Synthesis, spectroscopic characterisation, thermal analysis, DNA interaction and antibacterial activity of copper(I) complexes with N, N′- disubstituted thiourea

Article abstract of DOI:10.1016/j.molstruc.2015.10.010

copper(I) complexes [Cu(4MTU)₂Cl] (2), [Cu(4MTU) (B)Cl] (3), [Cu(6MTU)₂Cl] (5) and [Cu(6MTU) (B)Cl] (6) where 4MTU = 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (1) and 6MTU = 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea (4), B is a N

Full text of DOI:10.1016/j.molstruc.2015.10.010

Journal of Molecular Structure 1106 (2016) 352e365
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, spectroscopic characterisation, thermal analysis, DNA
interaction and antibacterial activity of copper(I) complexes with N,
N0- disubstituted thiourea
,
P.R. Chetana a *, B.S. Srinatha a, M.N. Somashekar a, R.S. Policegoudra b
a Department of Chemistry, Central College Campus, Bangalore University, Bangalore 560001, India
b Department of Pharmaceutical Technology, Defense Research Laboratory, Tezpur 784001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
copper(I) complexes [Cu(4MTU)2Cl] (2), [Cu(4MTU) (B)Cl] (3), [Cu(6MTU)2Cl] (5) and [Cu(6MTU) (B)Cl]
(6) where 4MTU ¼ 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (1) and 6MTU ¼ 1-Benzyl-3-(6-methyl-
pyridin-2-yl)-thiourea (4), B is a N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 3, 6), were
synthesized, characterized by various physico-chemical and spectroscopic techniques. The elemental
analysis suggests that the stoichiometry to be 1:2 (metal:ligand) for 2, 5 1:1:1 (metal:ligand:B) for 3, 6. X-
ray powder diffraction illustrates that the complexes have crystalline nature. IR data coupled with
electronic spectra and molar conductance values suggest that the complex 2, 5 show the presence of a
trigonal planar geometry and the complex 3, 6 show the presence of a tetrahedral geometry about the
Cu(I) centre. The binding affinity towards calf thymus (CT) DNA was determined using UV-Vis, fluores-
cence spectroscopic titrations and viscosity studies. These studies showed that the tested phen com-
Received 10 September 2015
Received in revised form
10 October 2015
Accepted 19 October 2015
Available online 3 November 2015
Keywords:
Copper(I) complexes
Thiourea
Antibacterial activity
DNA binding
plexes 3, 6 bind moderately (in the order of 105
M
ꢀ1) to CT DNA. The complex 2, 5 does not show any
Chemical nuclease activity
apparent binding to the DNA and hence poor cleavage efficiency. Complex 3, 6 shows efficient oxidative
cleavage of plasmid DNA in the presence of H2O2 involving hydroxyl radical species as evidenced from
the control data showing inhibition of DNA cleavage in the presence of DMSO and KI. The in vitro
antibacterial assay indicates that these complexes are good antimicrobial agents against various path-
ogens. Anti-bacterial activity is higher when thiourea coordinates to metal ion than the thiourea alone.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
obviously on the nature of the metal complex as well as in the
potential supramolecular interactions it can have with the DNA
In the past few years, chemical nucleases have been presented
as valuable tools in genomic research, as well as promising candi-
dates for application in chemotherapeutics [1e3]. In this context a
large number of transition metal complexes such as Cu, Fe, Pt, Ru,
Au, Pd etc. have been reported to mediate DNA reactions by
themselves or assisted by both oxidation or reducing agents or
without any assistant agents [4]. In this line lower oxidation state
transition metals have been used in combination of oxygen or other
forms of reactive oxygen such as hydrogen peroxide or alkylhy-
droperoxides to generate reactive oxygen species (ROS) that ulti-
mately may cleave DNA by direct strand scission or base
modification [5]. The reactivity of a metal complex will depend
[6,7]. The bio essential element copper, and its complexes are of
particular interest because they possess biologically accessible
redox potentials and they are typically used, upon association with
dioxygen or hydrogen peroxide, for efficient and, in some instances,
selective DNA cleavage through oxidative pathways [8e10].
Sigman and co-workers have reported that the bis-(1,10-
phenanthroline) copper(I) complex in presence of H2O2 acts as a
‘chemical nuclease’ that efficiently nicks DNA, and copper(I) com-
plexes showing efficient chemical nuclease activity [11]. Pt(II),
Pd(II), organoruthenium(II) and iridium thiourea complexes are
good DNA intercalative binders [12e16]. Active and well-defined
thiourea ligands containing thione, thiol (eNeCSeNe) groups are
considered as ‘privileged ligands’ because of their capability to
remarkably stabilize different metals in various oxidation states,
and their complexes are extensively studied due to sensitivity,
selectivity and synthetic flexibility towards a variety of metal ions
* Corresponding author.
E-mail address: pr.chetana@gmail.com (P.R. Chetana).
http://dx.doi.org/10.1016/j.molstruc.2015.10.010
0022-2860/© 2015 Elsevier B.V. All rights reserved.

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
353
[17e20]. It is thus the DNA cleavage through copper(I) complexes
2.2. Syntheses
associated to heteronuclear bases an object of large discussions in
the literature [11]. Essentially, these latter studies are set to
continue toward the synthesis and characterization of novel cop-
per(I) thiourea derivatives that are indeed anticipated to be much
more capable of being antimicrobial agents [21]. However, there is a
prerequisite of a thorough investigation relating to structure and to
the activity of copper(I) thiourea derivatives and hence their sta-
bility under physiological conditions, in order to design more
potent antimicrobial agents.
Along these lines, a set of four different copper(I) complexes
were synthesized in high yield form through a reaction of CuCl with
4MTU, 6MTU and heterocyclic base phen. These Cu(I) complexes
are stable and show appreciable solubility toward common polar
organic solvents. This study includes the synthesis, structure
2.2.1. Synthesis of 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea
(4MTU) (1), 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea (6MTU)
(4)
A solution of 2-amino-4-methyl pyridine (0.01 mol) (1), 2-
amino-6-methyl pyridine (0.01 mol) (4) in ethanol (15 ml) was
added to (0.01 mol) benzyl isothiocyanate in 10 ml ethanol and
refluxed with stirring for 2 h. The resulting white precipitate was
filtered off and washed with 5 mL cold ethanol and recrystallized
from ethanol, dried, Yield ~80%. The analytical data Calc. for
C
14H15N3S (1): C, 65.34; H, 5.87; N, 16.33; S, 12.46 found: C, 65.32;
H, 5.82; N, 16.32; S, 12.77%. IR, cmꢀ1 (KBr disc): 3226 m, 2981br,
2915w, 1615s, 1538vs, 1486s, 1326s, 1185s, 809s, 732s, 448vs (s,
strong; m, medium; w, weak; br, broad; vs, very strong). 1H NMR
10.58 (s,1H), 8.03 (s,1H), 7.34 (m, J ¼ 4.5 Hz, 2H), 7.27 (m, J ¼ 4.5 Hz,
2H),ꢁ6.99 (s, 2H), 6.87 (d, J ¼ 7 Hz, 2H), 4.90 (d, 2H), 2.26 (s, 3H), MP
142 C, ESI-MS in MeCN: m/z 257.94 [MþH]þ.
elucidation and spectroscopic properties of
a thiourea MTU
(Scheme 1) containing pyridyl group together with their respective
mononuclear Cu(I)-based complexes 2, 3, 5 and 6. This study ex-
plores the possibility of Cu(I) complexes being artificial nucleases
based on peculiar characteristics of metal-ligand coordination; (i)
the existence of planar aromatic and/or heterocyclic ring system
capable of being inserted or stacked between base pairs in the
hydrophobic interior of helical double stranded DNA, (ii) the
presence of nitrogen atoms that can establish hydrogen bonds with
the DNA and the capacity to yield cationic copper(I) complex where
the positive global charge could favor their electrostatic attraction
to the anionic phosphate backbone of DNA.
The analytical data Calc. for C14H15N3S (4): C, 65.34; H, 5.87; N,
16.33; S, 12.46 found: C, 65.12; H, 5.82; N, 16.22; S, 12.75%. IR, cmꢀ1
(KBr disc): 3188 m, 3037br, 2912w, 1603s, 1535vs, 1452s, 1299s,
1222s, 1185s, 1152s, 785s, 716s, 449vs (s, strong; m, medium; w,
weak; br, broad; vs, very strong). 1H NMR 10.6 (s, 1H), 8.05 (d,
J ¼ 5.5 Hz, 1H),7.34 (d, J ¼ 5 Hz, 2H), 7.26 (d, J ¼ 4 Hz, 2H), 7.04 ꢁ(s,
2H), 6.91 (m, 2H), 4.84 (d, J ¼ 6.5 Hz, 2H), 2.26 (s, 3H), MP 148 C,
ESI-MS in MeCN: m/z 257.94 [MþH]þ.
2.2.2. Synthesis of [Cu(4MTU)2Cl] (2) and [Cu(6MTU)2Cl] (5)
To a pre N2 purged acetonitrile solution (15 ml) of CuCl (0.049 g,
0.5 mmol), 1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (4MTU)
(0.257 g 1 mmol) (2), 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea
(6MTU) (0.257 g 1 mmol) (5) was added as solid with magnetic
stirring for a duration of 2 h. The resulting white precipitate was
filtered off and washed with diethyl ether, dried under vacuum.
Anal. Calcd. for C28H30N6S2CuCl (2): C, 54.80; H, 4.92; N, 13.69;
S,10.44 found: C, 54.26; H, 4.87; N, 13.55; S, 10.81%. IR, cmꢀ1 (KBr
disc): 3173br, 3000w, 2914w, 1619s, 1533vs, 1488s, 1345 m, 1207s,
936s, 815s, 695s, 444vs (s, strong; m, medium; w, weak; br, broad;
vs, very strong). 1H NMR in DMSO d6 10.66 (s, 1H), 9.31(S, 2H) 8.92
(d, J ¼ 8 Hz, 2H) 8.04(S,1H), 7.39e6.91 (m,12H), 4.90 (s, 4H), 2.75 (q,
J ¼ 7.5 Hz, 6H), [Yield: ~70%], MP 168 ꢁC, ESI-MS in MeCN: m/z
577.12 [MꢀCl]þ.
2. Materials and methods
2.1. Materials
All reagents and chemicals were obtained from Sigma Aldrich
and Fluka. Solvents were further purified by standard procedures
[22]. Super coiled pUC19 (cesium chloride purified) DNA was pur-
chased from Bangalore Genei (India). Agarose (molecular biology
grade), ethidium bromide (EB) and calf thymus DNA (CT DNA) were
obtained from Sigma (USA) and bacterial media were purchased
from Himedia. The four Salmonella typhimurium mutant strains
(histidine-dependant) TA98, TA100, TA1535 and TA1538 were
procured from IMTECH, Chandigarh, India.
Anal. Calcd. for C28H30N6S2CuCl (5): C, 54.80; H, 4.92; N, 13.69;
S,10.44 found: C, 54.36; H, 4.74; N, 13.33; S, 10.63%. IR, cmꢀ1 (KBr
disc): 3178br, 3125w, 3026w, 2912 m, 1675s, 1522vs, 1451s, 1226s,
1065 m, 784s, 695s, 627 m (s, strong; m, medium; w, weak; br,
broad; vs, very strong). 1H NMR in DMSO d6 10.71 (s, 1H), 9.41(S,
2H) 8.12 (d, J ¼ 9 Hz, 2H) 8.08(S, 1H), 7.72e6.77 (m, 12H), 4.85 (d,
J ¼ 6.5 Hz, 4H), 2.73(q, J ¼ 7.5 Hz, 46), [Yield: ~70%], MP 168 ꢁC, ESI-
MS in MeCN: m/z 577.12 [MꢀCl]þ.
TriseHCl buffer solution was prepared by using deionized,
sonicated triple distilled water.
2.2.3. Synthesis of [Cu(4MTU) (phen)Cl] (3) and [Cu(6MTU) (phen)
Cl] (6)
To an acetonitrile solution (15 ml) of CuCl (0.049 g, 0.5 mmol), 1-
Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea (4MTU) (0.1285 g,
0.5 mmol) (3), 1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea
(6MTU) (0.1285 g, 0.5 mmol) (6) was added as solid with magnetic
stirring for a duration of 0.5 h. To this an acetonitrile solution of
heterocyclic base [phen (0.09 g), 0.5 mmol] was added. The reaction
mixture was then stirred at room temperature for 2 h with con-
tinues purging of N2. The resulting deep red colored solid was
collected by filtration and washed with diethyl ether, and finally
dried in vacuum over phosphorus pentoxide, Anal. Calc. for
C
26H23N5SCuCl (3): C, 58.20; H, 4.32; N, 13.05; S, 5.98 found: C,
Scheme 1. Preparation of ligands and copper(I) complexes.
58.10; H, 4.67; N, 12.77; S, 6.06%. IR (KBr phase, cmꢀ1): 3159b,

354
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
3039w, 2904w, 1566 m, 1529vs, 1419 m, 1211s, 976s, 840s, 720s,
445 m (vs, very strong; s, strong; m, medium; w, weak; b, broad). 1H
NMR in DMSO d6 10.63 (s, 1H), 9.19 (s, 1H), 8.30 (d, J ¼ 6.5 Hz, 2H),
8.03 (d, J ¼ 6.5 Hz, 2H), 7.45e6.86 (m, 12H), 4.89 (d, J ¼ 7 Hz, 2H),
2.58 (t, J ¼ 2.5 Hz, 3H), [Yield: ~70%], MP 208 ꢁC, ESI-MS in MeCN:
m/z 500.05 [MꢀCl]þ.
ꢂ
ꢃ
ꢀ
ꢁ.ꢀ
ꢁ
ꢀ
ꢁ
.
1=2
εa ꢀ εf
εb ꢀ εf
¼
b ꢀ b2 ꢀ 2Kb2Ct½DNAꢃ=s
2KbCt
(1)
where b is (1 þ KbCt þ Kb [DNA]/2s), εa is the extinction coefficient
observed for the charge transfer absorption band at a given DNA
concentration, εf is the extinction coefficient of the complex free in
solution, εb is the extinction coefficient of the complex when fully
bound to DNA, Kb is the equilibrium binding constant, Ct is the total
metal complex concentration, [DNA] represents the DNA concen-
tration in nucleotides and s is the binding site size in base pairs. The
non-linear least-squares analysis was done using Origin Lab,
version 6.1 [25,26].
The apparent binding constant (Kapp) of the complexes 2, 5 and
3, 6 were also determined by fluorescence spectral technique using
ethidium bromide (EB) bound CT DNA solution in TriseHCl/NaCl
buffer (pH 7.2). The fluorescence intensities of EB at 600 nm
(546 nm excitation) with an increasing amount of the complex
concentration were recorded. Ethidium bromide was non-emissive
in Tris-buffer medium due to fluorescence quenching of the free EB
by the solvent molecules [27,28]. In the presence of DNA, EB
showed enhanced emission intensity due to its intercalative bind-
ing to DNA. A competitive binding of the copper complexes to CT
DNA could result in the displacement of EB or quenching of the
bound EB by the copper (I) species decreasing its emission
intensity.
Anal. Calc. for C26H23N5SCuCl (6): C, 58.20; H, 4.32; N, 13.05; S,
5.92 found: C, 58.84; H, 4.53; N, 13.20; S, 5.51%. IR (KBr phase,
cmꢀ1): 3193 m, 3041b, 2905 m, 1600s, 1531vs, 1446 m, 1221s, 784s,
714s, 690s, 455s (vs, very strong; s, strong; m, medium; w, weak; b,
broad). 1H NMR in DMSO d6 10.68 (s,1H), 8.83 (s,1H), 8.23e6.84 (m,
16H), 4.81 (d, J ¼ 6 Hz, 2H), 2.47 (t, J ¼ 2.5 Hz, 3H), [Yield: ~70%], MP
ꢁ
209 C, ESI-MS in MeCN: m/z 500.05 [MꢀCl]þ
The compounds showed stability in the solid state and good
solubility in common organic solvents other than hydrocarbons.
They were less soluble in water.
2.3. General methods
All compounds were systematically subjected to characteriza-
tion tools. Elemental analyses of carbon, nitrogen, hydrogen and
sulfur were performed with the Elementar varioMICRO V1.9.7
analyzer, copper and chloride with Energy dispersive spectroscopy
analysis of X-rays (EDAX). Powder XRD data were recorded on
Philips X'pert PRO X-ray diffractometer with graphite mono-
Viscometric titrations were performed with a SchottGerate AVS
310 Automated Viscometer. The viscometer was thermostated at
37 ꢁC in a constant temperature bath. The concentration of DNA
chromatized Cu K
a
radiation (
l
¼ 1.541 Å).
Thermogravimetric analysis (TGA) was carried out at
10 ꢁC minꢀ1 heating rate using SDT Q600 V20.9 Build 20 Module
DSC-TGA Standard under nitrogen up to 700 ꢁC with a closed
perforated aluminum pan. Conductivity measurements were made
using 10ꢀ3 M solutions in DMF on a model Control Dynamics (India)
conductivity meter and a dip-type cell, calibrated with a solution of
AnalaR potassium chloride. Magnetic susceptibility data at 298 K
for the polycrystalline samples of the compound were obtained
using Model 300 Lewis-coil-force magnetometer of George Asso-
ciates Inc. (Berkeley, USA) make. Hg [Co(NCS)4] was used as a
standard.
was 200
automated timer, and each sample was measured three times, and
an average flow time was calculated. Data were presented as (
mM in NP and the flow times were measured with an
h
/
1/3
h0
)
vs. [complex]/[DNA], where h is the viscosity of DNA in the
presence of complex and h0 that of DNA alone. Viscosity values
were calculated from the observed flowing time of DNA-containing
solutions (t) corrected for that of buffer alone (t0),
h
¼ (t ꢀ t0).
2.5. DNA cleavage
Spectroscopic measurements include: IR spectroscopy of sam-
ples in KBr pellets over the region 400e4000 cmꢀ1 in a Shimadzu
spectrometer, Far IR spectroscopy of samples in polythene discs
over the range 50e400 cmꢀ1 in a Thermo-Nicolate 6700 spec-
trometer. UV and visible absorption measurements of wavelengths
ranging from 200 to 800 nm using Shimadzu UV-3010 PC spec-
trophotometer, full scan mass spectra (MS mode) using MicroTOF
LC Bruker Daltonics spectrometer equipped with an electrospray
source operating in positive ion mode samples were dissolved in
DMF/MeOH (50/50) solution and were injected into the apparatus
by direct infusion. 1H NMR (400 MHz liquid state NMR spectrom-
eter, Bruker) was recorded in DMSO d6 solution using tetrame-
thylsilane (TMS) as internal standard.
The gel was prepared warming up 0.8% weight by volume of
agarose (from Sigma Aldrich) in 1ꢂ Tris acetate buffer (1ꢂ TAE,
which was obtained by dilution of 100 ml of 10ꢂ TAE, supplied by
SIGMA, in 1 L of water). The same 1ꢂ TEA buffer was used as
working buffer.
The extent of cleavage of supercoiled (SC) DNA in the presence
of the complex and oxidizing agent H2O2 was determined by
agarose gel electrophoresis. The gel electrophoresis was carried out
by using gel trays of 210e150 mm with a 16-toothed comb to
produce the sample wells. A Electrophoresis Power Supply Ban-
galore- Genei (India) system was used as a constant voltage supply
set to 60 V and 15 mA. In a typical reaction, supercoiled pUC19 DNA
(0.2 mg), taken in 50 mM Tris-HCl buffer (pH 7.2) containing 50 mM
NaCl, was treated with the complex. The extent of cleavage was
measured from the intensities of the bands using UVITEC Gel
Documentation System. Due corrections were made for the low
level of nicked circular (NC) form present in the original super-
coiled (SC) DNA sample and for the low affinity of EB binding to SC
compared to NC and linear forms of DNA [29]. The error range
observed in determining %NC form from the gel electrophoresis
experiments was 3e5%.
For mechanistic investigations, inhibition reactions were done
on adding the reagents prior to the addition of the complex. The
solutions were incubated for 1 h in a dark chamber at 37 ꢁC fol-
lowed by addition to the loading buffer containing 25%
2.4. Studies on DNA interaction
The UV absorbance at 260 and 280 nm of the CT-DNA solution in
5 mM TriseHCl buffer (pH 7.2) gave a ratio of ~1: 1.9, indicating the
DNA free of protein [23], where the free DNA concentration per
nucleotide was ε ¼ 6600 L molꢀ1 cmꢀ1 at 260 nm [24], with an ionic
strength 5 ꢂ 10ꢀ3 mol Lꢀ1 NaCl, and the pH 7.3.
The equilibrium binding constant (Kb) values for the interaction
of the compound with CT-DNA were obtained from the absorption
spectral titration data using the following equation:

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
355
bromophenol blue, 0.25% xylene cyanol, and 30% glycerol (2
and the solution was finally loaded on 0.8% agarose gel containing
1.0 g/ml ethidium bromide (EB).
m
L),
spectra of the complexes which display peaks due to
range 180e250 cmꢀ1. The
n
(CuS) in the
n
(CuS) at 250 cmꢀ1 indicates for Cu(I) in
m
three coordination mode [33] of the complex 2, 5 and the
n(CuS) at
220 cmꢀ1 indicates for Cu(I) in four coordination mode of the
complex 3, 6. Both the complexes displays peak due to n(CuCl),
2.6. Antibacterial activity
band ~175 cmꢀ1 indicating coordination of Cl to Cu(I) centre [34].
The antibacterial activity was tested against seven clinical iso-
lates like Bacillus subtilis, Bacillus mycodies, Staphylococcus aureus,
Yersinia enterocolitica, Proteus mirabilis, Pseudomonas aeruginosa,
Klebsiella pneumoniae. The test organisms were maintained on
nutrient agar slants. In-vitro antibacterial activity was determined
by agar well-diffusion method as described by Mukherjee et al.
[30]. The overnight bacterial culture was centrifuged at 8000 rpm
for 10 min. The bacterial cells were suspended in saline to make a
suspension of 105 CFU/mL and used for the assay. Plating was car-
ried out by transferring the bacterial suspension to a sterile Petri
plate, mixed with molten nutrient agar medium, and allowing the
3.2. Electronic absorption spectra
The electronic absorption spectra of compounds 2, 3, 5 and 6
(Fig. 2, Table 1) show two intense broad bands with maxima in the
range of ~270 and ~325 nm; the high energy band can be attributed
to ligand
p / p* transitions on the pyridine and benzene aromatic
rings, where as the lower energy band can be considered as a thi-
one originating CT transition at the C]S bond [35]. But they do not
register any band characteristic to Cu (II) ions in the visible region.
This confirms the observation that the compounds contain copper
in the þ1 oxidation state [36,37]. None of the thione bands is found
to express any significant shift upon coordination to copper. So that
the complexes adopt the trigonal planar geometry for 2, 5 and
tetragonal geometry around the copper(I) centre for 3, 6.
mixture to solidify. About 75
mL of the sample (2 mg/mL) was
placed in the wells. Plates were incubated at 37 ꢁC and activity was
determined by measuring the diameter of the inhibition zones. The
bactericidal assays were performed in triplicate.
The time dependent stability of complexes 2, 5 and 3, 6
measured over 5 h at pH 7.4, 25 ꢁC showed no significant changes in
UVeVis spectral line shapes, but more appropriately loss in signal
intensity of complex 2, 5 in the later part of the time scale [38]. The
rate of changes in the spectra for the complex 2, 5 and 3, 6 followed
an order, 3, 6 > 2, 5. A result of this clearly indicates the complexes
are stable in solution state to carry out the DNA binding and
cleavage studies of the compounds.
3. Results and discussion
A set of four different copper (I) complexes were synthesized in
high yield form through a reaction of CuCl with 4MTU, 6MTU and
heterocyclic base phen by the continuous purging of N2. The
elemental analysis closely corresponded to spectroscopic mea-
surements. The purity of compounds is reflected in elemental
analysis (complete data is given in synthesis part) and in their
appropriate spectral patterns. The elemental analysis suggests that
the stoichiometry to be 1:2 (metal:ligand) for 2, 5 and 1:1:1
(metal:ligand:B) for 3, 6. Presence of copper and chloride is
confirmed through the EDAX analysis of complexes.
3.3. 1H NMR spectra
The 1H NMR spectra of the thiourea ligand and its diamagnetic
Cu(I) complexes were recorded in DMSO d6 solution using tetra-
methylsilane (TMS) as internal standard. The chemical shifts of the
different types of protons of the ligand and its diamagnetic Cu(I)
complexes data are given in synthesis, 1H NMR chemical shifts of
ligand and its diamagnetic Cu(I) complexes were compared with
that of parent thiourea, (Fig. 3) in which 1H NMR signals of former
shift to lower fields upon binding to metal ions as expected to
observe at NeH and N0-H sites respectively at 10.63 and 9.41 ppm.
The NMR results are in good agreement with the FT-IR spectra
suggesting that the nitrogen is not involved in coordination with
Cu(I) in the complex 2, 5 and leaving the coordination occur
through C]S. In complex 3, 6 also NeH proton resonance is not
shifted indicating that the coordination through C]S of thiourea
and nitrogen of phen to Cu(I) center.
3.1. Infra-red spectroscopy
IR data of ligand and the complexes were recorded as KBr pallets
in the region 4000e400 cmꢀ1, the spectra of the complexes are
similar to that of uncoordinated N-heterocycles except for minor
shifts in the position of the peaks. selected IR spectroscopic vibra-
tion bands of 1e6 listed in Table 1 (Fig. 1), where the n(C]S) occurs
between 740 and 680 cmꢀ1 for the free ligand, is shifted toward
lower frequency upon complexation, as observed for the other
thione compounds [31] and n
(NH) around 3200 cmꢀ1 is slighly
shifted to higher wave numbers in Cu(I)-complex indicates the
existence of the thione form of the ligand in the solid state of the
complex. n
(CuN) band ~470 cmꢀ1 in compound 3, 6 indicates the
coordination via nitrogen of phen to copper centre [32]. The IR
spectral data of the complexes suggest that the ligands bind to
copper (I) via sulfur of thiourea. Further it is supported by far IR
3.4. ESI MS spectra
ESI mass spectra (Fig. 4) of complexes 2, 5 exhibits base peak at
Table 1
Physical and analytical data of the compounds.
Sl. No
Compound
na (NH)
n(C]S)
n(CuS)
n(CuN)
n(CuCl)
Lb
Electronic spectral data lmax (nm)
1
2
1-Benzyl-3-(4-methyl-pyridin-2-yl)-thiourea
[Cu(4MTU)2Cl]
3226
3173
732
695
e
e
e
e
e
315 (4800)
255 (24500)
315 (17500)
275 (26800)
330 (4500)
315 (25200)
239 (19120)
315 (25200)
270 (21600)
294 (23520)
248
178
23
3
[Cu(4MTU) (phen)Cl]
3159
720
220
445
175
32
4
5
1-Benzyl-3-(6-methyl-pyridin-2-yl)-thiourea
[Cu(6MTU)2Cl]
3188
3178
722
695
e
e
e
e
e
254
176
27
6
[Cu(6MTU) (phen)Cl]
3193
714
221
455
175
40

356
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
Fig. 1. IR spectra of compounds in KBr phase (a) 4MTU, (b) Cu(4MTU)2Cl, (c) Cu(4MTU)phen Cl. Far IR spectra of compounds in pressed Polythene disks (d) Cu(4MTU)2Cl & (e)
Cu(4MTU)phen Cl. IR spectra of compounds in KBr phase (f) 6MTU, (g) Cu(6MTU)2Cl, (h) Cu(6MTU)phen Cl. Far IR spectra of compounds in pressed Polythene disks (i) Cu(6MTU)2Cl
& (j) Cu(6MTU)phen Cl.

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
357
3.6. Interactions of Cu(I)-complexes with DNA
DNA Binding Properties of the compound 2, 3, 5 and 6 to the calf
thymus (CT) DNA, the electronic absorption spectral studies have
been performed. The panoply of absorption signals of the com-
pound 3, 6 as a function of increasing concentration of CT DNA is
shown in Fig. 6. It is observed from graph that the minor bath-
ochromic shift of 1e3 nm along with significant hypochromicity.
The bis ligand compounds does show weak binding to the DNA due
to less extended planarity compared to phen, which is consistent
with its trend in hypochromism (Table 3). The relatively higher
binding propensity of the phen compounds is possibly due to the
presence of extended planar aromatic ring in phen backbone
structure. Previous studies on bis-phen copper complex have
explored that this complex binds to DNA either by partial interca-
lation or binding of one phen ligand to the minor groove while the
other phen making favorable contacts within the groove [42,43].
The binding nature of the phen complex toward DNA is appeared to
be as similar as that has been observed in the case of bis-phen
species.
Fig. 2. UVeVis absorption spectra of complexes Cu(4MTU)2Cl (-), Cu(4MTU)phen Cl(-
-), Cu(6MTU)2Cl(
) and Cu(6MTU)phen Cl(-.-). in DMF at 25 ꢁC.
…
The emission spectral method is used to study the relative
binding of the compound to CT-DNA. The emission intensity of
ethidium bromide (EB) is used as a spectral probe as EB shows no
apparent emission intensity in buffer solution because of solvent
quenching and shows an enhancement of the emission intensity
when intercalatively bound to DNA [44]. The binding of the com-
pound to DNA decreases the emission intensity of EB. The relative
binding propensity of the compound to DNA is measured from the
extent of reduction of the emission intensity Fig. 7, Table 3. The
reduction of the emission intensity of EB on increasing the complex
concentration could be caused due to displacement of the DNA
bound EB by the copper(I) thiourea compound.
To investigate further the binding modes of the compound
viscosity measurements on solutions of CT-DNA incubated with the
compound were carried out. Because the viscosity of a DNA solu-
tion is sensitive to the addition of organic drugs and metal com-
pound bound by intercalation, we examined the effect on the
specific relative viscosity of DNA upon addition of compound. The
m/z 577.12 indicating the presence of complex cation [Cu(MTU)2]þ.
The ESI mass spectra of complexes 2, 5 also exhibits peak at m/z
320.12 which can be assigned to [Cu(MTU)]þ. ESI mass spectra of
complexes 3, 6 exhibits base peak at m/z 500.05 indicating the
presence of complex cation [Cu(MTU)(phen)]þ. The ESI mass
spectra of complexes 3, 6 also exhibits peak at m/z 283.91 which
can be assigned to [Cu(phen)þK]þ.
Molar conductance values of 10ꢀ3 M solutions of complexes fall
in the range of 23e40 Scm2 molꢀ1. These values are lower than that
expected for a 1:1 electrolyte in DMF, indicating that the com-
pounds act as non-electrolytes, i.e., the anions are coordinated to
the metal ions [39](Table 1).
3.5. TG and DTA
The thermal behavior of 4MTU and 6MTU Copper(I) compounds
recorded from ambient temperature up to 600 ꢁC in nitrogen flow
do not show any loss in weight up to 190 ꢁC, reveals that crystal
water molecules and coordinated water molecules are not present
in the compounds [40]. The TG curves in the 190e400 ꢁC range
suggest that the loss in weight for complexes 2, 5 corresponds to
elimination of 2 molecules of MTU ligand with a DTGmax 258 ꢁC.
The TG curves in the 190e285 ꢁC range suggest that the loss in
weight for complex 3, 6 corresponds to elimination of one molecule
of MTU ligand with a DTGmax 208 ꢁC and 285e560 ꢁC range sug-
gest that the loss in weight one molecule of phen with a DTGmax
396 ꢁC. In both cases the remaining residues are metal oxides.
These results are in accord with the composition of the compounds.
The thermograms for complexes 2, 5 & 3, 6 are given in Fig 5. The
stages of decomposition, temperature ranges, the temperature of
the greatest rate of decomposition (DTAmax), the evolved products,
as well as the found and calculated mass loss percentages of
complexes 2, 5 and 3, 6 are given in Table 2.
relative specific viscosity (h/h0), (h and h0 are the specific viscos-
ities of DNA in the presence and absence of a complex, respectively)
of DNA reflects the increase in contour length associated with the
separation of DNA base pairs caused by intercalation. The relative
viscosity of DNA and contour length follows the equation: (h/
h0) ¼ (L/L0)1/3, where L and L0 are the contour length of DNA in the
presence and absence of complex respectively [45]. A classical
intercalator such as EtBr could cause a significant increase in the
viscosity of DNA solutions, in contrast, a partial and/or non-classical
intercalation of the ligand could bend or kink DNA resulting in a
decrease in its effective length with a concomitant increase in its
viscosity [46]. The plots of relative viscosities with R ¼ [Com]/[DNA]
are shown in Fig. 8. The change in relative viscosity for the com-
pound 3 and 6 are more than that for the compound 2 and 5 sug-
gesting greater DNA binding propensity of the phen complexes in
comparison to the bis ligand complexes, but this change is less
compared to that of potential classical intercalators, e.g. ethidium
bromide. This is consistent with the observed trend shown by other
optical methods and suggests primarily an electrostatic and/or
groove binding nature of the complex.
3.5.1. Powder X-ray diffraction
To obtain further evidence about the structure of the metal
complexes X-ray diffraction was performed. The powder XRD pat-
terns indicate crystalline nature for the complexes [41]. It can be
easily seen that the pattern of the thiourea differs from its metal
complexes, which may be attributed to the formation of a well-
defined distorted crystalline structure.
3.7. DNA cleavage activity
DNA cleavage activity of the compounds 1e6 in the presence of
hydrogen peroxide has been studied using plasmid supercoiled (SC)
pUC19 DNA (33.3
mM, 0.2 mg) in 50 mM Tris-HCl buffer/50 mM NaCl
(pH 7.2). The extent of DNA cleavage, observed by gel

358
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
Fig. 3. H1 NMR spectra of compounds in DMSO d6 solution using tetramethylsilane (TMS) as internal standard (a) 4MTU, (b) Cu(4MTU)2Cl, (c) Cu(4MTU)phen Cl, (d) 6MTU, (e)
Cu(4MTU)2Cl & (f) Cu(4MTU)phen Cl.
electrophoresis, gives the order: 6 > 3 > 2 > 5. A 30
tration of 3 and 6 completely cleaves SC DNA into its nicked circular
(NC) form on reaction with 200 M H2O2 Fig. 9(a), (b), Tables 4a and
4b. The DNA cleavage activity order for 1e6 follows the relative
binding propensities of the compounds to DNA. Control experi-
ments using only H2O2 or the compounds alone do not show any
apparent cleavage of SC DNA under similar reaction conditions. The
reaction of SC DNA with CuCl in the presence of H2O2 does not show
m
M concen-
any cleavage activity. Addition of hydroxyl radical scavengers like
DMSO or KI significantly inhibits the cleavage. The inhibitory effect
is large with the DMSO as this reagent could reduce the effect of the
oxidizing agent by reacting with the externally added hydrogen
peroxide. The DNA cleavage reaction of the compound in the
presence of H2O2 probably proceeds through the hydroxyl radical
pathway and/or ‘‘copper-oxo’’ intermediate as the reactive species.
The pathways involved in the DNA cleavage reactions are believed
m

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
359
Fig. 3. (continued).
to be analogous to those proposed by Sigman and co-workers for
the chemical nuclease activity of bis(phen)copper species [11,43].
found to be highly active. Notably, they exhibited much more
antibacterial activity than the standard drug ciprofloxacin, while
compounds 1, 4 inactive against both Gram-positive and Gram-
negative bacteria. On chelation, the polarity of the metal ion will
be reduced to a greater extent due to the overlapping of the ligand
orbital and the partial sharing of the positive charge of the metal
ion with the donor groups. Further, it increases the delocalization of
3.8. Antibacterial activity
Gram-positive and Gram-negative bacteria can be differentiated
in the physical appearance of their cell envelopes. The compounds
were screened for their in-vitro antibacterial activity against both
Gram-positive and Gram-negative bacteria. In-vitro antibacterial
activity was determined by the agar well-diffusion method. The
percentage zone of inhibition data are represented in Table 5. The
data reviles that a series of novel compounds 2, 3, 5 and 6 were
p-electrons over the whole chelate ring and enhances the lip-
ophilicity of the complexes. This increased lipophilicity enhances
the penetration of the complexes into lipid membranes, interferes
with enzyme activity and may lead to cell apoptosis.

360
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
Fig. 4. ESI MS spectra of compounds in MeCN solution (a) Cu(4MTU)2Cl, (b) Cu(4MTU)phen Cl, (c) Cu(4MTU)2Cl & (d) Cu(4MTU)phen Cl.
4. Conclusion
2-yl)-thiourea has been synthesized and all the four complexes
structurally characterized. The spectral data are in consistent with
A series of novel Cu(I) complexes derived from 1-Benzyl-3-(4-
methyl-pyridin-2-yl)-thiourea and 1-Benzyl-3-(6-methyl-pyridin-
the proposed structure of ligands and its Cu(I) complexes. Amongst
the metal complexes explored, the phen complexes displays

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
361
Fig. 4. (continued).
significant binding propensity with CT DNA providing an order: 6,
3 >> 2, 5. The complexes 2, 5 does not show any measurable
binding interaction to the DNA and hence its poor cleavage effi-
ciency. Complexes 3, 6 show efficient oxidative cleavage of SC-DNA
in the presence of hydrogen peroxide involving hydroxyl radical
species. Pathways involving hydroxyl radicals in the DNA cleavage
reactions are proposed from control studies, which show inhibition
of the cleavage in the presence of the hydroxyl radical scavengers
DMSO and KI. The DNA cleavage activity of transition metal com-
plexes with bio-essential constituents like copper and active thio-
urea showing DNA cleavage have the potential for applications in
antibacterial agents. All complexes show a profound antibacterial
activity against both Gram-negative and Gram-positive bacteria
than ligands.

362
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
Fig. 5. TG and DTA curves of complexes (a) Cu(4MTU)2Cl, (b) Cu(4MTU)phenCl, (c) Cu(6MTU)2Cl, (d)Cu(6MTU)phenCl.
Table 2
Thermo analytical results for the investigated compounds.
Complex
Stage
TG results temperature range (ꢁ C)
DTA results temperature peak (ꢁC)
258 (Endo)
Weight loss (%)
Evolved moiety
Found
Calculated
2
3
Single
Residue
I
II
Residue
Single
Residue
I
190e400
>450
190e285
285e560
>567
190e400
>450
190e285
285e560
>567
83.92
12.07
47.68
28.35
23.20
79.69
18.60
47.17
28.9
83.52
12.97
47.90
33.00
22.0
83.52
18.59
47.90
33.00
21.32
MTU (2 mol)
CuO
MTU
208 (Endo)
307 (Exo)
Phen
CuO þ Cl
MTU (2 mol)
CuO þ Cl
MTU
Phen
CuO þ Cl
5
6
258 (Endo)
209 (Endo)
392 (Endo)
II
Residue
23.13
Fig. 6. Absorption spectral traces on addition of CT DNA to the solution 3 and 6 (shown by arrow). The inset shows the best least-squares fit of
and 6.
D
εaf/
Dεbf vs. [DNA] of compound 3

P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
363
Table 3
DNA binding data of the Cu(I) complexes.
Sl. No
Complex
Ka/Mꢀ1
Kb/Mꢀ1 [S]
2
3
[Cu(4MTU)2Cl]
[Cu(4MTU) (phen)Cl]
5.46 ꢂ 105
e
8.53 ꢂ 105
8.6 ( 0.7) ꢂ 105
[0.27 ( 0.01)]
e
5
6
[Cu(6MTU)2Cl]
[Cu(6MTU) (phen)Cl]
3.72 ꢂ 105
2.0 ꢂ 106
1.6 ( 0.6) ꢂ 106
[0.59 ( 0.02)]
Note: Ka ¼ Equilibrium binding constant from absorption spectroscopy.
Kb ¼ Apparent binding constant from EB displacement assay by emission spectroscopy.
Fig. 7. Emission spectral changes on addition of complexes 2, 3 and 5, 6 to the CT-DNA bound to ethidium bromide. The inset shows the effect of addition of increasing con-
centration of the complexes 2 (-) and 3 ( ), 5 (-) and 6 ( ) to an ethidium bromide bound CT-DNA solution in a 5 mM Tris-HCl buffer (pH, 7.2) at 25 ꢁC.
Fig. 9. Gel electrophoresis diagram showing the oxidative cleavage of SC pUC19 DNA
(0.2 mg, 30 mM b.p.) by the compounds 1e6 in the presence of H2O2 (176 mM) in 50 mM
Tris-HCl/NaCl buffer (pH, 7.2) containing 10% DMF. (a) lane-1, DNA control; lane-2,
DNA þ H2O2; lane-3, DNA þ CuCl; lane-4, DNA þ1; lane-5, DNA þ1 þ H2O2; lane-6,
DNA þ 4; lane-7, DNA þ4 þ H2O2; lane-8, DNA þ2; lane-9, DNA þ2 þ H2O2; lane-
10, DNA þ5; lane-11 þ H2O2; and (b) lane-1, DNA control; lane-2, DNA þ 3; lane-3,
DNA þ 3 þ H2O2; lane-4, DNA þ 3 þ H2O2 þ DMSO; lane-5, DNA þ 3 þ H2O2þ KI;
lane-6, DNA þ3 þ H2O2 þ NaN3; lane-7, DNA þ6; lane-8, DNA þ6 þ H2O2; lane-9,
Fig. 8. Change in relative specific viscosity of CT-DNA (200
mM) with addition of
complexes 2(-), 3( ), ( ), ( ) and ethidium bromide ( ) in 5 mM TriseHCl buffer
DNA
þ
6
þ
H2O2
þ
DMSO; lane-10, DNA
þ
6
þ H2O2þ KI; lane-11,
medium at 37 0.1 ꢁC.
DNA þ3 þ H2O2 þ NaN3.

364
P.R. Chetana et al. / Journal of Molecular Structure 1106 (2016) 352e365
Table 4a
Selected cleavage data of SC DNA pUC19 by ligands and complexes (2, 5).
Sl. No.
Condition
SC%
NC%
1
2
3
4
5
6
7
8
DNA control
98
96
97
96
95
97
96
95
76
94
82
2
4
3
4
5
3
4
5
24
6
DNA þ H2O2(2
m
m
M)
M)
DNA þ CuCl(50
DNA þ 1 (100
DNA þ 1 (100
DNA þ 4 (100
DNA þ 4 (100
DNA þ 2 (100
DNA þ 2 (100
DNA þ 5 (100
DNA þ 5 (100
m
M)
m
m
m
m
m
m
m
M) þ H2O2
M)
M) þ H2O2
M)
9
10
11
M) þ H2O2
M)
M) þ H2O2
18
Table 4b
Selected cleavage data of SC DNA pUC19 by complexes (3, 6) with controls compounds.
Sl. No.
Condition
SC%
NC%
1
2
3
4
5
6
7
8
DNA control
DNA þ3 (30
DNA þ 3 (30
DNA þ 3 (30
DNA þ 3 (30
DNA þ 3 (30
DNA þ 6 (30
DNA þ 6 (30
DNA þ 6 (30
DNA þ 6 (30
DNA þ 6 (30
98
96
12
96
94
20
96
5
2
4
88
4
6
80
4
95
6
9
mM)
m
m
M) þ H2O2
M) þ H2O2þ DMSO
M) þ H2O2þ KI
M) þ H2O2þ NaN3
M)
m
m
m
m
m
m
m
M) þ H2O2
9
10
11
M) þ H2O2þ DMSO
M) þ H2O2þ KI
M) þ H2O2þ NaN3
94
91
14
86
Table 5
Antibacterial activity of the compounds 1e6 of concentration 2 mg/mL: zone of inhibition values (in %).
Compounds
Gram-positive
Gram-negative
S. aureus
Bacillus mycoides
B. subtilis
Yersinia enterocolitica
P. aeruginosa
K. pneumoniae
Proteus mirabilis
(MTCC 3160)
(MTCC 645)
(MTCC 441)
(MTCC 4848)
(MTCC 741)
(MTCC 109)
(MTCC 743)
1
2
3
4
5
6
e
e
e
e
e
e
e
17
24
e
17
23
e
16
26
e
12
28
e
22
30
e
17
26
e
18
27
e
16
30
16
32
15
32
14
30
18
34
16
31
16
27
(b) P.R. Chetana, B.S. Srinatha, M.N. Somashekar, R.S. Policegoudra, R. Rao,
Acknowledgments
B. Maity, S.M. Aradya, Int. J. Pharm. Sci. Rev. Res. 21 (2013) 355e363.
ꢀ
[10] M.J. Fernandez, B. Wilson, M. Palacios, M.M. Rodrigo, K.B. Grant, A. Lorente,
Financial support received from the University Grants Com-
mission. (UGC, Ref No. F 39-754/2010(SR), Government of India, is
gratefully acknowledged. The authors gratefully acknowledge the
support of Prof. Akhil R. Chakravarty, Department of Inorganic and
Physical Chemistry, Indian Institute of Science, in providing facil-
ities and useful discussions.
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