Synthesis and characterisations of copper(II) complexes of 5-methoxyisatin thiosemicarbazones: Effect of N-terminal substitution on DNA/protein binding and biological activities

Article abstract of DOI:10.1016/j.ica.2019.04.019

The newly synthesized ligands (HL1-HL3) of 5-methoxyisatin thiosemicarbazone with different N-terminal substituents and their copper(II) complexes 1–3 respectively were exposed to spectral and structural characterization. The spectral studies reveal that

Full text of DOI:10.1016/j.ica.2019.04.019

InorganicaChimicaActa492(2019)131–141
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Inorganica Chimica Acta
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Research paper
Synthesis and characterisations of copper(II) complexes of 5-methoxyisatin
thiosemicarbazones: Effect of N-terminal substitution on DNA/protein
binding and biological activities
⁎
K.N. Aneesrahmana, K. Ramaiahb, G. Rohinia, G.P. Stefya, N.S.P. Bhuvaneshc, A. Sreekantha,
a Department of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India
b Department of Chemistry, National Institute of Technology, Warangal 506004, India
c Department of Chemistry, Texas A & M University, College Station, TX 77842, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The newly synthesized ligands (HL1-HL3) of 5-methoxyisatin thiosemicarbazone with different N-terminal
substituents and their copper(II) complexes 1–3 respectively were exposed to spectral and structural char-
acterization. The spectral studies reveal that the ligands show a tridentate mode of binding (ONS donor) with Cu
(II) to form square planar complexes. The crystal structure of the ligand HL1 and complex 1 were confirmed by
single crystal XRD. The compounds were subjected to DNA/BSA interaction studies using electronic and
fluorescence spectroscopic techniques. The binding studies showed that, the complexes (1–3) were able to bind
with DNA/BSA macromolecules with good binding constant. In which 1 (Kb = 0.83 × 105 M−1) shows good
interaction with the DNA while 3 shows better interaction with BSA (Kb = 2.4 × 105 M−1). The complexes (1–3)
were also tested for antimicrobial activity, radical scavenging activity and in vitro anti-proliferative activity.
Complex 3 have shown potent efficacy with broad spectrum antimicrobial activity against the microorganisms,
whereas the complex 1 has shown decent scavenging properties compared to the reference drug (ascorbic acid)
Isatin derivatives
Copper(II) complexes
DNA/BSA interaction
Antimicrobial
Antioxidant
In vitro anti-proliferative activity
with an IC50 value of 19.23
1 has exhibited broad spectrum activity against MCF-7, A549 and HeLa with IC50 values 14.83
17.88 0.16 μM and 6.89 0.42 μM, respectively compared to standard drug Doxorubicin.
1.05 µM. Further the, anti-proliferative activity study revealed that, the complex
0.45 μM,
1. Introduction
complexes are one of the better choices for the cellular level medicinal
applications as therapeutic agents. There were copper(II) complexes of
Schiff bases have been reported with good chemical nuclease activity
[4,5], Moreover, a number of copper complexes have been reported as
potential antitumor substances, proving significant anticancer activity
while maintaining lower toxicity than cisplatin [6–8].
Cisplatin being a successful model in the medical treatment of
several types of tumours, has made greater interest in developing metal-
based drugs. Even though the cisplatin is effective in healing varieties of
cancers, their limitations such as resistance, serious toxicity and other
side effects [1] forces the chemists to search for alternative metallo-
drugs with more efficient, target specific and less toxicity. As a result,
numerous metal complexes with deferent combinations of metals and
ligands were reported [2].
The earlier studies on platinum complexes showed the significance
of the ligands in tuning the activity and efficiency of metal complexes
[9,10]. Hence the choice of ligand is also important in the metal
complex based drugs. Literature shows that thiosemicarbazones (TSC)
are the class of ligands with remarkable biological activity and med-
icinal properties [11–13]. TSCs shows wide spectrum of biological ac-
tivities such as antitumour [14], antiviral [15], anti-HIV [16], anti-
fungal [17,18], antimalarial [19], antibacterial [20], anti-filarial
activities [21]. The biological activities of the TSCs can improve sig-
nificantly by complex formation with various metal ions. The thiose-
micarbazone complexes of transition metals with the successful
The selection of metal ion is one of the significant factors in the
development of metal-based therapeutic agent. The copper compounds
have been active in research since 1980s. Copper is known to be bio-
essential metal in the human body and is a catalytic and structural
centre of many metalloenzymes and proteins. Hence copper involved in
many redox active mechanisms such as antioxidative activity (SOD) and
energy metabolism [3]. Therefore biologically compatible copper(II)
⁎ Corresponding author.
E-mail address: sreekanth@nitt.edu (A. Sreekanth).
https://doi.org/10.1016/j.ica.2019.04.019
Received 8 November 2018; Received in revised form 1 March 2019; Accepted 10 April 2019
Availableonline11April2019
0020-1693/©2019PublishedbyElsevierB.V.

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
demonstration of anticancer activities have been reported [22,23].
The present work discusses copper complexes of 5-methoxyisatin
4N-substituted thiosemicarbazones ligands. Previous reports are sug-
gesting that, 4N-substitution can alter the activities of the thiosemi-
carbazones and its complexes [24–26]. Thiosemicarbazones are usually
less soluble water, which may reduce its bioavailability at the same
time, the hydrophobicity or lipophilic nature is an important factor for
a molecule its cell availability. Hence the right balance between a
molecule’s hydrophilicity, which promotes solubility and bioavail-
ability, and lipophilicity, which allows these molecules for the cell
membrane permeability through lipidic bilayer, is an essential re-
quirement for an efficient drugs and its cellular uptake [27]. Three
different terminal substituent have chosen for the present study, they
are pyrrolidine, morpholine and cyclohexyl ammine. When these mo-
lecules are fused into the TSCs, and considering the polar nature and
hydrogen bonding capability with the water molecules, pyrrolidine
substituent become more hydrophobic and cyclohexyl substituted TSC
become less hydrophobic due to the presence hydrogen at 4 N position
and possible hydrogen bonding interactions. On this basis, the study
also aim to look the influence various fragments on the terminal amino
group on various biological studies. The synthesised compounds were
explored for their in vitro antibacterial, antifungal, scavenging and anti-
proliferative studies along with DNA and Protein binding studies.
transfer (LMCT) transition, spreading in to the visible region [28]. The
spectra of the complexes in solution did not show the absorption band
responsible for the d–d transition. But the solid-state DRS spectra ex-
hibited a d–d band as a weak shoulder band in the range of 610–640 nm
[29–31].
In the FT-IR spectra, ligands showed bands in the range of
3376–3471,
3136–3281,
1691–1693,
1525–1593
and
1341–1349 cm−1, which were ascribed to the stretching vibrations of
indole NeH, azomethine NeH, C]O, C]N and C]S groups, respec-
tively (Figs. S3–S5). Both NeH vibrations were showed weak bands for
all ligands. In HL3, a weak band was observed at 3239 cm−1, which
was assigned to the terminal NeH. The intensities of carbonyl vibra-
tions was medium for HL1 while strong for HL2 and HL3. Complexa-
tion causes the shifting of these bands considerably (Figs. S6–S8). There
was a shift in the frequencies of carbonyl (1656–1663 cm−1), and
thiocarbonyl (1326–1332 cm−1) groups. Intensities of both bands were
reduced (strong to medium for ʋC]O and medium to weak for C]S).
Furthermore, the band at 3136–3278 cm−1 was disappeared, which
was due to the deprotonation of the azomethine NeH and resulted in
the appearance of new bands in the region with medium to strong in-
tensities at 1580–1628 cm−1 region, corresponding to the newly
formed N]C due to the coordination via thiol sulfur [28,32,33]. The
appearance of new bands in Far IR region of the spectra resultant to the
metal-ligand vibrations further confirms the formation of the complexes
(Figs. S9–S11). The ʋ(CueN) stretching frequencies for the azomethine
nitrogen are observed around 278–280 cm−1 region. The formation of
new band in the 260–263 cm−1 range was assigned to ʋ (CueS), is
another evidence for the involvement of sulfur coordination from the
ligands. All complexes showed strong ʋ(CueCl) vibrations at
319–320 cm−1 region indicating the CueCl bond in the complexes. The
presence of new bands at 460–469 cm−1 range corresponding to the ʋ
(CueO), proves the coordination of Oxygen from the carbonyl group of
isatin to the copper center [28,34,35]. Thus, it suggested that the
thiosemicarbazone coordinates to Cu(II) through an ONS donor atoms
from thiosemicarbazone ligands.
2. Results and discussion
2.1. Synthesis
The Schemes 1 and 2 represent the synthesis of ligands (HL1-HL3)
and their copper(II) complexes (1–3). Both the ligands and complexes
are air stable and non-hydrophobic. All the ligands (orange to orange
red) and complexes (chocolate) coloured powder. Both ligands and
complexes are insoluble in water and common organic solvents but
soluble in DMSO and DMF. The ligands are slightly soluble in methanol.
Mass and elemental analysis of the complexes suggested that the ligand
and metal ratio of the complex is 1:1 of the type [M(L)(Cl)]. Here the L
indicates the deprotonated ligands. The structure of HL1 and 1 was
further elucidated by single crystal X-ray diffraction method.
The NMR spectra were recorded for the ligands (HL1-HL3) in
DMSO‑d6 solvent and the detailed NMR data of each ligand are given in
the experimental section. In the 1H NMR spectra of the ligands, the
HeNeC]S proton resonates at 12.72–13.38 ppm. The signals of pyr-
rolidine, morpholine and cyclohexyl protons on the terminal N of
thiosemicarbazone appeared as multiplets in the region 4.01–1.09 ppm.
The amide NeH proton is in the range of 10.84–11.15 ppm. All the
aromatic protons are seen as multiplets around 6.87–7.09 ppm. The
methoxy substituent in the ligands showed signal as a singlet peak in
the range of 3.42–3.81 ppm. In HL3 the thiosemicarbazone terminal
NeH proton showed a singlet at 7.90 ppm. The 13C NMR spectra of the
ligands showed the chemical shift values at 175.8–179.9, 163.2–163.4,
155.7–155.8 ppm due to C]S, C]O and C]N groups, respectively.
The aromatic carbon was observed in the range, 136.9–106.9 ppm. The
2.2. Spectroscopy
Electronic absorption spectra (Figs. S1 and S2) of the ligands (HL1-
HL3) exhibited significant bands around 286–305 nm and 348–381 nm,
attributable to π–π* and n–π* transitions, respectively. Whereas, a slight
shift in energy of these bands were observed on complexation. The
spectra of the complexes (1–3) showed bands in the range 269–274 nm
and 346–361 nm, corresponds to π–π* and n–π* intra ligand transitions.
Additionally, a third band was observed for complexes in the range of
416–422 nm, as a result of the S → Cu(II) ligand to metal charge
Scheme 1. Synthesis of 5-methoxyisatinthiosemicarbazone derivatives (HL1-HL3).
132

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
Scheme 2. Synthesis of copper(II) complexes (1–3).
peak corresponding to methoxy carbon was seen at 56.09 ppm, and the
signals of the aliphatic carbon present on the terminal N of thiosemi-
carbazone appeared in the region of 66.19–24.12 ppm.
Table 1
EPR parameters of complexes 1–3.
Complex
g∥
g⊥
G
A∥(mT)
The ESR spectra of all the Cu(II) complexes were recorded in DMF
solution at liquid nitrogen temperature (Figs. 1 and S12). The spectral
data of all the complexes are given in Table 1. The ESR spectra of Cu(II)
complexes show two peaks, one of small intensity towards low field
region and the other of large intensity towards high field region. From
1
2
3
2.214
2.217
2.219
2.043
2.051
2.039
5.20
4.40
5.90
14.55
14.33
14.43
these peaks the vales of
g
and
g
have been calculated [36]. The g-va-
Hathaway and Billing [39], If the value of G is larger than 4, the ex-
change interaction is negligible while G values of less than 4 indicate
considerable interaction in solid complexes. The present G values are
found to be greater than 4, indicating that there is no exchange inter-
action in the solid state, further confirms the mononuclear nature of the
complexes.
lues of Cu(II) complexes can be used to derive the ground state. In
elongated octahedral and square planar complexes, the unpaired elec-
2
2
2
tron lies in the dx−y orbital giving B1g as the ground state with
g
>
g
. In a compressed octahedron, on the other hand, the unpaired
2
2
electron lies in dz orbital giving A1g as ground state with
g
<
g
.
From the observed values (Table 1) it is evident that the unpaired
electron lies predominantly in the dx−y2 orbital characteristic of square
2
planar geometry [35]. Recalling the complexes with dx−y2 ground state,
2
2.3. Single crystal X-ray crystallographic studies
strong interaction along Z axis is to be accompanied by increase in the
value of g∥. Strong axial bonding leads to an increase in the length of
the bond in the x-y plane which results in a decrease of both in plane
The crystal structures of the ligand HL1 and the complex 1 are
shown in Fig. 2. The crystal data and structure refinement parameters,
selected bond lengths and bond angles are listed in Tables 2, 3 and
S1–S3). The HL1 crystallized in the triclinic crystal system with space
group P−1 with two molecules in the unit cell. The molecule is in
thione form with trance configuration of the thiocarbonyl group and
isatin moiety with respect to N2eN3 bond (Fig. 2b). The bonds in the
eC]NeN]C(]S)eN skeleton of the molecules shows partial double
bond and single bond character (Table 2) indicates the delocalization of
π electrons over the eC]NeN]C(]S)eN chain. Structure of the
compound brings out quasi-coplanarity of the whole molecular skeleton
which was evidenced by torsional angles. The crystal structure further
reveals an intramolecular hydrogen bonding between hydrazinic hy-
drogen (N3–H) and the oxygen of indole carbonyl (C1]O1). Further,
there is an intramolecular hydrogen bonding between the hydrogen on
the indole (NeH) and the water molecule in the crystal lattice (Table
S2). The complex 1 was crystallised in a monoclinic crystal system with
a space group of P1211 and consists of two separate mononuclear Cu(II)
complexes in the unit cell (Fig. 2b). The Cu(II) centre is in a (4 + 1)
square-pyramidal coordination geometry, in which the thiosemicarba-
zone ligand (HL1) is coordinated in a tridentate manner through
oxygen (O1), nitrogen (N1) and sulphur (S1). The fourth coordination is
occupied by chloride ion, forming an equatorial plane of square-pyr-
amidal geometry. The DMSO molecule in the lattice is coordinated to
the copper centre via an oxygen atom (O3) at the axial position. In the
equatorial plane, the two angles S1eCu1eO1 and N1eCu1eCl1 be-
tween the trans-oriented ligator atoms are 162.68(17)° and 164.6(2)°,
respectively. Furthermore, the summation of the angles (Table 3) be-
tween the donor atoms around the Cu1 is 357° (360° ideal value for in-
plane angles), affirming distortion from perfect square planar geometry
[40]. The azomethane NeH proton of HL1 was deprotonated during the
covalency and the energy of dx−y2 transition. Both these effects tend to
2
increase the value of
g
. The
g
is the most sensitive function for in-
dicating the covalency, being 2.3 or more for ionic compounds and less
than 2.3 for covalent compounds [37,38]. It is clear from Table 1 that
obtained is less than 2.3 indicating covalent character of the metal–li-
gand bond. The axial symmetry parameter G defined as ( – 2.0023)/
g
g
(
g
– 2.0023) is shown to be a measure of the exchange interaction
between copper centres in the polycrystalline solids. According to
Fig. 1. X-band EPR spectrum of complex 1 in frozen DMF solution.
133

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
Fig. 2. (a) Thermal ellipsoid plot of HL1 and (b) Thermal ellipsoid plot of 1.
various drugs, especially for anticancer agents involving the metal ions,
hence the investigation of the interacting ability of metal complexes
with DNA is extremely significant for the development of therapeutic
agent with great medicinal value. Small molecules can interact with
DNA in different ways such as (i) intercalation to adjacent base pairs;
(ii) electrostatic binding to the sugar-phosphate backbone of the DNA
helix; (iii) groove binding (iv) the covalent binding of metal complexes
to the nitrogen atoms of bases pairs of DNA [41]. The type and extent of
binding of complexes (1–3) to CT-DNA were studied by different
techniques.
Table 2
Selected bond lengths (Å), angles (°) of ligand HL1.
Bond lengths (Å)
Bond angle (°)
S(1)–C(10)
O(1)–C(1)
N(2)–N(3)
N(2)–C(2)
N(3)–C(10)
N(4)–C(10)
C(1)–C(2)
1.673(2)
1.236(3)
1.349(3)
1.300(3)
1.375(3)
1.333(3)
1.505(3)
O(1)–C(1)–N(1)
127.2(2)
126.1(2)
127.0(2)
121.9(2)
123.67(18)
123.44(19)
O(1)–C(1)–C(2)
N(2)–C(2)–C(1)
N(2)–N(3)–C(10)
N(3)–C(10)–S(1)
N(4)–C(10)–S(1)
Torsional angle (°)
O(1)–C(1)–C(2)–N(2)
N(2)–N(3)–C(10)–S(1)
N(2)–N(3)–C(10)–N(4)
−3.1(4)
11.1(3)
2.4.1. Electronic absorption titration
−170.5(2)
The UV–Vis absorption spectroscopy is an effective way to analyse
the binding affinity and interaction mode of metal complexes with
DNA. The complexes (1–3) showed absorption bands in the range of
416–422 nm. Upon the titration of CT-DNA with the complexes 1–3, a
significant decrease in molar absorptivity (hypochromism) with a slight
towards longer wavelength (redshift) was observed (Figs. 3a and S13).
The above observations are generally associated with intercalative
mode of interaction involving a strong stacking interaction between the
base pairs of the DNA and the aromatic chromophore present in the
complexes [42]. Also, the strength of interaction of metal complexes
with DNA can be correlated to the observed hypochromism. For the
complexes 1–3, the order of hypochromism was observed as
1 > 2 > 3, which indicate that the complex 1 have higher affinity
and complex 3 showed the least interaction. In order to obtain the
coordination with Cu(II) ion and hence the thiosemicarbazone (HL1)
act as mono-anionic ONS pincer type ligand, further evidenced by an
increment of the C10eS1 bond length [1.673 (L1) and 1.728 (1)]. This
indicates the thiolate coordination of sulphur. The crystal data of 1
further shows an intermolecular hydrogen bonding between the indole
NeH of a molecule and carbonyl oxygen and chlorine of another mo-
lecule in the crystal lattice (Table S3).
2.4. DNA binding studies
Deoxyribonucleic acids (DNA) are the vital cellular components,
which contain the instructions in the form of base sequences for the life
process such as cell survival, proliferation and other biological pro-
cesses. Transitions of the sequences enable the synthesis of proteins
which are found in many forms (hormones, enzymes, structural pro-
teins, etc.), executing all body process. Hence any changes to the DNA
nature can alter the normal cell activities and can prevent the cell
multiplications. This makes the DNA as the pharmacological target for
quantitative comparison of DNA interaction, the binding constant (Kb
)
of the complexes were calculated by examining the UV–Vis spectral
data with Eq. (1) [43].
[DNA]/(
f ) = [DNA]/(
f ) + 1/Kb (
)
f
a
b
b
(1)
where
Table 3
Selected bond lengths (Å), angles (°) of complex 1.
Bond lengths (Å)
Bond angles (°)
Torsional angles (°)
Cu(1)–Cl(1)
Cu(1)–S(1)
Cu(1)–O(1)
Cu(1)–O(3)
Cu(1)–N(1)
S(1)–C(10)
O(1)–C(2)
N(1)–N(3)
N(1)–C(1)
N(3)–C(10)
N(4)–C(10)
2.241(2)
2.273(2)
2.103(6)
2.250(5)
1.989(7)
1.728(9)
1.258(10)
1.336(9)
1.292(11)
1.377(11)
1.323(10)
Cl(1)–Cu(1)–S(1)
Cl(1)–Cu(1)–O(3)
O(1)–Cu(1)–Cl(1)
O(1)–Cu(1)–S(1)
N(1)–Cu(1)–O(1)
O(1)–Cu(1)–O(3)
O(3)–Cu(1)–S(1)
N(1)–Cu(1)–Cl(1)
N(1)–Cu(1)–S(1)
N(1)–Cu(1)–O(3)
O(3)–Cu(1)–S(1)
100.98(9)
98.26(15)
91.39(16)
162.68(17)
82.1(3)
Cu(1)–S(1)–C(10)–N(3)
Cu(1)–S(1)–C(10)–N(4)
Cu(1)–O(1)–C(2)–N(2)
Cu(1)–O(1)–C(2)–C(1)
Cu(1)–N(1)–N(3)–C(10)
Cu(1)–N(1)–C(1)–C(2)
Cu(1)–N(1)–C(1)–C(4)
N(1)–N(3)–C(10)–S(1)
N(1)–N(3)–C(10)–N(4)
N(1)–C(1)–C(2)–O(1)
N(1)–C(1)–C(2)–N(2)
–4.3(8)
177.2(6)
–176.9(7)
3.8(9)
5.4(9)
84.2(2)
–3.4(9)
105.64(15)
164.6(2)
82.9(2)
178.4(8)
0.3(10)
178.9(7)
–0.5(11)
–180.0(7)
95.0(3)
105.64(15)
134

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InorganicaChimicaActa492(2019)131–141
Fig. 3. (a) Absorption spectra of complex 1 up on the titration of CT‐DNA. The arrow indicates the absorption intensity decrease upon incremental addition of DNA.
(b) Emission spectra of EB bound DNA system with the incremental addition of complex 1. (c) The plots of [DNA]/(
with the complexes 1–3. (d) Stern-Volmer plots of fluorescence titrations of the complexes (1–3) with CT-DNA.
) versus [DNA] for the titration of CT-DNA
a
b
[DNA] = is the concentration of DNA in base pairs
2.4.2. Fluorescence spectroscopic studies
a = the apparent molar absorption coefficient value obtained as
EB (Ethidium bromide) displacement test was also done to recognise
the mode of metal complexes-DNA interaction. EB emits intense fluor-
escence when it binds to CT-DNA due to the strong hydrophobic in-
teraction of EB through intercalation between the base pairs of DNA. If
a molecule competitively binds to DNA, the molecule will displace the
bound EB from base pairs, will causes quenching in the fluorescence of
EB. The magnitude of quenching of fluorescence reflects the extent of
interaction with the added molecule. The fluorescent intensity of DNA
bound EB was recorded in the absence and the incremental addition of
1–3 (Fig. 3b and S14). The addition of the complexes (1–3) caused a
noticeable reduction in emission intensity, which indicate the binding
of the complexes to DNA by replacing the EB. In order to find out the
relative binding ability complexes to CT-DNA with respect to EB, the
observed data were analysed with the help of the Stern-Volmer equa-
tion (Eq. (2)) [44].
A
(observed)/[complex]
= the molar absorption coefficient of the free compound
f
b = the molar absorption coefficient of the compound in the fully
bound form.
The intrinsic binding constants Kb for complexes were obtained as
the ratio of slope [1/(
f )] to the intercept [1/Kb (
f )] of the he
plot [DNA]/(
b) vebrsus [DNA] (Fig. 3c). The valuesbof (Kb) are given
a
in Table 4. The observed values of Kb revealed that the complex 1 shows
higher binding affinity than 2 and 3. It may be due to the fact that the
pyrrolidine substituted L1 of 1 is least polar and hence more hydro-
phobic compared to the L2 (2) and L3 (3), which has morpholine and
cyclohexyl substitution respectively. The oxygen atom on the mor-
pholine moiety of L2 which increase the polarity of the molecule and
the terminal N-H on the L3 which helps for hydrogen bonding will
enhance the hydrophilicity. Since the hydrophobic group interact more
strongly to the hydrophobic region (base pairs) of DNA complex 1
shows higher interaction.
F0/F = 1 + Kq [Q]
(2)
where F0 and
F
are the fluorescence intensities with and without pre-
senc of the complex respectively, Kq is the linear Stern-Volmer
quenching constant, and [Q] is the complex concentrations. The slope of
F0/F
[Q]
Kq
the plot of
versus
gave the
value. The Stern-Volmer plot
Table 4
(Fig. 3d) illustrate that, the fluorescnt quenching of EB-DNA system by
1–3 is linear in nature. From Kq values, it is clear that complex 1 have
higher quenching constant and the order of the magnitude of Kq is
1 > 2 > 3, and is similar to the result from absorption spectral stu-
dies. The apparent DNA binding constant (Kapp) values were also cal-
culated with the equation,
DNA binding constant (Kb), Stern-Volmer constant (Kq) and the apparent
binding constant (Kapp) for complexes 1–3.
Complex
Kb(M−1
)
Kq(M−1
)
Kapp(M−1
)
1
2
3
(0.82
(0.69
(0.18
0.09) × 105
0.06) × 105
0.02) × 105
(5.38
(4.33
(3.47
0.18) × 104
0.13) × 104
0.10) × 104
(5.03
(3.31
(2.61
0.19) × 106
0.23) × 106
0.14) × 106
KEB [EB] = Kapp [compound]
(3)
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InorganicaChimicaActa492(2019)131–141
Fig. 4. (a) Emission spectrum of BSA with the incremental addition of complex 1. (b) Synchronous spectra of BSA up on the titration of complex 1 and Δλ = 60 nm.
(c) Synchronous spectra of BSA up on the titration of complex 1 and Δλ = 15 nm. {For (a), (b) and (c) the [BSA] = 1 µM and [complex] = 0–20 µM}. (d) The
absorption spectra of BSA (1 µM) and BSA with 1–3 (5 µM).
where [compound] is the concentration complexes required to reduce
50% initial fluorescence intensity of EB, KEB = 1.0 × 107 M−1 and
[EB] = 5 μM. The quenching constant (KEB and Kapp) values are listed in
Table 4
emission spectra of BSA recorded in the range of 285–500 nm by ex-
citing the BSA (1 μM) at 280 nm with increasing concentration
(0–20 μM) of the complexes (1–3). Fig. 4a shows the representative
fluorescence emission spectra of BSA after the addition of complex 1. As
the figure shows, the incremental addition of the complexes 1–3 caused
a substantial decline in the fluorescence intensity of BSA at 345–350 nm
region with a blue shift of 6–8 nm for all complexes. The percentage of
decrease was found as 82%, 75% and 78% from the initial intensity of
BSA, respectively for 1, 2 and 3. The observed hypochromism and blue
shift suggesting the interactions of added metal complexes with BSA
and affecting its conformation which in turn causing the changes in the
local environment of the fluorophore. The extent of fluorescence
quenching was calculated in terms of quenching constant (Kq) is de-
scribed by the Stern-Volmer relation (Eq. (2), Fig. 5a). The binding
ability of complexes were also evaluated in terms of binding constant
2.5. Protein binding studies
Protein interaction studies of 1–3 were performed with Bovine
serum albumin (BSA). Intensity changes and shifts in the wavelength of
both UV–Vis and fluorescent spectra of BSA were analysed in the pre-
sence and the absence of complexes 1–3.
2.5.1. Absorption studies
UV–Visible absorption spectra of BSA was recorded with complexes
to understand the mechanism of quenching process (Fig. 4d). As shown
in Fig. 4d, there was no significant changes in absorption spectra of BSA
after the addition of complexes. This indicates that, the kind of inter-
action between Cu(II) complexes (1–3) and BSA was mainly a dynamic
quenching process [40].
(
Kb) using the Scatchard equation (Eq. (4)), which represent the equi-
librium among bound and free molecules of the system in which ligands
bind independently to a set of similar sites on a macromolecule.
log[(F0 F)/F] = logKb + nlog[Q]
(4)
2.5.2. Fluorescence studies
n
The number of binding sites ( ) and the binding constant (Kb) values
The intrinsic fluorescence of proteins is sensitive in nature due to
the high sensitivity of fluorophore to its local environment which may
change with conformational changes, association and dissociation of
subunits and denaturation. Hence the emission spectra of proteins can
provide useful information on their structure and mechanism and are
frequently utilised in protein folding and association reactions. Hence,
fluorescence spectroscopy is one of the key technique to investigate the
interactions of metal complexes with BSA
were calculated from the plot of log[(F0 F)/F] versus log[Q] (Fig. 5b).
n
The (Kq), (Kb) and ( ) values for the interactions of the 1–3 with BSA
are shown in Table 5. The result suggested that, all complexes showing
good interactions in which complex 1 have a higher binding affinity.
Based on the Kb and Kq values, the affinity of the complexes (1–3) to the
n
BSA follows the order 1–3 > 2. The value suggest the presence of
mono binding site in BSA for all the complexes.
To study the interactions of complexes Cu(II) (1–3) with BSA, the
136

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
Fig. 5. (a) Stern-Volmer plots of fluorescence titrations of complexes 1–3 with BSA. (b) Scatchard plots of fluorescence titrations of complexes 1–3 with BSA.
shown good inhibition against S. epidermidis (MIC: 5.37 µg/mL) and P.
vulgaris (MIC: 18.63 µg/mL). Complex 1 and HL1 shown moderate ac-
tivity against E. coli and S. aureus have respectively with respective MIC
values of 35.75 µg/mL and 25.19 µg/mL. Remaining compounds have
shown poor activity against all the tested strains.
Table 5
Binding constant (Kb), quenching constant (Kq) and number of binding sites (n)
for the interaction of complexes 1–3 with BSA.
Complex
Kb(M−1
)
Kq(M−1
)
n
1
2
3
(2.29
(1.13
(2.43
0.15) × 105
0.08) × 105
0.23) × 105
(9.47
(6.21
(7.18
0.31) × 104
0.17) × 104
0.33) × 104
1.09
1.06
1.13
2.6.2. Antifungal activity
All the compounds were evaluated against five selected strains such
as Aspergillus niger, Aspergillus flavus, Fusarium solani, Candida albicans
and Curvularia lunata. Nystatin was chosen as a standard drug. The MIC
(µg/mL) of the tested compounds results are presented in Table 7. The
antifungal activity results revealed that in all the compounds, complex
3 has shown notable activity against A. niger (MIC: 11.67 µg/mL), good
activity against A. flavus, C. albicans and C. lunata (MIC: 8.14 µg/mL,
17.56 µg/mL and 14.38 µg/mL, respectively) and poor activity against
F. solani. The complex 1 has shown good activity C. albicans and
moderate activity against A. flavus and F. solani (MIC: 16.94 µg/mL,
11.53 µg/mL and 16.52 µg/mL) compared to standard Nystatin. Re-
maining all the compounds have exhibited moderate to poor activity
with MIC ranging from 21.93 µg/mL to 71.53 µg/mL.
2.5.3. Synchronous fluorescence spectroscopic studies
Synchronous fluorescence spectroscopy (SFS) have a crucial role in
the analysis of the multicomponent sample. It has the significant ad-
vantage of improved selectivity, reduction in light scattering and
spectral simplification [45]. Hence SFS can be used for the specific
analysis of tryptophan and tyrosine fluorescence spectra of BSA to
better understand the changes in the microenvironment around this
fluorophore and hence the conformational changes to the BSA during
the addition of metal complexes. The constant-wavelength SFS (CWSFS)
was used here. In CWSFS, a wavelength difference (Δ λ ) between the
excitation (λex) and emission (λem) wavelength were kept constant
during the simultaneous scanning of excitation and emission mono-
chromators. The Δ λ echoes the nature of the chromophore [32]. Hence
the Δλ = 60 nm and Δλ = 15 nm is the characteristic of tryptophan and
tyrosine residue respectively. After the addition of the complexes, the
fluorescence intensity of both the tryptophan (at 350 nm) and tyrosine
(at 310 nm) was decreased (Fig. 4b, c, S16 and S17). Hence the SFS
studies suggested that the interaction of complexes (1–3) influenced the
local environment of both tryptophan and tyrosine of BSA, which in-
dicate the conformational changes of BSA during the interaction with
metal complexes 1–3.
2.6.3. Scavenging activity
The newly synthesized complexes (1–3), were subjected for their in
vitro radical scavenging activity in terms of hydrogen donating or ra-
dical scavenging ability by DPPH (1,1-diphenyl-2-picrylhydrazyl)
method [48]. The results were expressed in µM and ascorbic acid was
considered as reference drug (Table 8, Fig. 6). All the complexes
showed radical scavenging activity with IC50 values ranging from
19.23
complex 1 has shown good scavenging (IC50: 19.23
pared to standard ascorbic acid. Complex 2 has shown moderate ac-
tivity and complex shown poor activity with IC50 values of
1.05 µM to 35.17
0.55 µM. Among all the complexes,
1.05 µM) com-
3
2.6. Biological activities
27.01
1.93 µM and 35.17
0.55 µM. Based on the above results
the order of activity is 1 > 2 > 3.
2.6.1. Antibacterial activity
The in vitro antibacterial activity of HL1-HL3 and its metal com-
plexes 1–3 were tested against sensitive organisms such as
Staphylococcus Aureus, Bacillus Subtilis and Staphylococcus Epidermidis
(Gram-positive), Escherichia Coli, Pseudomonas aeruginosa and Proteus
Vulgaris (Gram-negative) bacterial strains by disc diffusion method
[46,47] and Ciprofloxacin as a positive control. The minimum in-
hibitory concentration (MIC) values for the compounds under test and
standard were reported in µg/mL and the results are tabulated in
Table 6. It is evident from Table 6 that the complex 3 has shown potent
efficacy with broad-spectrum activity against S. aureus (MIC: 8.56 µg/
mL), B. subtilis (MIC: 10.35 µg/mL), S. epidermidis (MIC: 6.50 µg/mL), P.
aeruginosa (MIC: 21.36 µg/mL) and P. vulgaris (MIC: 17.64 µg/mL), re-
spectively compared to the standard drug. Similarly, complex 2 has
2.6.4. In vitro anti-proliferative activity:
The in vitro anti-proliferative activity of the complexes (1–3) was
evaluated for different cancer [MCF-7 (breast), A549 (lung) and HeLa
(cervical)] cell lines. The percentage of cell viability versus con-
centration graph is shown in Fig. 7. The half minimum inhibitory
concentration (IC50) values of the complexes are tabulated (Table 9).
The activity results revealed that complex 1 has exhibited broad-spec-
trum activity against MCF-7, A549 and HeLa with IC50 value
14.83
0.45 μM, 17.88
0.16 and 6.89
0.42 μM, respectively
which was comparable to that of the standard Doxorubicin. Similarly,
complex
25.02
3
against MCF-7 shown moderate activity (IC50
:
0.51 μM) and the remaining complexes against all the cell
137

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
Table 6
Antibacterial activity of the synthesized compounds (MIC, µg/mL).
Compound
S. aureus
B. subtilis
S. epidermidis
E. coli
P. aeruginosa
P. vulgaris
L1
25.19
61.75
> 150
> 150
> 150
8.56
> 150
> 150
47.26
29.81
> 150
10.35
6.25
51.94
> 150
> 150
18.39
5.37
47.58
> 150
63.45
35.75
50
> 150
75.91
45.57
> 150
> 150
21.36
12.5
> 150
> 150
> 150
38.60
18.63
17.64
12.5
L2
L3
1
2
3
6.50
39.28
12.5
Ciprofloxacin
6.25
3.12
Table 7
The antifungal activity of the compounds (MIC, µg/mL).
Compound
A. niger
A. flavus
F. solani
C. albicans
C. lunata
L1
> 150
50
71.53
65.74
> 150
11.53
> 150
8.14
27.80
> 150
> 150
16.52
> 150
50
> 150
39.26
36.51
16.94
> 150
17.56
4.16
36.79
50
L2
L3
43.51
> 150
21.93
11.67
4.75
50
1
> 150
37.61
14.38
3.12
2
3
Nystatin
2.81
3.28
Table 8
Radical scavenging activity of the newly synthesized
compounds (1–3) by DPPH scavenging assay.
Compound
IC50 (µM)
1
19.23
27.01
35.17
1.05
1.93
0.55
2
3
Ascorbic acid
6.13
0.43
Fig. 6. Radical scavenging activity of the newly synthesized complexes (1–3).
lines were exhibited less activity IC50 ranging from 10.86 0.65 μM to
64.97
1.06 μM. We have also tested for anti-proliferative activity
against normal HEK-293 (Embryonic Kidney 293) cell line and the re-
sults are tabulated in Table 9.
Fig. 7. Dose response curves of complexes 1–3 against (a) MCF-7 cell lines, (b)
A-549 cell lines and (c) HeLa cell lines.
3. Conclusion
The thiosemicarbazone derivatives of 5-methoxy isatin, differ-
entiated by different N-terminal substitutions (HL1, Pyrrolidine; HL2,
Morpholine and HL3, Cyclohexyl) and their copper(II) complexes (1–3)
have been synthesised and characterised with an aim to assess their
biological applications and the effect of terminal N-substitution on
biological activity. The crystal data and molecular structure of the li-
gand HL1 and complex 1 was established by single crystal X-ray dif-
fraction method. The UV–Vis and fluorescent spectroscopic investiga-
tion on DNA interacting capability of complexes 1–3 showed the ability
of the complexes to interact with DNA, mainly through the intercalative
mode. The binding parameters such as
b, Kq, Kapp suggesting that, each
K
complexes have a different activity, in which complex 1 have higher
affinity. The complexes 1–3 were also showed their ability to interact
with BSA protein and influence its confirmation, again the effect of
complex 1 is better than that of complex 2 and 3. The higher activity of
1 can be attributed to its higher hydrophobic nature compare to 2 and
3, so the complex 1 can more effectively interact with the hydrophobic
138

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
Table 9
4.2. Synthesis of ligands
The in vitro anticancer activity of compounds 1–3 on human cancer cell lines
(IC50 in μM).a
Compound
Pyrrolidine-1-carbothiohydrazide,
morpholine-4-carbothiohy-
drazide, 4-Cyclohexyl-3-thiosemicarbazide were synthesized according
to the literature [49,50]. The ligands (HL1-HL3) were synthesized by
refluxing an ethanolic mixture of 5-methoxyindoline-2,3-dione and
appropriate carbothiohydrazide in the presence of 1 drop of conc.
H2SO4. The reaction was monitored by TLC. After 4 h, the resultant
product was filtered, washed and dried [51].
MCF-7
A549
HeLa
HEK 293
1
14.83
49.38
25.02
3.47
0.45
0.16
0.51
0.31
17.88
55.52
64.97
4.69
0.16
0.63
1.06
0.25
6.89
0.42
90.85
94.53
72.16
ND
2.31
1.22
1.25
2
28.79
10.86
1.29
0.32
3
0.65
Doxorubicin
0.17
a
Values are stated as mean
SEM. Cytotoxicity as IC50 for each cell line, is
the concentration of compound required to reduce the optical density of treated
cells to 50% with respect to untreated cells using the MTT assay. ND = Not
Done.
4.2.1. (Z)-N-(5-methoxy-2-oxoindolin-3-ylidene)pyrrolidine-1-
carbothiohydrazide (HL1)
Pyrrolidine-1-carbothiohydrazide (0.145 g, 0.001 mol), 5-methox-
yindoline-2,3-dione were (0.177 g, 0.001 mol) were used. Orange co-
loured powder, yield: 81%. M.p.: 245 °C. Anal. Calcd. C14H16N4O2S
(%): C, 55.25; H, 5.30; N, 18.41; S, 10.53. Found: C, 55.37; H, 5.41; N,
18.21; S, 10.28. UV–Vis (DMSO): λmax (nm) 287, 347. FT-IR (ATR): ʋ
(cm−1) 3453 (NeH isatin), 3200 (NeH), 1667 (C]N), 1693 (C]O),
1341 (C]S). 1H NMR (500 MHz, DMSO‑d6): δ, ppm 13.64 (s, 1H),
11.13 (s, 1H), 7.69–6.87 (m, 3H), 3.42 (s, 3H), 3.78–3.72 (d, 4H),
2.09–1.90 (m, 4H). 13C NMR (125 MHz, DMSO‑d6): δ, ppm 176.1 (C]
S); 163.57 (C = 0); 152.4 (C]N); 135.95, 134.85, 121.45, 117.44,
112.33, 105.72 (aromatic carbons); 56.10 (O–CH3); 53.70, 49.68,
centres of both DNA and BSA. For BSA interaction studies, both com-
plex 1 and 3 almost similar activity. I may be due to the possible hy-
drogen bonding interactions between the amino acids of proteins and
hydrogen present in 4N position of HL3 ligand. The antibacterial study
has shown that each complexes have a different range of activity. The
complex 3 has shown potent activity against three gram positive bac-
teria such as S. aureus, B. subtilis and S. epidermidis, two gram negative
bacteria P. aeruginosa and P. vulgaris while complex 2 showed activity
towards S. epidermidis and P. vulgaris. In case of antifungal activity the
complex 3 has shown promising activity against A. niger and A. flavus.
The radical scavenging study indicates that, all complexes showed the
scavenging activity and the order of activity is 1 > 2 > 3. The in vitro
anticancer activity of synthesized complexes on human cancer cell lines
(MCF-7, A549, HeLa) have also been investigated, the results are
showing that the complexes 1–3 have the ability to inhibit the growth
of cancer cells. Further the complexes showed some selective nature
towards various cell lines, in which complex 1 showed more potent
nature towards all three cancer cell lines. Complex 2 showed more
activity towards HeLa cell lines. The more active complex 1 was also
studied against HEK 293 as a demonstrative of normal cell line. The
result showed that, it is less toxic towards normal cell line. Compared to
DNA/BSA interaction studies, other biological studies did not follow a
particular order. Which indicates the possibilities of other mechanistic
pathways and factors, and need to be studied further in order to fully
understand the structural activity of different 4N-substituted thiose-
micarbazone and its complexes. Finally, the synthesised thiosemi-
carbazone series and their complexes which are only differed by N-
terminal substitution of thiosemicarbazone ligand showed different
level of activities towards various biological studies. So the whole
studies signifying the influence of the substitution at the N-terminal
position of thiosemicarbazones which gives us more option to further
tune and enhance the activity thiosemicarbazones and their metal
complexes.
26.31, 24.17 (aliphatic carbons). ESI-MS (m/z) = 305.12 [M+1]+
.
4.2.2. (Z)-N-(5-methoxy-2-oxoindolin-3-ylidene)morpholine-4-
carbothiohydrazide (HL2)
Morpholine-4-carbothiohydrazide (0.161 g, 0.001 mol), 5-methox-
yindoline-2,3-dione (0.177 g, 0.001 mol) were used. Orange-red-
coloured powder, yield: 80%. M.p.: 260 °C. Anal. Calcd. C14H16N4O3S
(%): C, 52.49; H, 5.03; N, 17.49; S, 10.01. Found: C, 52.72; H, 5.29; N,
17.37; S, 9.73. UV–Vis (DMSO): λmax (nm) 305, 369. FT-IR (ATR): ʋ
(cm−1) 3475 (NeH isatin), 3281(NeH), 1628 (C]N), 1701 (C]O),
1348 (C]S).1H NMR (500 MHz, DMSO‑d6): δ, ppm 13.30 (s, 1H), 11.15
(s, 1H), 7.05–6.37 (m, 3H), 3.41 (s, 3H), 4.01–3.75 (m, 8H).13C NMR
(125 MHz, DMSO‑d6): δ, ppm 179.82 (C]S); 163.18(C]O); 155.44
(C]N); 136.43, 135.16, 121.21, 117.92, 122.39, 105.94 (aromatic
carbons); 56.33 (O–CH3); 66.19, 50.42 (aliphatic carbons). ESI-MS m/
z = 321.10 [M+1]+
.
4.2.3. (Z)-N-cyclohexyl-2-(5-methoxy-2-oxoindolin-3-ylidene)
hydrazinecarbothioamide (HL3)
4-Cyclohexyl-3-thiosemicarbazid (0.173 g, 1 mmol), 5-methox-
yindoline-2,3-dione (0.177 g, 1 mmol) were used. Red coloured
powder, yield: 83%. M.p.: 252 °C. Anal. Calcd. C16H20N4O2S (%): C,
57.81; H, 6.06; N, 16.85; S, 9.64. Found: C, 57.98; H, 5.98; N, 16.69; S,
9.73. UV–Vis (DMSO): λmax (nm) 304, 381. FT-IR (ATR): ʋ(cm−1) 3373
(NeH isatin), 3204(NeH), 1622 (C]N), 1692 (C]O), 1349 (C]S).1H
NMR (500 MHz, DMSO‑d6): δ, ppm 12.71 (s, 1H), 10.34 (s, 1H), 8.29 (s,
1H), 7.27–6.30 (m, 3H), 3.81 (s, 3H), 2.56–1.24 (m, 12H). 13C NMR
(125 MHz, DMSO‑d6): δ, ppm 176.59 (C]S); 163.02 (C]O); 155.51
(C]N); 136.49, 132.18, 121.18, 117.14, 111.99, 106.90 (aromatic
carbons); 56.13 (O–CH3); 40.27, 32.15, 25.21(aliphatic carbons). ESI-
4. Experimental section
4.1. Materials and methods
Chemicals and solvents for the experiments were purchased from
Alfa Aesar/Merck/Sigma-Aldrich, and were used as received. Lab India
instrument was used to record the melting points of the compounds and
are uncorrected. Elemental analysis was carried out with a Vario EL-III
CHNS analyser. The FT-IR spectra of HL1-HL3 and 1–3 were recorded
MS (m/z) = 333.13 [M+1]+
.
4.3. Synthesis of copper(II) complexes (1–3)
by
the
PerkinElmer
Frontier
FT-IR/FIR
spectrometer.
The ethanolic solution of CuCl2·2H2O (1 mmol) was added into the
solution of appropriate ligands (1 mmol) in ethanol. The reaction
mixture was stirred for 5 h under reflux, and then the precipitate
formed was filtered, washed and dried.
Analytikjenaspecord S 600 UV–Vis spectrophotometer was used to
perform the electronic absorption studies. Emission spectra were re-
corded using Jasco FP–8300 Fluorescent spectrophotometer. NMR
studies were carried out in DMSO‑d6 solvent on a Bruker 500 MHz in-
strument. The ESI mass spectra of the ligands and complexes were re-
corded using THERMO exactive orbitrap mass spectrometer. EPR
spectra were recorded on a JES-X3 Series EPR instrument at liquid ni-
trogen temperature and operating at X-band frequency (9.1 GHz).
4.3.1. Bis[(Z)-N-(5-methoxy-2-oxoindolin-3-ylidene)pyrrolidine-1-
arbothiohydrazide] Cu(II) (1)
Brown coloured powder, yield: 80%. M.p.: 281 °C. Anal. Calcd.
C
14H15ClCuN4O2S (%): C, 41.79; H, 3.76; N, 13.92; S, 7.97. Found: C,
139

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
41.98; H, 3.81; N, 13.79; S, 7.59. UV–Vis (DMSO): λmax (nm) 269, 352,
416, 635(DRS). FT-IR (ATR): ʋ(cm−1) 3305 (NeH isatin), 1572 (C]N),
1656 (C]O), 1326 (C]S). EPR (LNT): ‘g’ values (g∥, g⊥) 2.214, 2.043.
ESI-MS (m/z) = 400.04 [M]+, 401.05 [M+1]+
(pH = 7.2). The stock solutions the complexes were prepared in DMSO-
phosphate buffer (5:95) solution. The titrations were done by the suc-
cessive addition of the solution of complexes 1–3 (0–20 µM) to a 2.5 mL
of BSA solution in the cuvette. The changes in protein nature was
monitored by observing the emission spectra of BSA at 345 nm with an
excitation wavelength of 280 nm. Synchronous fluorescence spectra
was also recorded using the same concentration of BSA and the com-
plexes as mentioned above with two different Δλ (difference between
the excitation and emission wavelengths of BSA) values of 15 and
60 nm.
4.3.2. Bis[(Z)-N-(5-methoxy-2-oxoindolin-3-ylidene)morpholine-4-
carbothiohydrazide] Cu(II) (2)
Brown coloured powder, yield: 82%. M.p.: 293 °C. Anal. Calcd.
C
14H15ClCuN4O3S (%): C, 40.19; H, 3.61; N, 13.39; S, 7.66. Found: C,
40.31; H, 3.71; N, 13.31; S, 7.37. UV–Vis (DMSO): λmax (nm) 265, 376,
417, 639(DRS). FT-IR (ATR): ʋ(cm−1) 3231 (NeH isatin), 1592 (C]N),
1649 (C]O), 1332 (C]S). EPR (LNT): ‘g’ values (g∥, g⊥) 2.217, 2.051.
4.7. Biological evaluation
ESI-MS (m/z) = 417.04 [M]+, 382.02 [M−Cl]+
.
4.7.1. Antimicrobial screening
4.3.3. Bis[(Z)-N-cyclohexyl-2-(5-methoxy-2-oxoindolin-3ylidene)
hydrazinecarbothioamide] Cu(II) (3)
The in vitro antimicrobial activity of the ligand and its metal com-
plexes were evaluated against gram-negative bacteria such as
Escherichia coli, Proteus vulgaris and Pseudomonas aeruginosa, gram po-
sitive bacteria like Staphylococcus aureus and Bacillus subtilis and
Staphylococcus epidermidis, and few fungal strains such as Aspergillus
niger, Aspergillus flavus, Fusarium solani, Candida albicans and Curvularia
lunata by disc diffusion method. Ciprofloxacin and Nystatin were
chosen as standards for antibacterial and antifungal activity, respec-
tively. Sterile antibiotic discs (6 mm in diameter, prepared using
Whatmann No. 1 paper) were placed over the nutrient agar medium.
100 µg of the compounds (initially dissolved in DMSO) were transferred
to each disc with the help of a micropipette. The samples were in-
cubated at 37 °C for 24 h (bacteria) and 25 °C for 48 h (fungi), respec-
tively. The plates were then observed and the inhibition zones were
recorded in terms of millimeter.
Brown coloured powder, yield: 85%. M.p.: 301 °C. Anal. Calcd.
C
16H19ClCuN4O2S (%): C, 44.65; H, 4.45; N, 13.02; S, 7.45. Found: C,
44.87; H, 4.53; N, 12.93; S, 7.21. UV–Vis (DMSO): λmax (nm) 267, 366,
417, 610(DRS). FT-IR (ATR): ʋ(cm−1) 3313 (NeH isatin), 1545 (C]N),
1663 (C]O), 1340 (C]S), . EPR (LNT): ‘g’ values (g∥, g⊥) 2.219, 2.039.
ESI-MS (m/z) = 429.07 [M]+, 430.06 [M+1]+
.
4.4. Single crystal X-ray diffraction studies
The suitable crystals for the diffraction studies were obtained from
DMSO/methanol solution.
The detailed explanations of the experiments are given in the sup-
plementary information file.
4.5. DNA interaction studies
4.7.2. In vitro cytotoxic bioassay
The synthesized compounds were evaluated for their in vitro antic-
ancer activity against five different human cancer cell lines such as
HeLa (cervical), MCF-7 (breast), A549 (lung), and HEK293 (embryonic
kidney). Cell viability in the presence of the test samples was measured
by the MTT-microcultured tetrazolium assay. This assay is a quantita-
tive colorimetric method for the determination of cell viability. The
assessed parameter is the metabolic activity of viable cells.
Metabolically active cells reduce pale yellow tetrazolium salt (MTT) to
a dark blue water-insoluble formazan, which can be directly quantified
after solubilisation with DMSO. The absorbance of the formazan di-
rectly correlates with the number of viable cells. MCF-7, A549, HeLa
and HEK293 cells were plated into a 96-well plate at a density of
1 × 104 cells/well. Cells were grown overnight in the full medium and
then switched to the low serum media. 1% DMSO was used as a control.
After 48 h of treatment with different concentrations of test compounds,
the cells were incubated with MTT (2.5 mg/mL) in the CO2 chamber for
2 h. The medium was then removed and 100 µL of DMSO was added
into each well to dissolve formazan crystals. After thoroughly mixing,
the plates were read at 570 nm for optical density, which is directly
correlated with cell quantity. The results were represented as percen-
tage of viability. All the experiments were carried out in triplicate.
4.5.1. Electronic absorption studies
The interaction studies of complexes (1–3) with CT-DNA were
performed in Tris HCl/NaCl buffer (pH 7.2). The buffer was prepared by
dissolving tris(hydroxymethyl) aminomethane (Tris, 5 mM) and sodium
chloride (50 mM) in double distilled water and adjusted to pH 7.2 with
diluted hydrochloric acid. The stock solution of CT-DNA was prepared
by dissolving the CT-DNA in Tris HCl/NaCl buffer. The purity of the calf
thymus DNA was verified by taking absorption ratio of CT DNA solu-
tions at λmax 260 and 280 nm, and the ratio was found to be 1.87, in-
dicating that DNA was sufficiently protein free [52]. The DNA con-
centration was decided using UV–Visible absorbance and monitoring
the molar absorption coefficient (6600 M−1cm−1) values at 260 nm
[53]. The solution of metal complexes with required concentration
were prepared in 5% DMSO/Tris HCl/NaCl solution. The absorption
titration was performed with incremental addition of CT-DNA
(5–50 µM) to a fixed concentration of (25 µM) complex solution in the
cuvette. An equal amount of DNA was also added to the reference so-
lution to avoid the absorbance of DNA itself.
4.5.2. Fluorescent titration studies
The quenching constant (Kq) and apparent binding constant (Kapp
)
values of the complexes (1–3) were determined by a ethidium bromide
(EB) displacement assay using fluorescence spectroscopy. Ethidium
bromide solution was prepared in Tris HCl/NaCl buffer (pH 7.2) solu-
tion and the DNA was pre-treated with ethidium bromide and then the
solutions of complexes were incrementally (0–50 µM) added to this
mixture of EB bound DNA solution, and the change in the fluorescence
intensities of EB was measured at 605 nm (520 nm excitation).
4.7.3. Antioxidant activity
Free radical scavenging capacity of the metal complexes were de-
termined by using DPPH (1,1-Diphenyl-2-picrylhydrazyl) free radical
method described in the literature. Ascorbic acid solution was used as
standard. 0.2 mM solution of DPPH was prepared in 100% methanol.
The required amount of ascorbic acid was dissolved in 100% methanol
to prepare 1 mM solution and the test compounds were dissolved in
0.2% DMSO and methanol to prepare 1 mM stock solutions. From the
stock solutions different concentrations (3, 10, 30 and 100 μM) of so-
lutions were prepared on diluting with methanol. 1 mL of each com-
pound solution was added to the 3 mL of DPPH solution. After 30 min,
the absorbance of the solutions at 517 nm (A1) was recorded using
UV–Visible spectrophotometer. As a control, the absorbance of the
4.6. Protein binding studies
Fluorescence spectroscopy method was employed to study the
binding of the copper thiosemicarbazone complexes (1–3) with BSA.
The stock solution of BSA was prepared in 50 mM phosphate buffer
140

K.N. Aneesrahman, et al.
InorganicaChimicaActa492(2019)131–141
blank solution of DPPH without test compound was also determined at
517 nm (A0). The following equation was used for the calculation of the
% of scavenging activity.
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where A0 is the absorbance of DPPH in the absence of antioxidant and
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KNA and RK thanks to the MHRD, Govt. of India for providing
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