Highly active copper(i) complexes of aroylthiourea ligands against cancer cells-synthetic and biological studies

Article abstract of DOI:10.1039/c8nj04246b

The reaction of copper(i) bromide with aroylthiourea ligands (L) in the molar ratio 1?:?3 resulted in the formation of [CuBr(L)₃]. The complexes were well characterized by analytical and spectroscopic (UV-visible, FT-IR, NMR and mass) technique

Full text of DOI:10.1039/c8nj04246b

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Haribabu, C. Balachandran, E. Narmatha, N. Bhuvanesh, S. Aoki and R. Karvembu, New J. Chem., 2019,
DOI: 10.1039/C8NJ04246B.
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Highly Active Copper(I) Complexes of Aroylthiourea Ligands Against Cancer
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Cells – Synthetic and Biological Studies
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Kumaramangalam Jeyalakshmi,a,b Jebiti Haribabu,a Chandrasekar Balachandran,c Eswaramoorthi
Narmatha,a Nattamai S. P. Bhuvanesh,d Shin Aoki,c and Ramasamy Karvembu*a
Abstract
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The reaction of copper(I) bromide with aroylthiourea ligands (L) in the molar ratio 1:3,
resulted in the formation of [CuBr(L)3]. The complexes were well characterized by analytical and
spectroscopic (UV-Visible, FT-IR, NMR and mass) techniques. Tetrahedral geometry of
complexes 1 and 3 was confirmed by single crystal X-ray diffraction study. The interaction of
complexes with biomolecules (DNA/protein) has been evaluated using spectrophotometric and
spectrofluorometric techniques. The compounds were screened for its in vitro cytotoxicity against
cancer and normal cell lines. Complexes 1 and 2 showed potent cytotoxic activity against HeLa
cells with IC50 value of 1.23 and 0.47 µM, respectively. The staining and colony formation studies
were also carried out for the most active complexes to predict the morphological changes and
colony formation ability respectively.
______________________________________________________________________________
aDepartment of Chemistry, National Institute of Technology, Tiruchirappalli 620015, India
bDepartment of Chemistry, M. Kumarasamy College of Engineering, Karur 639113, India
cFaculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510
Japan
dDepartment of Chemistry, Texas A & M University, College Station, TX 77842, USA
Electronic supplementary information (ESI) available: Graphs of the binding experiments.
Crystallographic data for the structures reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre (CCDC) as supplementary publication numbers CCDC
1850757-1850758. Copies of the data can be obtained free of charge from the CCDC (12 Union
Road, Cambridge CB2 1EZ, UK; Tel.: + 44-1223-336408; Fax: +44-1223-336003; e-mail:
deposit@ccdc.cam.ac.uk; web site: http://www.ccdc.cam.ac.uk).

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1. Introduction
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The research on the development of metal based drugs has attained a great significance
after the discovery of cisplatin as an anticancer drug. Copper is an essential trace element in the
biological systems, existing in the +2 and +1 oxidation states. It is employed as a structural and
catalytic cofactor, and consequently involved in many biological pathways.1-3 There are numerous
reports on the biological applications of copper(II) complexes. In contrast to copper(II) complexes,
reports on the biological applications of copper(I) complexes are scarce due to lower stability of
the +1 oxidation state and their tendency towards oxidation to Cu(II) compounds. However, there
are few reports which strongly evidenced the anticancer activity of copper(I) complexes. Copper(I)
complexes bearing tris(hydroxymethyl)phosphine ligands inhibited the tumor growth via G2/M
cell cycle arrest and paraptosis accompanied by the loss of mitochondrial transmembrane
potential.4 Mixed ligand Cu(I) complexes of triazolylborate and alkyl- or aryl-phosphines have
been found to be effective against A549 adenocarcinoma cells which are resistant to cisplatin.5
Binary Cu(I) complexes bearing 1,3,5-triaza-7-phosphaadamantane exhibited moderate
cytotoxicity against various cancer cell lines.6
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Copper(I) ion has a strong affinity to form bonds with ligands containing P or S donor
atoms.7-12 Such sulfur coordinated copper complexes acquire great importance due to its
resemblance to the active centers of some copper proteins, hemocyanin and tyrosinase, which
mediate redox and electron transfer reactions in biological systems. In this view, we have
synthesized copper(I) complexes containing aroylthiourea ligands. Aroylthiourea is a versatile
complexing agent capable of coordinating through O/S atom(s) by mono or bidentate fashion.
Thiourea and its metal complexes found significant interest due to its wide range of biological
activities such as antiviral, antibacterial, antifungal, antitubercular, antiinflammatory, anticancer,
herbicidal and insecticidal.13-17 Our group has explored the biological activities of Cu(II), Ni(II),
Pd(II) and Ru(II) complexes of aroylthiourea ligands.18-24 In continuation of our interest in
aroylthiourea complexes, we have synthesized the copper(I) complexes of aroylthiourea ligands.
The binding propensity of synthesized complexes with DNA/protein was investigated. The
compounds were also subjected to MTT assay to assess its cytotoxicity against various cancer and
normal cell lines. Staining and colony formation assays were also carried out to support the
cytotoxic ability of complexes.

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2. Results and discussion
2.1. Synthesis
The ligands were prepared from thiophene-2-carbonyl chloride/furoyl chloride, potassium
thiocyanate and aniline/1-napthyl amine (Scheme 1). The ligands on reaction with CuBr in acidic
media (3:1 ratio) yielded complexes with the general formula [CuBr(L)3] (Scheme 2). The ligands
and their Cu(I) complexes were characterized by UV-Visible, FT-IR, NMR and single crystal X-
ray diffraction techniques.
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KSCN, Acetone
70 oC, 1 h
RNH2, Acetone
27 oC, 3 h
H
Cl
NCS
NHR
N
X
X
X
O
O
O
S
X = S or O
X = S or O, R = C6H5 or C10H7
L1: X = S, R = C6H5 L2: X = S, R = C10H7
L3: X = O, R = C6H5 L4: X = O, R = C10H7
Scheme 1 Synthesis of the aroylthiourea ligands
X
R
HN
HN
O
H
N
R
NH
NHR
CH3CN, 0.5% HCl
27 oC, 24 h
N
X
S
S
CuBr
H
X
Cu
S
O
S
O
Br
R
O
N
H
N
H
X
X = S or O,
R = C6H5 or C10H7
1: X = S, R = C6H5 2: X = S, R = C10H7
3: X = O, R = C6H5 4: X = O, R = C10H7
Scheme 2 Synthesis of the copper(I) complexes
2.2. Spectroscopy
The UV-Visible spectra of ligands showed two bands around 261-263 and 283-296 nm,
which corresponded to →* and n→* transitions respectively. The spectra of complexes were
similar to those of uncoordinated ligands with minor shift in the position of the peaks. There was
no shift in the UV-Visible spectral bands of the complexes when it was recorded in DMF after 24

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h. This confirms the stability of complexes in solution. The bands observed in the FT-IR spectra of
ligands at 3121-3657, 1663-1669 and 1263-1275 cm-1 were assigned to (N−H), (C=O) and
(C=S) respectively. The decrease in the stretching frequency of C=S confirmed the coordination
of sulphur to copper(I) ion.
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1H NMR spectra of L4 and complexes (1-4) are shown in figures S1-S5. In the 1H NMR
spectra of Cu(I) complexes, the carbonyl- and thiocarbonyl- attached N−H and thiocarbonyl-
attached N−H protons were observed around 10.45-11.49 and 12.51-12.87 ppm, respectively.
Signals due to protons of aromatic rings in 1-4 appeared at 7.78-8.19 ppm. The 1H NMR spectra
of ligand L2 and complex 2 are shown in figure 1. On complexation, there was a significant shift
in the signal due to amide N−H proton. In the 13C NMR spectra of complexes, the signals appeared
around 113.2-149.0 ppm, which were due to the aromatic carbons (Figs. S6-S10). The resonances
due to C=O and C=S carbons were observed around 162.8-163.6 and 178.3-180.8 ppm,
respectively.25
S
HN
HN
O
NH
N
H
S
S
S
Cu
S
O
Br
O
2
N
a
H
N
H
b
S
b
a
O
S
a
N
N
H
H
S
b
L2
a
b
Fig. 1 1H NMR spectra of ligand L2 and complex 2.

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2.3. Structure
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The molecular structures of complexes along with crystallographic numbering schemes are
depicted in figures 2 and 3. Crystal data and selected inter atomic bond lengths and angles are
tabulated (Tables 1 and 2). The crystals of complexes 1 and 3 were obtained from the slow
evaporation of their chloroform/methanol solution. Complexes 1 and 3 crystallized in triclinic
crystal system with space group P-1. The crystal structures confirmed tetrahedral geometry of
copper(I) complexes where three coordination sites were occupied by sulphur from the
aroylthiourea ligands and bromide occupied the fourth coordination site. The Cu–S bond distances
were in the range of 2.2694-2.3034 Å and the average Cu–S distance was 2.2821 and 2.2804 Å for
complexes 1 and 3 respectively. The S–Cu–S angles ranged from 108.15 to 111.37° in 1, and
107.02 to 111.35° in 3. The Br–Cu–S angles were in the range of 107.72-110.41° (1) and 107.04-
112.21° (3), and their average value was close to that of ideal tetrahedral geometry. The crystal
structures depicted the existence of intramolecular hydrogen bonding between NH and carbonyl
O (NH···O) in complexes 1 and 3.23,26 In complex 3, there was also an intramolecular hydrogen
bonding between NH and coordinated Br ion (NH···Br).
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Table 1 Crystal data and structure refinement for complexes 1 and 3
1
3
C36H30Br0.72Cl0.29CuN6O6S3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
C36H30BrCuN6O3S6
870.19
110.15
0.71073
Triclinic
P-1
930.47
110.15
0.71073
Triclinic
P-1
Space group
Unit cell dimensions

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9.942(2)
10.631(2)
19.827(4)
100.087(2)
97.217(2)
111.760(2)
1874.1(7)
2
a (Å)
10.105(3)
11.067(3)
19.715(5)
100.277(3)
97.167(3)
112.992(3)
1950.6(8)
2
b (Å)
c (Å)
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 (°)
 (°)
 (°)
Volume (Å3)
Z
1.542
Density (calculated) Mg/m3
Absorption coefficient (mm-1)
F(000)
1.584
1.592
1.950
886
944
0.336×0.245×0.099
1.067 to 27.479
Crystal size (mm3)
0.485×0.244×0.068
Theta range for data collection (°) 2.064 to 27.458
13<=h<=13,
12<=h<=12,
13<=k<=13,
25<=l<=25
Index ranges
14<=k<=14,
25<=l<=25
21812
8455
Reflections collected
22623
8769
Independent reflections [R(int)]
Completeness to theta= 25.242°
99.7 %
99.6%
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Absorption correction
0.7456 and 0.6002
Max. and min. transmission
Refinement method
0.7456 and 0.4769
Full-matrix least-
Full-matrix least-squares on

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squares on F2
F2
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8455/768/613
1.026
Data / restraints / parameters
8769/0/478
1.032
Goodness-of-fit on F2
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R1 = 0.0423,
wR2 = 0.0857
R1=0.0492,
wR2=0.0985
Final R indices [I>2sigma(I)]
R1=0.0849,
wR2=0.1125
R1 = 0.0653,
wR2 = 0.0948
R indices (all data)
Largest diff. peak and hole (e.Å-3)
0.615 and 0.514
0.534 and 1.033
Table 2 Selected bond lengths (Å) and angles (°) of complexes 1 and 3
1
3
2.4959(7)
2.2709(12)
2.2720(11)
2.470(4)
Br(1)Cu(1)
Cu(1)S(1)
2.2694(9)
2.2969(9)
Cu(1)S(3)
2.3034(11) 2.2749(9)
Cu(1)S(5)/S(2)
110.41(4)
111.37(4)
110.56(4)
108.52(4)
108.15(4)
107.72(4)
112.21(8)
107.02(3)
S(1)Cu(1)Br(1)
S(1)Cu(1)S(3)
S(1)Cu(1)S(5)/S(2)
S(3)Cu(1)Br(1)
S(3)/S(2)Cu(1)S(5)/S(3)
S(5)/S(2)Cu(1)Br(1)
C(1)S(1)Cu(1)
C(13)/C(25)S(3)Cu(1)
110.07(3)
107.04(10)
111.35(3)
109.12(9)
109.10(14) 112.08(10)
112.31(14)
109.11(11)

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Fig. 2 Molecular structure of 1.
Fig. 3 Molecular structure of 3.

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2.4. DNA interaction studies
2.4.1. Electronic spectroscopic studies
The binding mode of metal complexes with DNA can be effectively interpreted by
electronic absorption spectroscopy.27-29 The electronic absorption spectra of Cu(I) complexes 1-4
exhibited a band around 293-297 nm which corresponded to intraligand transition. On titration of
the complexes (1.0×10−5 M) with CT DNA, the absorption intensity decreased, resulting in
hypochromism with a small red shift. The binding ability of complexes influences extent of shift
and hypochromicity, and it normally correlates with the intercalative binding strength. The
magnitude of hypochromism (4>2>1>3) reflected the DNA binding affinity of the complexes.
(Figs. 4 and S11).
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From the observed spectral changes, binding constant of the complexes with CT DNA (Kb)
was obtained. The binding constant was calculated by plotting [DNA]/(εa−εf) against [DNA]
according to the equation [DNA]/(εa−εf) = [DNA]/(εb−εf) + 1/Kb (εb−εf) where [DNA] is the
concentration of DNA in base pairs, εa is the apparent extinction coefficient value found by
calculating A(observed)/[complex], εf is the extinction coefficient for the free compound, and εb
is the extinction coefficient for the compound in the fully bound form. Each set of data, when fitted
into the above equation, gave a straight line with a slope of 1/(εb−εf) and an y-intercept of 1/Kb
(εb−εf), and the value of Kb was determined from the ratio of slope to intercept (Fig. 5). The
magnitudes of intrinsic binding constant (Kb) are given in Table 3. The observed values of Kb
revealed that the Cu(I) complexes bound to DNA via intercalative mode.30 The Kb values were
found to be in the range of 9.5850×103 - 2.1046×104 M-1. The enhanced binding ability of
complexes 2 and 4 was due to the presence of planar naphthyl ring.

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Fig. 4 Absorption spectra of complex 2 in Tris-HCl buffer upon addition of CT DNA. [Complex]
= 1.0×10−5 M, [DNA] = 0-50 µM. Arrow shows that the absorption intensities decrease upon
increasing DNA concentration.
Fig. 5 Plots of [DNA]/(εa−εf) versus [DNA] for the titration of complexes with CT DNA.
2.4.2. Fluorescence spectroscopic studies
The complexes did not exhibit fluorescence property at room temperature in solution or in
the presence of CT DNA. So binding of the complexes with DNA could not be directly predicted
through the emission spectra. Hence, competitive binding study was done to confirm the mode of
DNA binding with the complexes.31-33 Ethidium bromide (EB) is a classical intercalator and

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therefore it emits intense fluorescence in the presence of CT DNA. On titration of the complexes
with DNA bound EB, there was a quenching in the emission of DNA bound EB at 605 nm (Figs.
6 and S12). The quenching proved that the complexes replaced EB and bound more strongly with
DNA. The extent of quenching of CT DNA-EB reflects the extent of interaction with the added
molecule. Fluorescence quenching is explained by the Stern-Volmer equation34 Fo/F = 1 + Kq [Q]
where Fο and F are the fluorescence intensities in the absence and presence of complex
respectively, Kq is a linear Stern-Volmer quenching constant, and [Q] is the concentration of
complex. The slope of the plot of Fo/F versus [Q] gave Kq (Fig. 7). The apparent DNA binding
constant (Kapp) values were calculated by using the equation KEB [EB] = Kapp [complex], where
[complex] is the complex concentration at 50% reduction in the fluorescence intensity of EB, KEB
= 1.0107 M-1 and [EB] = 5 µM. The quenching constant Kq and Kapp values are listed in Table 3.
The observed binding constants are comparable with the previously reported copper(I)
complexes.22
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Fig. 6 Fluorescence quenching curves of EB bound to DNA in the presence of 2. [DNA] = 5 µM,
[EB] = 5 µM and [complex] = 0-50 µM.

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Fig. 7 Stern-Volmer plots of fluorescence titrations of the complexes with CT DNA.
Table 3 DNA binding constant (Kb), Stern-Volmer constant (Kq) and the apparent binding constant
(Kapp) for complexes 1-4
Complex
Kb (M-1)
9.98×103
2.10×104
9.58×103
1.67×104
Kq (M-1)
1.87×104
2.13×104
1.78×104
2.11×104
Kapp (M-1)
9.35×105
1.06×106
8.94×105
1.05×106
1
2
3
4
2.5. Protein binding studies
2.5.1. Absorption and emission spectroscopic studies
The electronic absorption titration of BSA with the complexes (1-4) was carried out to
validate the type of quenching process. On addition of the complexes to BSA, there was an increase
in BSA absorption intensity at 280 nm without any red/blue shift. This clearly indicated a static
type of interaction between the Cu(I) complexes and BSA.35 The UV-Visible titration spectra are
shown in Fig. 8.

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Fig. 8 The absorption spectra of BSA (10 µM) and BSA with 1-4 (4 µM).
Figure S13 shows the fluorescence emission spectra of BSA after the addition of
complexes (1-4). On incremental addition of the complexes to the fixed quantity of BSA, there
was a decrease in the fluorescence intensity of BSA at 345 nm. The percentage decrease in
fluorescence intensity was 81.0, 69.2, 75.22 and 86.3 % for complexes 1-4 respectively, with
hypsochromic shift of 1, 2 and 2 nm for complexes 1, 2 and 3 respectively. The observed spectral
changes predicted that the complexes interacted hydrophobically with BSA.36 The fluorescence
quenching is described by the Stern-Volmer relation Fo/F = 1 + Kq [Q] where Fo and F demonstrate
the fluorescence intensities in the absence and presence of quencher, respectively. Kq is a linear
Stern-Volmer quenching constant, and [Q] is the quencher concentration. The quenching constant
(Kq) can be calculated using the plot of Fo/F versus [Q] (Fig. S14). When small molecules bind
independently to a set of equivalent site, on a macromolecule, the equilibrium between free and
bound molecules is represented by the Scatchard equation37,38 log[(FoF)/F] = logKb + n log[Q]
where Kb is the binding constant of complex with BSA and n is the number of binding sites. From
the plot of log[(FoF)/F] versus log[Q] (Fig. S15), the number of binding sites (n) and the binding
constant (Kb) values have been calculated. The quenching constant (Kq), binding constant (Kb) and
number of binding sites (n) for the interaction of the Cu(I) complexes with BSA are shown in
Table 4. The binding, quenching constants correlates with the previously reported copper(I)
complexes.22 In all the complexes, the number of binding sites is one. Results revealed that
complexes 1 and 4 interacted strongly with BSA compared to 2 and 3.

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Table 4 Protein binding constant (Kb), quenching constant (Kq) and number of binding sites (n)
for complexes 1-4
Complex
Kb (M-1)
1.75×106
5.39×104
6.64×105
3.65×106
Kq (M-1)
4.46×105
2.12×105
3.09×105
6.61×105
n
1
2
3
4
1.27
0.87
1.07
1.16
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2.5.2. Synchronous fluorescence spectroscopic studies
Synchronous fluorescence spectroscopy provides information about the molecular
microenvironment, particularly in the vicinity of the fluorophore functional groups.39,40 The
fluorescence property of BSA is due to the presence of tyrosine, tryptophan and phenylalanine
residues. The difference between the excitation and emission wavelengths (Δλ) reflects the nature
of the chromophore. The large Δλ value, such as 60 nm is characteristic of tryptophan residue, and
small Δλ value, such as 15 nm is characteristic of tyrosine. The synchronous fluorescence spectra
of BSA with various concentrations of the Cu(I) complexes (1-4) were recorded at Δλ = 15 and
60 nm. The incremental addition of complexes decreased the fluorescence intensity of tyrosine
residue at 300 nm in the magnitude of 86.4, 64.3, 70.5 and 87.3 % for complexes 1-4 respectively
(Fig. S16). Similarly, the decrease observed in the intensity of tryptophan residue at 340 nm was
80.0, 66.5, 72.7 and 85.6 % for complexes 1-4 respectively (Fig. S17). The results showed that all
the complexes affected the microenvironments of both tyrosine and tryptophan residues and the
effect was more pronounced towards tyrosine than tryptophan.
2.6. Cytotoxic property of the compounds
The compounds (L1-L4, CuBr and 1-4) were screened for their cytotoxic property against
a panel of five human cancer (A549, HeLa S3, PANC-1, MIA PaCa2 and HeLa) and one normal
(IMR90) cell lines. The graph of percentage of cell viability versus concentration is shown in Figs.
9, S18 and S19. The half minimum inhibitory concentration (IC50) values calculated for the
compounds are tabulated (Table 5). Complexes 1-4 showed promising cytotoxic activity against
HeLa cancer cells. Interestingly complexes 1, 2 and 3 exhibited excellent activity against HeLa
cell line with IC50 value of 1.23, 0.47 and 1.84 µM respectively, which are comparable with the
previously reported copper(I) complexes.41 Besides, complexes 1, 2 and 3 showed potent ability

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to kill the MIA PaCa2 cells with IC50 value of 4.32, 4.96 and 3.01 µM respectively. Complexes 1
and 2 were active against PANC-1 cell line with IC50 value of 7.3 and 8.09 µM respectively. The
present complexes also exhibited appreciable activity (IC50 = 12.2 - 29 µM) against A549 and
HeLa S3 cell lines. The cytotoxic results of ligands (> 100 µM) against A549 and HeLa S3 cell
lines clearly indicated that complexation played a key role in enhancing the activity. It is
noteworthy to mention that the present complexes are more active than the well-known anticancer
agents such as gemcitabine and cisplatin.42 There is hardly a correlation between DNA/protein
binding results and cytotoxicity, which reveals that the enhanced cytotoxicity of complexes is not
due to DNA/protein binding; there might be a different cytotoxic mechanism.22,43
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Fig. 9 Cytotoxic effects of complexes 1-4 against PANC-1, MIA PaCa2 and HeLa cells. The data
were expressed in SD±mean with three replications.
Table 5 IC50 values (µM) of the compounds against A549, HeLa S3, HeLa, PANC-1, MIA
PaCa2 and IMR90 cells after 24 h.
Complex
A549
>100
>100
>100
>100
>100
17.7
HeLa S3
>100
>100
>100
>100
>100
12.6
HeLa
NT
PANC-1
NT
MIA PaCa2
NT
IMR90
NT
L1
L2
L3
L4
CuBr
1
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
1.23
0.47
1.84
7.3
4.32
4.96
3.01
26.3
10
2
18.1
12.2
8.09
14.34
3
25.9
21.9
52.5

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13.4
8.14
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12.32
>25
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Gemcitabine
Cisplatin44
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>25
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NT-Not Tested
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2.7. Acridine orange/ethidium bromide staining
Acridine orange/ethidium bromide staining was carried out to view the morphological
changes associated with apoptotic cell death. Acridine orange (AO) is a dye that could stain nuclear
DNA and RNA with an intact cell membrane, and ethidium bromide (EB) could only stain cells
that had lost membrane integrity.45 The morphological changes in the HeLa cancer cell line after
treatment with 5 µM concentration of 1 and 2 are shown in figure 10. The control cells exhibited
normal green nucleus with intact membrane. The cells treated with complexes 1 and 2 displayed
remarkable changes in the morphology such as irregularity in shape and membrane blebbing.
Fig. 10 Effect of 1 and 2 against HeLa cells. Images are taken under fluorescence microscope. AO
indicates live cells and EB indicates dead cells.
The early apoptotic cells were characterized by greenish yellow colour with condensed chromatin
whereas the late apoptotic cells were indicated by orange red nucleus with chromatin condensation
and nuclear fragmentation. Staining studies confirmed that the complexes induced apoptosis in the
cancer cell lines.

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2.8. Colony formation studies
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Colony formation study is a technique used to evaluate the effectiveness of a cell to
proliferate effectively, which consequently retains its reproductive ability to form colonies.
Complexes 1 and 2 were treated with HeLa cells to assess the colony formation ability (Fig. 11).
The results confirmed that complex 1 showed complete inhibition of colony formation at 4 µM
concentration. Complex 2 also inhibited 80% of colony formation at 4 µM concentration.
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Fig. 11 Effects of 1 and 2 on colony formation studies. The data were obtained by using ImageJ
software under colony area with three independent experiments. **** p < 0.0001, *** P<0.001
and ** P<0.001 (n = 3), significance was compared with treated and untreated groups.
3. Conclusion
A series of copper(I) complexes containing aroylthiourea ligands has been synthesized and
characterized by analytical and spectroscopic techniques. The Cu(I) complexes adopt a tetrahedral
geometry which was confirmed by crystallographic technique. The binding ability of the
complexes with DNA/BSA was validated using spectroscopic techniques. In vitro cytotoxic assay
revealed appreciable activity of the complexes against A549, HeLa S3, HeLa, PANC-1 and MIA
PaCa2 cancer cell lines. Complex 2 showed phenomenal activity with least IC50 value (0.47 µM)

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against HeLa cell line. Further the most active complexes were subjected to staining and colony
formation experiments. The staining studies proved the apoptosis mechanism of cell death and
colony formation experiments demonstrated the ability of the complexes to inhibit the colony
formation effectively. On conclusion, the Cu(I) complexes interacted with DNA/BSA appreciably,
exhibited excellent cytotoxicity against tested cancer cell lines. Further investigation may lead to
the development of potential anticancer agent.
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4. Experimental section
4.1. Materials and methods
All the chemicals were purchased from Sigma Aldrich / Merck and used as received.
Solvents were purified according to standard procedures. The melting points were determined on
Lab India instrument and are uncorrected. The elemental analyses were performed using a Vario
EL−III CHNS analyzer. FT-IR spectra were obtained as KBr pellets using a Nicolet-iS5
spectrophotometer. UV-Visible spectra were recorded using a Shimadzu-260 spectrophotometer.
Emission spectra were measured on a Jasco V-630 spectrophotometer using 5 % DMF in buffer
as a solvent. NMR spectra were recorded in CDCl3 by using TMS as an internal standard on a
Bruker 400/500 MHz spectrometer. A549, HeLa S3, HeLa, PANC-1, MIA PaCa2 and IMR90 cells
were purchased from the Riken BRC, Japan.
4.2. Synthesis of the ligands
The ligands were synthesized by following the reported procedure. L1, L2 and L3 were
already reported.23 L4 was synthesized from the reaction between furoyl chloride, potassium
thiocyanate and 1-naphthylamine.
4.2.1. N-(naphthalen-1-ylcarbamothioyl)furan-2-carboxamide (L4)
Furoyl chloride (1.4057 g, 10 mmol) and 1-napthylamine (1.4319 g, 10 mmol) were used.
Yield: 80 %. White solid. M.p.: 195 °C. Anal. calcd. for C16H12N2O2S: C, 64.85; H, 4.08; N, 9.45;
S, 10.82. Found: C, 64.13; H, 3.98; N, 9.28; S, 10.18. UV-Vis (CHCl3): max, nm (, dm3mol-1cm-1)
269 (7533), 294 (9933). FT-IR (KBr, cm−1): 3657 (m; ν(amide N−H)), 3218 (s; ν(thiourea N−H)),
1
1663 (s; ν(C=O)), 1270 (s; ν(C=S)). H NMR (500 MHz, CDCl3) δ, ppm 12.47 (s, 1H), 9.39 (s,
1H), 8.01 (dd, J = 13.4, 7.9 Hz, 2H), 7.90 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.62 (d, J
13
= 0.9 Hz, 1H), 7.60-7.50 (m, 3H), 7.42 (d, J = 3.2 Hz, 1H), 6.64 (dd, J = 3.6, 1.7 Hz, 1H). C

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NMR (126 MHz, CDCl3) δ, ppm 179.6, 157.0, 146.6, 145.0, 134.2, 133.5, 128.6, 128.5, 128.0,
127.0, 126.4, 125.2, 123.9, 121.6, 119.1, 113.5.
4.3. Synthesis of the copper(I) complexes
7
8
9
Copper(I) bromide (28.6 mg, 0.2 mmol) dissolved in acetonitrile (5 mL) was added to the
ligand (0.6 mmol) in acetronitrile (10 mL). Then 0.5 % HCl (2 mL) was added, which turned the
reaction mixture to green colour. The resultant solution was stirred at room temperature for 24 h.
The pale yellow solid was filtered off, washed with hexane and dried in vacuum. The solid was
recrystallized in chloroform/methanol mixture (1:9) to get suitable crystals for X-ray diffraction.
4.3.1. [CuBr(L1)3] (1)
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L1 (157.4 mg, 0.6 mmol) was used. Yield: 80 %. Pale yellow solid. M.p.: 210 °C. Anal.
calcd. for C36H30BrCuN6O3S6: C, 46.47; H, 3.25; N, 9.03; S, 20.68. Found: C, 46.13; H, 3.06; N,
9.17; S, 20.18. UV-Vis (DMF): max, nm (, dm3mol-1cm-1) 267 (50600), 293 (58900). FT-IR (KBr,
cm−1): 3265 (m; ν(amide N−H)), 3129 (s; ν(thiourea N−H)), 1663 (s; ν(C=O)), 1155 (s; ν(C=S)).
1H NMR (400 MHz, CDCl3): δ, ppm 12.61 (s, 1H), 10.56 (s, 1H), 8.54 (dd, J = 3.9, 0.9 Hz, 1H),
7.72 (dd, J = 5.0, 0.9 Hz, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.37 (t, J = 7.7 Hz, 2H), 7.29 (d, J = 7.4
Hz, 1H), 7.17 (dd, J = 4.9, 4.0 Hz, 1H). 13C NMR (101 MHz, CDCl3): δ, ppm 178.3, 162.8, 136.7,
135.8, 134.9, 133.4, 129.0, 128.7, 127.6, 125.1.
4.3.2. [CuBr(L2)3] (2)
L2 (187.4 mg, 0.6 mmol) was used. Yield: 78 %. Pale yellow solid. M.p.: 231 °C. Anal.
calcd. for C48H36BrCuN6O3S6: C, 53.35; H, 3.36; N, 7.78; S, 17.80. Found: C, 53.63; H, 3.06; N,
7.67; S, 17.18. UV-Vis (DMF): max, nm (, dm3mol-1cm-1) 266 (23000), 297 (30300). FT-IR (KBr,
cm−1): 3434 (m; ν(amide N−H)), 3213 (s; ν(thiourea N−H)), 1656 (s; ν(C=O)), 1211 (s; ν(C=S)).
1H NMR (400 MHz, CDCl3): δ, ppm 12.87 (s, 1H), 11.10 (s, 1H), 8.77 (d, J = 3.7 Hz, 1H), 7.88
(dd, J = 8.8, 3.6 Hz, 2H), 7.85 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 4.9 Hz, 1H), 7.65 (d, J = 7.3 Hz,
1H), 7.55-7.50 (m, 2H), 7.50-7.45 (m, 1H), 7.24 (d, J = 5.0 Hz, 1H). 13C NMR (101 MHz, CDCl3):
δ, ppm 180.5, 163.7, 135.3, 134.2, 128.8, 128.5, 127.3, 126.6, 125.1, 124.8, 121.8.
4.3.3. [CuBr(L3)3] (3)
L3 (147.7 mg, 0.5 mmol) was used. Yield: 85 %. Pale yellow solid. Anal. calcd. for
C36H30BrCuN6O6S3: C, 49.01; H, 3.43; N, 9.53; S, 10.90. Found: C, 48.63; H, 3.56; N, 9.67; S,
10.18. M.p.: 179 °C. UV-Vis (DMF): max, nm (, dm3mol-1cm-1) 270 (51200), 293 (46300). FT-

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IR (KBr, cm−1): 3260 (m; ν(amide N−H)), 3215 (s; ν(thiourea N−H)), 1675 (s; ν(C=O)), 1247 (s;
1
ν(C=S)). H NMR (500 MHz, CDCl3): δ, ppm 12.53 (s, 1H), 10.45 (s, 1H), 7.86 (d, J = 2.9 Hz,
1H), 7.67 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 7.7 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.3 Hz,
1H), 6.61 (d, J = 1.6 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ, ppm 178.4, 158.2, 147.6, 144.5,
136.7, 129.0, 127.65, 125.1, 120.1, 113.0.
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4.3.4. [CuBr(L4)3] (4)
L4 (177.8 mg, 0.6 mmol) was used. Yield: 73 %. Pale yellow solid. Anal. calcd. for
C48H36BrCuN6O6S3: C, 55.84; H, 3.51; N, 8.14; S, 9.32. Found: C, 55.63; H, 3.16; N, 8.67; S, 9.18.
M.p.: 216 °C. UV-Vis (DMF): max, nm (, dm3mol-1cm-1) 274 (54400), 294 (57900). FT-IR (KBr,
cm−1): 3447 (m; ν(amide N−H)), 3224 (s; ν(thiourea N−H)), 1679 (s; ν(C=O)), 1249 (s; ν(C=S)).
1H NMR (500 MHz, DMSO-d6): δ, ppm 12.51 (s, 1H), 11.49 (s, 1H), 8.19-8.16 (m, 1H), 8.09 (d,
J = 7 Hz, 1H), 8.02 (d, J = 4 Hz, 3H), 7.82 (d, J = 6 Hz, 1H), 7.66 (d, J = 6 Hz, 3H), 6.88-6.86 (m,
13
1H). C NMR (126 MHz, DMSO-d6): δ, ppm 180.8, 158.1, 149.0, 145.2, 134.5, 134.2, 129.1,
128.8, 128.1, 127.2, 126.8, 125.9, 123.2, 122.7, 119.5, 113.2.
4.4. X-ray crystallography
A BRUKER APEX 2 X-ray (three-circle) diffractometer was employed for crystal
screening, unit cell determination, and data collection. Integrated intensity information for each
reflection was obtained by reduction of data frames with APEX2.46 SADABS was employed to
correct the data for absorption effects.47 A solution was obtained readily by using
SHELXT (XT).48,49 Hydrogen atoms were placed in idealized positions and were set riding on the
respective parent atoms. All the non-hydrogen atoms were refined with anisotropic thermal
parameters. Absence of additional symmetry and / or voids was confirmed by using PLATON.
Olex2 was employed for final data presentation and structure plots.50
4.5. DNA binding experiments
For absorption spectral studies, CT-DNA was dissolved in 50 mM NaCl/5 mM Tris-HCl
(pH 7.2) solution. The DNA solution displayed a UV absorbance ratio (A260/A280) of 1.9:1,
indicating that the DNA was sufficiently in protein free form. Further, dilution of DNA solution
was made in such a way that its maximum absorbance should be 260 nm with the absorption
coefficient value of 6600 cm-1M-1 per nucleotide.51,52 The synthesized complexes were dissolved
using 5 % DMF/Tris-HCl/NaCl buffer.

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There was an urge for the ethidium bromide (EB) displacement study due the non-emissive
property of the copper(I) complexes. The competitive binding study has been carried out using
fluorescence spectroscopic technique to find out the efficacy of the complexes to displace EB from
DNA-EB complex. The experiment was carried out by fixing the excitation wavelength at 510 nm
and the spectra were recorded from 520 to 800 nm. In this experiment, DNA-EB concentration was
fixed and the changes in the emission intensity were measured by titrating with the complexes in
aliquots of 5 µM.53
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4.6. Protein binding experiments
Binding efficacy of the Cu(I) complexes with Bovine Serum albumin (BSA) was analyzed
using fluorescence spectra recorded at a fixed excitation wavelength corresponding to BSA (280
nm) and monitoring the emission at 345 nm. The excitation and emission slit widths and scan rates
were constantly maintained for all the experiments. The stock solution of BSA was prepared using
50 mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use. The stock solutions
of complexes were prepared using DMF-phosphate buffer (5:95) and were further diluted to the
required concentration using phosphate buffer. 2.5 mL of BSA solution was titrated by successive
additions of 10−6 M stock solution of the complexes using a micropipette. Synchronous spectra
were also recorded using the same concentration of BSA and complexes as mentioned above at two
different Δλ (difference between the excitation and emission wavelengths of BSA) values of 15 and
60 nm.
4.7. In vitro cytotoxicity evaluation by MTT assay
Cytotoxicity of the compounds was tested against human lung (A549 and IMR90-normal),
human cervical (HeLa S3 and HeLa), human pancreatic epitheloid (PANC-1) and human
caucasian pancreatic (MIA PaCa2) cell lines (2x104/well). Standard 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cell viability.54-57 The
detailed experimental procedure is provided in the supporting information.
4.8. Fluorescence images
HeLa cells were seeded in 30 mm plates, treated with complexes 1 and 2 and incubated for
24 h. After treatment, cells were stained with AO/EB (acridine orange/ethidium bromide) for 15
minutes. The images were captured under EVOS (20x) microscope.

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4.9. Colony formation studies
1×103 HeLa cells/well were cultured in 12 well plates and treated with different
concentrations of 1 (2, 4 and 8 µM) and 2 (1, 2 and 4 µM) for 24 h. After 24 h treatment, cells
were washed twice with PBS (phosphate buffer saline) and incubated with fresh DMEM
(Dulbecco's Modified Eagle Medium) for 10 days. The cells were washed twice with distilled H2O
and stained with crystal violet for 10 min. After incubation, cells were washed with distilled H2O
and dried. The data were obtained by ImageJ under colony area.58
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4.10. Data analysis
Data were analyzed (SD±mean) with three independent experiments using Graphpad Prism
6. Significance was calculated by Tukey's multiple comparisons test (p<0.05).
Acknowledgement
K. J. thanks the Department of Science and Technology, Ministry of Science and
Technology, Government of India for doctoral fellowship under DST-INSPIRE programme. J. H.
thanks the University Grants Commission for the fellowship (F1-17.1/2012-13/RGNF-2012-13-
ST-AND-18716). R. K. gratefully acknowledges DST for the financial support. Authors also thank
Prof. Toshiyuki Kaji and Dr. Eiko Yoshida, Faculty of Pharmaceutical Sciences, Tokyo University
of Science, Japan for providing cell lines for carrying out the anticancer studies.
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Cu(I) complexes bearing aroylthiourea ligands were synthesized and their biological properties were
evaluated.
64x39mm (300 x 300 DPI)