Synthesis and spectral characterization of Schiff base complexes of Cu(II), Co(II), Zn(II) and VO(IV) containing 4-(4-aminophenyl)morpholine derivatives: Antimicrobial evaluation and anticancer studies

Article abstract of DOI:10.1016/j.saa.2013.07.101

Metal(II) chelates of Schiff bases derived from the condensation of 4-morpholinoaniline with substituted salicylaldehyde have been prepared and characterized by ¹H NMR, IR, electronic, EPR, and magnetic measurement studies. The complexes are of

Full text of DOI:10.1016/j.saa.2013.07.101

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis and spectral characterization of Schiff base complexes of
Cu(II), Co(II), Zn(II) and VO(IV) containing 4-(4-aminophenyl)
morpholine derivatives: Antimicrobial evaluation and anticancer studies
K. Dhahagani a, S. Mathan Kumar a, G. Chakkaravarthi b, K. Anitha c, J. Rajesh d, A. Ramu e, G. Rajagopal a,
⇑
a Department of Chemistry, Government Arts College, Melur, Madurai 625 106, India
b Department of Physics, CPCL Polytechnic College, Chennai 600 068, India
c School of Physics, Madurai Kamaraj University, Madurai 625 021, India
d Department of Chemistry, Sethu Institute of Technology, Kariapatti 626 106, India
e School of Chemistry, Madurai Kamaraj University, Madurai 625 021, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of novel morpholinoaniline
based Schiff bases.
Reporting structurally solved
morpholinoaniline for the first time.
Complexes having potential
antibacterial and antifungal activity.
Good to moderate anticancer activity
against HepG2.
Zinc complex exhibits a good
inhibitory activity against the human
gastric cancer cells.
Untreated HepG2 cell
Treated HepG2 cell
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2013
Received in revised form 23 July 2013
Accepted 31 July 2013
Available online 7 August 2013
Metal(II) chelates of Schiff bases derived from the condensation of 4-morpholinoaniline with substituted
salicylaldehyde have been prepared and characterized by 1H NMR, IR, electronic, EPR, and magnetic mea-
surement studies. The complexes are of the type M(X-MPMP)2 [where M = Cu(II), Co(II)), Zn(II), or
VO(IV); MPMP = 2-[(4 morpholinophenyl imino) methyl] 4-X-phenol, X = Cl, (L1H), X = Br (L2H)]. Single
crystal X-ray crystallography studies confirm the structure of newly synthesized Schiff bases. The Schiff
bases act as bidentate monobasic ligands, coordinating through deprotonated phenolic oxygen and azo-
methine nitrogen atoms. The free ligands and metal complexes are screened for their biopotency. Metal
complexes exhibit better activity than ligands. Anticancer activity of ligands and their metal complexes
are evaluated in human heptocarcinoma(HepG2) cells. The preliminary bioassay indicates that the Schiff
base and its zinc complex exhibit inhibitory activity against the human gastric cancer cell lines.
Ó 2013 Elsevier B.V. All rights reserved.
Keywords:
Schiff bases
Morpholinoaniline
Anticancer
Antimicrobial
HepG2 cells
Crystal studies
Introduction
and phenolic group or via its azomethine or phenolic groups [1–4].
The chemistry of Schiff base ligands and their metal complexes
Schiff bases, a class of chelators are capable of forming coordi-
nate bonds with many metal ions through both azomethine group
have attracted a lot of interest due to their facile synthesis and
wide range of applications including antifungal, antibacterial,
anticancer and catalytic fields [5–8]. The design, synthesis and
structural characterization of salicylaldimine complexes are a sub-
ject of current interest [9]. Due to their interesting and versatile
⇑
Corresponding author. Tel./fax: +91 452 2415671.
E-mail address: rajagopal18@yahoo.com (G. Rajagopal).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.07.101

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K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
structural, magnetic, spectral, and catalytic and redox properties,
they are used as models for metalloenzymes and various theoreti-
cal problems of chemistry [10].
approximately 104 to 106 CFU (colony forming unit) per mL are
spread on the surface of Muller Hinton Agar plates. Wells are cre-
ated in medium with the help of a sterile metallic bores and nutri-
ents agar media (agar 20 g + beef extract 3 g + peptones 5 g) in
1000 mL of distilled water (PH 7.0), autoclaved and cooled down
to 45 °C. Then, it is seeded with 10 mL of prepared inocula to have
106 CFU/mL. Petri plates are prepared by pouring 75 mL of seeded
nutrient agar. The activity is determined by measuring the diame-
ter of the inhibition zone (in mm). The growth inhibition is calcu-
lated according to Ref. [38].
4-Phenyl-morpholine derivatives are reported to possess anti-
microbial [11,12], anti-inflammatory [13–15] and central nervous
system activities [16–20]. Linezolide (commercially available anti-
microbial) also possess a 4-phenyl-morpholine substituent. It is
observed that 4-phenyl-morpholine is a potential substituent to
impart significant antimicrobial property to quinazoline moiety
[21]. These observations led to the conception that Schiff bases of
4-(4-aminophenyl)-morpholine would possess potential antimi-
crobial properties.
Antifungal screening
Cancer has overtaken heart disease as the world’s top killer by
2010, part of a trend that should be more than double global cancer
cases and deaths by 2030 [22]. Hepatocellular carcinoma is the
most common type of liver cancer and causes more than 600,000
deaths in china each year [23]. Metal-based pharmaceuticals
emerging from the interface of inorganic chemistry, pharmacology,
toxicology and biochemistry have witnessed spectacular successes
[24,25]. The discovery of the cytotoxic properties of cisplatin has
provided enormous impetus for research into the use of metal
complexes in the fight against cancer [26]. A broad array of medic-
inal applications of these compounds has been investigated, and
some of them are found to be useful both as clinical diagnostic
agents and in chemotherapeutic applications [27–36]. Though sev-
eral Schiff base complexes are reported in the literature, morphol-
inoaniline Schiff base complexes have not been published. In
continuation of our work in the area of Schiff base complexes
[37], the synthesis, characterization, crystal structure determina-
tion and biological screening of metal (II) complexes of morphol-
inoaniline Schiff bases are described here.
The anti-fungal activity of the ligands and their metal com-
plexes are studied by paper disc method [38]. Aspergillusniger, Pen-
cilliumchrysogenum and Candida albicans are used as test
organisms. Solution of desired concentration (1 mg/mL) was ob-
tained by dissolving 2, 4, 6 mg of each compound in DMSO and
added to Potato Dextrose Agar (PDA) medium in sterile Petri
dishes. The sterilized medium with the added sample solution is
poured into sterile Petri plates and allowed to solidify. Filter paper
discs of 5 mm diameter are prepared prior to the experiment. The
filter paper discs are placed on nutrient medium mixed with fungal
strains. These Petri dishes are incubated at 35 °C for 48 h. The per-
cent reduction in the radial growth diameter over the control is
calculated. The growth is compared with dimethylsulfoxide as
the control and Ketoconazole as a standard drug.
Cell viability test
The viability of cells is assessed by MTT assay using mononu-
clear cells. The assay is based on the reduction of soluble yellow
tetrazolium salt to insoluble purple formazan crystals by metabol-
ically active cells. Only live cells are able to take up the tetrazolium
salt. The enzyme (mitochondrial succinate dehydrogenase) present
in the mitochondria of the live cells is able to convert internalized
tetrazolium salt to formazan crystals, which are purple in color.
Then, the cells are lysed and dissolved in DMSO solution. The color
developed is then determined in an ELISA reader at 570 nm.
The Hepatocellular carcinoma cells (HepG2 cells) are plated sep-
arately in 96 well plates at a concentration of 1 ꢁ 105 cells/well.
Experimental
Materials
5-Chlorosalicylaldehyde, 5-Bromosalicyladehyde and 4-Mor-
pholinoaniline are procured from Sigma–Aldrich. Copper acetate,
cobalt acetate, zinc acetate, vanadyl (IV) sulfate, N,N0-dimethylfor-
maide, N,N-dimethylsulfoxide, acetonitrile, chloroform, ethanol
and methanol are purchased form Merck.
After 24 h, cells are washed twice with 100
ium and starved for an hour at 37 °C. After starvation, cells are trea-
ted with different concentrations of test compound (50–300 g/ml)
ll of serum-free med-
Physical measurements
l
for 24 h. At the end of the treatment period, the medium is aspi-
rated and serum free medium containing MTT (0.5 mg/ml) is added,
Then it is incubated for 4 h at 37 °C in a CO2 incubator.
Crystal data collection APEX2 (Bruker, 2004); cell refinement:
SAINT (Bruker, 2004), 1H NMR spectra are recorded on a Bruker
300 MHz spectrometer. IR spectra are recorded in KBr disks with
a Perkin Elmer FT-IR spectrophotometer. UV–Vis spectra of solu-
tion are recorded on a Shimadzu 1700 series spectrometer. Bio-
Analytical Systems (BAS) model CV 50 electrochemical analyzer
is used for cyclic voltammetric experiments. Magnetic parameters
are measured with Lakeshore VSM 7304 with a maximum field of
20 KOe. ESR spectra are recorded on a Varian E-112 spectrometer.
The MTT containing medium is then discarded and the cells are
washed with PBS (200
ll). The crystals are then dissolved by add-
ing 100 l of DMSO and this is mixed properly by pipetting up and
l
down. Spectrophotometrical absorbance of the purple blue forma-
zan dye is measured in a micro-plate reader at 570 nm [39].
Synthesis of Schiff base ligands
Anti-bacterial screening
An ethanolic solution of 4-morpholinoaniline (5 mmol) is mag-
netically stirred in a round bottom flask followed by dropwise
addition of appropriate substituted salicylaldehyde (5 mmol) con-
taining 2–3 drops of glacial aceticacid. The reaction mixture is then
refluxed for 3 h and upon cooling to 0 °C, crystalline solid precipi-
tates from the mixture are separated out. Crystalline products are
washed with ice cold ethanol and dried in vacuo over anhydrous
CaCl2. Single crystals suitable for the X-ray diffraction are obtained
by slow evaporation of a solution of the title compound in DMF at
room temperature (Supplementary files, Scheme 1).
Antibacterial activities are investigated using agar well diffu-
sion method. The activity of the free ligand, its metal complexes
and standard drug Amikacin are studied against the Chromobacte-
rium vialacium, Staphylococcus aureus (as grampositive bacteria),
Staphylococciaureus, Pseudomonasaeruginosa, and Shigella byogenes
(as gram negative bacteria). The solution of 2 mg/mL of each
compound (free ligand, its metal complexes and standard drug
Amikacin) in DMSO is prepared for testing against bacteria. Centri-
fuged pelletes of bacteria from a 24 h old culture containing

K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
89
Synthesis of Cu(II), Co(II), Zn(II) and VO(IV) complexes
An inversion related hetero dimer is found with H bonds
C15AH15ꢂ ꢂ ꢂCl1 and C15AH15ꢂ ꢂ ꢂBr1 and these hydrogen bonds
form a motif R22ð8Þ. The molecules are packed as ridges in the struc-
ture along Y-axis. The packing of the molecules looks like a net
across XY plane. The angle of inclination between the ridges is
64.54° in Br-MPMP and 66.48° in Cl-MPMP.
All the complexes are prepared using the following general pro-
cedure. Methanolic solutions (10 mL) of the Schiff base (1 mmol)
and the metal acetate (0.5 mmol) (10 mL) are mixed thoroughly
and boiled under reflux for 4–6 h, and then cooled to room temper-
ature. The resulting precipitate is filtered, washed in the ice cold
methanol and dried ‘in vacuo’ (Supplementary files Scheme 2).
NMR spectra
The 1H NMR spectra of the ligand and the zinc complex are re-
corded in CDCl3 and DMSO-d6-solvent respectively (Supplemen-
tary files Figs. S2 and S3). Singlet peaks observed in the 1H NMR
spectra of the ligands at ꢃ13.55 and 8.54 ppm are due to phenolic
(AH) and azomethine(ACH@N) protons of ligands respectively
(Table 4). The peak due to the phenolic proton is absent, while
the peak due to the azomethine proton is shifted to 8.26 ppm in
the spectrum of the Zn(II) complex. This observation clearly sup-
ports the involvement of the AOH chromophore and azomethine
nitrogen in coordination. The aromatic ring protons are observed
as multiplets in the region 6.74–7.48 ppm in the spectrum of
ligand. These peaks are shifted slightly in the spectrum of the Zn(II)
complex (Fig. 2). Multiplets observed in 3.19–3.22 ppm and
3.79–3.89 ppm ranges are due to aliphatic-CH2 protons.
Results and discussion
Analytical, color and magnetic susceptibility data of all metal
complexes are given in Table 1 and are in good agreement with
proposed composition.
Crystal structure of Cl-MPMP and Br-MPMP
The crystals of present compounds belong to P21/C space group.
The structures have been solved by direct method. The hydrogen
positions have been fixed by a three dimensional difference Fourier
synthesis. The Position coordinates and their temperature factors
have been refined by least square method.
The geometric parameters like bond length and bond angle are
listed in Table 2. Inter and intra molecular hydrogen bonding geo-
metric parameters are listed in the Table 3. These parameters are
similar in both the molecular structures. Most of the hydrogen
bonding features are similar in both the molecular structures.
The intra molecular and intermolecular interactions can be consid-
ered in structure analysis. The morphology of hydrogen bonded ar-
rays present in these structures can be defined by the graph set
assignments. The morpholine ring has found to assume a chair
conformation in both the structures. Rings (C5ꢂ ꢂ ꢂC10) and
(C12ꢂ ꢂ ꢂC17) are planar in both the structures and inclined about
an angle 15.58° in Cl-MPMP. But both the rings are nearly in the
same plane in the structure Br-MPMP. An intra molecular hydro-
gen bond O2AH2ꢂ ꢂ ꢂN2 forms S(6) motif in graph set notation
(Fig. 1. and Supplementary files Fig. S1). Due to the lack of more
hydrogen bonds CAHꢂ ꢂ ꢂO hydrogen bonds determine the crystal
packing. The H bond C2AH2Aꢂ ꢂ ꢂO2 forms a linear chain with graph
set motif C(3) along X-axis. The H bond C13AH13ꢂ ꢂ ꢂO1 forms a
srew related zig–zag chain in C(3) graph set motif along Y-axis.
Infrared spectra
The IR spectral data for the ligands and their metal complexes
are given in Table 5. The spectrum of free ligands has showed a
band in 1655 cmꢄ1 region characteristics of the tC
@ stretching
N
mode indicating the formation of the Schiff base product. This band
is shifted towards lower frequencies in the spectrum of its metal
complexes (1615–1613 cmꢄ1) and is compared with the above
Schiff bases indicating the involvement of the azomethine nitrogen
in coordination with metal ion. The CAO (phenolic) is observed in
the range 1352–1301 cmꢄ1. Both the ligands in the present inves-
tigation exhibit a broadband centered at 3410–3450 cmꢄ1. This
suggests the presence of a strongly hydrogen bonded OH group.
It also suggests that the ligands exist in enol form in the solid state
[40]. The coordination mode of ligand is supported by the disap-
pearance of these bands in the spectra of complexes. [41,42]. Bands
due to M–N appear in 647–669 cmꢄ1 region.
Table 1
Analytical and physical data of the ligand and its metal complexes.
Compound
Empirical formula (formula weight)
Color
Elemental analysis(found)
Magnetic moment (lB)
C
H
N
L1-Cl-MPMP (1)
L2-Br-MPMP (2)
Cu(L1)2 (3)
C
C
17H17N2O2Cl (316.81)
17H17N2O2Br (361.23)
Pale yellow
Pale yellow
Brown
64.46
(64.31)
56.52
(56.64)
58.75
(59.89)
52.09
(52.33)
59.14
(58.89)
52.4
(52.25)
58.46
(58.66)
51.86
5.41
(5.51)
4.74
(4.54)
4.64
(4.49)
4.11
(4.21)
4.67
(4.54)
4.14
(4.23)
4.62
(4.39)
4.1
8.84
(8.67)
7.75
(7.98)
8.06
(7.68)
7.15
(6.89)
8.11
(7.89)
7.19
(6.79)
8.02
(7.89)
7.12
–
–
C34H32N4O4Cl2Cu (695.09)
C34H32N4O4Br2Cu (784)
C34H32N4O4Cl2Co (690.48)
C34H32N4O4Br2Co (779.38)
C34H32N4O5Cl2V (698.49)
C34H32N4O5Br2V (789.49)
C34H32N4O4Cl2Zn (696.94)
C34H32N4O4Br2Zn (785.84)
1.86
1.81
4.72
4.56
1.87
1.92
–
Cu(L2)2 (4)
Brown
Co(L1)2 (5)
Red
Co(L2)2 (6)
Red
VO(L1)2 (7)
VO(L2)2 (8)
Zn(L1)2 (9)
Brown
Brown
(52.34)
58.59
(58.67)
51.97
(4.28)
4.63
(4.42)
4.1
(7.27)
8.04
(7.82)
7.13
Dark yellow
Dark yellow
Zn(L2)2 (10)
–
(51.75)
(4.37)
(7.42)

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K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
Table 2
Crystal data and structure refinement for Cl-MPMP and Br-MPMP.
Empirical formula
Formula weight
C17H17ClN2O2
316.78
C17H17BrN2O2
361.24
Temperature
Wavelength
T = 293(2) K
0.71073 Å
T = 293(2) K
0.71073 Å
Crystal system, space group
Unit cell dimensions
Monoclinic, P21/c
Monoclinic, P21/c
a = 10.2620(6) Å
r
= 90°
a = 10.2981(7) Å
r = 90°
b = 8.0531(5) Å b = 99.465 (2)°
b = 8.1334(6) Å b = 99.673(2)°
c = 18.6368 (11) Å
1519.20 (16) Å3
4,1.385 Mg/m3
0.260 mmꢄ1
664
c
= 90°
c = 18.7663(13) Å
1549.49(19) Å3
8, 1.549 Mg/m3
2.662 mmꢄ1
736
c = 90°
Volume
Z, Calculated density
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Limiting indices
0.30 ꢁ 0.20 ꢁ 0.20 mm
0.30 ꢁ 0.20 ꢁ 0.20 mm
h = 2.2–26.00°
q = 2.20–26.00°
h = ꢄ12 ? 12
ꢄ17 6 h 6 17,
k = ꢄ9 ? 9
ꢄ11 6 k 6 11,
l = ꢄ21 ? 22
ꢄ20 6 l 6 20
Reflections collec/unique
Completeness to
15,357/2976 [Rint = 0.0285]
99.90%
15,323/3028 [R(int) = 0.0291]
99.50%
D = 26.29
Max. and min. transmission
Refinement method
0.9498 and 0.9260
Full-matrix least-squares on F2
2976/0/200
0.6181 and 0.5023
Full-matrix least-squares on F2
3028/0/200
Data/restraints/parameters
Goodness-of-fit on F2
1.05
1.076
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.031, wR2 = 0.0837
R1 = 0.038, wR2 = 0.0895
R1 = 0.0257, wR2 = 0.0744
R1 = 0.0361, wR2 = 0.0805
ꢄ0.16 (e Åꢄ3
)
ꢄ0.250 (e Åꢄ3
)
influenced by the ligand factors such as nature of donor atoms
and their structural arrangement around the copper ion [52]. Cop-
per complexes show an irreversible redox couple with a cathodic
and anodic peak potential at Ecp = ꢄ650 mV and Epa = ꢄ950 mV
respectively. Complexes 3 and 4 show the negative reduction po-
tential, which is typically seen in many other simple square planar
complexes [53,54].
Table 3
Hydrogen bonds for Cl-MPMP and Br-MPMP (A and °).
DAHꢂ ꢂ ꢂA
O(2)AH(2)ꢂ ꢂ ꢂN(2)
O(2)AH(2)ꢂ ꢂ ꢂN(2)
d(DAH)
d(Hꢂ ꢂ ꢂA)
1.83
1.84
d(Dꢂ ꢂ ꢂA)
2.5604(16)
2.569(2)
<(DHA)
0.82
0.82
147.5
147.7
Symmetry transformations used to generate equivalent atoms.
EPR spectra
Electronic spectra
The X-band EPR spectra of copper complexes are recorded in
acetonitrile solution. The EPR spectral parameters derived from
The electronic spectra are recorded in CHCl3. The ligand field
spectra of the copper complex in CHCl3 show a band around
their spectra are presented in Table
6 (Supplementary files
2
2
2
2
700 nm due to a combination of B1g ! A1g and B1g ! B2g tran-
sitions [43]. For a vanadyl complex, in a square pyramidal environ-
ment, three transitions are expected [44]. In the present case, an
intense charge transfer band masks the higher energy bands and
hence only one band is observed. In chloroform solution, a band
at 655 nm is observed which is assigned to the transition
Fig. S4a). The EPR spectra of copper complexes in solution exhibit
a set of four well resolved peaks in the high field region. The ‘‘g’’
tensor values of the copper(II) complexes can be used to derive
the ground state [50,55–57]. In axially elongated octahedral and
square planar complexes, the unpaired electron occupies the
dx2
orbital with 2B1g ground state resulting in gk > g?. However,
ꢄy2
2B2 ? 2E (
t1) [45–49].
in a compressed octahedron the unpaired electron occupies the dz2
orbital with 2A1g ground state having g? > gk. The observed ‘‘g’’ val-
ues suggest that the unpaired electron lies predominantly in the
Magnetic moment
dx2
orbital.
ꢄy2
The magnetic susceptibility measurements in the solid state
show that the present complexes are paramagnetic at room tem-
perature. The leff. value of the copper(II) and oxovanadium(IV)
complexes fall in the range of 1.80–1.92 B.M. corresponding to
one unpaired electron [50,51]. For Co(II) complexes, the observed
magnetic moment values suggest a high spin octahedral structure.
For all vanadyl complexes, the ESR. spectra are recorded in tol-
uene/acetonitrile solutions at RT and LNT. The solution spectra
consist of eight hyperfine lines (Supplementary files Fig. S4b).
The observed spin Hamiltonian parameters A|| and g|| (Table 6)
are in good agreement with the values generally expected for a
vanadyl complex with square pyramidal geometry having a VON2-
O2 chromophore [58,59]. From the ‘g’ values, it is evident that the
unpaired electron is in dxy orbital.
Electrochemistry
Cyclic voltammograms of the present complexes are recorded
in CH3CN and scanned in the cathodic direction at a rate of
100 mV/s using tetrabutylammonium perchlorate as the support-
ing electrolyte in the potential +1.6 V to ꢄ1.6 V range. The repre-
sentative cyclic voltammogram of the complex is given in Fig. 3.
In general, redox data have been used to identify the model sys-
tem among the synthetic complexes. In the copper complexes, the
redox couple Cu(II)/Cu(I) is important and is known to be
Antimicrobial activity
The synthesized ligand and the complexes are tested for their
in vitro antimicrobial activity. They are tested against the bacteria
C.vialacium, S. Flexinari, S.aureus, P.aeruginosa, S.byogenes, P. chrys-
ogenum, A. niger, and C. albicans. The minimum inhibitory concen-
tration (MIC) values of the investigated compounds against
bacteria and fungi are summarized in Tables 7 and 8 respectively.

K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
91
Fig. 1. Crystal structure and unit cell packing diagram for L1-(Cl-MPMP).
Table 4
increased activity of the complexes can be explained on the basis
of the Tweedy’s chelation theory [60]. Chelation reduces the polar-
ity of the metal ion considerably because of the partial sharing of
1NMR spectral data of the ligand and its metal complexes (in ppm).
Compound
Br-MPMP
ꢀ(OAH) ꢀ(CH@N) Aromatic
Aliphatic
its positive charge with the donor group and also due to p-electron
13.55
13.52
–
8.545
8.551
8.26
6.89–6.95(m,3H)
7.21–7.48(m,4H)
6.92–6.96(m,3H)
7.26–7.34(m,4H)
6.74–7.32(m,14H) 3.79–3.81(m,4H)
3.08–3.11(m,4H)
3.86–3.89(m,4H)
3.19–3.22(m,4H)
3.86–3.89(m,4H)
3.19–3.22(m,4H)
delocalization on the whole chelate ring. The lipids and polysac-
charides are some important constituents of the cell wall and
membranes which are preferred for metal ion interaction. Apart
from this, the cell walls also contain many phosphates, carbonyl
and cystenyl ligands which maintain the integrity of the mem-
brane by acting as a diffusion barrier and also provide suitable sites
for binding. Furthermore, the reduction in polarity increases the
lipophilic character of the chelates and an interaction between
the metal ion and the lipid is favored. This may lead to the
breakdown of the permeability barrier of the cell resulting in
Cl-MPMP
Zn(Cl-MPMP)
Representative inhibitory images are presented in Supplementary
files Figs. S5 and S6 respectively. A comparative study of MIC val-
ues of ligand and the complexes indicate that the metal complexes
exhibit higher antimicrobial activity than the free ligand. Such

92
K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
Fig. 2. 1H NMR spectrum of Zn (Cl-MPMP)2 in CDCl3.
Table 6
Table 5
EPR spectral data of copper and vanadium complexes.
IR spectral data of the ligand and its metal complexes (in
cmꢄ1).
Compounds
g||
g
A|| (ꢁ10ꢄ4 cmꢄ1
)
A\ (ꢁ10ꢄ4 cmꢄ1
)
?
[Cu(L1)2] (3)
[Cu(L2)2] (4)
[VO(L2)2] (8)
2.231
2.240
1.951
2.059
2.067
1.989
154.7
153.6
164.7
15.5
16.4
56.5
Compound
t
(C@N)
t(CAO)
L1-Cl-MPMP (1)
L2-Br-MPMP (2)
Cu(L1)2 (3)
Cu(L2)2 (4)
Co(L1)2 (5)
1655
1655
1615
1612
1604
1610
1610
1613
1606
1614
1352
1342
1308
1306
1320
1296
1304
1307
1321
1301
bial property. The synthesized Schiff base ligand has moderate
inhibitory effects on the growth of tested microorganism. This is
due to the presence of azomethine groups which have chelating
properties. These properties may be used in metal transport across
the bacterial membranes or to attach to the bacterial cells at a spe-
cific site from which it can interfere their growth. Ligands exhibit
MIC in the range of 3–15 and 3–16 2 mg/mL for all the bacteria
and 9–17 and 11–18 2 mg/mL for all the fungi, respectively. The
antimicrobial activity of zinc complex shows greater bactericidal
and fungicidal activities against P. aerugonisa and C. albicans as
compared to their corresponding Schiff base. The present investi-
gated antimicrobial activity data indicate that all the newly syn-
Co(L2)2 (6)
VO(L1)2 (7)
VO(L2)2 (8)
Zn(L1)2 (9)
Zn(L2)2 (10)
thesized complexes exhibit
a slightly different antimicrobial
activity as compared to that of the control drugs [60].
Cytotoxic activity
The in vitro cytotoxic activities of the synthesized Schiff base
and its complexes are studied on hepatoma cell lines (HepG2) by
applying the MTT colorimetric assay (Table 9). The calculated
IC50 values, that is, the concentration (lg/mL) of a compound able
to cause 50% of cell death with respect to the control culture, are
presented in Figs. S7–S9 (Supplementary files). Cyclophosphamide
is used as a reference compound. The HepG2 cells are sensitive to
Fig. 3. Cyclic voltammogram of [Cu(L1)2].
the OAN Schiff base with the IC50 value of 860
complex is cytotoxic to the gastric cancer cell lines with IC50 value
of 706
g cmꢄ3 (Table 10). Although the Schiff base and zinc com-
l
g cmꢄ3. The Zn(II)
interference with the normal cell processes. Besides this, the
complexes may also indulge in the formation of hydrogen bonded
interaction through the coordinated anions and azomethine group
with the active centers of the cell constituents. Factors capable of
increasing lipophilic nature are expected to enhance the antimicro-
l
plex are both less effective than cyclophosphamide, the Zn(II) com-
plex is more effective than the Schiff base ligand towards cancer
cell lines. The complexation of metal ions enhances the anticancer
behavior as is evidenced by the lower IC50 values of the complex

K. Dhahagani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 87–94
93
Table 7
Minimum inhibition concentration (MIC) data of the synthesized compounds against growth of bacteria.
Compound
Microorganism (MIC) values
Chromobacterium
Vialacium
Shigella
Flexinari
Staphylococci
aureus
Pseudomonas
aeruginosa
Streptococci
byogenes
L1-Cl-MPMP (1)
L2-Br-MPMP (2)
Cu(L1)2 (3)
Cu(L2)2 (4)
Co(L1)2 (5)
Co(L2)2 (6)
VO(L1)2 (7)
VO(L2)2 (8)
Zn(L1)2 (9)
Zn(L2)2 (10)
Amikacin
11
12
13
12
15
7
12
8
14
13
20
3
R
3
R
5
R
R
10
R
3
3
17
3
6
R
8
5
15
16
12
18
14
12
12
10
7
R
R
R
8
R
R
R
5
15
16
18
R
3
18
R
20
14
17
investigated in terms of the mechanism of action of these com-
pounds at the cellular level [61].
Table 8
Minimum inhibition Concentration (MIC) data of the synthesized compounds against
growth of fungi.
Conclusions
Compound
% Inhibition of spore germination
Pencillium
chrysogenum
Candida
albicans
Aspergillus
niger
The present study describes the synthesis of new Schiff bases
derived from morpholinoaniline and mutisubstituted salicylalde-
hydes. The X-ray crystallography study confirms the structure of
newly synthesised Schiff bases. The spectral data show that the
Schiff bases act as monobasic bidentate NO chelating agents coor-
dinating the metal ion via the azomethine nitrogen atom and the
phenolic oxygen atom. Further, the promising results have been
observed for the antimicrobial screening especially for the metal
complexes against both the fungi and bacteria and it is attributed
to the fact that the metal complexes are potentially active against
bacterial cells than fungi cells. The free ligand and its metal com-
plexes show anticancer activity against HepG2 cells. In view of
the biological activity, the ligands and the complexes have shown
a moderate activity.
2
9
4
6
2
4
6
2
4
6
L1-Cl-MPMP (1)
13
15
14
13
17
14
17
13
14
18
18
15
17
16
16
19
16
19
16
19
21
18
14
11
15
10
15
12
16
12
12
14
17
16
13
16
13
16
16
19
14
15
16
17
17
16
18
16
18
19
21
17
13
17
17
13
14
12
11
13
14
13
13
11
15
20
15
16
14
14
15
17
14
15
13
19
20
17
18
15
17
16
18
15
19
15
21
20
L2-Br-MPMP (2) 12
Cu(L1)2 (3)
Cu(L2)2 (4)
Co(L1)2 (5)
Co(L2)2 (6)
VO(L1)2 (7)
VO(L2)2 (8)
Zn(L1)2 (9)
Zn(L2)2 (10)
Ketoconazole
14
11
15
10
15
11
12
16
18
Table 9
Percentage of cell viability and death analytsis in duplicate study model.
Acknowledgments
Samples
50
lg
100
lg
200
lg
300 lg
L1(Cl-MPMP)
Cu(L1)
Co(L1)
VO(L1)
Zn(L1)
91.9
91
94.1
76.2
92.2
100
66.2
95.2
94.5
93.8
69.1
100
64.2
90.6
86.8
91.8
65.3
100
70.6
87.5
93.7
89.7
54.6
100
Financial support from the Department of Science and Technol-
ogy, New Delhi [Grant No: SR/FTP/CS-40/2007] and University
Grants Commission, New Delhi [Grant No: F. No. 38-70/2009(SR)]
are gratefully acknowledged.
Cyclophosphamide
Appendix A. Supplementary material
Table 10
CCDC 848959 and 848960 contain the supplementary crystallo-
graphic data for Br-MPMP and Cl-MPMP respectively. These data
can be obtained free of charge via http://www.ccdc.cam.ac.uk/con-
ts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223
336 033; or e-mail: deposit@ccdc.cam.ac.uk.
Further, the schemes and figures related to this work are avail-
able in the Supplementary files. Supplementary data associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.saa.2013.07.101.
IC50 values of the ligand and the metal complexes.
Compounds
Values in(l )
g cmꢄ3
P.C
N.C
1002
897
860
2920
3321
1486
706
L1-(Cl-MPMP)
Cu(L1)
Co(L1)
Vo(L1)
Zn(L1)
P.C = Positive control (cyclophosphamide).
N.C = Negative control (untreated cell).
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