DNA/protein binding and cytotoxicity studies of copper(ii) complexes containing N,N′,N′′-trisubstituted guanidine ligands

Article abstract of DOI:10.1039/c4ra01459f

A series of N,N′,N′′-trisubstituted guanidine ligands (L1-L5) and their copper(ii) complexes (1-5) [Cu(ii){C₄H ₃SCONC(NHR)NC₆H₅}₂] [where R = p-tolyl (1), phenyl (2) benzyl (3), butyl (4) and cyclohexyl (5)] were synthesized and characterized by elemental analyses and UV-visible, FT-IR, ¹H & ¹³C NMR/EPR and mass spectroscopic techniques. The molecular structure of L1-L5, 3, and 5 was confirmed by single crystal X-ray crystallography. The single crystal X-ray structure of the complexes reveals the square planar geometry. The interaction of calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) with the copper(ii) complexes was investigated using UV-visible and fluorescence spectrophotometric methods. Spectral evidence shows the intercalative mode of DNA binding (in the order of 10⁴ M ^-1) with the complexes. The Stern-Volmer quenching constant (K _q) values were found from competitive binding studies and found to be in the range of 1.07-1.30 × 10⁵ M^-1 for the complexes. Spectral evidence also shows good binding properties of the complexes with the protein. Complexes 3 and 4 showed significant cytotoxicity against human breast (MCF7) and lung cancer (A549) cell lines.

Full text of DOI:10.1039/c4ra01459f

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Page 1 of 39
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DNA/protein binding and cytotoxicity studies of copper(II) complexes
containing N, N′, N″ꢀtrisubstituted guanidine ligands
Kumaramangalam Jeyalakshmi,a Nagamani Selvakumaran,a Nattamai S. P. Bhuvanesh,b
Anantharaman Sreekantha and Ramasamy Karvembua*
A series of N, N′, N″ꢀtrisubstituted guanidine ligands (L1ꢀL5) and their copper(II)
complexes (1ꢀ5) [Cu(II){C4H3SCONC(NHR)NC6H5}2] [where R = pꢀtolyl (1), phenyl (2)
benzyl (3), butyl (4) and cyclohexyl (5)] were synthesized and characterized by elemental
1
analyses and UVꢀvisible, FTꢀIR, H & 13C NMR / EPR and mass spectroscopic techniques.
The molecular structure of L1ꢀL5, 3, and 5 was confirmed by single crystal Xꢀray
crystallography. The single crystal Xꢀray structure of the complexes reveals the square planar
geometry. The interaction of calf thymus DNA (CTꢀDNA) and bovine serum albumin (BSA)
with the copper(II) complexes was investigated using UVꢀVisible and fluorescence
spectrophotometric methods. Spectral evidences show intercalative mode of DNA binding (in
the order of 104 Mꢀ1) with the complexes. The Stern–Volmer quenching constant (Kq) values
were found from competitive binding studies and found to be in the range of 1.07ꢀ1.30 × 105
Mꢀ1 for the complexes. Spectral evidences also show good binding property of the complexes
with the protein. Complexes 3 and 4 showed significant cytotoxicity against human breast
(MCF7) and lung cancer (A549) cell lines.
___________________________________________________________________________
a
Department of Chemistry, National Institute of Technology, Tiruchirappalli ꢀ 620 015,
India. Eꢀmail address: kar@nitt.edu (R. Karvembu); Fax: +91 431 2500133; Tel: +91 431
2503631
b Department of Chemistry, Texas A & M University, College Station, TX 77842, USA
Electronic supplementary information (ESI) available: Crystallographic data for the
structures reported in this paper have been deposited with the Cambridge Crystallographic
Data Centre (CCDC) as supplementary publication numbers (CCDC 984605, CCDC 984606,
CCDC 984604, CCDC 984603, CCDC 984607, CCDC 984602, CCDC 984601 for L1, L2,
L3, L4, L5, 3 and 5 respectively). Copies of the data can be obtained free of charge from the
CCDC (12 Union Road, Cambridge CB2 1EZ, UK; Tel.: + 44ꢀ1223ꢀ336408; Fax: + 44ꢀ1223ꢀ
336003; eꢀmail: deposit@ccdc.cam.ac.uk ; Web site http://www.ccdc.cam.ac.uk).

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1. Introduction
There is a widespread interest in the identification and development of transition
metal based compounds for biological applications. It is well known that DNA is an
important cellular receptor. The interaction of metal complexes with DNA has recently
gained much attention because it indicates that the complexes may have potential biological
activity and their activity depends on the mode and affinity of the binding with DNA.1–3 Even
though many transition metal complexes were reported as antitumour agents and some of
them were under clinical trials, developing copperꢀbased metallodrug is of special importance
because of its biocompatibility. Copper complexes have proved to be the best candidates
towards the search of the metal complexes of biological importance.4ꢀ6 Synthetic copper(II)
complexes have been reported as potential anticancer and cancer inhibiting agents7,8 and
number of copper complexes9,10 have been found to be active both in vitro and in vivo.
Similarly, protein was also established as one of the main molecular targets in the action of
anticancer agents.11 The interaction between protein and drugs provides useful information on
the structural features that determine the therapeutic effectiveness of drugs and also to study
the pharmacological response of drugs.12,13 Nowadays interaction of the proteins with the
metal complexes become important in the search of new drug molecules.
The guanidine molecule, (NH2)2C=NH, is an important ingredient of both organic and
inorganic chemistry. It is used to synthesize a number of biologically and pharmaceutically
relevant compounds.14ꢀ17 Guanidine derivatives are very useful pharmacophores in medicinal
chemistry due to their capacity to interact with functional groups present in enzymes or
receptors through hydrogen bonds and electrostatic interactions. The guanidine group also act
as a inhibitor of urokinase which plays a vital role in tumor metastasis which implicated in a
large number of malignancies, including breast, lungs, bladder, stomach, cervix, kidney, and
brain cancers.18,19 Various guanidine compounds have been synthesized and tested for their
antitumor activity.20,21 Copper complexes containing guanidine ligands are also found to have
many biological applications.22 For instance, copper(II) complexes of bis(2ꢀ
pyridylmethyl)amine ligand with guanidinium pendant groups had an enhanced ability to
cleave plasmid DNA and it can also act as RNA mimic.23 Copper(II) complexes with
guanidine ligands have been recently reported as urease inhibitors.24 These stem a great
interest to develop guanidine based copper(II) complexes for biological applications. Herein
we report the synthesis and characterization of copper(II) complexes containing trisubstituted
guanidine ligands. The interaction of the copper(II) complexes with CTꢀDNA and BSA was

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studied using spectrometric methods. We have also tested the in vitro cytotoxicity of the
copper(II) complexes against MCF7 and A549 cancer cell lines.
2. Results and discussion
2.1. Synthesis
The guanidine ligands (L1–L5) were synthesized by a mercuryꢀpromoted guanylation
reaction from NꢀthiophenecarbonylꢀN′ꢀphenylthiourea (Scheme 1). The copper(II) complexes
were synthesized using Cu(CH3COO)2ꢁH2O as the precursor (Scheme 2). All the ligands and
their copper(II) complexes were characterized by elemental analyses and/or various
spectroscopic techniques. The molecular structure of L1ꢀL5, 3 and 5 were confirmed by
single crystal Xꢀray diffraction studies.
Scheme 1 Synthesis of Nꢀphenyl ꢀN′ꢀ(aryl/alkyl)ꢀN″ꢀthiophenecarbonylguanidine
Scheme 2 Synthesis of copper(II) complexes
2.2. Spectroscopy
The electronic spectra of the ligands showed two bands around 260 and 300 nm,
which correspond to πꢀπ* and nꢀπ* transitions respectively. The spectra of the complexes
exhibited three bands. Two bands appeared around 254ꢀ306 nm, which correspond to

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intraligand transitions. The broad band in the region 601ꢀ626 nm corresponds to dꢀd
transition.
In the FTꢀIR spectra of the ligands, two bands (strong and weak) were observed for
N−H group in the range of 3367ꢀ3150 cmꢀ1. The weak band is due to the hydrogen bonded
N−H. The C=N stretching frequency of the ligands was observed at 1588ꢀ1568 cmꢀ1. This is
an intermediate value between double and single bonds, which shows the resonance between
all the three nitrogen atoms in the guanidine moiety. The C=O stretching frequency appeared
around 1632ꢀ1608 cmꢀ1 in the spectra of the ligands was shifted to a lower value in the
complexes, showing a single bond behaviour due to bonding with the Cu(II) ion. Further, the
weak N−H band was disappeared, which indicates the coordination of N atom after
deprotonation. The strong N−H band was appeared in the spectra of the complexes with
minor shift towards higher value.
1
In the H NMR spectra of the ligands (L1 and L2), signals of both the N−H protons
were found in the same region (10.09ꢀ11.00 ppm) since both the N−H are attached to an
aromatic ring. However, in the case of ligands L3, L4 and L5, one of the N−H protons gave
signal in the up field range (4.40ꢀ5.25 ppm) and another in the down field range (11.82ꢀ11.69
ppm). Chemical shift of all other aromatic and aliphatic protons was observed in the expected
regions. 13C NMR spectra of the ligands showed resonance due to C=O and C=N in the
regions 171.8ꢀ173.4 and 156.4–158.3 ppm respectively.25
Xꢀband EPR spectra of the Cu(II) complexes were recorded at room temperature as
well as at liquid nitrogen temperature in solid and solution states. The observation of quartet
hyperfine structure on the parallel component is due to the interaction of unpaired electron of
Cu(II) with Cu having nuclear spin I = 3/2.26,27 All the complexes showed well resolved
quartet hyperfine splitting typical of square planar Cu(II) system (Fig. 1), which was
confirmed by single crystal XRD technique. For all the complexes g|| > g suggesting that the
system is axial. The trend in the g value (g|| > g > 2.00) and the value of exchange
interaction term (G > 4.0) suggested that the unpaired electron of Cu(II) ion is present in the
dx2−y2 orbital (Table 1).
2.3. Single crystal Xꢀray crystallographic studies
Thermal ellipsoid plots of ligands (L1ꢀL5) and complexes (3 and 5) with the atomic
labelling schemes are shown in the Figs. 2ꢀ8. Crystal data and selected inter atomic bond

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lengths and angles are given in the Tables 2ꢀ5. The crystal structures of the ligands showed
the existence of an intra molecular hydrogen bond between N−H and carbonyl oxygen. The
thiophene ring was oriented in two opposite directions in the structures of L3ꢀL5. Slightly
elongated thermal parameters of the thiophene groups (S1, C1ꢀC4) indicated a possible
disorder, which was successfully modelled with a ratio of 90:10. The –C(O)N=C(NH–)(NH–)
core exhibited a large amount of delocalization due to the Yꢀaromaticity, as can be observed
by the C−N bond lengths [1.334ꢀ1.355 Å (L1), 1.332ꢀ1.353 Å (L2), 1.333ꢀ1.351 Å (L3),
1.329ꢀ1.350 Å (L4) and 1.329ꢀ1.356 Å (L5)].
Structures of 3 and 5 confirmed the square planar geometry of the complexes.
Complex 3 crystallized in triclinic Pꢀ1 space group with Z of 1 and complex 5 crystallized in
monoclinic C2/c space group with Z of 4. The Cu−N bonds are longer than the Cu−O bonds
[Cu(1)−O(1) 1.9087 Å, Cu(1)−N(1) 1.9589 Å (3) and Cu(1)−O(1) 1.9069 Å Cu(1)−N(1)
1.9688 Å (5)], which are in the expected range for guanidine complexes.28ꢀ31 Two guanidine
ligands are coordinated to Cu(II) ion in a trans fashion. There is an increase in the C−O bond
length and decrease in the C−N bond (involved in coordination) length in 3 and 5 compared
to L3 and L5, respectively.
2.4. DNA binding studies
2.4.1. Electronic absorption titration
The Cu(II) complexes (1ꢀ5) showed absorption band at 291ꢀ298 nm, which was
assigned to πꢀπ* transition. Upon the incremental addition of CT DNA to the complexes, the
intensity of absorption decreases resulting in hypochromism (ꢂε, 15ꢀ28%) with a small red
shift. Intercalative mode of binding due to the strong stacking interaction between an
aromatic chromophore and the base pairs of DNA usually results in hypochromism along
with or without a small red or blue shift.32 The extent of shift and hypochromism are normally
found to correlate with the intercalative binding strength. The magnitude of hypochromism is
in the order of 5>3>2>1>4, which reflects the DNA binding affinities of the complexes. The
absorption spectra of the complexes (1ꢀ5) in the presence and absence of CT DNA are shown
in Fig. 9.
The binding constant of the complexes with CT DNA (Kb) was obtained from the
ratio of slope to intercept in plots [DNA]/(εa
−
ε
f) versus [DNA] according to the equation33
[DNA]/(εa f) = [DNA]/(εb
−
ε
−ε
f) + 1/Kb (εb
−εf)

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where [DNA] is the concentration of DNA in base pairs, εa is the apparent extinction
coefficient value found by calculating A(observed)/[complex], f is the extinction coefficient
ε
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 yꢀintercept of 1/Kb(εb − εf) and the value of Kb was determined
from the ratio of slope to intercept (Fig. 11). The magnitudes of intrinsic binding constants
(Kb) are given in Table 6. The observed values of Kb revealed that the Cu(II) complexes bind
to DNA via intercalative mode.34 The Kb values were found to be in the range of 1.20ꢀ2.41 ×
104 Mꢀ1. Complex 5 showed better DNA binding affinity compared to the other complexes.
The Kb value of 1, 2, 3 and 4 differs only by a small value. In complex 5, the cyclohexyl ring
is in the molecular plane, which might be the reason for its enhanced DNA binding ability
compared to other complexes (1ꢀ4).
2.4.2. Fluorescence spectroscopic studies
Fluorescence property has not been observed for the complexes at room temperature
in solution or in the presence of CT DNA. So the binding of the complexes with DNA could
not be directly predicted through the emission spectra. Hence, competitive binding study was
done to understand the mode of DNA interaction with the complexes.35ꢀ37 Ethidium bromide
(EB) emits intense fluorescence in the presence of CT DNA because of strong intercalation of
the planar EB phenanthridine ring between adjacent base pairs in the double helix; therefore,
EB has been considered as a typical indicator of intercalation.38 If another molecule which
can bind to DNA more strongly than EB was added, the molecule will replace the bound EB
and there was a quenching in the DNA induced EB emission. The extent of quenching of CT
DNAꢀEB reflects the extent of interaction with the added molecule. On adding Cu(II)
complexes (0ꢀ25 ꢃM) to CT DNAꢀEB, the quenching in the emission of DNA bound EB
takes place (Fig. 10). Fluorescence quenching is explained by the SternꢀVolmer equation39
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 SternꢀVolmer quenching constant, and [Q] is the concentration of
complex. The slope of the plot of Fo/F versus [Q] gave Kq (Fig. 12). The apparent DNA
binding constant (Kapp) values were calculated by using the equation
KEB [EB] = Kapp [complex]

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where [complex] is the complex concentration at 50% reduction in the fluorescence intensity
of EB, KEB = 1.0 ×107 Mꢀ1 and [EB] = 5 ꢃM. The quenching constant Kq and Kapp values are
listed in Table 6.
2.5 Protein binding studies
2.5.1. Absorbance and fluorescence studies
Fig. 13 shows the fluorescence emission spectra of BSA after the addition of
complexes (1ꢀ5). When increasing amount of complex solution was added to a fixed quantity
of BSA, there observed a decrease in the fluorescence intensity of BSA at 345 nm, upto 86.2,
88.5, 78.3, 74.6 and 83.5% for complexes 1ꢀ5 respectively, with bathochromic shift of 4, 3
and 1 nm for complexes 1, 2 and 3 respectively. There was no appreciable shift in the case of
complexes 4 and 5. The observed hypochromicity has revealed that the complexes interact
hydrophobically with the BSA protein.40
UVꢀVisible absorption titration of BSA with complexes (1ꢀ5) was done to predict the
type of quenching process. Addition of the complex to BSA lead to an increase in BSA
absorption intensity without affecting the position of absorption band. This indicates that the
type of interaction between Cu(II) complexes and BSA was mainly a static quenching
process.41 The representative absorption titration spectrum is shown in Fig. 14. The
fluorescence quenching is described by the SternꢀVolmer 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 SternꢀVolmer quenching constant, and [Q] is the
quencher concentration. The quenching constant (Kq) can be calculated using the plot of log
(Fo/F) versus log [Q] (Fig. 15). 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 equation42, 43
log[(Fo
−F)/F] = log Kb + n log[Q]
where Kb is the binding constant of the complex with BSA and n is the number of binding
sites. From the plot of log[(Fo−F)/F] versus log[Q] (Fig.16), the number of binding sites (n)
and the binding constant (Kb) values have been obtained. The quenching constant (Kq),
binding constant (Kb) and number of binding sites (n) for the interaction of the Cu(II)
complexes with BSA are shown in Table 7. In all the complexes, only one binding site is
available to interact with BSA. Results showed that complexes 1 and 2 interact strongly with
BSA compared to 3, 4 and 5.

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2.5.2 Characteristics of synchronous fluorescence spectra
Synchronous fluorescence spectroscopy provides information about the molecular
microenvironment, particularly in the vicinity of the fluorophore functional groups.44
Tyrosine, tryptophan and phenylalanine residues are responsible for the fluorescence property
of BSA. The difference between the excitation and emission wavelength (ꢂλ) reflects the
nature of the chromophore.45 The large ꢂλ value, such as 60 nm, is characteristic of
tryptophan residue and a small ꢂλ value, such as 15 nm, is characteristic of tyrosine. The
synchronous fluorescence spectra of BSA with various concentrations of Cu(II) complexes
(1ꢀ5) were recorded at ꢂλ = 15 nm and ꢂλ = 60 nm. On addition of the complexes, the
fluorescence intensity of tryptophan residue at 340 nm decreased in the magnitude of 82.6,
88.9, 79.2, 71.0 and 80.3% for complexes 1, 2, 3, 4 and 5 respectively (Fig. 17). Similarly,
there was also decrease in the intensity of tyrosine residue at 300 nm. The magnitude of
decrease was 86.6, 88.0, 80.0, 74.0 and 85.1% for complexes 1ꢀ5 respectively (Fig. 18). The
synchronous fluorescence spectral studies clearly suggested that the fluorescence intensities
of both the tryptophan and tyrosine were affected with increasing concentration of the
complexes. The results indicate that the interaction of complexes with BSA affects the
conformation of both tryptophan and tyrosine microꢀregion.46
2.6 Cytotoxicity assay
The cytotoxicity of the Cu(II) complexes (1ꢀ5) toward MCF7 (human breast cancer
cells) and A549 (human lung cancer cells) cells has been examined by using MTT assay and
compared with cyclophosphamide [IC50 = 6.58 ꢃM (MCF7) and 22.36 ꢃM (A549)] under
identical conditions.47 Figs. 19 and 20 show the cytotoxicity of the compounds (1ꢀ5) after 24
h incubation on MCF7 and A549 cancer cell lines, respectively. Complexes 3 and 4 exhibited
cytotoxicity with IC50 values of 76.05 and 61.08 ꢃM, respectively, against MCF7 cell line.
The same complexes showed IC50 values of 91.68 and 68.8 ꢃM against A549 cell line.
Complexes 1 and 5 possess a moderate cytotoxicity. The IC50 values of 1 and 5 were found to
be 128.67 and 145.0 ꢃM, respectively, against MCF7. The same complexes showed IC50
values of 241.58 and 164.01 against A549. Complex 2 exhibited least activity than the other
complexes. The IC50 values of all the complexes are listed in Table 8.

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3. Conclusion
Five Cu(II) complexes with trisubstituted guanidine ligands have been synthesized.
Single crystal Xꢀray diffraction studies revealed that the Cu(II) complexes have square planar
geometry. The DNA binding of the complexes was investigated using absorption and
fluorescence spectrometric techniques. The results supported the interaction of the complexes
with CT DNA through nonꢀcovalent intercalation. The good protein binding ability of the
complexes was revealed from fluorescence measurement. In vitro cytotoxicity results showed
that the complexes have moderate activity against MCF7 and A549 cancer cell lines. The
result of DNA binding does not correlate with that of the in vitro cytotoxic studies. This
clearly states that the mechanism involved in cytotoxic activity of the complexes is different.
Further studies are needed to study the relation between the DNA binding and cytotoxic
activity of the complexes.
4. Experimental
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. FTꢀIR spectra were obtained as KBr pellets using a Nicoletꢀ
iS5 spectrophotometer. UVꢀVisible spectra were recorded using a Shimadzuꢀ2600
spectrophotometer. Emission spectra were measured on a Jasco Vꢀ630 spectrophotometer
using 5% DMF in buffer as the solvent. NMR spectra were recorded in CDCl3 by using TMS
as an internal standard on a Bruker 400 MHz spectrometer. EPR spectra were recorded on a
JEOL EPR spectrometer at room temperature and liquid nitrogen temperature, operating at
Xꢀband frequency (9.1 GHz).
4.2. Synthesis of N, N′, N″ꢀtrisubstituted guanidines
The guanidine ligands were synthesized from NꢀthiophenecarbonylꢀN
′ꢀphenylthiourea
by a guanylation method.48 The thiourea was mixed with the desired substituted amine in
DMF in an equimolar ratio with two equivalents of triethylamine. The temperature was
maintained below 5 °C using an ice bath and one equivalent of mercuric chloride was added
to the reaction mixture with vigorous stirring. The ice bath was removed after 30 minutes,
while the stirring continued overnight. The progress of the reaction was monitored using TLC

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until all the thiourea was consumed. 20 mL of chloroform was added to the reaction mixture
and the suspension was filtered through a sintered glass funnel to remove the HgS residue.
The solvents were evaporated under reduced pressure and the solid residue was dissolved in
20 mL of CH2Cl2, then washed with water and the organic phase was dried over anhydrous
Na2SO4. The residue obtained after evaporation of the solvent was recrystallized from ethanol
to get crystals of the title compounds.
4.2.1. NꢀPhenyl ꢀN′ꢀ(4ꢀmethylphenyl)ꢀN″ꢀthiophenecarbonylguanidine (L1)
Yield: 79%. Colourless solid. M.p.: 130 °C. UV−Vis (5% CHCl3): λmax, nm (ε,
dm3molꢀ1cmꢀ1) 265 (23300), 301 (26900). FTꢀIR (KBR, ν cmꢀ1): 3279, 3203 (N−H), 1611
1
(C=O), 1568 (C=N). H NMR (400 MHz, DMSOꢀd6): δ, ppm 2.26 (s, 3H), 7.44ꢀ7.011 (m,
9H), 7.65 (d, J = 3.6 Hz, 1H,), 7.58 (d, J = 2.4 Hz, 1H), 7.08 (t, J = 3.6 Hz, 1H), 10.46 (s,
1H), 10.09 (s, 1H). 13C NMR (100 MHz, DMSOꢀd6): δ, ppm 21.0 (aliphatic CH3), 123.9,
124.2, 125.1, 128.4, 129.9, 130.8, 132.1, 134.7. 135.1, 138.1, 144.4 (aromatic C), 156.4
(C=N), 171.8 (C=O). HRMS Calcd for C19H17N3OS: 335.4228 Found: 335.4197.
4.2.2. N,N′ꢀDiphenylꢀN″ꢀthiophenecarbonylguanidine (L2)
Yield: 78%. Colourless solid. M.p.: 120 °C. UV−Vis (5% CHCl3): λmax, nm (ε,
dm3molꢀ1cmꢀ1) 266 (13500), 302 (16800). FTꢀIR (KBR): ν, cmꢀ1 3394, 3165 (N−H), 1602
1
(C=O), 1571 (C=N). H NMR (400 MHz, CDCl3) δ, ppm: 7.09 (t, J = 4 Hz, 1H), 7.07ꢀ7.46
(m, 11H), 7.79 (d, J = 3.6 Hz, 1H), 10.30 (s, 2H). 13C NMR (100 MHz, CDCl3): δ, ppm
124.1, 126.1, 127.7, 129.5, 130.9, 131.1, 136.3, 144.4 (aromatic C), 156.1 (C=N), 173.3
(C=O). HRMS Calcd for C18H15N3OS: 321.3962 Found: 321.4001.
4.2.3. NꢀPhenyl ꢀN′ꢀbenzylꢀN″ꢀthiophenecarbonylguanidine (L3)
Yield: 82%. Colourless solid. M.p.: 100 °C. C19H17N3OS (335.42). UV−Vis (5%
CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 262 (15000), 296 (28500). FTꢀIR (KBR): ν, cmꢀ1 3267,
3202 (N−H), 1611 (C=O), 1569 (C=N). 1H NMR (400 MHz, CDCl3): δ, ppm 4.72 (s, 2H),
5.25 (s, 1H), 7.07 (t, J = 3.6 Hz, 1H), 7.43ꢀ7.23 (m, 11H), 7.80 (d, J = 2.4 Hz, 1H), 11.82 (s,
1H,). 13C NMR (100 MHz, CDCl3): δ, ppm 45.2 (aliphatic, CH2), 125.7, 127.2, 127.7, 127.8,
128.8, 130.2, 130.5, 130.7, 135.8, 144.8 (aromatic C), 158.2 (C=N), 173.4 (C=O). HRMS
Calcd for C19H17N3OS: 335.4228 Found: 335.4194.

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4.2.4. NꢀPhenyl ꢀN
′
ꢀbutylꢀN
″
ꢀthiophenecarbonylguanidine (L4)
Yield: 74%. Colourless solid. M.p.: 80 °C. C16H19N3OS (301.40). UV−Vis (5%
CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 264 (9400), 296 (24300). FTꢀIR (KBR): ν, cmꢀ1 3341,
3230 (N−H), 1607 (C=O), 1556 (C=N). 1H NMR (400 MHz, CDCl3): δ, ppm 0.92ꢀ0.95 (t, J =
5.6 Hz, 3H), 1.340ꢀ1.38 (q, J = 5.6 Hz, 2H), 1.564ꢀ1.550 (t, J = 5.6 Hz, 2H), 3.46 (s, 2H),
4.96 (s, 1H), 7.68ꢀ7.05 (m, 8H), 11.69 (s, 1H). 13C NMR (100 MHz, CDCl3): δ, ppm 13.9,
20.1, 31.9, 41.2 (aliphatic), 125.6, 127.1, 130.2, 130.3, 130.5, 135.9, 140.5 (aromatic C),
158.3 (C=N), 172.8 (C=O); HRMS Calcd for C16H19N3OS: 301.4066 Found: 301.4001.
4.2.5. NꢀPhenyl ꢀN′ꢀcyclohexylꢀN″ꢀthiophenecarbonylguanidine (L5)
Yield: 71%. Colourless solid. M.p.: 131 °C. C18H21N3OS (327.44). UV−Vis (5%
CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 263 (7700), 296 (17300). FTꢀIR (KBR): ν, cmꢀ1 3307,
1
3207 (N−H), 1608 (C=O), 1553 (C=N). H NMR (400 MHz, CDCl3): δ, ppm 2.05ꢀ1.15 (m,
11H), 4.80 (s, 1H), 7.07 (t, J = 3.6 Hz, 1H), 7.41ꢀ7.23 (m, 6H), 7.77 (s, 1H), 11.71 (s, 1H).
13C NMR (100 MHz, CDCl3): δ, ppm 24.9, 25.6, 33.1, 50.4 (aliphatic C), 125.3, 126.8, 127.1,
127.7, 130.2, 136.1, 145.1 (aromatic C), 157.3 (C=N), 172.7 (C=O); HRMS Calcd for
C18H21N3OS: 327.4438 Found: 327.4397.
4.3. Synthesis of copper(II) complexes (1ꢀ5)
The methanolic solution of Cu(CH3COO)2⋅H2O (1 mmol) was added into the solution
of an appropriate guanidine (2 mmol) in methanol at room temperature. The reaction mixture
was stirred for 6 h under an inert atmosphere, and then the precipitate formed was filtered and
washed with methanol. The suitable crystals of 3 and 5 for Xꢀray diffraction were grown
from CHCl3/nꢀhexane mixture (1:1).
4.3.1. Bis(NꢀphenylꢀN′ꢀ(4ꢀmethylphenyl)ꢀN″ꢀthiophenecarbonylguanidinato)copper(II)
(1)
Yield: 74%. Light blue solid. M.p.: 212 °C. Anal. Calcd. for C38H32CuN6O2S2
(732.38): C, 62.32; H, 4.40; N, 11.48; S, 8.76. Found: C, 61.93; H, 3.91; N, 11.50; S, 6.73.
UV−Vis (5% CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 267 (40300), 298 (30000), 604 (147). FTꢀ
IR (KBR): ν, cmꢀ1 3394 (N−H), 1594 (C=O), 1560 (C=N). EPR (300 K): ‘g’ values 2.286,
2.047. EPR (LNT): ‘g’ values 2.220, 2.038.

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4.3.2. Bis(N,N
′
ꢀdiphenylꢀN
″ꢀthiophenecarbonylguanidinato)copper(II) (2)
Yield: 85%. Light blue solid. M.p.: 215 °C. Anal. Calcd for C36H28CuN6O2S2
.
(704.32): C, 61.39; H, 4.01; N, 11.93; S, 9.1. Found: C, 61.17; H, 3.45; N, 12.10; S, 8.61.
UV−Vis (5% CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 269 (14500), 306 (25600), 601 (88). FTꢀ
IR (KBR): ν, cmꢀ1 3402 (N−H), 1593 (C=O), 1556 (C=N). EPR (300 K, ‘g’ value): 2.385,
2.047 EPR (LNT, ‘g’ value): 2.223, 2.046
4.3.3. Bis(Nꢀphenyl ꢀN
′
ꢀbenzylꢀN
″
ꢀthiophenecarbonylguanidinato)copper(II) (3)
for C38H32CuN6O2S2 (732.38): C,
Yield: 83%. Blue solid. M.p.: 202 °C; Anal. Calcd
.
62.32; H, 4.40; N, 11.48; S, 8.76. Found: C, 62.66; H, 3.72; N, 11.80; S, 7.97. UV−Vis (5%
CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 254 (13100), 298 (29000), 603 (154). FTꢀIR (KBR): ν,
cmꢀ1 3421 (N−H), 1557 (C=O), 1533 (C=N). EPR (300 K, ‘g’ value): 2.204, 2.043 EPR
(LNT, ‘g’ value): 2.217, 2.042
4.3.4. Bis(Nꢀphenyl ꢀN
′
ꢀbutylꢀN
″
ꢀthiophenecarbonylguanidinato)copper(II) (4)
for C32H36CuN6O2S2
Yield: 71%. Light blue solid. M.p: 172 °C. Anal. Calcd
.
(664.34): C, 57.85; H, 5.46; N, 12.65; S, 9.65. Found: C, 57.50; H, 5.44; N, 12.48; S, 9.88.
UV−Vis (5% CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 254 (12900), 298 (28900), 626 (298). FTꢀ
IR (KBR): ν, cmꢀ1 3418 (N−H), 1560 (C=O), 1537 (C=N). EPR (300 K, ‘g’ value): 2.203,
2.022 EPR (LNT, ‘g’ value): 2.218, 2.042
4.3.5. Bis(Nꢀphenyl ꢀN
′
ꢀcyclohexylꢀN
″
ꢀthiophenecarbonylguanidinato)copper(II) (5)
for C36H40CuN6O2S2
Yield: 73%; Light blue solid. M.p.: 210 °C. Anal. Calcd
.
(716.42): C, 60.35; H, 5.63; N, 11.73; S, 8.95; Found: C, 59.91; H, 5.05; N, 11.69; S, 8.63.
UV−Vis (5% CHCl3): λmax, nm (ε, dm3molꢀ1cmꢀ1) 254 (4600), 299 (16600), 608 (66). FTꢀIR
(KBR): ν, cmꢀ1 3418 (N−H), 1566 (C=O), 1532 (C=N). EPR (300 K, ‘g’ value): 2.283, 2.047
EPR (LNT, ‘g’ value): 2.217, 2.049
4.4. Single crystal Xꢀray diffraction studies
A Bruker APEX2 Xꢀray (threeꢀcircle) diffractometer was employed for crystal
screening, unit cell determination, and data collection. The Xꢀray radiation employed was
generated from a Mo sealed Xꢀray tube (Kα = 0.70173 Å with a potential of 40 kV, 40 mA)
fitted with a graphite monochromator in the parallel mode (175 mm collimator with 0.5 mm

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pinholes). Sixty data frames were taken at widths of 0.5°. These reflections were used in the
autoꢀindexing procedure to determine the unit cell. A suitable cell was found and refined by
nonlinear least squares and Bravais lattice procedures. The unit cell was verified by
examination of the h k l overlays on several frames of data by comparing with both the
orientation matrices. No superꢀcell or erroneous reflections were observed. After careful
examination of the unit cell, a standard data collection procedure was initiated using omega
scans. Integrated intensity information for each reflection was obtained by reduction of the
data frames with the program APEX2.49 The integration method employed a three
dimensional profiling algorithm and all data were corrected for Lorentz and polarization
factors, as well as for crystal decay effects. Finally, the data were merged and scaled to
produce a suitable data set. The absorption correction program SADABS50 was employed to
correct the data for absorption effects. Systematic reflection conditions and statistical tests of
the data suggested the space group. Solution was obtained readily using SHELXTL (XS).51
Hydrogen atoms were placed in idealized positions and were set riding on the respective
parent atoms. All nonꢀhydrogen atoms were refined with anisotropic thermal parameters. The
structure was refined (weighted least squares refinement on F2) to convergence.51, 52 Olex2
was employed for the final data presentation and structure plots.52
4.5. DNA binding studies
The interaction of metal complexes with CT DNA was carried out in Tris HCl/NaCl
buffer (pH 7.2). The bulk solution of CT DNA was prepared by diluting the CT DNA using
Tris HCl/NaCl buffer followed by stirring at 4 °C for three days, and kept at 4 °C for not
more than a week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and
280 nm (A260/A280) of 1.89, indicating that the DNA was sufficiently free of proteins. The
bulk DNA solution was further diluted to 10 folds to show maximum absorbance at 260 nm.
The absorption coefficient of CT DNA was 6600 cmꢀ1 Mꢀ1 per nucleotide.53 Cu(II) complexes
of required concentration were prepared by dissolving the calculated amount of the
complexes in 5% DMF/Tris HCl/NaCl. Complex solution of concentration 15 ꢃM was taken
in cuvette and CT DNA of equivalent concentration (5−40 ꢃM) was added each time and the
significant absorbance change was noted.
The competitive binding of each complex with EB has been investigated by
fluorescence spectroscopic technique in order to examine whether the complex can displace
EB from its CT DNAꢀEB complex. Ethidium bromide solution was prepared using Tris
HCl/NaCl buffer (pH 7.2). The test solution was added in aliquots of 2.5 ꢃM concentration to

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DNAꢀEB and the change in fluorescence intensities at 596 nm (450 nm excitation) was noted
down.
4.6 Protein binding studies
The binding of copper(II) complexes (1ꢀ5) with BSA was studied using fluorescence
spectra recorded at a fixed excitation wavelength corresponding to BSA at 280 nm and
monitoring the emission at 335 nm. The excitation and emission slit widths and scan rates
were constantly maintained for all the experiments. Stock solution of BSA was prepared in 50
mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use. Concentrated
stock solutions of each test compound were prepared by dissolving them in DMF–phosphate
buffer (5:95) and diluted with phosphate buffer to get required concentrations. 2.5 ml of BSA
solution was titrated by successive additions of a 10−6 M stock solution of the complexes
using a micropipette. For synchronous fluorescence spectra measurements, the same
concentration of BSA and the complexes were used and the spectra were measured at two
different ꢂλ (difference between the excitation and emission wavelengths of BSA) values of
15 and 60 nm.
4.7. Cytotoxic studies
Cytotoxicity studies of the copper complexes were carried out on human breast
(MCF7) and lung (A549) cancer cell lines. Cell viability was carried out using the MTT assay
method.54 The nonꢀsmall lung adenocarcinoma cells (A549 cells) and human breast cancer
cells (MCFꢀ7) were plated separately in 96 well plates at a concentration of 1 × 105
cells/well. Complexes (1ꢀ5) of concentration ranging from 10ꢀ200 ꢃM dissolved in DMSO
were seeded to the wells. DMSO was used as the control. It is important to mention here that
complexes (1ꢀ5) are stable in DMSO. After 24 h, the wells were treated with 20 ꢄL MTT [5
mg/ml phosphate buffered saline (PBS)] and incubated at 37 °C for 4 h. The purple formazan
crystals formed were dissolved in 200 ꢄL DMSO. The absorbance of the solution was
measured at a wavelength of 570 nm using a Beckmann Coulter Elisa plate. Triplicate
samples were analyzed for each experiment. The percentage inhibition was calculated using
the formula.

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Acknowledgements
K. J. thanks DST for fellowship under DST INSPIRE programme. We thank
Sophisticated Analytical Instrumentation facility (SAIF), Indian Institute of Technology ꢀ
Madras and Indian Institute of Technology ꢀ Bombay for NMR and EPR analyses
respectively. R. K. gratefully acknowledges DST for the financial support under FIST
programme.

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Fig. 1 EPR spectrum of complex 1 in frozen DMF solution. Microwave power, 0.98 mW;
microwave frequency, 9.1 GHz.
Fig. 2 Thermal ellipsoid plot of L1.

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Fig. 3 Thermal ellipsoid plot of L2.
Fig. 4 Thermal ellipsoid plot of L3.

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Fig. 5 Thermal ellipsoid plot of L4.
Fig. 6 Thermal ellipsoid plot of L5.

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Fig. 7 Thermal ellipsoid plot of 3.
Fig. 8 Thermal ellipsoid plot of 5.

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Fig. 9 Absorption spectra of complexes (1ꢀ5) in TrisꢀHCl buffer upon addition of CT DNA.
[Complex] = 1.5 × 10−5 M, [DNA] = 0ꢀ40 ꢃM. Arrow shows that the absorption intensities
decrease upon increasing DNA concentration.

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Fig. 10 Fluorescence quenching curves of EB bound to DNA in the presence of 1ꢀ5. [DNA] =
5 ꢃM, [EB] = 5 ꢃM and [complex] = 0ꢀ25 ꢃM.

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Fig. 11 Plot of [DNA]/(εa − εf) versus [DNA] for the titration of the complexes with CT
DNA.
Fig. 12 SternꢀVolmer plot of fluorescence titrations of the complexes with CT DNA.

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Fig.13 Fluorescence quenching curves of BSA in the absence and presence of 1ꢀ5.
[BSA] = 1 ꢃM and [complex] = 0ꢀ50 ꢃM.

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Fig. 14 The absorption spectra of BSA (10 ꢃM) and BSA with 1ꢀ5 (4 ꢃM).
Fig. 15 SternꢀVolmer plot of the fluorescence titrations of the complexes with BSA.

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Fig. 16 Scatchard plot of the fluorescence titrations of the complexes with BSA.

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Fig. 17 Synchronous spectra of BSA (1 ꢃM) as a function of concentration of 1ꢀ5 (0ꢀ50 ꢃM)
with ꢂλ = 60 nm.