Journal of Saudi Chemical Society (2018) 22, 416–426

King Saud University

Journal of Saudi Chemical Society

www.ksu.edu.sa
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ORIGINAL ARTICLE

Synthesis, spectral characterization, theoretical,

antimicrobial, DNA interaction and in vitro
anticancer studies of Cu(II) and Zn(II) complexes
with pyrimidine-morpholine based Schiff base
ligand
M. Sankarganesh a, J. Rajesh a, G.G. Vinoth Kumar a, M. Vadivel a, L. Mitu b,*,
R. Senthil Kumar c, J. Dhaveethu Raja a,*

a
Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai-623 806, Ramanathapuram (Dist), Tamil
Nadu, India
b
Department of Nature Sciences, University of Pitesti, Pitesti 110040, Romania
c
Department of Pharmaceutical Chemistry, Swami Vivekanandha College of Pharmacy, Elayampalyam, Trichengode-637
005, Namakkal (Dist), Tamilnadu, India

Received 22 May 2017; revised 30 August 2017; accepted 30 August 2017

Available online 11 September 2017

KEYWORDS Abstract Novel Cu(II) (1) and Zn(II) (2) complexes with 4-(1-(4-morpholinophenyl)ethylidenea
Pyrimidine; mino)pyrimidine-5-carbonitrile) (L) have been synthesized and characterized by various spectro-
Morpholine; scopic and analytical techniques. DFT (density functional theory) studies result confirms that,
DFT; LMCT mechanism have been done between L and M(II) ions. The antimicrobial studies indicate
Antimicrobial; that the ligand L and complexes 1 & 2 exhibit higher activity against the E. coli bacteria and
DNA binding; C. albicans fungi. The groove binding mode of ligand L and complexes 1 & 2 with CT-DNA have
Anticancer studies been confirmed by electronic absorption, competitive binding, viscometric and cyclic voltammetric
studies. The electronic absorption titration of ligand L and complexes 1 & 2 with DNA have been
carried out in different buffer solutions (pH 4.0, 7.0 & 10.0). The Kb values of ligand L and com-
plexes 1 & 2 are higher in acidic buffer at pH 4.0 (Kb = 2.42
105, L; 2.8
105, 1; 2.65
105, 2)
and the results revealed that, the interaction of synthesized compounds with DNA were higher in
the acidic medium than basic and neutral medium. Furthermore, CT-DNA cleavage studies of
ligand L and complexes 1 & 2 have been studied. The in vitro anticancer activities results show that
* Corresponding authors.
E-mail address: jdrajapriya@gmail.com (J. Dhaveethu Raja).
Peer review under responsibility of King Saud University.

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Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies
complexes 1 & 2 have moderate cytotoxicity against cancer cell lines and low toxicity on normal cell
line than ligand L.
Ó 2017 King Saud University. Production and hosting by Elsevier B.V. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Transition metal complexes are playing an important role in
the field of bioinorganic chemistry. The transition metal com-
plexes contain several biological activities like wise DNA bind-
ing [1], anticancer [2], antidiapetic [3] and antimicrobial [4]
activities. Many literature reports suggest that the transition
metal complexes have been employed in the probes of DNA
structure and chemotherapeutic drugs [5–8]. The consequence
of Cu(II) and Zn(II) ions in nature make enthusiastic interest
to investigate the biological application of their new drugs.
In reality, the Cu(II) and Zn(II) ion are present in hemocyanin
and peptidase enzymes which is a good evidence for their bio-
logical application [9–12]. Moreover, these metal complexes
achieved from the Schiff base ligands which are due to their
various applications in pharmaceutical, antimicrobial, anticar-
cinogenic reagents, industrial and analytical fields [13]. On the
other hand, the ligand obtained from the pyrimidine and mor-
pholine based drugs. However, pyrimidine and morpholine are
nitrogen containing heterocyclic compounds which are present
in anticancer and antibiotic drugs such as gefitinib and line-
zolid [14,15].
In this research work, Cu(II) 1 and Zn(II) 2 complexes of
pyrimidine and morpholine based Schiff base ligand were syn-
thesized and characterized by various spectroscopic tech-
niques. And also, theoretical, antimicrobial, DNA
interaction and in vitro anticancer studies have been carried
out.
2. Experimental
2.1. Materials and methods
All the chemicals and the reagents were purchased from Sigma
Aldrich and Alfa Aesar Company. The electronic spectra and
absorption spectral titration were carried out on UV-1800
spectrophotometer (Shimadzu). The FTIR spectra were
explored on IR affinity-1 spectrometer (Shimadzu).
2.2. Synthesis of ligand L
An ethanolic solution of 4-morpholinoacetophenone (1 mM)
was added to the solution of 4-amino-5-
pyrimidinecarbonitrile (1 mM) in ethanol were stirred and
refluxed for 2 h. Then, the above mixture was evaporated on
a water bath and washed with cold ethanol for several times.
The resulting yellow solid was filtered and recrystallized from
hot ethanol (Scheme 1).
2.3. Synthesis of complexes 1 & 2
A solution of metal(II) perchlorates in methanol (1 mM) was
stirred for 1 h and refluxed with an hot methanolic solution
417
of ligand L (1 mM) for 2 h. Then the solution was concen-
trated to one-third. The solid complex precipitated was filtered
and washed thoroughly with cold methanol and dried in vacuo.
All the complexes are soluble in DMSO (Scheme 2).
2.4. Antimicrobial studies
The antibacterial and antifungal activity of ligand L and com-
plexes 1 & 2 were tested against five different bacterial species,
Escherchia coli (E. coli), Klebsiella pneumonia (K. pneumonia),
Pseudomonas fluorescens (P. fluorescens), Shigella sonnei (S.
sonnei) & Staphylococcus aureus (S. aureus) and five different
fungal species, Aspergillus niger (A. niger), Candida albicans
(C. albicans), Candida tropicalis (C. tropicalis), Mucor indicus
(M. Indicus) & Rhizopus. The streptomycin (antibacterial) and
amphotericin (antifungal) were used as standard drugs. The
procedure as followed from reported literatures [16,17].
2.5. DNA interaction studies
The DNA interactions of ligand L and complexes 1 & 2 were
measured by electronic absorption spectra, fluorescence spec-
tra, viscometric and cyclic voltammetry experiments using pre-
viously reported literatures [18–22].
2.6. DNA cleavage studies
The DNA cleavage activity of ligand L and complexes 1 & 2
were monitored by Agarose gel electrophoresis technique
[23–25].
2.7. In vitro anticancer studies
In vitro anticancer ability of ligand L and complexes 1 & 2
were assessed by MTT assay [26]. Here, three different cancer
cell lines (MCF-7, HeLa and HEp2) and one normal (NHDF)
cell line were used to analyze the anticancer ability of synthe-
sized compounds.
3. Results and discussion
The ligand L is soluble in ethanol, methanol, acetone, acetoni-
trile, DMSO but complexes 1 & 2 are soluble in DMSO and
DMF. The metal-ligand stoichiometry ratio of complexes 1
& 2 support 1:1 which is confirmed from the analytical report
(Table S1).
3.1. 1H NMR spectra
The 1H NMR spectra of ligand L and complex 2 were carried
out in DMSO solution, expressed the following signals (SI-1
and Fig. 1(a & b)): The free ligand L, pyrimidine proton
(AN‚CHANA) at 8.61 ppm (s, 1H), pyrimidine proton
418 M. Sankarganesh et al.

Scheme 1 Synthetic route of ligand L.

Scheme 2 Proposed structure of complexes 1 & 2.

1
Fig. 1 H NMR spectra of ligand L and its complex. (a) L; (b) 2.
Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies
(AN‚CHACA) at 8.55 ppm (s, 1H), AN‚CACH3 proton at
2.09 ppm (s, 3H), aromatic CAH protons at 7.00 ppm (d, 2H)
and 7.98 ppm (d, 2H), morpholino-O-CH2- proton at
3.75 ppm (t, 4H), morpholino-N-CH2- proton (t, 4H)
3.30 ppm. After the complexation, the AN‚CACH3 proton
and pyrimidine proton AN‚CHACA signals in ligand at
2.09 and 8.55 ppm have been shifted to down field region
2.2 ppm (s, H), 8.8 (s, 1 H) in complex 2 which indicates imine
and nitrile groups of nitrogen atoms are one of the coordina-
tion sites of the ligand L around the central metal atom.
3.1. FTIR spectra
The IR spectra of the complexes 1 & 2 were studied by con-
trasting with the IR spectra of ligand L to discover the neces-
sary approach of metal and ligand (SI-1 and Fig. S1). In IR
spectra of the free ligand L, a sharp intensity m(C‚N) band
appeared at 1597 cm1 [27]. In complexes 1 & 2 the imine
nitrogen peaks appeared at 1572 and 1580 cm1 which
revealed that the imine nitrogen atom takes part in the com-
plexation. A band is appeared at 2225 cm1 due to the nitrile
group of nitrogen in ligand L. This band shifted towards lower
frequency about 2205 and 2206 cm1 in complexes 1 & 2 indi-
cating the nitrile group of nitrogen is coordinated to the cen-
tral metal atom. This fact is promote sustained by the
appearance of 403–416 cm1 assignable to m(M-N) vibrations
[28,29]. A band appeared at 1087 cm1 in the spectra of com-
plexes 1 & 2 indicates the presence of perchlorates around the
central metal atom.
3.2. Electronic absorption spectra
The electronic spectra of the ligand L and complexes 1 & 2
were recorded in DMSO at room temperature (SI-1,
Table S2 and Fig. S2). The UV spectra of ligand L exhibit
two intense bands in 255 and 318 nm which are assigned to
p-p* and n-p* transitions respectively. In absorption spectra
of complexes 1 & 2, display parallel absorption spectra of
ligand L which are shifted towards lower or higher wave-
lengths. It confirms the coordination through imine nitrogen
atom. On the other hand, the LMCT band appeared at 250–
312 nm which confirms the complexation. The electronic
spectra of complex 1 exhibit a broad band at 669 nm and
the band assignment is 2B1g ? 2E1g and 2B1g ? 2B2g transi-
tions in a square planar environment [30]. The spectral data
reveals that the complex 1 adopt square planar geometry
around the central metal ions. The electronic configuration
for the complex 2 has d10 which confirms the absence of d-
d transition, so the complex 2 exhibit INCT band at 250
and 312 nm.
3.3. ESR spectra
The ESR spectrum of complex 1 was recorded in DMSO at
300 and 77 K (Fig. S3). The g-tensor value of the complex 1
is gk > g? > ge (2.0023) indicate that the unpaired electron
present in the dx2  dy2 orbital and also suggesting a square
planar geometry around the copper(II) ion [31]. The gk =A?
value of complex 1 is 135.52 cm1 showed that the geometry
of the complex 1 adopt square planar environment [32]. The
419
value of exchange interaction term G was calculated from
g 2:0023
the equation, G ¼ g?k 2:0023. G-value obtained from complex 1
is greater than four, indicate the absence of exchange interac-
tion between Cu(II) centers in the solid state [33]. The observed
value for exchange interaction parameter for the complex 1
have higher G value (>4) suggested that the local tetragonal
axes are aligned parallel or only slightly misaligned and there
are no interaction between the copper centre in DMSO [34].
3.4. Mass spectra
The ESI mass spectra of the ligand L and complex 1 & 2
recorded at room temperature were used to calculate their sto-
ichiometry composition (SI-1 and Fig. S4 (a-c) & Fig. S5 (a &
b)). The ligand L showed a molecular ion peak at m/z 307 and
fragmented ions are C5H2N+ 4 (1 1 8), C12H15NO
+
(1 8 9),
+
C13H9N4 (2 2 1), C16H17N4O (2 8 1), C16H16N5O+ (2 9 1),
+
C17H17N5O+ (3 0 7). The molecular ion peak for the complex
1 at m/z 568 and the fragmented ions are C17H17N5OCu
(ClO4)+ (5 6 8), C17H17N5OCu2+ (3 7 0), C17H17N5O+
2
(3 0 7), C16H16N5O+ (2 9 1), C16H17N4O+ (2 8 1), C13H9N+ 4
(2 2 1), C12H15NO+ (1 8 9) and C5H2N+ 4 (1 1 8). The molecu-
lar ion peak for the complex 2 at m/z 568 and the fragmented
ions are C17H17N5OZn(ClO4)+ 2 (5 6 8), C17H17N5OZn2+
+
(3 7 0), C17H17N5O (3 0 7), C16H16N5O+ (2 9 1),
C16H17N4O+ (2 8 1), C13H9N+ (2 2 1), C12H15NO+ (1 8 9)
4
and C5H2N+ 4 (1 1 8). These observed results confirm that,
the stoichiometry of metal chelates as ML type.
3.5. Theoretical studies
Density Functional Theory (DFT) calculation was executed by
using B3LYP/6-31G and B3LYP/LANL2DZ basis set using
Gaussian 09 program [35] respectively. From the optimized
structure of ligand L, the bond length of N„C and CH‚N
was determined to be 1.17 Å and 1.40 Å. After the chelation
of Cu(II) with ligand L in complex 1, the bond length of
N„C and CH‚N was shortened to 1.08 Å and 1.29 Å. Sim-
ilarly in complex 2, the bond length was calculated to 0.98 Å
and 1.22 Å. These information obviously exposed that the
coordination complex formation of Cu(II) and Zn(II) with
ligand L in complex 1 and 2, respectively. Besides, the HOMO-
LUMO band gap of complexes 1 & 2 was obviously
decreased and dipole moment was increased as compared with
ligand L as depicted in Table 1. The computed HOMO and
LUMO diagrams of ligand L and complexes 1 & 2 are repre-
sented in Fig. 2. In ligand L, the electron clouds of HOMO
were spread over the morpholine group and aromatic moiety
whereas LUMO spread over the richer in morpholine group
and slightly in aromatic compounds. Interestingly, after the
complexation with Cu(II), electron clouds of HOMO and
LUMO were spread over the whole molecule of the frame
work with concerned over metal centre. Correspondingly, in
complex 2, the electron clouds of HOMO were spread on the
morpholine group and aromatic compounds where as the elec-
tron clouds of LUMO was spread only in the metal centre. The
above result shows that, the complex 1 has involved in the
LMCT mechanism rather than complex 2. Also, DFT calcula-
tions were theoretically supported by the proposed mecha-
nism. The theoretically calculated absorption spectral data of
420 M. Sankarganesh et al.

Table 1 The evaluation of calculation results for optimized structures of ligand L and complexes 1 & 2.
Entry Calculation results Ligand L Complex 1 Complex 2
1 Basis set B3LYP/6–31G B3LYP/LANL2DZ B3LYP/LANL2DZ
2 Dipole moment(Debye) 5.59 22.21 14.66
3 HOMOLUMO 3.98 eV 1.85 eV 1.36 eV
4 Electronegativity (v) 3.79 eV 5.95 eV 5.65 eV
5 Hardness (c) 1.99 eV 2.45 eV 0.68 eV
6 Chemical potential (p) 3.79 eV 5.95 eV 5.65 eV
7 Softness (d) 0.50 eV 0.41 eV 1.47 eV

Fig. 2 DFT computed HOMO and LUMO diagrams of ligand L and complexes 1 & 2.

Table 2 DFT calculated the excitation energies for the lowest transition (eV, nm), composition in terms of molecular orbital
involvement and absorption experimental maxima.
Compounds State Composition Theoretical (nm) Experimental (nm)
L S1 H ? L (98.1%) 312 318
2 S1 H ? L (75.8%) 507 669
H = HOMO, L = LUMO.

ligand L and complex 1 are more reliable with the experimental
data as illustrated in Table 2.
3.6. Antimicrobial activity
Antibacterial activity of ligand L and complexes 1 & 2 were
screened against E. coli, K. pneumonia, P. fluorescens, S. sonnei,
S. aureus using Muller-Hindon agar medium by well diffusion
method using DMSO as solvent and streptomycin (control
drug) (Fig. 3a & Table 3). These results show that ligand L
and complexes 1 & 2 are having good antibacterial activity
against all five different bacterial species in a dose dependent
manner. Furthermore, the zone of inhibition results have been
suggested that, complex 1 having greater antibacterial activity
than ligand L and complex 2. Also, the synthesized com-
pounds have significant antibacterial activity against E. coli,
bacteria (ligand L, 7; complex 1, 9 & complex 2, 8 mm) as com-
pared to other bacterial species.
Antifungal activity of ligand L and complexes 1 & 2 against
A. niger, C. albicans, C. tropicalis, M. Indicus, Rhizopus by
Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies 421

Fig. 3 Antimicrobial studies of ligand L and complexes 1 & 2 (a) antibacterial; (b) antifungal.

Table 3 Zone inhibition values (mm) of ligand L and complexes 1 & 2 against five different bacterial species.
Compounds Zone of inhibition (mm)
E. coli K. pneumonia P. fluorescens S. sonnei S. aureus
1 9 7 7 8 7
2 8 5 5 5 6
L 7 4 3 3 3
Streptomycin 10 18 14 16 16

paper disc method using DMSO as solvent and control drug
amphotericin (Fig. 3b). The zone of inhibition values of
amphotericin, ligand L and complexes 1 & 2 are against the
five different fungal species are depicted in Table 4. The result
shows that complex 1 has higher antifungal activity than
ligand L and complex 2. And also, the inhibitory activity of
synthesized compounds against C. albicans (ligand L, 10; com-
plex 1, 18 & complex 2, 10 mm) has higher than other fungal
strains.
3.7. DNA interaction studies
3.7.1. Absorption titration
The binding mode of ligand L and complexes 1 & 2 with CT-
DNA were studied by using the electronic absorption spectra
in different buffer solutions (sodium-acetate, Tris-HCl & sodi-
umbicarbonate) at multiple pH media (4.0, 7.0 & 10.0). In
general, hypochromism can be examined for the groove bind-
ing mode between compound and DNA, results show that the
position of the absorption band is do not change. It can be cor-
related with the degradation of the DNA double helix struc-
ture [36,37]. The absorption of spectra of the complexes 1 &
2 in the presence and absence of DNA are shown in Fig. 4.
The addition of DNA increases to the rigid concentration of
ligand L and complexes 1 & 2 results shows that hypochro-
mism (20.26%, L; 85.91%, 1; 42.73%, 2 at pH 4.0) to the
red shift (3 nm). The results confirmed that the interaction of
DNA with synthesized ligand L and complexes 1 & 2 owing
to the presence of pyrimidine-morpholine moieties. Due to
the presence of methyl group [38] and not sufficient planarity
of the structure of ligand L and complexes 1 & 2 which induces
the groove binding mode of interaction not intercalation. The
intrinsic binding constant (Kb) of ligand L and complexes 1 & 2
at various pH media (4.0, 7.0 & 10.0) were calculated using the

Table 4 Zone inhibition values (mm) of ligand L and complexes 1 & 2 against five different fungal species.
Compounds Zone of inhibition (mm)
A. niger C. albicans C. tropicalis M. Indicus Rhizopus
1 8 18 11 8 8
2 7 17 8 6 7
L 4 10 7 5 5
Amphotericin 18 17 31 9 24
422 M. Sankarganesh et al.

Fig. 4 Absorption spectra of ligand L and complexes 1 & 2 with CT-DNA in different buffer solution containing multiple pH media. pH
4.0 (a, L; b, 1 & c, 2), pH 7.0 (d, L; e, 1 & f, 2) and pH 10.0 (g, L; h, 1 & i, 2).

formula (Table 5), ½DNA=ea  ef ¼ ½DNA=eb  ef
þ½Kb ðeb  ef Þ1 ; where, [DNA] is the concentration of base
pairs of DNA. The apparent absorption coefficients ea, ef
and eb correspond to Aobs./[M], the extinction coefficient for
the free complex and extinction coefficient for the complex in
the fully bound form respectively. The Kb values of present
compounds are higher in acidic medium (Kb = 2.42
105, L;
2.8
105, 1; 2.65
105, 2 (pH 4.0)). From the Kb values, the
ligand L and complexes 1 & 2 are potential binding ability in
acidic medium than neutral and basic medium. The Kb values
of complexes 1 & 2 have good binding affinity to DNA than
that of ligand L. Moreover, the Kb values of these synthesized
compounds were lower than those of classical intercalator
(ethidium bromide, 1.4
106 M1) [39]. The decrease of the
binding constants could be explicated by the presence of
methyl group and the absence of sufficient planarity in the
ligand framework and thus encouraging a groove binding
mode for the prepared compounds.
3.7.2. Competitive binding studies
The fluorescence spectra of the EB bound DNA in the pres-
ence of the ligand L and complexes 1 & 2 were depicted in
Fig. 5. Here, the ligand L and complexes 1 & 2 were act as sec-
ondary molecules when added to the EB bound DNA. Fig. 5
shows that the incremental concentration of complexes 1 & 2
to the EB bound DNA, the fluorescence intensity of complex
1 decrease gradually to attained the 50% reduction than
ligand L and complex 2. From this observation, the interaction
of DNA with complex 1 is more potent than ligand L and com-
plex 2. The above results revealed that the competitive interac-

Table 5 Electronic absorption spectral properties of interaction of ligand L and complexes 1 & 2 with CT-DNA with different pH
media ranging from 4 to 10.
Compounds Binding constants (Kb) (Different pH) (M1) Hypochromism (H%)
4.0 7.0 10.0 4.0 7.0 10.0
L 2.42
105
5.14
104
1.37
104
20.26 17.35 16.89
1 2.80
105 2.00
105 1.86
105 85.91 45.99 42.13
2 2.65
105 1.49
105 2.22
104 42.73 25.73 21.09
Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies 423

Fig. 5 Emission spectra of DNA bound EB in the presence of ligand L (a) and complexes 1 (b) & 2 (c). The arrow indicates the changes
in the emission intensity. Insert: The plot of I0 =I vs ½Q.

tions between compounds and EB bound DNA due to the

replacement of EB. The binding nature of the ligand L and
complexes 1 & 2 with DNA bound EB were precised by
Stern-Volmer equation, [40], I0 =I ¼ 1 þ KSV ½Q, where, I and
I0 are emission intensities in the presence and absence of
quenchers respectively. KSV is linear Stern-Volmer quenching
constant and [Q] is the concentration of quencher. KSV value
attained from the slope of the plot I0 =Ivs½Q (Fig. 5). The
Ksv values of the present compounds are following the order:
1 (1.89
105) > 2 (3.94
103) > L (3.3
103) (Table 4).
Moreover, the binding affinity ðKapp Þ of ligand L complexes
1 & 2 in contrast of ethidium bromide was calculated by using
the following equation [41], KEB ½EB ¼ Kapp ½Complex, where,
[complex] is the concentration of the compounds necessary
for the 50% reduction in the fluorescence intensity of EB
and KEB = 1.0
107 M1 (Table 6). The apparent binding
constants ðKapp Þ at room temperature are given as follows: 1
(1.89
106) > 2 (1.11
106) > L (1.25
105). The calculated
Fig. 6 Effect of increasing amount of ligand L and complexes 1
values of KSV and Kapp provide a good concurrence of the dis-
& 2 on the relative viscosities of CT-DNA in Tris-HCl buffer
placement of ligand L and complexes 1 & 2 in DNA bound
solution.
EB. The competitive binding results were compared to elec-
tronic absorption results and they suggested that the interac-
tion between DNA and ligand L and complexes 1 & 2 3.7.4. Electrochemical studies
through groove binding mode.
The cyclic voltammogram of complexes 1 & 2 in the absence
and the presence of different concentration of DNA are shown
3.7.3. Viscosity measurements
Fig. 7. The addition of DNA increases to the complexes 1 & 2
To promote the clarification of the mode of binding in the causes the cathodic (1) and anodic (2) peak current decreases
ligand L and complexes 1 & 2 with CT-DNA were analyzed of the complexes and viceversa. The incremental concentration
by viscosity measurements. The plots of relative viscosity ver- of DNA to the complex 1 & 2 causes the cathodic and anodic
sus [complex]/[DNA] (Fig. 6) shows that the viscous flow of peak potential shifted towards the negative directions. These
DNA is disorderly increasing the concentration of ligand L results confirmed that the complexes 1 & 2 are interacting with
and complexes 1 & 2. These results have been established that DNA. The spectrophotometric, spectroflurometric and visco-
interaction of ligand L and complexes 1 & 2 with DNA via metric results are correlated to the cyclic voltammogram
groove binding mode which is also confirmed from the results results, the complexes 1 & 2 can be bind with DNA via groove
of electronic and competitive binding studies. binding mode.

3.7.5. DNA cleavage studies

Table 6 The quenching constant (Ksv) and binding affinity The prospective of ligand L and complexes 1 & 2 to cleave CT-
(Kapp) of the interaction of ligand L and complexes 1 & 2 with DNA assayed with the help of gel electrophoresis in the
CT-DNA. absence and presence of external agents such as hydrogen per-
Compounds Ksv (M1) Kapp (M1) oxide (H2O2), 3-mercapto propionic acid (MPA) and photo-
induced light are shown in Fig. 8(a-d). The fastest migration
L 3.3
103
1.25
105
1 1.89
105 1.66
106
was observed for the open circular form (Form I). If one
2 3.94
103 1.11
106 strand is cleaved a slower-moving linear form (Form-II) was
observed [42].
424 M. Sankarganesh et al.

Fig. 7 The cyclic voltammogram of complexes 1 & 2 in Tris-HCl buffer at 25 °C in the presence of increasing amount of DNA: (a) 1; (b)
2. Arrow indicates the changes in voltammetric peak current and potentials upon increasing the DNA concentration.

The chemical nuclease activity of the ligand L and com-

plexes 1 & 2 were monitored in the presence of oxidizing and
reducing agents. This result suggests that complex 1 is found
highly active in the presence of oxidizing and reducing agents

Fig. 9 In vitro anticancer studies of ligand L and complexes 1 &

2 on human cancer (MCF-7, HeLa, HEp2) cell lines and normal
(NHDF) cell line.

than ligand L and complex 2. Control experiments using only

MPA and H2O2 did not show appreciable DNA cleavage.
The chemical nuclease activity of the ligand L and com-
plexes 1 & 2 were monitored in the absence of external reagents
and under illuminated conditions using UV source at 360 nm
(8 W). The ligand L and complexes 1 & 2 do not show any
appreciable activity in the absence of external agents and
under illuminated conditions due to their low redox stability
in the absence of any relevant external agents.

3.8. In vitro anticancer studies

The in vitro anticancer activities of the newly synthesized com-

pounds on three different human cancer cell lines (MCF-7
Fig. 8 Agarose gel diagram showing cleavage studies of ligand L (human breast adenocarcinoma), HeLa (human cervical),
and complexes 1 & 2 with CT-DNA at RT. Lane 1 DNA control; HEp-2 (human laryngeal)) and one normal (NHDF normal
Lane 2 DNA + L; Lane 3 DNA + 1; Lane 4 DNA + 2; (a-d). human dermal fibroblasts) cell line were performed by using
Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies
Table 7 In vitro anticancer activities of ligand L and complexes 1 & 2 against cancer cell lines and normal cell line.
Compounds IC50 values (mg/mL)*
MCF-7
L 104.88
1 62.21
2 75.46
MTT assay (Fig. 9) [32]. The IC50 (mg/mL) values of synthe-
sized compounds against cancer cell lines and normal cell line
are shown in Table 7. From IC50 values results, ligand L does
not affect both the cancer as well as normal cell line where as
complexes 1 & 2 can moderately affect the cancer cell lines and
least affect the normal cell lines. As a result, newly synthesized
complexes 1 & 2 are having moderate cytotoxic ability on can-
cer cell lines and low toxic ability on normal cell line as com-
pared to ligand L. And also, the IC50 values of synthesized
compounds have been suggested that, the ligand L and com-
plexes 1 & 2 are highly targeted to kill the MCF-7 cancer cell
line than other cancer cell lines.
4. Conclusion
In summary, the new Schiff base ligand L and complexes 1 & 2
were successfully prepared from 4-morpholinoacetophenone
and 4-amino-5-pyrimidinecarbonitrile. The synthesized com-
pounds were characterized by multiple spectroscopic tech-
niques. The spectral results supports the proposed structure
of the complexes 1 & 2 adopt may be square planar geometry.
The LMCT mechanism between ligand L and M(II) in com-
plex 1 was theoretically confirmed by DFT calculation. Com-
plexes 1 & 2 have potent antimicrobial activity than ligand L.
The interaction studies of the ligand L and complexes 1 & 2
with DNA results suggest that they were involved in groove
binding interaction. Chemical nuclease activity of ligand L
and complexes 1 & 2 results have been established that com-
plex 1 is more potent cleavage ability than ligand L and com-
plex 2. The in vitro anticancer studies result have been
suggested that, the complexes 1 & 2 have moderate cytotoxic
ability against cancer cell lines and low toxic effect on normal
cell lines. Further studies are needed to prove the chemothera-
peutic properties of pyrimidine-morpholine based Cu(II) and
Zn(II) complexes in future.
Acknowledgements
The authors acknowledge the DST-SERB (Ref. No.: SR/FT/
CS-117/2011 dated 29.06.2012), Government of India, New
Delhi for the financial support. The authors also express their
sincere and heartfelt thanks to Managing Board, Dean, Princi-
pal, Head and staff members, Chemistry Research Centre,
Mohamed Sathak Engineering College, Kilakarai for constant
encouragement and providing laboratory facilities.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jscs.2017.
08.007.
425
HeLa HEp2 NHDF
105.67 107.55 108.33
69.35 71.08 106.12
80.81 86.28 108.57
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