Synthesis, structure and cytotoxicity evaluation of complexes of N1-substituted-isatin-3-thiosemicarbazone with copper(I) halides

Article abstract of DOI:10.1016/j.ica.2016.05.013

Synthesis of Isatin-N¹-methyl-thiosemicarbazone (H₂itsc-N¹-Me, H₂L¹) and isatin-N¹-ethyl-thiosemicarbazone (H₂itsc-N¹-Et, H₂L²) has been carried out and the effect of substituents (at N¹ atom of isatin-3-thiosemicarbazones) on nuclearity of copper(I) halide complexes has been investigated. Reactions of copper(I) halides (X = I, Br, Cl) with H₂L¹ and H₂L² using Ph₃P as co-ligand in 1:1:1 (M:L:PPh₃) molar ratio in acetonitrile yielded complexes of stoichiometry [CuX(H₂L¹)(Ph₃P)] (X = I, C1; Br, C2; Cl, C3) and [CuX(H₂L²)(Ph₃P)] (X = I, C4; Br, C5; Cl, C6) respectively. All these complexes have been characterized using analytical and spectroscopic data (IR, ¹H NMR and ESI mass). The single crystal structure has been solved for H₂L¹ and C2. The complex C2 has distorted tetrahedral geometry around copper(I) and isatin-N¹-methyl-thiosemicarbazone coordinated to metal center as neutral, bidentate, N³, S-chelating ligand. Elemental analysis suggested the presence of one acetonitrile molecule in complexes C3 and C6 and half CH₃CN in complexes C2 and C4 as solvent of crystallization. MTT assay, supported by docking studies have revealed the cytotoxic nature of the compounds C1-C6.

Full text of DOI:10.1016/j.ica.2016.05.013

Accepted Manuscript
Research paper
Synthesis, structure and cytotoxicity evaluation of complexes of N1-substituted-
isatin-3-thiosemicarbazone with copper(I) halides
Ashiq Khan, Jerry P. Jasinski, Victoria A Smoleaski, Kamaldeep Paul,
Gurpinder Singh, Rekha Sharma
PII:
S0020-1693(16)30242-0
http://dx.doi.org/10.1016/j.ica.2016.05.013
ICA 17049
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 February 2016
6 May 2016
6 May 2016
Please cite this article as: A. Khan, J.P. Jasinski, V.A. Smoleaski, K. Paul, G. Singh, R. Sharma, Synthesis, structure
and cytotoxicity evaluation of complexes of N1-substituted-isatin-3-thiosemicarbazone with copper(I) halides,
Inorganica Chimica Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.05.013
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1
Synthesis, structure and cytotoxicity evaluation of complexes of N1-
substituted-isatin-3-thiosemicarbazone with copper(I) halides
Ashiq Khana, Jerry P. Jasinskib, Victoria A Smoleaskib, Kamaldeep Paulc, Gurpinder Singha,
Rekha Sharmaa∗
aDepartment of Chemistry, Lovely Professional University, Phagwara, Punjab, India
bDepartment of Chemistry, Keene State College, 229 Main Street, Keene, NH 03435–2001, USA
cSchool of Chemistry and Biochmistry, Thapar University, Patiala-147004, Punjab, India
∗Corresponding author: R. Sharma (E-mail: rekha.14537@lpu.co.in)
Abstract
Synthesis of Isatin-N1-methyl-thiosemicarbazone (H2itsc-N1-Me, H2L1) and isatin-N1-ethyl-
thiosemicarbazone (H2itsc-N1-Et, H2L2) has been carried out and the effect of substituents (at N1
atom of isatin-3-thiosemicarbazones) on nuclearity of copper(I) halide complexes has been
investigated. Reactions of copper(I) halides (X = I, Br, Cl) with H2L1 and H2L2 using Ph3P as co-
ligand in 1 : 1 : 1 (M : L : PPh3) molar ratio in acetonitrile yielded complexes of stiochiometry
[CuX(H2L1)(Ph3P)] (X = I, C1; Br, C2; Cl, C3) and [CuX(H2L2)(Ph3P)] (X = I, C4; Br, C5; Cl,
C6) respectively. All these complexes have been characterized using analytical and
spectroscopic data (IR, 1H NMR and ESI mass). The single crystal structure has been solved for
H2L1 and C2. The complex C2 has distorted tetrahedral geometry around copper(I) and isatin-

2
N1-methyl-thiosemicarbazone coordinated to metal center as neutral, bidentate, N3, S- chelating
ligand. Elemental analysis suggested the presence of one acetonitrile molecule in complexes C3
and C6 and half CH3CN in complexes C2 and C4 as solvent of crystallization. MTT assay,
supported by docking studies have revealed the cytotoxic nature of the compounds C1-C6.
Keywords: isatin-N1-methyl-thiosemicarbazone; isatin-N1-ethyl-thiosemicarbazone; copper(I)
halide; X-ray crystallography; cytotoxicity; docking.
1. Introduction
Isatin and schiff bases of isatin derivatives possess a wide range of biological properties, like
antibacterial1-3, antifungal4-6, antiviral7–10
,
anti-HIV11–13
,
antiprotozoal14,15 and anti-
helminthic16,17 activities. Isatin on condensation with thiosemicarbazide forms three types of
thiosemicarbazones: isatin-3-thiosemicarbazone (I), isatin-2-thiosemicarbazone (II) and isatin-
2,3-bis(thiosemicarbazone) (III) (Chart 1).
S
H2N
NH2
S
S
NH
HN
N
O
N
HN
NH2
N
O
H
N
N
NH2
N
N
N
R
R
R
S
R = H or CH3
(II)
(I)
(III)
Chart 1
Out of these three, isatin-3-thiosemicarbazone has been of interest as 1-methylisatin-3-
thiosemicarbazone was found to be active in the treatment of smallpox.18-20 Moreover, a number
of substituted isatin-3-thiosemicarbazones are structurally characterized.21-31 Isatin-3-
thiosemicarbazones are good O, N, S- donors and form complexes with metals. 32-42 They show
variable bonding modes like, η1-S, terminal (A)36, N3, S- chelation (B)37, N3, S, O- chelation
(C)37, N3, S, O-chelation-cum-O-bridging (D)38 (Chart 2).

3
N3
R
N3
N3
S
S
O
M
O
M
S
M
M
M
S
( A )
( B )
( C )
( D )
Chart 2
Recently, the chemistry of copper(I) halides with thiophene-2-carbaldehyde thiosemicarbazones
and furan-2-carbaldehyde thiosemicarbazones have been reported and it was observed that
substituents at C2 and N1 atoms of thiosemicarbazone influenced the bonding and nuclearity of
copper(I) halide complexes.42-45 Thus, we were interested in finding the similar effect in case of
isatin-3-thiosemicarbazone. Un-substituted isatin-3-thiosemicarbazone reacted with copper(I)
halides and Ph3P in 1: 1: 2 molar ratio to form tetrahedral monomers.36, 46 In present work, isatin-
3-thiosemicarbazone with methyl (H2itsc-N1-Me) or ethyl group (H2itsc-N1-Et) group at N1 atom
have been synthesized (Chart 3) and the effect of substituent at N1 atom on the nuclearity of their
copper(I) complexes has been investigated. The synthesized complexes have been characterized
using elemental analysis, spectroscopic studies (IR, ESI mass, 1H NMR) and single crystal x-ray
crystallography (Complex C2). These compounds were further investigated for their cytotoxic
evaluations.
R
NH
S
N
N
H
N
H
O
R= CH3 (H2L1); C2H5 (H2L2)
Chart 3
Cancer figures among the leading cause of morbidity and mortality worldwide, with
approximately 14 million new cases and 8.2 million cancer related deaths in 2012.47 It is
expected that annual cancer cases will rise from 14 million in 2012 to 22 within the next 2
decades.47 These rising and alarming figures have prompted the discovery of some important
anticancer drugs, currently used in the therapy and tremendous efforts are being taken towards
identifying new efficient drug molecules.48 Towards this end, we evaluated the cytotoxicity

4
potency of copper(I) complexes and further investigated the mechanism of action by molecular
modelling studies.
2. Experimental
2.1. Materials and Methods
Potassium chloride, potassium bromide, potassium iodide and triphenylphosphine were
purchased from Loba Pvt. Ltd, whereas isatin, 3-methylthiosemicarbazide and 3-ethyl
thiosemicarbazides were procured from Aldrich Chemicals Ltd. Copper(I) iodide, bromide and
chloride were prepared by the reduction of CuSO4⋅5H2O using SO2 in the presence of KI, KBr or
KCl in water.49 C, H and N analyses were obtained with a Thermoelectron FLASHEA1112
CHNS analyzer. Infrared spectra were recorded from KBr pellets in the range 4000-200 cm-1 on
a SHIMADZU FTIR 8400S spectrophotometer. Melting points were determined with an
1
electrically heated Gallenkamp apparatus. H NMR were recorded on an AV500 FT
spectrometer operating at a frequency of 500 MHz using d6-DMSO/CDCl3 as the solvent with
TMS as the internal standard. Crystal snaps were taken from OLYMPUS Magnus MLX Camera
(40x and 100x). Mass spectra, ESI-methods, were recorded on Bruker Daltonics Esquire 300 mass
spectrometer.
2.2. Synthesis of N1-substituted-isatin-3-thiosemicarbazones
Isatin-N1-methyl thiosemicarbazone (H2L1) and Isatin-N1-ethyl thiosemicarbazone (H2L2) were
prepared by refluxing isatin with their respective thiosemicarbazides in methanol for 8-10 h.
2.3. Procedure for Synthesis of Copper(I) Complexes C1-C6
[CuI(η2-N3, S-H2itsc-N1-Me)(Ph3P)] C1
To a solution of CuI (0.050 g, 0.26 mmol) in 15 ml of acetonitrile was added, solid Ph3P (0.068
g, 0.26 mmol) and stirred for 2-3 hrs. The white ppt. of [CuI(Ph3P)] were formed. The
acetonitrile was decanted off from the reaction and precipitates were dissolved in 15 ml
chloroform. To it was added, solid H2itsc-N1-Me (0.061 g, 0.26 mmol) and the reaction mixture
was stirred for 3-4 h. Yellow color clear solution thus obtained was filtered and kept for
crystallization on slow evaporation at room temperature. The product obtained was recrystallized
from acetonitrile. Yield: 0.061 g, 68%; m.p. 218-220 ºC. Elemental analysis: Found: C, 48.5; H,

5
3.7; N, 8.04; S, 4.36. C28H26N4PSOCuI requires, C, 48.9; H, 3.8; N, 8.16; S, 4.66%. Main IR
peaks (KBr, cm−1), υ(N–H), 3468s, 3402s; υ(–NH–) 3217m; (NH, isatin) 3142s; υ(C–HPh),
3051s; υ(C–HMe), 2814; υ(C=O) 1687s; δ(NH2) + υ(C=N) + υ(C–C), 1597, 1554s; υ(C=S) 826s
1
(thioamide moiety), υ(P–CPh), 1095s. H NMR (DMSO, δ ppm): 12.64 s (1H NHisatin), 11.23s
(1H, N2H), 7.76 s (1H N1H), 7.67-6.93 m (19H, C5,7,8,6H + Ph3P), 2.50 m (3H, CH3). m/z (ESI)
707.5 (M++ Na), 684.9 (M+).
Complexes C2-C6 were prepared by a similar method albeit with their respective metal salts.
[CuBr(η2-N3, S-H2itsc-N1-Me)(Ph3P)] C2.
Yield: 0.080 g, 76%; m.p. 182-185 ºC. Elemental analysis, Found: C, 53.5; H, 4.0; N, 9.76; S,
4.39. C28H26N4SOPCuBr⋅0.5CH3CN requires: C, 53.0; H, 4.2; N, 9.54; S, 4.84. Main IR peaks
(KBr, cm−1), υ(N–H), 3306s, 3207s, 3051s, 3026s; (NH, isatin) 2983m; υ(C–HMe), 2935s; υ(C–
HPh), 3026; υ(C=O) 1707s; δ(NH2) + υ(C=N) + υ(C–C), 1614s, 1556m, 1479m, 1464m; υ(C=S)
826s (thioamide moiety), υ(P–CPh), 1095s. 1H NMR (DMSO, δ ppm): 11.25 s (1H, NHisatin), 9.50
s (1H, N2H), 7.80 s (1H N1H), 7.39-6.89 m (19H C5,7,8,6H+Ph3P), 3.06 m (3H, CH3). m/z (ESI)
660 (M++ Na), 637 (M+).
[CuCl(η2-N3, S-H2itsc-N1-Me)(Ph3P)] C3.
Yield: 0.115 g,72 %; m.p. 182-185 ºC. Elemental analysis: Found: C, 56.2; H, 4.4; N, 11.00, S,
4.99. C28H25N4SOPCuCl⋅CH3CN requires, C, 56.6; H, 4.4; N, 11.26; S, 5.03. Main IR peaks
(KBr, cm−1), υ(N–H), 3470s, 3402s, 3215m; υ(–NH–) 3132m; (NH, isatin) 2982m; υ(C–HMe),
2893, 2823; υ(C=O) 1687s; δ(NH2) + υ(C=N) + υ(C–C), 1653, 1620s, 1543s, 1467s; υ(C=S)
1
827s (thioamide moiety), υ(P–CPh), 1103s. H NMR (DMSO, CDCl3, δ ppm): 11.23 s (1H,
NHisatin) 9.42 s (1H, N2H), 7.78 s (1H, N1H), 7.39-6.91 m (19H, C5,7,8,6H + Ph3P), 3.10 m (3H,
CH3). m/z (ESI) 616 (M++ Na), 593 (M+).

6
[CuI(η2-N3, S-H2itsc-N1-Et)(Ph3P)] C4.
Yield: 0.065 g, 71%; m.p. 197-200 ºC. Elemental analysis, Found: C, 49.5; H, 3.8; N, 7.96; S,
4.53. C29H28N4SOPCuI requires, C, 49.6; H, 3.9; N, 7.98; S, 4.56. Main IR peaks (KBr, cm−1),
υ(N–H), 3306s, 3273s; υ(–NH–) 3180m; (NH, isatin) 3053s; υ(C–HPh), 2982w, 2929w; υ(C–
HEt), 2829; υ(C=O) 1685s; δ(NH2) + υ(C=N) + υ(C–C), 1651s, 1591m, 1543s; υ(C=S) 827s
(thioamide moiety), υ(P–CPh), 1094s. 1H NMR (DMSO, δ ppm): 11.24 s (1H, NHisatin), 9.5 s(1H,
N2H), 7.8 s (1H, N1H), 7.46-6.9 m (19H, C5,7,8,6H+Ph3P), 2.5 t (3H, CH3), 1.206 m (2H, CH2).
m/z (ESI) 722 (M+ + Na), 699 (M+).
[CuBr(η2-N3, S-H2itsc-N1-Et)(Ph3P)] C5.
Yield: 0.087 g, 74%; m.p. 182-185 ºC. Elemental analysis, Found: C, 53.4; H, 4.0; N, 9.17; S,
4.81. C29H28N4PSOCuBr⋅0.5CH3CN requires, C, 53.5; H, 4.4; N, 9.36; S, 4.75. Main IR peaks
(KBr, cm−1), υ(N–H), 3308s; υ(–NH–) 3176s; (NH, isatin) 3049s, 3012s 2980m; υ(C–HPh),
2928s; υ(C–HEt), 2889; υ(C=O) 1705s; δ(NH2) + υ(C=N) + υ(C–C), 1614s, 1570m, 1543s,
1
1523m, 1458s; υ(C=S) 828s (thioamide moiety), υ(P–CPh), 1094s. H NMR (DMSO, δ ppm):
11.23 s (1H, NHisatin), 9.56 s (1H, N2H), 7.9 s (1H, N1H), 7.34-6.90 m (19H, C5,7,8,6H + Ph3P),
2.5 t (3H, CH3), 1.179 m (2H, -CH2-). m/z (ESI) 674 (M+ + Na), 651 (M+).
[CuCl(η2-N3, S-H2itsc-N1-Et)(Ph3P)] C6.
Yield: 0.018 g, 72%; m.p. 182-184 ºC. Elemental analysis, Found: C, 57.2; H, 4.3; N, 10.07; S,
4.62. C29H28N4PSOCuCl⋅CH3CN requires, C, 57.3; H, 4.8; N, 10.78; S, 4.93. Main IR peaks
(KBr, cm−1), υ(N–H), 3306m; υ(–NH–) 3161m; (NH, isatin) 2982m; υ(C–HEt), 2929; υ(C=O)
1687s; δ(NH2) + υ(C=N) + υ(C–C), 1653, 1620s, 1543s, 1467s; υ(C=S) 827s (thioamide
1
moiety), υ(P–CPh), 1093s. H NMR (DMSO,CDCl3 δ ppm): 11.23 s (1H, NHisatin), 9.46 s (1H,
N2H), 7.80 s (1H, N1H),7.38-6.91 m (19H, C5,7,8,6H+Ph3P), 2.53 t (3H, CH3), 1.18 m (2H, CH2).
m/z (ESI) 630.1 (M+ + Na), 607.1 (M+).

7
2.4. X-ray Crystallography
A single crystal was used for data collection with Agilent, Eos, Gemini (C2) and X’calibur CCD
(H2L1), equipped with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å). Crystal data
was collected at 173 (2) (C2) and 293(2) (H2L1) K. Data was processed with CrysAlisPro,
Agilent Technologies (C2), CrysAlisPro, Oxford Diffraction Ltd (H2L1) (data collection) and
50
CrysAlisPro RED (cell refinement, data reduction). For structure solution program SHELXS-
51
97 (H2L1)
was used, whereas structure refinement was done by SHELXL (C2)52 and
SHELXL-97 (H2L1). Atomic scattering factors taken from “International Tables for
Crystallography”.53
2.5. Cytotoxicity studies
2.5.1. Cell culture
L123 (human lung cells) and HepG2 (Human hepatocellular carcinoma cells) was cultured in
Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (heat inactivated), 100 units
o
mL-1 penicillin, 100 µg mL-1 streptomycin, and 2.5 µg mL-1 amphotericin B, at 37 C in a
saturated humidity atmosphere containing 95% air/5% CO2.54 The cell lines were harvested when
they reached 80% confluence to maintain exponential growth.
2.5.2. MTT assay
The MTT assay is a standard colorimetric assay, in which mitochondrial activity is measured by
splitting tetrazolium salts with mitochondrial dehydrogenases in viable cells only.55 For viability
testing, MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide, M2128 from
Sigma) cell proliferation assay was carried out. The cell monolayers in exponential growth were
harvested using 0.25% trypsin and single-cell suspensions were obtained by repeated pipetting.
Only viable cells were used in the assay. Exponentially growing cells were plated at 1.2 × 104
cells per well into 96-well plates (Costar, Corning, NY, USA) and incubated for 48 h before the
addition of drugs to achieve the maximum confluency of the cells. Stock solutions were prepared
by dissolving the compounds in 10% (v/v) DMSO and further diluted with fresh complete
medium to achieve 1M concentration. Cells were incubated with different concentrations of

8
cisplatin (reference drug) and test compounds for 48 h at 37 oC in 5% CO2 humidified incubator
together with untreated control sample. At appropriate time points, cells were washed in PBS,
treated with 50 µL MTT solution (5 mgmL-1, tetrazolium salt) and incubated for 4 h at 37oC. At
the end of the incubation period, the medium was removed and pure DMSO 150 µL was added
to each well. The metabolized MTT product dissolved in DMSO was quantified by measuring
the absorbance at 570 nm on an Microplate reader (iMark, BIORAD) with a reference
wavelength of 655 nm. All assays were performed in triplicate and repeated thrice.
The percentage inhibition was calculated, from the data, using the formula given below, and IC50
values were calculated using nonlinear regression analysis.
Mean OD of untreated cells (control) ˗ Mean OD of treated cells × 100
Mean OD of untreated cells (control)
The IC50 concentration was determined as the dose that needs to be required to kill 50% of the
cells.
2.6. Molecular docking study
Docking studies
Compounds were built using the builder tool kit of the software package Argus Lab 4.0.1.23 and
energy minimized with semi-empirical quantum mechanical method PM3. Crystal coordinates of
Hodgkin lymphoma enzyme and DNA were downloaded from protein data bank and in the
molecule tree view of the software, the monomeric structures of the crystal co-ordinate was
selected and the active site was defined as 15 Å around the ligand. Validation of the docking
programme was checked by docking the known inhibitors of the respective enzymes in their
binding sites. The molecule to be docked in the active site of the enzyme was pasted in the work
space carrying the structure of the enzyme. The docking programme implements an efficient grid
based docking algorithm which approximates an exhaustive search within the free volume of the
S19 binding site cavity. The conformational space was explored by the geometry optimization of
the flexible ligand (rings were treated as rigid) in combination with the incremental construction
of the ligand torsions. Thus, docking occurs between the flexible ligand parts of the compound
and enzyme. The docking was repeated several times (approx. 10000 iterations) until no change
in the position of the ligand and a constant value of the binding energy was observed. The ligand
orientation was determined by a shape scoring function based on Ascore and the final positions

9
were ranked by lowest interaction energy values. H-bond between the respective compound and
enzyme were explored.
3. Results and Discussion
3.1. Chemistry
Reaction of un-substituted isatin-3-thiosemicarbazone (H2itsc) with copper(I) halides and Ph3P in
1 : 1 : 1 (M : L : PPh3) molar ratio formed insoluble orange colored compounds. To solubilize
them and to get crystalline product, one mole of Ph3P had been further added, which formed
tetrahedral monomeric complexes, [CuX(η1-S-H2itsc)(Ph3P)2] (X = I, Cl, Br).36, 46 However, with
substituted isatin-3-thiosemicarbazone (H2itsc-N1-R) (R = Me, Et), the similar reaction in 1 : 1 :
2 molar ratio formed three types of crystals (i) Red crystals of stoichiometry,
[CuX(H2itscR)(Ph3P)] (A), (ii) white crystals of formula, [CuX(Ph3P)3] (B) and (iii) yellow
needles of free ligand (C). The whole reaction sequence is given in Scheme 1, the
characterization data of B and C is provided in supplementary information.
+ 2H itsc-N1-R + 4Ph P
+
2CuX
2
3
+
R = Me, Et
(C)
(B)
(A)
Scheme 1
Compounds of stoichiometry, [CuX(H2itscR)(Ph3P)] (R=Me, H2L1; Et, H2L2) were of our
interest and were obtained by another route. Copper(I) halides (X = I, Br, Cl) were reacted with
Ph3P in 1 : 1 molar ratio to form white solid of composition, CuX(Ph3P) in acetinitrile.
Acetonitrile was then removed and precipitates were dissovled in chlorofrom and one mole of
H2L1 and H2L2 was added to get complexes of stiochiometry, [Cu(η2-N3, S-H2itsc-N1-Me)(Ph3P)]
(X = I, C1; Br, C2; Cl, C3) and [CuX(η2-N3, S-H2itsc-N1-Et)(Ph3P)] (X = I, C4; Br, C5; Cl, C6)
respectively (Scheme 2).

10
Scheme 2
The ν(N-H) bands in IR spectra of complexes can be divided into three categories: (i) bands
between 3490–3200 cm-1 due to (symmetric and asymmetric stretching mode) amino group, (ii)
band in the ranges, 3051–3081 cm-1, due to NH of isatin ring, (iii) band in the ranges, 3132-3149
cm-1 due to amide (-NH-) group. The presence of all these bands in the complexes C1-C6,
suggest that no deprotonation occurs at any nitrogen atom and ligand is coordinating in neutral
form. The characteristic ν(C=S) band in H2L1 and H2L2 appeared at 835 and 837 cm-1
respectively and showed shift to lower region (826-828 cm-1 C1-C6) in their complexes. This
shift indicates the binding of metal ion with thione sulfur. The presence of ν(P-CPh) in the range,
1093-1103 cm-1 ensures the coordination of triphenylphosphine to the copper center.36, 46
In 1H NMR spectra of ligands, –N2H- proton appeared at δ11.22 (H2L1) and δ11.21 ppm
(H2L2), which showed an upfield shift in complexes (C2-C6) (δ9.33 - 9.56 ppm). Similarly, the
NH proton of isatin ring and N1H proton appeared at δ12.60 (H2L1) and at δ12.55 (H2L2). Both
these signals also showed an upfield shift in complexes C2- C6 vis-à-vis free ligand and no
change was observed in complex C1. The presence of all these protons indicate that no
deprotonation take place during complexation and isatin thiosemicarbazone ligands coordinate to
the copper center in neutral form. The shift in the position of these protons also ensure
complexation. The C5H, C6H, C7H and C8H ring protons of thiosemicarbazone ligands appeared
in range, δ7.67-6.89 ppm in the complexes. The CH3 group at N1H gives a doublet (in range δ

11
3.17-2.50 ppm, C1-C3) and due to ethyl group a triplet (at δ2.5 ppm) and a multiplet (in the
range δ1.18-1.20 ppm, C4-C6).
3.2. Structure of N1-methyl-isatin-3-thiosemicarbazone (H2L1) and Complex C2
The molecular structure of ligand N1-methyl-isatin-3-thiosemicarbazone (H2L1) and Complex
C2 is given in Figure 1 and Figure 2 respectively. The crystallographic data and important bond
parameters are given in Tables 1 and 2 respectively.
Structure of H2L1
The ligand N1-methyl-isatin-3-thiosemicarbazone crystallizes in monoclinic crystal system with
space group P21/c. The two rings of isatin moiety (five and six membered) are nearly planer and
the dihedral angle between mean plane of both rings is 2.08(5)°, which is close to that of N1-
ethyl-isatin-3-thiosemicarbazone, (H2itsc-N1-Et, 2.1°) and N1-hexamethyleniminyl isatin-3-
thiosemicarbazone, (H2itsc-N-Hm, 0.5°).33 The angle describing plane of the thiosemicarbazone
moiety, N(1)−N(2)−C(3)−S(1) and N(1)−N(2)−C(3)−N(3) is 1.8 and 1.6° respectively.
Figure 1. ORTEP view of H2itsc-N1-Me (H2L1), showing the atom-labeling scheme
H2itsc-N1-Me, show Z – configuration about C(8)=N(1) double bond due to
intramolecular H-bonding between N(2)H of thiosemicarbazone and O(7) of isatin similar to
unsubstituted and substituted isatin-3-thiosemicarbazones (Table 2).34 The formation of five

12
membered ring due to H-bonding between N(4)H and N(1) leads to E-configuration about
N(2)-C(3) similar to H2itsc and H2itsc-N1-Et, but opposite to Z-configuration in case of H2itsc-
N1-Hm, where no such H-bonding is possible.33
Structure of [CuBr(η2-N3, S-H2itsc-N1-Me)(Ph3P)] C2:
Complex C2 crystallizes in triclinic crystal system with space group P-1. In this complex,
phosphorous atom from triphenylphosphine ligand, one bromide atom, imino nitrogen (N3) and
thione sulfur of thio-ligand formed corners of tetrahedron with copper(I) in center. N1-methyl-
isatin-3-thiosemicarbazone coordinate as neutral ligand binding to copper(I) in N3, S- chelation
mode. The Cu−S bond distance, 2.3274(10) Å is close to that of 2.3566(10) Å in [CuBr(η1-S-
H2itsc)(Ph3P)2]46, but shorter than, 2.4156(6) Å in [Cu(η2-N3, S-Hitsc)Ph3P)2]⋅NO3.56 Cu−Br
bond length in C2, 2.4219(6) Å is less than sum of ionic radii of Cu and Br (Cu+, Br-, 2.73 Å). 57
This Cu−Br bond length is also shorter than that of 2.5459(6) Å in [CuBr(η1-S-H2itsc)(Ph3P)2]46
.
Cu-N bond distance, 2.108(3) Å is close to that found in literature. 56, 58-59 The C−S bond length,
1.689(3) Å is close to 1.6880(15) Å in free ligand (H2L1) as well in literature56, 58-59, which
indicates binding of ligand to metal center in thione (C=S) form. The bond angles around copper
in range, 84.64(8)-121.35(3)° reveal distorted tetrahedral geometry. The maximum distortion of
121.35(3)° is found to be in P−Cu−Br angle. The Cu-S-C angle, 95.61(11)° in C2 is close to
96.91(12)° in [(Ph3P)Cu(µ-Br)(µ3-S, N3–Hcptsc)CuBr(PPh3)], where cyclohexanone
thiosemicarbazone (Hcptsc) binds to metal in N3,S-chelation-cum S-bridging mode.60 This angle
is much shorter than 113.30(12)° in [CuBr(η1-S-H2itsc)(Ph3P)2], where the thiosemicarbazone
was acting as terminal monodentate ligand.46

13
Figure 2. ORTEP view of C2 showing the atom-labeling scheme
In C2, inter-molecular H-bonding beween pyrrole hydrogen (N4H) of one molecule and
bromide atom of other molecule, N4H⋅⋅⋅Br, 2.778Å leads to formation of H-bonded dimer. An
additional CH⋅⋅⋅HC, 2.322 Å interaction between phenyl ring of triphenylphosphine of two
molecules is also present. These interactions are repeated to form 1D chain. Inter-molecular H-
bonding between prrrole nitrogen of one chain and Phenyl hydrogen of triphenylphosphine
molecule of second chain, HN4⋅⋅⋅HCPh, 2.733 Å lead to formation of 2D sheet (Figure 3).

14
Figure 3. Packing diagram of C2 showing H-bonding and various interactions
Table 1: Crystallographic data for H2itsc-N1-Me (H2L1)
H2L1
C2
Crystal description
Crystal colour
Block shaped
Orange
Prism
Red
Crystal size
0.30 x 0.20 x 0.20 mm
C10H10N4OS.H2O
252.30
0.34 x 0.24 x 0.12 mm
C28H25BrCuN4OPS
640.00
Empirical formula
Formula weight
Radiation, Wavelength
Unit cell dimensions
Mo Kα, 0.71073 Å
Mo Kα, 0.71073 Å
a = 8.9306(2), b = 13.0538(3), a = 9.8769(7), b =
11.2821(8), c =
c = 10.2347(2) Å, β =
93.105(2)o
14.1915(11) Å, α =
93.964(6), β = 102.013(6),
γ = 105.636(6)o
Triclinic
Crystal system
Monoclinic

15
Space group
P21/c
P-1
Unit cell volume
1191.39(4) Å3
1476.3(2) Å3
No. of molecules per unit cell, 4
Z
2
Temperature
293(2) K
0.268 mm-1
173(2) K
2.245 mm-1
648
Absorption coefficient
F(000)
528
Scan mode
ω scan
ω scan
θ range for entire data
collection
3.48 <θ< 24.99 º
3.000<θ< 32.743º
Range of indices
h= -10 to 10, k= -15 to 15, l= - h= -14 to 14, k= -16 to 15,
12 to 12
Reflections collected / unique 24272 / 2083
l= -21 to 21
18759 / 9734
6849
Reflections observed (I >
1798
2σ(I))
Rint
0.0404
0.0171
0.0320
0.0835
0.0998
0.0671
0.0998
0.1677
Rsigma
Final R
wR(F2)
Table 2: Selected bond distance (Å) and bond angles (°) for H2itsc-N1-Me (H2L1)
H2L1
1.6880(15)
1.2906(19)
1.3561(18)
1.364(2)
120.41(13)
116.97(14)
125.32(12)
117.71(11)
123.80(16)
127.44(15)
S(1) − C(3)
N(1) − C(8)
N(1) − N(2)
N(2) − C(3)
C(3) − N(4)
N(4) − C(5)
N(1) − N(2) − C(3)
N(4) − C(3) − N(2)
N(4) − C(3) − S(1)
N(2) − C(3) − S(1)
C(3) − N(4) − C(5)
O(7) − C(7) − N(6)
1.312(2)
1.455(2)

16
1.236(2)
1.507(2)
116.55(13)
126.07(14)
106.48(14)
126.13(14)
C(7) − O(7)
O(7) − C(7) − C(8)
N(6) − C(7) − C(8)
N(1) − C(8) − C(9)
C(7) − C(8)
C(8) − N(1) − N(2)
[CuBr(η2-N3, S-H2itsc-N1-Me)(Ph3P)] C2:
2.4219(6)
2.3274(10)
2.2300(10)
2.108(3)
121.35(3)
113.66(4)
105.57(8)
111.37(3)
114.24(8)
84.64(8)
Cu(1) − Br(1)
Cu(1) − S(1)
Cu(1) − P(1)
Cu(1) − N(3)
N(3) − N(2)
C(1) − N(1)
C(1) − S(1)
P(1) − Cu(1) − Br(1)
P(1) − Cu(1) − S(1)
P(1) − Cu(1) − N(3)
S(1) − Cu(1) − Br(1)
Br(1) − Cu(1) − N(3)
N(3) − Cu(1) − S(1)
Cu(1) − S(1) − C(1)
1.361(4)
1.330(5)
1.678(4)
96.91(12)
3.3. Cytotoxicity and docking studies
MTT assay was used to assess the % cell viability of the newly synthesized compounds against
L123 (human lung cells) and HepG2 (Hepatocellular carcinoma cells) cell lines to check their
cytotoxicity profile. Cisplatin was used as a reference drug. A sub-confluent population of
L123and HepG2 cells was treated with increasing concentration of these compounds and the
number of viable cells was measured after 48 h by MTT cell viability assay. The concentration
range of all the compounds was 1.55-50 µM. The cytotoxicity of complexes
C1-C6 was found to be concentration-dependent. The data reported in Table 3 suggest that all the
complexes showed different levels of cytotoxicity. IC50 value for Complexes C1-C6 fall in range,
8.31-25.50 µM (L123) and 18.65-22.50 µM (HepG2) (Table 3).

17
Table 3: Cytotoxicity profile of synthesized compounds and reference drug Cisplatin*.
Compounds→ (IC50µM)a
Cell lines↓
L123
C2
C5
C6 Cisplatin
C1
C3
C4
18.31 25.86 22.50 18.01 24.54 22.45
22.56 25.50 18.65 20.54 22.65 18.65
8.5
HepG2
9.65
aIC50 = the concentration of compound that inhibits 50% of cell growth.
*Each cytotoxic activity was performed in triplicate and average result is given with an error range of 0.05
3.4 Molecular modeling studies
Based on the in vitro anticancer activity to the Hodgkin lymphoma cell-line L1236, one of the
active complex 2 was selected to evaluate the putative binding mode with amino acids in the
active site of the enzyme using molecular docking technique. To confirm the binding affinity or
probable binding site, docking study of cisplatin was first carried out with Hodgkin lymphoma
cell line (PDB ID 1QOK)61 using builder tool kit of software package ArgusLab 4.0.1
(www.arguslab.com).62 It was observed that NH2 groups of cisplatin binds with the cysteine 184
(d = 2.51 Å) and 248 (d = 2.76 Å) amino acid residues through hydrogen bonding. Moreover, it
also showed binding interactions with histidine 194 (d = 1.03 Å) and glutamine 249 (d = 2.62 Å)
amino acid residue. Encouraging from the results of cisplatin with Hodgkin lymphoma cell line
and finding out the probable binding sites from these interactions, docking was then performed
by placing complex 2 into the binding site of target-specific region (where cisplatin showed its
interactions) of the Hodgkin lymphoma cell-line (PDB ID 1QOK). The docked model of
cisplatin and complex 2 with Hodgkin lymphoma enzyme was shown in Figure 4. Complex 2 is
surrounded by side chains of various amino acids such as F196, H194, Y192 and Y231,
indicating that the molecule is well fitted in the active pocket. The docking results revealed that
the interaction of complex 2 with Hodgkin lymphoma is dominated by hydrogen bonding
interactions with tyrosine (Y247), glutamine (Q249) and cysteine (C248 and C184) amino acid
residues. As shown in Figure 4, NH of complex 2 showed hydrogen bonding interactions with S
of cysteine (C248 and C184) amino acid residues and N of tryptophan (W195) of 1QOK (bond

18
distance: NH…S = 2.96 Å in case of C248, NH…S = 2.73 Å in case of C184 and NH…N = 2.81
Å of W195). NH group of indole ring of complex 2 also showed hydrogen bonding interactions
with oxygen of glutamine (Q249) and tyrosine (Y247) amino acid residue (bond distance:
NH…O = 2.41 Å in case of Q249 and NH…O = 1.98 Å in case of Y247). Therefore, docking of
complex 2 in the active site of enzyme indicated the probable mode of action for anticancer
activity.
A
B
Figure 4 Docking of cisplatin (A) and complex 2 (B) in the active site of Hodgkin lymphoma
(PDB ID 1QOK)
Molecular docking is an interesting tool to predict the possible drug–DNA interactions
because the mode of action of cisplatin is directly associated with their binding to DNA. Thus,
cisplatin showed interactions with DNA, crosslinked DNA in several different ways, interfering
with cell division by mitosis and kill cells. To explore the most feasible binding site or binding
affinity, docking studies have been performed on cisplatin and complex 2 with DNA (PDB ID:
1BNA). It has been demonstrated that both cisplatin and complex 2 showed interactions with
DNA, the probable mode of action for anticancer activity (Supporting Information).
Conclusion

19
Previous reports have suggested that un-substituted isatin-3-thiosemicarbazone formed
mononuclear tetrahedral complexes with copper(I) halides whereas the structure of complex C2
revealed that substituent at N1 (methyl) of isatin-3-thiosemicarbazone has changed the binding
mode of ligand from terminal to N3, S- chelation of its copper (I) bromide complex. The
analytical data supports same result for other complexes also. The in vitro cytotoxicity evaluation
showed moderate activity for these compounds. Docking study of C2 reveal that the molecule is
well fitted in the active pocket of Hodgkin lymphoma enzyme and the interaction of complex C2
with Hodgkin lymphoma for the binding site is dominated by hydrogen bonding interactions
with Y247, Q249, C248 and C184 amino acid residues.
Acknowledgements:
The authors thank Lovely Professional University for providing all the facilities to carry out the
experimental work. JPJ acknowledges the NSF–MRI program (Grant No. CHE-1039027) for
funds to purchase the X-ray diffractometer.
Conflict of interest
The authors declare no conflict of interest.
Supplementary material
Supplementary data is available from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
Fax: + 44-1223-336033 email: deposit@ccdc.cam.ac.uk) on request quoting the deposition
number CCDC 842975 for structure of H2L1 and 1448643 for C2.
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Compounds were constructed and docked with builder tool kit of software
package ArgusLab 4.0.1 (www.arguslab.com).

25
Graphical Abstract (Pictorial):
C2

26
Synopsis
Unsubstituted isatin-3-thiosemicarbazone formed mononuclear tetrahedral complexes with
copper(I) halides whereas the structure of complex C2 revealed that substituent at N1 (methyl) of
isatin-3-thiosemicarbazone has changed the binding mode of ligand from terminal to N3, S-
chelation of its copper (I) bromide complex.

27
Highlights
1. Substituent at N1 (methyl) of isatin-3-thiosemicarbazone has changed the binding mode
of ligand from terminal to N3, S- chelation of its copper (I) bromide complex.
2. The in vitro cytotoxicity evaluation of complexes showed moderate activity for these
compounds.
3. Docking study of C2 reveal that the molecule is well fitted in the active pocket of
Hodgkin lymphoma enzyme.