Cu(II) and Zn(II) complexes from a thiosemicarbazone derivative: Investigating the intermolecular interactions, crystal structures and cytotoxicity

Article abstract of DOI:10.1016/j.molstruc.2019.127319

Two complexes of Cu(II) and Zn(II) were prepared from a thiosemicarbazone derivative (H₂esct) using their corresponding metal acetates and 2,2′-bipyridine as base. The complexes were characterized by elemental analyses, UV–visible, FT-IR and NM

Full text of DOI:10.1016/j.molstruc.2019.127319

Journal Pre-proof
Cu(II) and Zn(II) complexes from a thiosemicarbazone derivative: Investigating the
intermolecular interactions, crystal structures and cytotoxicity
Nimya Ann Mathews, Jinsa Mary Jacob, P.M. Sabura Begum, M.R. Prathapachandra
Kurup
PII:
S0022-2860(19)31428-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127319
MOLSTR 127319
Reference:
To appear in: Journal of Molecular Structure
Received Date: 18 August 2019
Revised Date: 26 October 2019
Accepted Date: 30 October 2019
Please cite this article as: N.A. Mathews, J.M. Jacob, P.M. Sabura Begum, M.R.P. Kurup, Cu(II) and
Zn(II) complexes from a thiosemicarbazone derivative: Investigating the intermolecular interactions,
crystal structures and cytotoxicity, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
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Cu(II) and Zn(II) complexes from a thiosemicarbazone derivative:
Investigating the intermolecular interactions, crystal structures and
cytotoxicity
Nimya Ann Mathewsa, Jinsa Mary Jacobb, P.M. Sabura Beguma and M.R. Prathapachandra
Kurupa,c*
aDepartment of Applied Chemistry, Cochin University of Science and Technology, Kochi 682
022, Kerala, India
bDepartment of Chemistry, Bharata Mata College, Thrikkakara, Kochi, 682021,Kerala, India
cDepartment of Chemistry, School of Physical Sciences, Central University of Kerala, Tejaswini
Hills, Periye, Kasaragod, 671 320, India
Graphical Abstract
Hirshfeld Surface
Cu(II) complex
In vitro cytotoxicity
Thiosemicarbazone
Zn(II) complex
*Corresponding author at: Department of Chemistry, School of Physical Sciences, Central University of Kerala,
Tejaswini Hills, Periye, Kasaragod, 671 320, India, E-mail address: mrpcusat@gmail.com (M.R.P. Kurup).

Cu(II) and Zn(II) complexes from a thiosemicarbazone derivative:
Investigating the intermolecular interactions, crystal structures and
cytotoxicity
Nimya Ann Mathewsa, Jinsa Mary Jacobb, P.M. Sabura Beguma and M.R. Prathapachandra
Kurupa,c*
aDepartment of Applied Chemistry, Cochin University of Science and Technology, Kochi 682
022, Kerala, India
bDepartment of Chemistry, Bharata Mata College, Thrikkakara, Kochi, 682 021,Kerala, India
cDepartment of Chemistry, School of Physical Sciences, Central University of Kerala, Tejaswini
Hills, Periye, Kasaragod, 671 320, India
Abstract
Two complexes of Cu(II) and Zn(II) were prepared from a thiosemicarbazone derivative (H2esct)
using their corresponding metal acetates and 2,2’-bipyridine as base. The complexes were
characterized by elemental analyses, UV-visible, FT-IR and NMR spectroscopy. The structure
of the thiosemicarbazone derivative and its five coordinated complexes were also determined by
ꢀ
the single crystal X-ray diffraction study. Copper complex got crystallized in triclinic P1 space
group and zinc complex in monoclinic P21/c space group. The crystal structures of the
complexes exhibited different types of intermolecular interactions, which have been further
analyzed by Hirshfeld surface analysis and fingerprint plots. The in vitro cytotoxicity against
Dalton lymphoma ascites cell lines showed that the Zn(II) and Cu(II) complexes are more
cytotoxic than their corresponding proligands.
Keywords: Cu(II) complex, Zn(II) complex, Hirshfeld surface, Cytotoxicity
*Corresponding author at: Department of Chemistry, School of Physical Sciences, Central University of Kerala,
Tejaswini Hills, Periye, Kasaragod, 671 320, India, E-mail address: mrpcusat@gmail.com (M.R.P. Kurup).
1

Introduction
A range of complexes with totally different donor atoms coordinating to metals forming different
nuclearities is the versatility of thiosemicarbazones [1]. They have shown significant biological
activities, such as in vitro molecular imaging, metal ion-sensing properties, analytical and
catalytic applications [2]. Copper with its oxidative nature and bio-essential activity, has
attracted various inorganic chemists to address Cu(II) complexes with medical applications [3–
6]. Copper(II) complexes containing heterocyclic bases have been extensively explored in virtue
of their strong interactions with DNA and cytotoxic activity [7–9].
The quantification of intermolecular forces such as weak and strong hydrogen bonds, halogen-
halogen interactions, π⋯ π contacts, van der Waals forces, which can help us to acquire more
information on the crystal structure and stability plays an important role in the study of metal
complexes. An important method which is a convenient tool for the investigation of
intermolecular interactions is the molecular Hirshfeld surface [10]. A Hirshfeld surface is a 3D
surface which is different for each crystal and gives an insight into structural aspects of crystal
systems. It also helps us to generate a 2D fingerprint plot which can deconstruct the atom pair
contacts and quantitatively gives all the intermolecular interactions [11].
An extensive interest has been developed in metallodrugs and radiopharmaceuticals ever since
the discovery of the cisplatin anticancer drug [12], which is being used for the treatment of
cancer patients. One of the first examples of thiosemicarbazone is 3-aminopyridine-2-
carboxaldehyde which undergo sophisticated stage of clinical trials as an antineoplastic agent
[13,14]. Water-soluble copper complexes of thiosemicarbazones containing proline ligands
exhibited anti-proliferative activity against ovarian carcinoma cells and cause inhibition for
Topoisomerase IIα [15]. Along with the significant pharmacological properties of the
thiosemicarbazones, they also exhibit interesting and versatile mode of chelation in their
complexes.
Copper is a biologically important metal element that plays a vital role in the structural and
catalytic co-factor of various enzymes and proteins, and consequently it is involved in many
biological functions in energy metabolism (cytochrome c oxidase) and anti oxidative activity
[16]. In this admiration, many copper complexes developed had the potential for the treatment
of cancers
2

and many other diseases [17]. The second most abundant transition metal in the body is zinc and
is the only metal ion which is able to simplify the rewinding of DNA [18]. Many zinc
complexes are reported to have anticancer activity [19]. Additionally, zinc is involved in the
cellular metabolism and has various catalytic, structural or regulatory role in many
metalloenzymes [20].
Many studies have been reported on the study of metal complexes with bipyridyl ligands
[21]. 2,2’-Bipyridine is a conjugated system having planarity, with which it can easily insert into
the adjacent DNA base pairs. It is a potent antitumor agent [22], which normally acts as
ancillary ligand which strengthens the binding ability of a complex with the planarity of the
molecule. Thus the complexes having 2,2’-bipyridine have drawn much attention due to their
high nucleolytic efficiency [23]. So here in, we report a study of a thiosemicarbazone derivative
and its two new Cu(II) and Zn(II) complexes with 2,2’-bipyridine as a coligand.
2. Experimental
2.1. Materials and Instruments
Elemental analyses of all the compounds were collected on a Vario EL III CHNS elemental
analyzer. IR spectra were recorded using KBr pellets on a JASCO FT-IR-4100 spectrometer in
the 4000-400 cm-1 range using KBr pellets. The electronic spectra of the compounds were
recorded on a Thermo Scientific Evolution 220 model UV-Vis spectrophotometer in the 200-800
nm range using solutions in DMF. The 1H NMR spectra of the compounds were recorded using
Bruker AMX 400 FT-NMR spectrometer. The single crystal X-ray diffraction studies were
carried out using Bruker SMART APEXII CCD diffractometer. The programs SAINT and
XPREP were used for data reduction and APEX2 and SAINT were used for cell refinement [24].
Absorption corrections were carried out using SADABS based on Laue symmetry using
equivalent reflections [25]. The structure was solved by direct methods and refined by full matrix
least-squares refinement on F2 using SHELXL-2018/7 [24] as well as WinGX software packages
[26]. The molecular and crystal structures were plotted using ORTEP-3 [22] and DIAMOND
version 3.2 g [27].
Anisotropic refinements were performed for all non-hydrogen atoms and all H atoms on C atoms
were placed in calculated positions, guided by difference maps, with C–H bond distances of
0.93–0.96 Å. H atoms were assigned as Uiso = 1.2 Ueq (1.5 for Me). The reflections (0 0 1), (1
0 0), (1 0 0) and (0 1 0) in 1 and (0 1 1), (1 1 0), (1 0 0), in 2 were omitted owing to bad
3

agreement. Compound 2 is having a disordered DMF solvent molecule. The anisotropic
displacement parameters of the disordered DMF molecule were restrained with the SIMU
command.
2.2 Synthesis of metal complexes
Synthetic routes to the complexes are given in Scheme 1.
Synthesis of 3-ethoxysalicylaldehyde-cyclohexylthiosemicarbazone (H2esct)
The synthesis includes a two step process as per an earlier report [28]
1. Synthesis of cyclohexylthiosemicarbazide
Cyclohexyl isothiocyanate (2.18 g, 15 mmol) in methanol and hydrazine hydrate (4.50 g,
90 mmol) in methanol were mixed and the resulting solution was stirred for an hour. The white
product, cyclohexylthiosemicarbazide formed was filtered, washed with methanol and dried.
2. Synthesis of 3-ethoxysalicylaldehyde-cyclohexylthiosemicarbazone (H2esct)
In this step, N4-cyclohexylthiosemicarbazide (0.17 g,
1
mmol) and
3-ethoxysalicylaldehye (0.16 g, 1 mmol) in acetonitrile was refluxed for 1 h after adding a drop
of glacial acetic acid. The white product was washed with methanol and dried. Colorless crystals
were obtained from the recrystallization from DMF.
Colorless product Yield: 0.18 g (56.25%). IR (KBr, cm-1): 3370 (–OH), 1526 (C=N), 1223 (C–
1
O), 1452(C=S), 1143(N–N); UV (λ, nm):292, 343; H NMR (CDCl3 ∂ ppm); 11.30 ppm (s,1H),
9.12 (s,1H), 5.5 ppm (s, 1H) 7.44-7.93 ppm (m), 1.22-1.87 ppm (m). Anal. Calcd. for:
C16H23N3O2S (321.44) C.59.78; H.7.21; N.13.07; S.9.98; Found: C.59.84; H.7.27; N.13.13;
S.9.95%.
2.2.1 Synthesis of [Cu(esct)bipy] (1)
3-Ethoxysalicylaldehyde-cyclohexylthiosemicarbazone (0.30 g, 1 mmol) is dissolved in DMF-
methanol mixture. 2,2’-Bipyridine (0.15 g,1 mmol) and copper acetate (0.19 g, 1 mmol) was
added to this solution and was refluxed for about 4 h. The solution was allowed to stand for slow
evaporation. Dark green colored crystals were obtained within two weeks. It was filtered,
washed with methanol and dried over P4O10 in vacuo. Yield: 0.320 g (59.47%). Color. Dark
green. IR (KBr, cm-1): 1447 (C=N), 1214 (C–N), 1440 (C=S), 1214 (N–N); UV–Vis (λ, nm):
4

290, 330, 400; Anal. Calc. for C26H28CuN5O2S (538.13): C, 57.92; H, 5.44; N, 12.99;S, 5.95.
Found: C, 58.89; H, 5.65; N, 13.48; S, 5.93%.
2.2.2 Synthesis of [Zn(esct)bipy] DMF (2)
To a DMF- methanolic solution of H2esct (0.30 g,1 mmol) prepared in the above mentioned way,
a methanolic solution of zinc acetate (0.21 g, 1 mmol) and 2,2’ bipyridine (0.15 g, 1 mmol) was
added and was allowed to reflux for 4 h. The solution is kept for slow evaporation. Yellow
single crystals were obtained which was washed and dried over P4O10 in vacuo. Yield: 0.37 g
(62.41%). Color. Yellow. IR (KBr, cm-1): 1482 (C=N), 1210 (C–N), 1435 (C=S), 1210 (N–N);
UV–Vis (λ, nm): 325, 390; Anal. Calc. for C29H36ZnN6O3S (596.95): C, 57.95; H, 5.79; N,
1
14.52; S, 5.93%. Found: C, 57.89; H, 5.95; N, 14.25; S, 5.96%. H NMR (DMSO, ppm): 9.0
(1H, s), 8.4 (1H, s), 6.7-7.9 (m), 1.5-1.8 (m).
2.3. Hirshfeld surface analysis
Hirshfeld surface (HS) analyses and 2D fingerprint calculations were used to investigate the
intermolecular contacts in the crystals of compounds and were performed using Crystal Explorer
17.5 [29].
2.4. In vitro cytotoxicity
The thiosemicarbazone derivative and the metal complexes were evaluated for their short term in
vitro cytotoxicity using Dalton’s lymphoma ascites cells (DLA) [30]. The tumour cells from the
peritoneal cavity of the mice (tumour bearing) were washed three times with PBS (Phosphate
Buffered Saline) or normal saline. The method adopted for checking the cell viability was
Trypan blue exclusion method. Viable cell suspension (1 x 106 cells in 0.1 mL) was added to
tubes containing different concentrations of the test compounds which is dissolved in DMSO and
the final volume was made up to 1 mL using PBS control tube which contains only the cell
suspension. These mixtures were then incubated for 3 hours at 37 ºC. The cell suspension was
finally mixed with 0.1 mL of 1% trypan blue and kept for 2-3 minutes in haemocytometer. The
blue color of trypan blue indicates the dead cells while live cells do not take up the dye. The
total numbers of stained and unstained cells were counted separately by using haemocytometer
and the % cytotoxicity was calculated using the equation below.
5

ꢁꢂꢃꢄꢅꢆ ꢇꢈ ꢉꢅꢊꢉ ꢋꢅꢌꢌꢍ
% Cytotoxicity = ꢁꢂꢃꢄꢅꢆ ꢇꢈ ꢌꢎꢏꢅ ꢋꢅꢌꢌꢍ ꢐꢁꢂꢃꢄꢅꢆ ꢇꢈ ꢉꢅꢊꢉ ꢋꢅꢌꢌꢍ × 100
The results obtained have been compared with a standard molecule, cyclophosphamide. It is a
most broadly used cytostatic and is a component of several polychemotherapy regimens [31]. It
is used in the treatment of lymphoma, ovarian cancer, leukemia, small cell lung cancer, breast
cancer etc.
3. Results and Discussion
The thiosemicarbazone (H2esct) derivative is soluble in aprotic solvents such as DMF and
DMSO and insoluble in protic solvents. The reaction in the presence of DMF-methanol mixture
yielded Cu(II) and Zn(II) complexes of the same thiosemicarbazone with 2,2’-bipyridine as co-
ligand. The complexes are readily soluble in DMSO and DMF and are stable in room
1
temperature. The thiosemicarbazone derivative and its complexes were characterized by IR, H
NMR and UV-Vis spectroscopic techniques.
3.1. IR spectral studies
IR spectral analysis gave an insight into the structure of the compounds by providing valuable
information regarding the various functional groups present. It is tabulated in Table S1. On
coordination of the azomethine nitrogen in the ligand to the complex, v(C=N) is shifted to a
lower wavenumber from 1526 cm-1 to 1447 cm-1 and 1482 cm-1 in the spectra of complexes [32].
The v(N−N) of the thiosemicarbazone derivative is at 1143 cm-1 and there is an increase in this
frequency in the spectra of the complexes due to the increase in the bond strength and thereby
confirming the coordination via azomethine nitrogen [33].
3.2. Electronic spectral studies
The electronic spectra of the complexes were recorded in DMF in the 200-800 nm region at
room temperature. The electronic spectra of the Zn(II) complex showed intraligand transitions
around 320 nm, along with a new metal to ligand charge transfer transition (MLCT) band at 390
nm (5,360 ϵ/M-1 cm-1). Except this, the complexes show no appreciable absorptions in the region
below 450 nm in DMF solution, in accordance with the d10 electronic configuration of the Zn(II)
ion. The Cu(II) complex showed intraligand transitions around 290 (7,330 ϵ/M-1 cm-1) and 330
nm (15,482 ϵ/M-1 cm-1) which corresponded to π→ π* and n → π* transitions respectively.
Along with the intraligand transitions LMCT band is observed around 400 nm (8,510 ϵ/M-1 cm-
6

1). (Table S2). The broadness in peak is due to the combination of N→Cu and O→Cu transitions
[34]. For complex 1, three transitions, dx2-y2 →dz2, dx2-y2→ dxy and dx2-y2→ dxz, dyz are possible for
2
its square pyramidal geometry with a dx -y2 ground state. The four d orbitals lie very close to
each other so that the each transition cannot be distinguished by its energy and it is difficult to
resolve them into separate components [34] (Fig.S1-S3).
3.3. 3.3. Conductance measurements
The molar conductivity values for all the complexes which is taken in 10−3 M DMF solutions
were in the range of 2–10 ꢀ−1 cm2 mol−1 which is much less than the value of 65–90 ꢀ−1 cm2
mol−1 reported for a 1:1 electrolyte in the same solvent [35]. So the nonelectrolytic nature of
complexes is evident from conductance measurements in DMF.
3.3. Crystal structure of 3-ethoxysalicylaldehyde-cyclohexyl thiosemicarbazone (H2esct)
A thiosemicarbazone derivative crystallized in monoclinic P21/c space group is obtained by the
slow evaporation of mother liquor. According to molecular structure (Fig. 1) the pro ligand
adopts an E configuration with respect to C(8)–N(2) bond. The torsion angle N(1)–N(2)–C(8)–
S(1) is -178.2(156)°, confirms the existence in E conformation. The C(8)–S(1) bond distance
[1.68(22) Å] is closer to the C=S bond length [1.60 Å] than to C–S bond length which confirms
its existence in thioamido form [36]. The azomethine bond formation is confirmed by the C(7)–
N(1) bond distance which is similar to C=N [1.28 Å] double bond [33,34]. The ring puckering
analysis and least square calculations show that the Cg(2) ring comprising of C(9), C(10),
C(11), C(12), C(13), C(14) is having puckering amplitude (Q)= 0.570(3) Å, θ =180.0(3)°, φ =
114(17)° and confirmed the chair conformation.
Fig.1.
The crystal packing involves two intermolecular hydrogen bonding interactions between O(1)–
H(1')...S(1) and N(2)–H(2')...O(1) with H–A distance of 2.47(3) Å and 2.27(2) Å (Fig.2.).
Further stabilization is also provided by the C–H…π interaction [C(15)–H(15B)…Cg(1) : 2.90 Å]
(Table S3 and S4) (Fig.3).
Fig. 2.
Fig. 3.
3.4 Crystal structure of metal complexes
7

ꢀ
The compound [Cu(esct)bipy] (1) crystallized in a triclinic P1 space group with Z=2 whereas the
compound [Zn(esct)bipy] DMF (2) crystallized in a monoclinic P21/c space group with Z=4.
The ORTEP plots representing the molecular structure of the complexes are given in Figs. 4 and
5. The coordination geometry around the complexes showed a distorted square pyramidal
environment with ONS chromophore contributed by dideprotonated tridentate thiosemicarbazone
derivative through phenolate oxygen, azomethine nitrogen and iminolate sulfur atoms and fourth
and fifth sites are occupied by nitrogen atoms of the bipyridine molecule. The distortion of the
geometrical environment is confirmed by the so-called τ5 index. This parameter measures the
distortion between a perfect trigonal bipyramidal (τ5 = 1) and a perfect square pyramidal
geometry (τ5 = 0) with the formula: τ5 = ꢑꢒ ꢓ in which α and β being the two largest angles
ꢔꢕ
around the central metal in the complex. The τ5 value is 0.564 for zinc center and 0.273 for the
copper center. These values confirmed the coordination geometry of complex 1 as distorted
square pyramidal geometry and complex 2 as distorted trigonal bipyramidal geometry around the
metal center [34]. In complex 1, the equatorial positions of the geometry was satisfied by three
donor atoms of the dideprotonated thiosemicarbazone derivative and one nitrogen atom N(4) of
the bipyridine moiety while the remaining nitrogen atom N(5) of bipyridine completes the axial
position. One of the reasons for the deviation from an ideal stereochemistry is the restricted bite
angle imposed by both the esct and bipy ligands. The bite angle around the metal viz, N(4)–
Cu(1)–N(5) is 77.43(10)º for [Cu(esct)bipy] and 76.67(9)º for [Zn(esct)bipy] DMF which may
be considered normal, when compared with an average value of 77º cited in the literature
[33,34]. The O(2)–Cu(1)–N(4) bond angle, 99.55(10)º or O(2)–Zn(1)–N(4) bond angle, and
S(1)–Cu(1)–N(4) bond angle, 101.26(7)º, indicate a slight tilting of the apical Cu(1)–N(4) bond
in the direction of the O(2)–Cu(1) bond and away from S(1)–Cu(1) bond.
Fig.4.
Fig.5.
In [Cu(esct)(bipy)], intermolecular hydrogen bonding interactions, N(3)–H(3’)···S(2) and N(8)–
H(8’)···S(1) existing between the two molecules of the asymmetric unit give rise to
centrosymmetric dimers in the crystal lattice (Fig. 6). Further stabilization is also provided by
the C–H…π interaction [C(14)–H(14B)→Cg(5): 2.93 Å]. {Cg(5): N(5), C(22), C(23), C(24),
C(25), C(26)} (Fig. 7).
8

In [Zn(esct)bipy] DMF, a strong classical intermolecular hydrogen bond, N(3)–H(3’)···O(3) is
present in the crystal structure in which the oxygen atom of the solvent DMF acts as the acceptor
(Fig. 8). Various non-classical hydrogen bonds are also present in the crystal structure.
Stabilization is also provided by the C–H…π interaction [C(17) -H(17) →Cg(6) : 2.57 Å] {Cg(6):
C(1), C(2), C(3), C(4), C(5), C(6)} (Fig. 9).
Fig.6.
Fig.7.
Fig.8.
Fig.9.
3.4. Hirshfeld surface analysis
The in-depth visualization of crystal packing and the supramolecular arrangements in a crystal
lattice is analyzed using Hirshfeld surface analysis [37]. Recently, the method has received wide
attention as a graphical analyzer for studying the intermolecular interactions and is applied in
studying all the interactions in different molecular and ionic compounds [38]. Surfaces in 3D
(dnorm) that have been mapped over a range of -0.3981 to 1.4852 Å for H2esct, − 0.1794 to 1.7474
Å for 1, and − 0.5472 to 1.7544 Å for 2, shape index (− 1.0 to 1.0 Å) and curvedness (− 4.0 to
0.4 Å). The asphericity (ꢀ) [39] and globularity values [40] indicates the degree in which the
anisotropy of objects and its deviation from spherical surface, respectively are also shown in
Table S5. The expanded 2D fingerprint plots [41] are displayed in the range of 0.6–2.8 Å
viewed with the de and di distance scales displayed on the graph axes where, de is the nearest
distance to the nuclei from outside the surface and di is the nearest distances to the nuclei from
inside the surface to the Hirshfeld surface. A dnorm surface enables an easy comparison of
intermolecular contacts relative to van der Waals radii with the help of a simple red-white-blue
color scheme. The red spots exhibit short contacts, it has distances shorter than the sum of van
der Waals radii. White areas represent contacts with distances equal to the sum of the van der
Waals radii, and blue spots do not have any close contacts [42] (Fig. 10).
Fig.10
All contact distances on the HS can be represented in the form of 2D fingerprint plots whose
shapes are distinctive of certain close-contact environments [43]. The 2D fingerprint plots of the
9

interactions in ligand and complexes are shown in Fig. 11. Each 2D fingerprint plot can be split
into the respective close contacts, and their contributions can be expressed as percentages [44].
The relative contributions of various interactions for ligand and complexes 1-2 are presented in
Fig. 12. The 2D fingerprint plots and relative contributions in ligand and complexes 1-2 indicate
the importance of the four categories of interactions. The packing of the molecules is primarily
driven by C/C, O/H, H/H interactions. However, the C/C, O/H contacts in 1 and 2 exhibit
minimal contributions of 5.2, 2.7, 3.8, 1.4, 2.9, and 3.5% and respectively. The contribution by
C/C interaction is represented by bow-tie pattern in the shape index curve. The large percentage
of H/H contacts in H2esct, 1 and 2 complexes (60.6%, 56.1% and 56.5%) suggest that the vdW
and hydrogen-bonding interactions play a major role in the crystal packing.
Fig.11
Fig.12
3.5. In vitro cytotoxicity
In vitro cytotoxicity of the thiosemicarbazone derivative and its zinc and copper complexes were
evaluated using Dalton’s lymphoma ascites cell line (DLA). The studied complexes
demonstrated a dose-dependent (10, 25, 50,100 and 200 ꢁg/mL) cytotoxicity against the cell line
under study. The high toxicity of copper complex as compared to zinc complex is evident from
the percentage of cytotoxicity. Thus the [Cu(esct)bipy] (1) becomes more effective complex in
controlling the growth of DLA cell lines. The difference in cytotoxicity may be attributed to the
difference in the metal ion which is present. The cytotoxicity of Cu(II) complex is higher as
compared to the other metal complexes such as Mo(VI) reported from our group [45]. Further
the in vitro cytotoxicity of the complexes were compared with a standard molecule
cyclophosphamide, which is a well-known medicine used for chemotherapy and to subdue the
immune system. The results showed that the Cu(II) complex showed much higher cytotoxicity
than the standard molecule as shown in Table 5. The results support the work by Palaniandavar
et al. which stated that the Cu(II) complexes are the best replacements to cisplatin [46]. The
solubility and the capability to cross cell membranes of the drug candidate are governed by
hard/soft nature of metal ion and the lipophilic/hydrophilic balance. Copper can also exist in two
different oxidation states in cells and provides a therapeutic opportunity to target tumours. It was
also reported that the copper(II) complexes can interact better with nucleic acid by intercalation
when they are integrated with planar aromatic rings like 2,2’-bipyridine [47].
10

Conclusion
Zn(II) and Cu(II) complexes of a thiosemicarbazone derivative derived from 3-
ethoxysalicylaldehyde and cyclohexylthiosemicarbazide were successfully synthesized and
1
spectrally characterized (FT-IR, UV–Vis, H NMR). Conductivity measurements showed the
non-electrolytic nature of the metal complexes. The crystal structures of the synthesized
compounds were confirmed from single crystal X-ray diffraction methods. SCXRD studies
shows that the complexes are monomeric in nature and it binds in tridentate dideprotonated form
to the thiosemicarbazone derivative. The present exploration gives perfect substantiation for the
significance of hydrogen bonding and weak non-covalent forces in the development of the
supramolecular architectures. The different types of intermolecular interactions in these crystal
systems have been supported by Hirshfeld surface analysis and their fingerprint plots. Finally
the complexes displayed a concentration dependent cytotoxic profile against DLA cell lines. The
highest activity was exhibited by complex 1 with the copper ion acting as the central metal and
the cytotoxicity reported was even higher than the standard molecule cyclophosphamide.
Acknowledgements
Nimya Ann Mathews is thankful to UGC, New Delhi, India for providing financial assistance
under UGC-JRF (111132 /June 2017). The authors are thankful to the Sophisticated Analytical
Instrumentation Facility, Cochin University of Science and Technology, Kochi, India for
1
elemental analyses, H NMR spectra and single crystal X-ray diffraction studies. The authors
acknowledges the Amala Cancer Research center for in vitro cytotoxicity studies.
Appendix A. Supplementary data
Crystallographic data for the structural analysis has been deposited with the Cambridge
Crystallographic Data Center as CCDC 1920139, 1941134 and 1941135 for H2esct,
[Cu(esct)bipy] (1), [Zn(esct)bipy] DMF (2) respectively. Copies of this information may be
obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html or from the Director,
11

CCDC, 12 Union Road, Cambridge, CB2, 1EZ, UK (fax: +44-1223-336-033; e-mail:
deposit@ccdc.cam.ac.uk).
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14

NCS
H
N
H
N
Stir
H2O
+
H2N NH2
H2N
S
H
N
H
O
H
N
H
H
N
N
N
H
H
Acetonitrile
H+
H2N
+
S
S
OH
OH
O
O
CH3
CH3
H2esct
Scheme 1. Synthetic pathway to synthesize thiosemicarbazone derivative (H2esct).
15

Scheme 2. Synthetic pathway to complexes 1 and 2.
Table 1 Crystal data and structure refinement parameters for 1 and 2
Parameters
Empirical formula
H2esct
C16H22N3O2S
[Cu(esct)bipy)] (1)
C26H28CuN5O2S
[Zn(esct)bipy] DMF (2)
C28.30H34.36N5.77O2.77SZn
320.42
Formula weight
538.13
596.95
ꢀ
Monoclinic, P21/c
Crystal system, space
group
Monoclinic, P21/c
Triclinic, P1
Unit cell dimensions
16.8161(15)
6.2796(5)
17.803(2)
90
117.002(3)
90
a (Å)
b (Å)
c (Å)
α (°)
9.4190(7)
11.4262(10)
12.1235(11)
92.988(4)
90.130(3)
96.386(3)
1294.87(19)
2
14.9271(8)
12.7747(6)
16.7185(9)
90
105.239(2)
90
β (°)
γ (°)
Volume (Å3)
Z
3075.9(3)
4
1675.0(3)
4
1.271
Calculated density
(Mg/m3)
1.380
1.289
0.204
Absorption coefficient,
0.956
0.903
ꢁ (mm-1)
Crystal size (mm3)
0.35 x 0.30 x 0.30
0.35 x 0.30 x 0.30
0.60 x 0.30 x 0.20
684
F(000)
560
1251
1.359 to 28.475
θ range for data
2.663 to 28.408
2.525 to 28.323
collection(°)
-22≤h≤22, -7≤k≤8,
-22≤l≤23
Limiting indices
-11≤h≤12, -
15≤k≤15, -
16≤l≤16
-12≤h≤19, -16≤k≤16, -22≤l≤22
13228
Reflections collected
10489
24151
4171
[R(int) =0.0349]
Unique reflections
(R(int))
6106
[R(int) = 0.0361]
7654
[R(int) = 0.0455]
16

25.242 (99.9 )
Completeness to θ
25.242 (98.3 %)
25.242 (99.9 %)
Semi-empirical from
equivalents
Absorption correction
Semi-empirical from
equivalents
Semi-empirical from equivalents
0.960 and 0.887
Max. and min.
transmission
0.8030 and 0.7740
0.8030 and 0.7740
Full-matrix least-squares on F2
7548 / 24 / 366
Full-matrix least-
squares on F2
Refinement method
Full-matrix least-
squares on F2
Data / restraints /
parameters
Goodness-of-fit on F2
4171 / 0 / 203
6504 / 1 / 321
0.988
1.033
1.009
R1 = 0.0503,
wR2 = 0.1439
R1 = 0.0532,
wR2 = 0.1525
Final R indices
[I>2σ(I)]
R1 = 0.0566,
wR2 = 0.1430
R indices
(all data)
R1 = 0.0855,
wR2 = 0.1773
0.759 and -0.300
R1 = 0.0708,
wR2 = 0.1693
1.056 and -0.629
R1 = 0.1154,
wR2 = 0.1691
0.555 and -0.475
Largest diff. peak and
hole
2
2 2 1/2
R1 = Σ||Fo|-|Fc|| / Σ|Fo|, wR2 = [Σw(Fo -Fc2)2 / Σw(Fo ) ]
Table 2. Selected bond lengths [Å] and angles [°] for H2esct, complexes 1 and 2
Bond lengths (Å)
H2esct
1
2
1.312(4)
1.373(4)
1.288(5)
1.755(4)
1.314(5)
O(1)–C(1)
N(1)–N(2)
N(1)–C(7)
S(1)–C(8)
N(2)–C(8)
N(3)–C(9)
1.353(2)
1.370(2)
1.273(2)
1.686(2)
1.355(2)
1.466(2)
O(1)–C(1)
N(1)–N(2)
N(1)–C(7)
S(1)–C(8)
N(2)–C(8)
N(3)–C(9)
O(1)–C(1)
N(1)–N(2)
N(1)–C(7)
S(1)–C(8)
N(2)–C(8)
N(3)–C(9)
1.311(3)
1.394(3)
1.292(4)
1.748(3)
1.309(4)
1.478(4)
1.976(2)
1.348(5)
1.959(2)
O(1)–Cu(1)
S(1)–Cu(1)
O(1)–Zn(1)
S(1)–Zn(1)
2.293(8)
2.342(12)
2.091(3)
2.136(2)
2.136(3)
N(1)–Cu(1)
N(4)–Cu(1)
1.944(2)
2.218(3)
N(1)–Zn(1)
N(4)–Zn(1)
N(5)–Cu(1)
2.016(2)
N(5)–Zn(1)
17

Bond angles (°)
H2esct
1
2
C(7)–N(1)–N(2)
C(8)–N(2)–N(1)
C(8)–N(3)–C(9)
N(3)–C(8)–N(2)
N(3)–C(8)–S(1)
C(7)–N(1)–N(2)
N(1)–Cu(1)–O(1)
N(5)–Cu(1)–N(4)
N(1)–Cu(1)–S(1)
O(1)–Cu(1)–N(5)
115.0(5)
119.5(5)
128.4(6)
113.7(8)
127.4(5)
115.0(5)
114.3(3)
113.6(3)
121.8(3)
117.5(3)
117.4(2)
114.3(3)
93.03(9)
77.43(10)
85.06(8)
90.63(9)
116.7(3)
112.2(3)
124.3(4)
118.0(4)
114.6(3)
116.7(3)
89.33(11)
76.67(9)
81.55(9)
109.15(11)
Table 5 In vitro cytotoxicity of the thiosemicarbazone derivative and its Cu(II) and Zn(II)
complexes
Drug
Percent Cell Death (DLA) (%)
Conc.(µg/ml)
Ligand
[Cu(esct)(bipy)] [Zn(esct)bipy] DMF Cyclophosphamide
H2esct
18
7
(1)
60
52
42
30
20
(2)
22
10
6
4
0
200
100
50
25
10
45
40
26
15
10
2
0
0
18

Fig. 1. ORTEP diagram of the 3-ethoxysalicylaldehyde-cyclohexylthiosemicarbazone (H2esct).
Displacement ellipsoids are drawn with 30% probability.
Fig. 2. Hydrogen bonding interaction in 3-ethoxysalicylaldehyde cyclohexylthiosemicarbazone
(H2esct).
19

Fig. 3. C–H…π interaction present in 3-ethoxysalicylaldehyde cyclohexylthiosemicarbazone
(H2esct).
Fig. 4. ORTEP diagram of [Cu(esct)bipy] (1). Displacement ellipsoids are drawn with 30%
probability.
20

Fig. 5. ORTEP diagram of [Zn(esct)bipy] DMF (2). Displacement ellipsoids are drawn with
30% probability.
Fig. 6. Hydrogen bonding interactions in [Cu(esct)bipy] (1).
21

Fig. 7. C–H…π interaction present in [Cu(esct)bipy] (1).
Fig. 8. C–H…π interaction present in [Zn(esct)bipy] DMF (2)
22

Fig. 9. Diagram depicting major hydrogen bonding interactions in [Zn(esct)bipy] DMF (2)
H2esct
[Cu(esct)bipy)]
[Zn(esct)bipy] DMF
Fig. 10. a) Hirshfeld surface mapped with dnorm (color scale from -0.1794 to 1.7474 (for 1); color
scale from -0.5472 to 1.7544 (for 2)), (b) shape index and (c) curvedness for the compounds 1-2.
23

Fig. 11. 2D fingerprint plots with de and di ranging from 1 to 2.8 Å for H2esct, 1 and 2 complexes
and their major decomposition plots.
24

Relative contribution to the Hirshfeld Surface
3
2
1
0
10
20
30
40
50
60
70
80
C-C H-H O-H
Fig. 12. Percentage contributions to the Hirshfeld surface area for the various close
intermolecular contacts for molecules (1: H2esct, 2: [Cu(esct)bipy], 3: [Zn(esct)bipy] DMF)
25

Cu(II) and Zn(II) complexes from a thiosemicarbazone derivative:
Investigating the intermolecular interactions, crystal structures and
cytotoxicity
Nimya Ann Mathewsa, Jinsa Mary Jacobb, P.M. Sabura Beguma and M.R. Prathapachandra
Kurupa,c*
aDepartment of Applied Chemistry, Cochin University of Science and Technology, Kochi 682
022, Kerala, India
bDepartment of Chemistry, Bharata Mata College, Thrikkakara, Kochi, 682021,Kerala, India
cDepartment of Chemistry, School of Physical Sciences, Central University of Kerala, Tejaswini
Hills, Periye, Kasaragod, 671 320, India
Highlights
1. A new thiosemicarbazone ligand and some of its transition metal (II) complexes were
synthesized.
2. The title compounds were characterized using spectroscopic techniques such as IR, UV-
Vis, NMR etc.
3. Single crystal X-ray analysis of Zn(II) and Cu(II) complexes.
4. In all complexes, the ligand acts as tridentate ONS donor forming five membered
geometry.
5. Visual analysis of short contacts was achieved through Hirshfeld surface analysis.
6. Cu(II) complex exhibited greater cytotoxicity as compared to a standard drug molecule.
*Corresponding author at: Department of Chemistry, School of Physical Sciences, Central University of Kerala,
Tejaswini Hills, Periye, Kasaragod, 671 320, India, E-mail address: mrpcusat@gmail.com (M.R.P. Kurup).

There are no conflicts of interest