Molecular docking, PASS analysis, bioactivity score prediction, synthesis, characterization and biological activity evaluation?of a functionalized 2-butanone thiosemicarbazone ligand and its complexes

Article abstract of DOI:10.1007/s12154-017-0167-y

2-Butanone thiosemicarbazone ligand was prepared by condensation reaction between thiosemicarbazide and butanone. The ligand was characterized by ¹H NMR, ¹³C NMR, FT-IR, mass spectrometry and UV spectroscopic studies. Docking studies were performed to study inhibitory action against topoisomerase II (Topo II) and ribonucleoside diphosphate reductase (RR) enzymes. Inhibition constants (K_i) of the ligand were 437.87 and 327.4?μM for the two enzymes, respectively. The ligand was tested for its potential anticancer activity against two cancer cell lines MDA-MB-231 and A549 using MTT assay and was found to exhibit good activity at higher doses with an IC₅₀?=?80?μM against human breast cancer cell line MDA-MB-231. On the other hand, no significant activity was obtained against the lung carcinoma cell line A549. Antibacterial activity of the ligand was tested against Staphylococcus aureus and E. coli using the disc diffusion method. Ligand did not exhibit any significant antibacterial activity. Four complexes of Co(III), Fe(II), Cu(II), and Zn(II) were prepared with the ligand and characterized by various spectroscopic studies. Low molar conductance values were obtained for all complexes displaying non-electrolyte nature except in Co(III) complex. As expected, complexation with metal ions significantly increased the cytotoxicity of the ligand against the tested cell lines viz. IC₅₀ values of <20?μM for Co, Fe, and Zn complexes and approx. 80?μM against MDA cells versus IC₅₀ value of <20?μM for Co and Cu complexes and that of 30 and 50?μM for Fe and Zn complexes, respectively, against A549 cells. The Cu complex was found to be active against E. coli and S. aureus with MIC values in the range of 6–10?mg/mL. Other than Cu, only Co complex was found to possess antibacterial activity with MIC values of 5–10?mg/mL when tested against S. aureus. Bioactivity score and Prediction of Activity Spectra for Substances (PASS) analysis also depicted the drug-like nature of ligand and complexes.

Full text of DOI:10.1007/s12154-017-0167-y

J Chem Biol
DOI 10.1007/s12154-017-0167-y
ORIGINAL ARTICLE
Molecular docking, PASS analysis, bioactivity score prediction,
synthesis, characterization and biological activity evaluation of
a functionalized 2-butanone thiosemicarbazone ligand and its
complexes
Tahmeena Khan1,2 & Shalini Dixit3 & Rumana Ahmad4 & Saman Raza2 & Iqbal Azad1
Seema Joshi2 & Abdul Rahman Khan1
&
Received: 10 December 2016 /Accepted: 9 March 2017
#
Springer-Verlag Berlin Heidelberg 2017
Abstract 2-Butanone thiosemicarbazone ligand was prepared
prepared with the ligand and characterized by various spectro-
scopic studies. Low molar conductance values were obtained for
all complexes displaying non-electrolyte nature except in Co(III)
complex. As expected, complexation with metal ions significant-
ly increased the cytotoxicity of the ligand against the tested cell
lines viz. IC50 values of <20 μM for Co, Fe, and Zn complexes
and approx. 80 μM against MDA cells versus IC50 value of
<20 μM for Co and Cu complexes and that of 30 and 50 μM
for Fe and Zn complexes, respectively, against A549 cells. The
Cu complex was found to be active against E. coli and S. aureus
with MIC values in the range of 6–10 mg/mL. Other than Cu,
only Co complex was found to possess antibacterial activity with
MIC values of 5–10 mg/mL when tested against S. aureus.
Bioactivity score and Prediction of Activity Spectra for
Substances (PASS) analysis also depicted the drug-like nature
of ligand and complexes.
by condensation reaction between thiosemicarbazide and
1
butanone. The ligand was characterized by H NMR, 13C
NMR, FT-IR, mass spectrometry and UV spectroscopic studies.
Docking studies were performed to study inhibitory action
against topoisomerase II (Topo II) and ribonucleoside diphos-
phate reductase (RR) enzymes. Inhibition constants (Ki) of the
ligand were 437.87 and 327.4 μM for the two enzymes, respec-
tively. The ligand was tested for its potential anticancer activity
against two cancer cell lines MDA-MB-231 and A549 using
MTT assay and was found to exhibit good activity at higher
doses with an IC50 = 80 μM against human breast cancer cell
line MDA-MB-231. On the other hand, no significant activity
was obtained against the lung carcinoma cell line A549.
Antibacterial activity of the ligand was tested against
Staphylococcus aureus and E. coli using the disc diffusion meth-
od. Ligand did not exhibit any significant antibacterial activity.
Four complexes of Co(III), Fe(II), Cu(II), and Zn(II) were
.
.
Keywords Thiosemicarbazone Topoisomerase II
.
.
.
.
Ribonucleotide reductase Docking PASS Anticancer
Antibacterial
Electronic supplementary material The online version of this article
(doi:10.1007/s12154-017-0167-y) contains supplementary material,
which is available to authorized users.
Introduction
* Abdul Rahman Khan
arahman@iul.ac.in
Metal complexes provide a rich platform for being used as ther-
apeutic agents. Transition metals show variable oxidation states
and an array of coordination geometries with different ligands.
The ligands can alter the reactivity of the metal and also play a
significant role in determining the nature of secondary coordina-
tion sphere interactions which involve recognition of biological
targets such as enzymes, protein receptors, and DNA [25].
Schiff-based thiosemicarbazones are a versatile class of ligands,
and their complexes have been used as therapeutic agents since
long [30, 36]. Thiosemicarbazone-based metal complexes have
1
Department of Chemistry, Integral University, Dasauli, P.O. Bas-ha,
Kursi Road, Lucknow, UP 226026, India
2
Department of Chemistry, Isabella Thoburn College, 7, Faizabad
Road, Lucknow, UP 226007, India
3
Department of Analytical Chemistry, CSIR-Central Institute of
Medicinal and Aromatic Plants, P.O.-CIMAP, Near Kukrail Picnic
Spot, Lucknow, UP 226015, India
4
Department of Biochemistry, Era’s Lucknow Medical College &
Hospital, Sarfarazganj, Hardoi Road, Lucknow, UP 226003, India

J Chem Biol
been used as antineoplastic, antibacterial, antiviral, and antifungal
agents [12, 20]. They are usually prepared by the condensation
reaction between aldehydes or ketones with thiosemicarbazide
[5]. Thiosemicarbazones as ligands show polydentate nature giv-
ing rise to a great variety of coordination modes [4]. The coor-
dination ability of the basic ligand moiety can be enhanced by
adding additional functional groups making them suitable for
chelation [24]. Medical applications of thiosemicarbazones be-
gan to appear in the 1950s against tuberculosis and leprosy. In the
1960s, their antiviral properties were discovered, and after a con-
siderable amount of research, methisazone and Marboran® were
commercialized to treat smallpox [3, 23, 27]. Enhancement in
activity can be obtained by optimization of the structure, which
can result in groundbreaking discovery of new drug compounds
[22]. It has been observed that coordination to a metal ion almost
systematically increases the activity or contributes to mitigate the
side effects of the organic parent compounds [14]. The major
known effects related to the anticancer activity of
thiosemicarbazone complexes include inhibition of ribonucleo-
tide reductase (RR) [6], reactive oxygen species (ROS) produc-
tion [38], topoisomerase II inhibition [17], mitochondrial disrup-
tion [43], and more recently, a multidrug resistance protein
(MDR1) inhibition [31, 41]. The gray zone between chemistry
and molecular biology further needs to be elucidated.
EI Deluxe conductivity meter, Model-601, in DMSO (1.0 × 10−
3 mol). For biological activity evaluation, 0.4% Trypan blue, PBS
(pH = 7.2, 1×), 0.25% trypsin-EDTA (1×), DMEM/F-12 (1×)
(Dulbecco’s modified Eagle’s medium), and antibiotic/
antimycotic solution (100×) were obtained from Gibco, Life
Technologies, whereas fetal bovine serum (FBS) and MTT were
from HiMedia. DMSO was purchased from Calbiochem. MDA-
MB-231 (human breast carcinoma, ER−, tumorigenic and inva-
sive), abbreviated as MDA cells, and A549 (lung carcinoma
epithelial cell line) were obtained from the National Centre for
Cell Science (NCCS), Pune, India. All other chemicals used in
the biological activity study were of analytical grade.
Molecular docking studies
The synthesized ligand and metal complexes were subjected
to molecular docking studies using the Auto Dock Tools
(ADT) version 1.5.6 and Auto Dock version 2 docking pro-
grams (interactive molecular graphics programs) to under-
stand the drug molecule interaction with topoisomerase II
and ribonucleotide diphosphate reductase (RR) enzymes to
investigate the potential binding mode and energy.
Toxicity potential assessment
Materials and methods
Toxicity risk assessment gives an idea about the probable side
effects of synthesized compounds that may be used for further
processing in drug development and discovery. The mutagen-
ic, tumorigenic, irritant, and reproductive toxicities were mea-
sured by means of precomputed set of structural fragments.
The prediction of different properties of molecules in the early
stage is a vital step in the drug discovery and development
process. Toxic parameters of the ligand and complexes were
generated by OSIRIS Data Warrior Software.
Molecular docking was performed using Auto Dock Tools
(ADT) version 1.5.6 and Auto Dock version 2.0. The structures
of receptors topoisomerase II (PDB ID: 5IWI) and ribonucleo-
side diphosphate reductase (PDB ID: 5CI3) were downloaded
from protein data bank (www.rcsb.org/pdb). IR spectra were
recorded on a Bruker vertex 70 IR spectrophotometer in the
frequency range 4000–500 cm−1. The samples were run as
1
their KBr pellets. H NMR spectra were recorded on a Bruker
spectrophotometer on 500.1 MHz at 295 K in the range of 0–10 δ
using tetramethylsilane (TMS) as an internal standard. The sam-
ples were run in CDCl3 and dimethyl sulfoxide (DMSO)-d6, and
chemical shifts were given relative to TMS (Merck) in ppm. UV
spectra were recorded on a SpectraMax-5 spectrophotometer
(Molecular Devices) in the range 200–400 nm. Mass spectrum
of ligand was recorded on FAB-MS, JEOL SX-102. The condi-
tions applied were as follows: ion source: ESI, positive; temper-
ature 350 °C; nebulizer pressure 45 psi (N2); fragmenter voltage
130 V; capillary voltage 4000 V, and scan range m/z 100–700
(cycle time 800 ms). For chemical syntheses, all chemicals and
solvents were of analytical grade and purchased from commer-
cial sources. Hydrated salts of metals, i.e., CuSO4.5H2O, ZnSO4.
7H2O, CoCl2.6H2O and FeSO4.7H2O, were used for complex
formation. Melting points were assessed in Ambassador electri-
cal melting point apparatus up to 400 °C by the open glass
capillary method. Conductivity measurements were made on an
Bioactivity score prediction
Drug score values indicate overall potential of a compound to
be a drug candidate. Mol inspiration is a web-based tool used
to predict the bioactivity score of the synthesized compounds
against regular human receptors such as GPCRs, ion channels,
kinases, nuclear receptors, proteases and enzymes [34].
Evaluation of drug likeliness based on Lipinski’s rule
of five
Lipinski’s rule of five is helpful in describing molecular prop-
erties of drug compounds required for estimation of important
pharmacokinetic parameters such as absorption, distribution,
metabolism, and excretion. The rule is helpful in drug design
and development [13, 28, 40].

J Chem Biol
Chemistry
[Co(C5H11N3S)2Cl2]+
Synthesis of 2-butanone thiosemicarbazone
Yield 46%; MW 420; MP (°C) 210; FTIR (KBr) (cm−1) 1570
(C=N), 700, 1050 (C=S); 1H NMR (DMSO) (ppm) 1.1 (t-3H),
2.2 (s,3H), 2.5 (q, 2H), 7.6 (br s, 1H,NH), 8.3 (br s,2H,NH2);
solubility DMSO, DMF; λmax (nm) 320; molar conductance
105.04 Ω−1 cm−1 mol−1.
The ligand was prepared using a previously reported procedure
with some modifications [26]. Reaction was monitored by TLC
in CHCl3, Rf value (0.514). Mass spectrometry data confirmed
the structure of ligand as indicated by molecular ion peak (M+1)
corresponding to its molecular formula C5H11N3S. Yield 90%;
MW 145; MP (°C) 99 °C; FTIR (KBr) (cm−1) 1609 (C=N), 761,
1077 (C=S); 1H NMR (MeOD) (MHz) 1.1 (t-3H), 1.9 (s, 3H),
2.3 (q, 2H), 3.3 (s, 1H,NH), 4.9 (s, 2H,NH2); 13C NMR (DMSO)
(δ) 10.70 (CH3), 18.72 (CH3), 31.62 (CH2), 155.5 (C=N) and
178.72 (C=S); ESI MS (m/z) 146.1 M+; solubility: ethanol,
DMSO, DMF; λmax (nm) 267.
Biological activity evaluation
MDA-MB-231 and A549 cell lines were maintained by
subculturing and passaging as monolayers in 25- and 75-cm2 cell
culture flasks (Nest, Tarsons) at 37 °C in Tissue and Cell Culture
Lab, Era’s Medical College, Lucknow, in a 5% CO2 incubator at
95% humidity for producing HCO3 buffering capacity as report-
ed earlier [21]. The cells were maintained at pH 7.4 in DMEM
containing phenol red as a pH indicator and supplemented with
5% FBS [21]. The medium, prior to being used in cell culture
experiments, was vacuum filtered using a Corning filtration sys-
tem (Corning®, Sigma-Aldrich).
Synthesis of metal complexes
Hydrated AR-grade metal salts were used for the preparation
of metal adducts. The complexes were synthesized in 1:2
(metal-ligand) molar ratios through a general preparatory
route. After refluxing the ethanolic solution of ligand with
the aqueous solution of metal salts, colored precipitate was
obtained which was filtered under vacuum. The precipitate
was washed with ethanol and dried in a desiccator. The
Co(II) salt was first oxidized to (III) state by using a few drops
of conc. HNO3 as an oxidizing agent.
Anticancer activity
Solutions (0.1 M) of the ligand and its four complexes were
prepared in 1.0 mL 50% DMSO solution. The solutions were
diluted ten times in DMEM to give 0.01 M (104 μM) solu-
tions. In separate experiments, cells were trypsinized and cul-
tured in a six-well plates (0.5 × 105 cells/well) initially for
24 h, so as to allow the cells to adhere. After 24 h of incuba-
tion, the cells were exposed to 20–100 μM of the ligand and
its complexes for the next 48 h. Suitable untreated controls
(containing 50% DMSO as vehicle) were also concomitantly
employed. Each dose was tested in at least three replicate
wells. For morphological analysis, cells in six-well plates
were observed under a phase contrast microscope (Nikon
Eclipse Ti, Japan) and photographed.
[Cu(C5H11N3S)2SO4]
Yield 38%; MW 450; MP (°C) 195; FTIR (KBr) (cm−1) 1562
1
(C=N), 705, 1058 (C=S); H NMR (DMSO) (ppm) 0.31 (t,
3H), 2.3 (s, 3H), 2.5 (q, 2H), 6.6 (br s, 1H,NH), 7.8 (br s,
2H,NH2); Solubility DMSO, DMF; λmax (nm) 299; molar
conductance 12.78 Ω−1 cm−1 mol−1.
[Fe(C5H11N3S)2SO4]
Cell viability/cytotoxicity analysis
Yield 23%; MW 442; MP (°C) 190; FTIR (KBr) (cm−1) 1562
(C=N), 705, 1050 (C=S); H NMR (DMSO) (ppm) not ob-
1. Cell viability analysis using Trypan blue dye exclusion
assay
1
tained as 1H NMR spectrum of the Fe(II)-thiosemicarbazone
complex system is difficult to detect due to high spin state of
Fe(II) in its complex [9, 15, 37]; solubility: DMSO, DMF;
To determine the cell viability, cells exposed to varying
concentrations of the ligand and its synthesized complexes
were subjected to Trypan blue assay. Briefly, cell suspen-
sions were made at a suitable dilution (105cells/mL) in
PBS. Respective cell suspensions (50 µL) were taken and
mixed with an equal volume of 0.4% Trypan blue, mixed
thoroughly and allowed to stand for 5 min at room temper-
ature. Solution (50 µL)was transferred to a hemocytometer
and viable cells were counted as clear cells and dead cells
as blue ones. The number of live cells in both treated (li-
gand and its complexes) and control (cells and vehicle)
λ
max (nm) 300; molar conductance 19.68 Ω−1 cm−1 mol−1.
[Zn(C5H11N3S)2SO4]
Yield 62%; MW 452; MP (°C) 250; (KBr) (cm−1) 1570
1
(C=N), 705, 1050 (C=S); H NMR (DMSO) (ppm) 1.1 (t,
3H), 1.9 (s, 3H), 2.5 (q, 2H), 7.3 (br s, 1H,NH), 7.5 (br s,
2H,NH2); solubility: DMSO, DMF; λmax (nm) 300; molar
conductance: 8.08 Ω−1 cm−1 mol−1.

J Chem Biol
Table 1 Docking results with Topo II (PDB ID: 5IWI)
wells was used for calculating the percentage cytotoxicity
as % Cytotoxicity = Live Cell No. in Treated Wells − Live
Cell No. in Control Wells/Live Cell No. in Control
Wells × 100).
S. Compound
No.
Binding
energy
Inhibition Interacting amino
constant acid(s)
(kcal/mol) (Ki)
1
C5H11N3S
−4.58
437.87µM G41, L42, K43,
H46, Q95, Y99,
L103, N166,
2. Methyl tetrazolium-MTT assay
MTT was performed as per published protocol [33] in
96-well microtiter tissue culture plates (Linbro, MP
Biomedicals). For MTT assay, 104 MDA cells were seed-
ed in 200 μL of medium in a 96-well microtiter tissue
culture plate and cultured in a humidified 5% CO2 incu-
bator at 37 °C for 24 h. Defined concentrations (20–
100 μM) of the ligand and its complexes in 50% DMSO
were freshly prepared in culture media by serial dilution.
Serial dilution was carried out in cell culture media in
such a way that the final concentration of DMSO in the
well did not exceed 0.5% (v/v). Three control wells con-
taining the medium alone to serve as blanks were also
included. After 24 h of incubation, in separate
N170, G171,
A172
2
[Fe(C5H11N3S)2SO4] −5.08
188.07µM P36, G41, L42,
K43, H46, R92,
M93, Y99,
L103, N166,
N170, G171,
A172, S173
3
[Zn(C5H11N3S)2SO4] −3.37
3.38mM P36, G41, L42,
H46, S98, Y99,
L103, N166,
L167, N170,
G171, A172,
S173, F266
(a)
(b)
(c)
Fig. 1 Docking pose of ligand (a), its Fe complex (b), and Zn complex (c) in the active site of Topo II

J Chem Biol
Table 2 Docking results with RR (PDB ID: 5 CI3)
in triplicates for 48 h. Equal volumes of 50% DMSO (in
cell culture media) were used as vehicle controls. At the
end of treatment, cell culture medium containing varying
amounts of ligand/complexes was removed and 20 μL of
MTT (stock made in PSS at 5.0 mg/mL) reagent was
added to each well and incubated for 4 h. Thereafter,
MTT was removed and formazan crystals were dis-
solved in 200 μL of DMSO. The plates were read in a
Bio-Rad PW41 ELISA plate reader at a wavelength of
570 nm with a reference wavelength of 630 nm.
Percentage cell viability (Y-axis) was calculated from
absorbance and plotted against concentration in micro-
molar (X-axis).
S. Compound
No.
Binding
energy
Inhibition Interacting amino
constant acid(s)
(kcal/mol) (Ki)
1
2
3
C5H11N3S
−4.75
327.4µM T81, Q80, D84,
H118, S119,
S121, T123,
I125, F208, I234
[Fe(C5H11N3S)2SO4] −3.43
[Zn(C5H11N3S)2SO4] −4.42
3.08mM R89, S90, V93,
A94, I96, I153,
S154, Y157,
D158, I161
571.48µM I86, R89, S90,
V93, I153,
S154, Y157,
D158, I161
% Cell survival was calculated as = {(AT − AB)/
(AC − AB)} × 100 where
AT = Absorbance of treatment well.
AB = Absorbance of blank.
experiments, cells were treated with the previously men-
tioned concentrations of the ligand and its four complexes
Ac = Absorbance of control well.
% cell inhibition = 100 − Cell Survival.
(a)
(b)
(c)
Fig. 2 Docking pose of ligand (a), its Fe complex (b), and Zn complex (c) in the active site of RR

J Chem Biol
IC50 values were obtained from the graph as the con-
centration, which decreased cell by viability 50%.
Data interpretation and statistical analysis
Absorbance values that were lower than the control wells
indicated a reduction in the rate of cell proliferation.
Conversely, a higher absorbance value indicated an increase
in cell proliferation. Rarely, an increase in proliferation might
be offset by cell death; evidence of cell death was inferred
from morphological analysis. Results were expressed as
mean SD of experiments done in duplicates.
Antibacterial activity
The in vitro antibacterial activity of the ligand and its
complexes was evaluated against Staphylococcus aureus
(Gram-positive) and Escherichia coli (Gram-negative)
bacteria by the disc diffusion method [1] using Mueller-
Hinton agar (MHA) medium. The bacteria were
subcultured in the agar medium and were incubated for
24 h at 37 °C. The discs (sterile filter paper discs,
Whatman No. 1.0), having a diameter of 5 mm, were then
soaked in the test solutions with the appropriate equiva-
lent amounts of the ligand and its four complexes dis-
solved in sterile 50% DMSO at concentrations of 2–
10 mg/disc and placed on lawn culture of the respective
microbial organism and stored in an incubator for the
previously mentioned period of time. Formation of inhi-
bition zone (if any) around each disc was measured, and
the results were recorded in the form of inhibition zones
as a function of diameter (mm). To clarify any effect of
DMSO (used as a vehicle for the dissolution of the ligand
and its complexes) on biological screening, 50% DMSO
was used as negative control where it showed no activity
against any bacterial strains. Tetracycline was used as a
positive control.
Results and discussion
Computational docking is an extremely useful tool to gain an
understanding about synthesized compounds and their interac-
tions with biological drug targets, which is very important in drug
discovery research. The Molecular Docking software predicted
the amino acids in active site region of the studied target proteins.
Docking with topoisomerase II
Calculated binding energies and inhibition constants (Ki)
of 2-Butanone Thiosemicarbazone and its Fe(II) and Zn(II)
complexes with respect to the enzyme and the interacting
amino acids in its active site have been summarized in
Table 1. Fe (II) complex was found to have minimum
binding energy (-5.08 kcal/mol) released on interaction
with target protein (Topo II) and also the lowest Ki
n= +1 in case of Co (III) complex, in case of sulfate as anion the donor atoms would occupy two adjacent
coordination site or Cl-
Fig. 3 Synthesis scheme

J Chem Biol
Table 3 Toxicity potential of the ligand and its complexes
S. No.
Compound
Toxicity potential (Ki or IC50 in nmol/L)
Mutagenic
Tumorigenic
Reproductive
Irritant
Molecular shape index
Spherical < 0.5 < linear
Molecular flexibility
Low < 0.5 < High
1
2
3
4
5
C5H11N3S
Low
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
0.77778
0.47368
0.47368
0.47368
0.47368
0.46893
0.84037
0.84037
0.84037
0.84037
[Fe(C5H11N3S)2SO4]
[Zn(C5H11N3S)2SO4]
[Cu(C5H11N3S)2SO4]
[Co(C5H11N3S)2Cl2]+
None
None
None
None
value (188.07 µM) as compared to the Zn (II) complex and
ligand. The docking interactions of the ligand and its Fe(II)
and Zn(II) complexes with Topo II have been shown in
Fig. 1a–c.
ion and nitrogen. In the 13C spectrum of ligand, the thiol
carbon peak (C=S, 178.72) and the carbon attached to nitro-
gen (C=N, 155.5) were found to be the most downfield be-
cause of electronegativity difference. The 1H NMR spectrum
of the Fe(II) thiosemicarbazone complex (t2g4, eg system)
2
Docking with ribonucleotide diphosphate reductase
was difficult to detect due high spin state of the Fe(II) com-
plex. In order to characterize d6 Fe-thiosemicarbazone com-
plex, the signal of protons centered at 0 to 10 ppm was found
to be very broad and difficult to distinguish from the baseline,
thereby making it not useful for monitoring the existence of
the Fe(II) complex [9, 15, 37]. Mass spectrum data gave a
molecular ion peak (M+1) at 146 corresponding to its molec-
ular weight of 145. The bonding mode of the ligand to metal
ion was elucidated by analysis of the IR data. By comparing
the frequencies of the ligand with the metal complexes, it was
concluded that ligand coordinates with the metal centre
through the azomethene nitrogen [11, 19] and the thione sulfur
[7, 42], hence indicating the bidentate nature of the ligand
because the C=N and C=S frequencies decrease in case of
metal complex formation. The ligand may show thione-thiol
tautomerization because it has a thiomide functional group.
However, the absence of v (S–H) stretching in 2500–2600-
cm−1 region of the IR spectrum of the ligand indicated that the
ligand retained its thione form in the solid state. In addition,
the band at 1119 cm−1 found in ligand was found to be missing
in complexes. Appearance of new bands at 1058 cm−1 (Cu (II)
complex), 1050 cm−1 (Co (III) complex, 1050 cm−1 (Fe (II)
complex), and 1050 cm−1 (Zn (II) complex) confirmed the fact
Calculated binding energies and inhibition constants (Ki) of the
ligand and its Fe(II) and Zn(II) complexes with respect to the
enzyme RR and the interacting amino acids in its active site
have been summarized in Table 2. The interactions between the
drug compound and the active sites on the target receptors may
be through non-covalent interactions like hydrogen bonding,
hydrophobic, and van der Waals interaction [10]. Zn (II) com-
plex had a lower binding energy (-4.42 kcal/mol) and lower Ki
value (571.48 µM) with respect to RR as compared to Fe (II)
complex.The docking interactions of the ligand and its Fe(II)
and Zn(II) complexes with RR have been shown in Fig. 2a–c.
Characterization and complex formation
Preparatory scheme of the ligand and metal complexes is giv-
en in Fig. 3. Spectral analysis confirmed complex formation.
1H NMR spectrum of the ligand revealed that NH and NH2
protons were observed downfield due to higher electronega-
tivity of nitrogen making the attached protons de-shielded. On
complexation, the frequencies further got down field (7–
8 ppm) due to electronegativity difference between the metal
Table 4 Bioactivity score of the ligand and its complexes
S. No. Compound
Parameters of Bioactivity score
GPCR
ligand
Ion channel modulator Kinase
Nuclear receptor ligand Protease
inhibitor
Enzyme
inhibitor
inhibitor
1
2
3
4
5
C5H11N3S
−3.65
−3.31
−0.17
−0.09
−0.17
−0.17
−3.77
−0.15
−0.15
−0.15
−0.15
−3.80
−0.54
−0.54
−0.54
−0.54
−3.32
−0.29
−0.29
−0.29
−0.29
−2.34
−0.11
−0.11
−0.11
−0.11
[Fe(C5H11N3S)2SO4] −0.39
[Zn(C5H11N3S)2SO4] −0.34
[Cu(C5H11N3S)2SO4] −0.39
[Co(C5H11N3S)2Cl2]+ −0.39

J Chem Biol
that there was a contribution of C=S vibration in this region.
Bands higher than 1080 cm−1 disappeared in complexes [16].
1H NMR data provided information about the different posi-
Toxic parameters of the complexes
The parameters predict the probable side effects of the com-
pounds. The synthesized ligand and complexes were predict-
ed to have no side effects as shown in Table 3.
1
tions of protons. In the HNMR spectra of the complexes, a
high-frequency shift of the methyl hydrogen atoms (C–CH3),
vis-à-vis ligand spectrum, gave evidence that coordination
occurred through the azomethine nitrogen atom [39]. High
melting points of the complexes were another indication of
coordination. λmax of the ligand (267 nm) corresponded to
π–π* transition [2], whereas in complexes, the absorption
maximum between 285 and 385 nm was assigned to n–π*
transition [8, 18]. Increase in absorbance (hyperchromic shift)
in all the complexes was attributed to complexation behavior
of ligand towards metal ions confirming the coordination of
the ligands to the metal ions.
Bioactivity score prediction
The pharmacological activity describes the beneficial ef-
fects of drugs in living beings. The drug is supposed to
bind with a biological target. Biological targets are the
most common proteins such as enzymes, ion channels,
and receptors. The biological target is also referred to as
drug target. The bioactivity scores of the synthesized com-
plexes were calculated for different parameters such as
binding to G protein-coupled receptor (GPCR) ligand and
nuclear receptor ligand, ion channel modulation, kinase
inhibition, protease inhibition, and enzyme activity inhibi-
tion. All the parameters were calculated with the help of
online software Molinspiration (www.molinspiration.com)
, which predicted moderate biological activity for the
synthesized complexes. The bioactivity score is given in
Table 4. It is known that for metal complexes, if the
bioactivity score is more than 0.0, then the complex is
active; if it is between −5.0 and 0.0, then the complex is
moderately active, and if the bioactivity score is less than
−5.0, then it is inactive. As seen in Table 4, the bioactivity
scores of the ligand as well as all the four complexes were
between −5.0 and 0.0, which clearly indicate that they
possess such properties as are required for the complexes
to act as potential drugs with some modifications in
chemical structure [10].
Conductivity measurements can predict the mode of coor-
dination of counter ions to metal complexes. They provide a
method of testing degree of ionization of the complexes. The
larger the number of ions outside the coordination sphere, the
greater would be the molar conductance [35]. Low molar con-
ductivities of the Cu(II), Zn(II), and Fe(II) butanone
thiosemicarbazone complexes were indicative of non-
electrolytic nature indicating the bonding of sulfate ions to
the metal ions [32]. Co(III) complex showed higher molar
conductance of 105.04 Ω−1 cm−1 mol−1, which may be due
to positive charge over the coordination sphere. The Fe, Cu,
and Zn complexes dissolved in DMSO and DMF with time
whereas Co complex dissolved readily further confirming its
electrolytic nature. The solubility difference can be explained
on the basis of different salts used for the synthesis having
sulfate and chloride as anions.
n ¼ þ1 in case of Co ðIIIÞ complex; A ¼ SO42−or Cl−
Table 5 Physicochemical properties of the synthesized ligand and its complexes
Physicochemical Properties
S.
Compound
Lipinski's Parameters
No.
%
Topological polar MW M log Hydrogen bond
Hydrogen bond
Number of
Lipinski’s
violations
Absorptiona surface area (Å)2
(TPSA)b
Pc
donors (nOHNH) acceptors (nON) rotatable bonds
1
2
3
4
5
C5H11N3S
91.6085
50.41
82.11
82.11
82.11
82.11
<500 1.08
<500 −6.02
<500 −5.88
<500 −5.99
<500 −5.93
3
6
6
6
6
3
6
6
6
6
3
2
2
2
2
0
1
1
1
1
[Fe(C5H11N3S)2SO4] 80.6720
[Zn(C5H11N3S)2SO4] 80.6720
[Cu(C5H11N3S)2SO4] 80.6720
[Co(C5H11N3S)2Cl2]+ 80.6720
a Percentage absorption (% absorption) was calculated by % Absorption = 109 − [0.345 × Topological Polar Surface Area]
b Topological polar surface area (defined as a sum of surfaces of polar atoms in a molecule)
c Logarithm of compound partition coefficient between n-octanol and water

J Chem Biol
PASS Analysis: Lipinski’s parameters
than one violation of the given criteria. In the present study,
the synthesized ligand and its complexes were found to be in
good agreement with the given criteria and can be said to
possess good oral bioavailability (Table 5).
Lipinski’s rule of five (RO5) is used to evaluate drug
likeliness of a chemical compound possessing properties
that would make it a likely or potential drug in humans
[29]. The oral activity of a drug compound is predicted by
calculating certain molecular parameters like log P (partition
coefficient), polar surface area, number of hydrogen bond
donors, number of hydrogen bond acceptors, and molecular
weight. The rule states that most metal complexes with good
membrane permeability have log P ≤ 5, number of hydrogen
bond acceptors ≤10, and number of hydrogen bond donors
≤5 [10]. In general, an orally active drug has no more
Biological evaluation of ligand and complexes
1. Morphological analysis
Suppl. Figs. 1-10 (a-l) respectively depict the dose-
dependent effect of the ligand and its four complexes on MDA
and A549 cells using phase contrast microscopy. Ligand was
found to be active against MDA cells in the tested range. No
Dose (µM)
(a)
Dose (µM)
Dose (µM)
(b)
(c)
Dose (µM)
Dose (µM)
(d)
(e)
Fig. 4 a–e Dose-dependent effect of ligand (a), its Co (b), Fe (c), Cu (d), and Zn (e) complexes on viability of MDA cells in vitro using MTTassay. Final
concentration of DMSO in each well did not exceed 0.5% (v/v). Results were expressed as mean SD of experiments done in triplicates.

J Chem Biol
significant activity of the ligand was detected in the tested range
against A549 cells. However, all complexes were found to pos-
sess significant activity against both cell lines with IC50 values of
<50 μM (refer to Suppl. data).
activity against A549 cells whereas its complexes displayed
significant cytotoxicity against both the tested cell lines.
Results were expressed as mean SD of experiments done
in duplicates (refer to Suppl. data).
2. Cell viability analysis
3. MTT assay
Suppl. Tables 1-5 present the dose-dependent cytotoxicity
of the ligand and its complexes when tested in the range of 20–
100 μM against breast cancer cells MDA-MB-231 and lung
carcinoma cells A549 cells using the Trypan blue dye exclu-
sion assay. As mentioned before, ligand did not exhibit any
Figures 4 and 5 respectively depict the dose-dependent
effect of the ligand (Figs. 4a and 5a), its Co (Figs. 4b and
5b), Fe (Figs. 4c and 5c),Cu (Figs. 4d and 5d) and Zn
(Figs. 4e and 5e) on MDA and A549 cells using the MTT
assay. Ligand showed 50% cytotoxicity against MDA at
Dose (µM)
(a)
Dose (µM)
Dose (µM)
(b)
(c)
Dose (µM)
Dose (µM)
(d)
(e)
Fig. 5 a–e Dose-dependent effect of ligand (a), its Co (b), Fe (c), Cu (d), and Zn (e) complexes on viability of A549 cells in vitro using MTT assay.
Final concentration of DMSO in each well did not exceed 0.5% (v/v). Results were expressed as mean SD of experiments done in triplicates.

J Chem Biol
Fig. 6 Antibacterial activity of
ligand (a), its Co (b), Fe (b), Cu
(c), and Zn (d) complexes against
S. aureus using disc diffusion
method
(a)
(b)
(c)
(d)
(e)
Table 6 Comparison of MIC
values (in mg/mL) of ligand and
its complexes and standard
antibiotic tetracycline against
S. aureus
Ring diameter (mm)
Compound
Tetracycline (30 mg/mL)
DMSO
Compound dose (mg/mL)
2
4
5
6
8
10
C5H11N3S
12
14
10
21
21
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6
ND
8
ND
8
ND
ND
ND
9
[Co(C5H11N3S)2Cl2]+
[Fe(C5H11N3S)2SO4]
[Cu(C5H11N3S)2SO4]
[Zn(C5H11N3S)2SO4]
ND
ND
ND
ND
8
ND
9
ND
ND
ND
ND not detected

J Chem Biol
Fig. 7 Antibacterial activity of
ligand (a), its Co (b), Fe (b), Cu
(c), and Zn (d) complexes against
E. coli using disc diffusion
method
(a)
(b)
(c)
(d)
(e)
Table 7 Comparison of MIC
values (in mg/mL) of ligand and
its Co and Fe complexes and
standard antibiotic tetracycline
against E. coli
Ring diameter (mm)
Compound
Tetracycline (30 mg/mL)
DMSO
Compound dose (mg/mL)
2
4
5
6
8
10
C5H11N3S
5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4
ND
ND
ND
5
[Co(C5H11N3S)2Cl2]+
[Fe(C5H11N3S)2SO4]
[Cu(C5H11N3S)2SO4]
[Zn(C5H11N3S)2SO4]
13
10
14
8
ND
ND
ND not detected

J Chem Biol
to Mr. Mohsin Ali Khan, Secretary, EET and Chairman Research, Era’s
Lucknow Medical College & Hospital, Lucknow, for providing the nec-
essary support and cooperation for carrying out the biological activity
evaluation studies and to the R&D wing of Integral University,
Lucknow, for providing support and communication number (IU/R&D/
2016-MCN008IU/R&D/2016-MCN008) for the manuscript.
80 μM (IC50 value approx. 80 μM). As mentioned earlier, the
ligand did not show any significant activity against A549 cells
within the tested range. However, complexation with metals
significantly increased the cytotoxicity of the ligand against
both cell lines.
Author’s contributions Tahmeena Khan carried out the chemical syn-
theses, analyzed and interpreted data, and drafted the manuscript. Shalini
Dixit carried out spectroscopic studies and analyzed data. Rumana
Ahmad carried out biological activity evaluation experiments, analyzed
and interpreted the data, and edited and revised the manuscript. Saman
Raza coanalyzed and interpreted the chemical data and revised the man-
uscript. Iqbal Azad carried out the computational analyses, docking stud-
ies, and data interpretation. Seema Joshi and Abdul Rahman Khan con-
ceived and conceptualized the overall study design and coordination and
revised the manuscript. All authors read and approved the final
manuscript.
Antibacterial activity
The ligand together with its complexes was also evaluated for
their potential antibacterial activity against S. aureus and
E. coli. The ligand did not exhibit any significant activity
against both species. However, Co and Cu complexes exhib-
ited mild antibacterial activities against S. aureus (Fig. 6,
Table 6), whereas Cu complex alone showed moderate activ-
ity against E. coli (Fig. 7, Table 7).
Compliance with ethical standards No permission was sought from
the Institutional Ethics Committee, Era’s Lucknow Medical College,
Lucknow, as the work did not involve human or animal subjects.
Conclusion
Conflict of interest The authors declare that they have no competing
interests.
The overall activity spectrum of biologically active com-
pounds can be predicted by computational analyses like mo-
lecular docking with probable receptors or by prediction of
bioactivity score and physicochemical properties. In this pa-
per, anticancer and antibacterial activities of ligand 2-
butanaone thiosemicarbazone and its four metal complexes
have been reported against two cancer cell lines and two spe-
cies of bacteria. While the ligand displayed mild anticancer
activity against MDA cell line (80 μM) and none against
A549 cell line, its four complexes displayed significant anti-
cancer activities against both the tested cell lines with IC50
<50 μM for Co, Fe, and Zn complexes. However, only Cu
complex exhibited appreciable activity against S. aureus and
E. coli while Co complex was found to be mildly active
against S. aureus only. Molecular docking studies showed
significant binding of the ligand with selected targets.
Bioactivity score and PASS analysis showed that the metal
complexes can have potential inhibitory action against the
selected cell lines. The study has shed light on the role of
different anions on the overall solubility of the complexes,
which can also affect their conductivity. Likewise, the oxida-
tion state of metal significantly influences its affinity for a
particular ligand working on the hard-soft acid-base concept.
This phenomenon would be explored in more detail in future.
In future, the complexes of ligand would be tested against
other cancer as well as normal cell lines and would also be
evaluated for their probable anticancer and antibacterial activ-
ities in vivo. The complexes would also be analyzed for their
potential antioxidant and DNA cleavage activity.
References
1. Al-Amiery AA, Mohammed A, Ibrahim H, Abbas A (2010) J
Biotechnol Res Cent 4:5
2. Barnum W (1961) J Inorg Nucl Chem 21:221
3. Bavin EM, Rees RJW, Robson JM, Seiler M, Seymour DE,
Suddaby D (1950) J Pharm Pharmacol 2:764
4. Beraldo H, Gambino D (2004) Med Chem 4:31
5. Bjelogrlic S, Todorovic T, Bacchi A, Zec M, Sladic D, Srdic-Rajic
T (2010) J Inorg Biochem 104(673):12–1339
6. Brockman RW, Sidwell RW, Arnett G, Shaddix S (1970)
Heterocyclic thiosemicarbazones: correlation between structure, in-
hibition of ribonucleotide reductase, and inhibition of DNAviruses.
Proc Soc Exp Biol Med 133:609–614
7. Chandra S, Sangeetika RAJ (2001) Saudi Chem Soc 5:175
8. Cotton FA, Wilkinson CW (1972) Advanced inorganic chemistry,
3rd edn. Interscience Publisher, New York
9. Crans DC, Yang L, Khan EGR, Jin W, Simonis U (2003)
Applications of Paramagnetic NMR Spectroscopy for Monitoring
Transition Metal Complex Stoichiometry and Speciation.
Ameriacan Chemica Society: 304–326
10. Dar AM, Khan MA, Mir S, Gatoo MA (2016) DNA binding, cleav-
age activity, molecular docking, cytotoxicity and genotoxicity stud-
ies of newly synthesized copper based metal complexes. Pharm
Anal Acta 7:464
11. Deepa KP, Aravindakshan KK (2000) Synth React Inorg Met Org
Chem: 3
12. Dhar DN, Taploo CL (1982) J Sci Ind Res 41:501
13. Ertl P, Rohde B, Selzer P (2000) Fast calculation of molecular polar
surface area as a sum of fragment- based contributions and its ap-
plication to the prediction of drug transport properties. J Med Chem
43(20):3714–3717
Acknowledgements The authors gratefully acknowledge Dr. E.S.
Charles, President and Dr. M. Harrison, Head, Department of
Chemistry,Isabella Thoburn College, Lucknow, for providing necessary
facilities to carry out the lab work. The authors are also thankful
14. French FA, Blanz EJJ (1965) The carcinostatic activity of
alpha-(N) heterocyclic carboxaldehyde thiosemicarbazones.
I. Isoquinoline-1-carboxaldehyde thiosemicarbazone. Cancer
Res 25:1454–1458

J Chem Biol
15. Fryzuk MD, Leznoff DB, Ma ESF, Rettig SJ, Young VG (1998)
Synthesis, structure, and reactivity of paramagnetic iron(II) and
iron(III) amidodiphosphine complexes. Organometallics 17:2313–
2323
16. Gingras BA, Sdomorjai RL, Bayley CH (1961) Can J Chem 39:973
17. Hall IH, Lackey CB, Kistler TD (2000) Cytotoxicity of copper and
cobalt complexes of furfural semicarbazone and thiosemicarbazone
derivatives in murine and human tumor cell lines. Pharmazie 55:
937–941
and permeability in drug discovery and development settings. Adv
Drug Deliv Rev 46:3–26
30. Lovejoy DB, Richardson DR (2002) Blood 100:666
31. Ludwig JA, Szakács G, Martin SE (2006) Selective toxicity of
NSC73306 in MDR1-positive cells as a new strategy to circumvent
multidrug resistance in cancer. Cancer Res 66:4808–4815
32. Mohamed GG, Ibrahim NA, Attia HAE (2009) Spectrochim. Acta
72:610
33. Mosmann T (1983) J Immunol Meth 65:55
34. Proudfoot JR (2002) Drugs, leads, and drug-likeness: an analysis of
some recently launched drugs. Bioorg Med Chem Lett 12:1647–
1650
18. Holm RH, Cotton FA (1958) J Am Chem Soc 80:5658
19. Joseph M, Sreekanth A, Suni V and Kurup MRP (2006)
Spectrochim Acta A: 637–641
20. Jutten P, Schumann W, Hartl A, Heinisch L, Grafe U, Warner W,
Uldrich T (2002) BIoorg Med Chem Lett 12:1339
21. Khajah MA, Almorhi I, Mathew PM, Lugmani YA (2013) PLoS
One 8:e76327
22. Khan T, Ahmad R, Joshi S, Khan AR (2015) Anticancer potential
of metal thiosemicarbazone complexes: a review. Der Chemica
Sinica 6(12):1–11
23. Koch O, Stuttgen G, Schmiedebergs N (1950) Arch Exp Pathol
Pharmakol 210:409
24. Kowal CR, Eichinger R, Jakupec MA (2007) Effect of metal ion
complexation and chalcogen donor identity on the antiproliferative
a c t i v i t y o f 2 - a c e t y l p y r i d i n e N , N -
dimethyl(chalcogen)semicarbazones. J Inorg Biochem 101:1946–
1957
25. Kumar D, Singh VK (2014) Application of metal complexes of
Schiff base with special reference to thiosemicarbazone: a review.
Journal of Drug Discovery and Therapeutics 2(13):24–32
26. Kumar S, Kumar N (2013) Int Research Journal of Pharmacy 4(1):
177–181
35. Rafat MS (2007) J Mol Struct 842:24
36. Richardson DR (2002) Crit Rev Oncol/Hematol 42:267
37. Roger C, Hamon P, Toupet L, Raba H, Saillard JY, Hamon J,
Lapinte C (1991) Halo- and alkyl(pentamethylcyclopentadienyl)
(1,2-bis(diphenylphosphino)-ethane)iron(III) 17-electron com-
plexes: synthesis, NMR and magnetic properties, and EHMO cal-
culations. Organometallics 10:1045–1054
38. Shao J, Zhou B, Di Bilio AJ (2006) A ferrous-triapine complex
mediates formation of reactive oxygen species that inactivate hu-
man ribonucleotide reductase. Mol Cancer Ther 5:586–592
39. Sharma B, Kothari R (2015) Int J Pharm Bio Sci 6 (1), (B): 1154–
1169
40. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple
KD (2002) Molecular properties that influence the oral bioavailabil-
ity of drug candidates. J Med Chem 45(12):2615–2623
41. Wu C, Shukla S, Calcagno AM, Hall MD, Gottesman MM,
Ambudkar SV (2007) Evidence for dual mode of action of a
thiosemicarbazone, NSC73306: a potent substrate of the multidrug
resistance linked ABCG2 transporter. Mol Cancer Ther 6:3287–
3296
27. Kune GA (1964) Br Med J 2:621
28. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Adv
Drug Deliv Rev 23(1–3):3–25
29. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001)
Experimental and computational approaches to estimate solubility
42. Youssef NS, Hegab KH (2005) Synth React Inor Met Org Chem 35:
391–399
43. Yuan J, Lovejoy DB, Richardson DR (2004) Novel di-2-pyridyl-
derived iron chelators with marked and selective antitumor activity:
in vitro and in vivo assessment. Blood 104:1450–1458