) 383–388.
[
45] R.A. Ahmadi, F. Hasanvand, G. Bruno, H. Amiri Rudbari, S. Amani,
Synthesis, Spectroscopy and Magnetic Characterization of Copper(II) and Cobalt(II)
Complexes with 2-Amino-5-bromopyridine as Ligand, J. Inor. Chem. (2013) 1- 7.
[
[
46] S.K. Verma, V.K. Singh, One pot three component synthesis of mononuclear
M(1,1-dithiolato)2] {Mn(II), Co(II), Zn(II) and Cd(II)} complexes, spectral characterization,
fluorescence, optical and thermo gravimetric study, Polyhedron 76 (2014) 1–9.
47] S. Sharma, F. Athar, M.R. Maurya, F. Naqvi, A. Azam, Novel bidentate complexes of
[
Cu(II) derived from 5-nitrofuran-2-carboxaldehyde thiosemicarbazones with antiamoebic
activity against E. histolytica, Eur. J. Med. Chem. 40 (2005) 557– 562.
1
6

[
48] B. G. Tweedy, “Plant extracts with metal ions as potential antimicrobial agents,
Phytopath. 55 (1964) 910–914.
49] Y. Zhang, L. Zhang, L. Liu, J. Guo, D. Wu, G. Xu, X. Wang, D. Jia, Anticancer
activity, structure, and theoretical calculation of N-(1-phenyl-3-methyl-4-propyl-pyrazolone-
[
5
2
)-salicylidene hydrazone and its copper(II) complex, Inorg. Chim. Acta. 363 (2010) 289–
93.
II
50] S. Tabassum, M. Afzal, F. Arjmand, New heterobimetallic Cu -Sn
IV
[
2
complex as
potential topoisomerase I inhibitor: In vitro DNA binding, cleavage and cytotoxicity against
human cancer cell lines, J. Photochem. Photobiol., B 115 (2012) 63-72.
[
51] I. Iakovidis, I. Delimaris, S.M. Piperakis, Copper and Its Complexes in Medicine: A
Biochemical Approach, Mol. Biol. Int. Rev. (2011) 1- 13.
52] F. Arjmand, F. Sayeed, M. Muddassir, Synthesis of new chiral heterocyclic Schiff base
[
modulated Cu(II)/Zn(II)complexes: Their comparative binding studies with CT-DNA,
mononucleotides and cleavage activity, J. Photochem. Photobiol., B 103 (2011) 166–179.
[
53] J. Liu, T. Zhang, L. Tongbu, Q. Lianghu, H. Zhou, Q. Zhang, J. Liangnian, DNA-
binding and cleavage studies of macrocyclic copper(II) complexes, J. Inorg. Biochem. 25
2002) 269-276
(
Scheme 1 Formation and suggested structure of tetraiminetetraamide ligand and their
complexes of the type [MLX ] and [CuL]X [M = Mn(II), Co(II), Ni(II) and Zn(II) ; X = Cl
or NO ].
2
2
3
Table Caption
Table 1. Elemental analyses, m/z values, colors, yields, molar conductance, melting points
and magnetic moments (µeff.) of the complexes.
-1
Table 2. IR spectral data of the complexes (cm ).
-
1
Table 3. Electronic spectral bands (cm ) with their assignments and EPR data of the
complexes.
Table 4. MIC and MBC results of ligand and its complexes with positive control
ciprofloxacin.
-1
Table 5. The IC50 values (µmol L ) of the macrocyclic ligand and its complexes.
Figure caption
1
7

Figure 1. Mass spectra of (a) ligand and their complexes: (b) [CoL(NO
CuL](NO
3 2
) ] and (c)
[
3 2
)
1
Figure 2. H- NMR spectra of (a) DPEDDH, (b) [ZnL(NO
3
)
2
] and (c) [ZnLCl
2
].
1
Figure 3. C- NMR spectra of mononuclear complexes: (a) Ligand (L), (b) [ZnL(NO ) ] and
3
3
2
2
(c) [ZnLCl ].
Figure 4. (a) EPR spectra of the [CuL](NO
spectra of the [CuL]Cl complex at room temperature.
Figure 5. TGA/DTA of the mononuclear complexes: (a) [MnL(NO
c) [CuL]Cl
3 2
) complex at room temperature; (b) EPR
2
3 2 2
) ] , (b) [CoLCl ] and
(
2
.
Figure 6. Zone of inhibition (in mm) of ligand and its complexes tested against S. aureus
and E. coli.
Figure 7 Antibacterial activity of ligand (a) (L), and its complexes (b) [MnLCl
CoLCl ], (d) [NiL(NO ] and (e) [CuL]Cl shown by well-diffusion method.
Figure 8 Dose-dependent effects of (a) Ligand (L), (b) [MnLCl ], (c) [CoLCl
and (e) [CuL]Cl complexes on different human cancer cell lines i.e., HeLa, MCF7 and
Hep3B.
2
], (c)
[
2
3
)
2
2
2
2 2
], (d) [NiLCl ],
2
1
8

1
9

(a)
m/z
(b)
m/z
2
0

(c)
m/z
Figure 1. Mass spectra of (a) ligand and their complexes: (b) [CoL(NO
CuL](NO
3
)
2
] and (c)
[
3 2
)
2
1

(a)
ppm
(b)
ppm
2
2

(c)
1
Figure 2. H- NMR spectra of (a) DPEDDH mononuclear complexes, (b)[ZnL(NO
3 2
) ] and
(
c) [ZnLCl2].
2
3

(a)
(b)
2
4

(c)
13
Figure 3. C-NMR spectra of mononuclear complexes: (a) Ligand (L),
(b) [ZnL(NO3)2]and (c) [ZnLCl2].
2
5

Figure 4. (a) EPR spectra of the [CuL](NO ) complex at room temperature; (b) EPR spectra
3
2
of the [CuL]Cl complex at room temperature.
2
2
6

Figure 5. TGA/DTA of the mononuclear complexes; (a) [MnL(NO
CuL]Cl
3 2 2
) ], (b) [CoLCl ] and
[
2
2
7

S. aureus
E. coli
27
24
21
18
15
12
9
6
3
0
Ligand and its complexes
Figure 6. Zone of inhibition (in mm) of ligand and its complexes tested against S. aureus
and E. coli.
2
8

Figure7. Antibacterial activity of ligand (a) (L), and its complexes (b) [MnLCl
2
],
(
c) [CoLCl ], (d) [NiL(NO ) ] and (e) [CuL]Cl shown by well-diffusion method.
2
3
2
2
2
9