Spectroscopic, electrochemical and DNA binding studies of some monomeric copper(II) complexes containing N2S(thiolate)Cu core and N4S(disulfide)Cu core

Article abstract of DOI:10.1016/j.ica.2016.10.045

We report the synthesis, characterization and spectroscopic results of the Cu(II) complexes [Cu(pabt)(OH₂)](ClO₄) (1), [Cu(pabt)(Imz)](ClO₄) (2), [Cu(pabt)(N-MeImz)](ClO₄) (3), [Cu(pabt)Cl] (4), [Cu(pma)Cl] (5), [Cu(pdta)Cl]Cl (6) and [Cu(reduced-pdta)Cl]Cl (7), (Hpabt = N-(2-mercaptophenyl)-2′-pyridylmethylenimine, Hpma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2′-di(pyridyl-2-methyleneimine)diphenyl disulfide, reduced-pdta = 2,2′-di(pyridyl-2-methylamine)diphenyl disulfide, Imz = imidazole, N-MeImz = N-methylimidazole). Electronic spectra of all these compounds display strong LMCT bands in the visible region mainly associated with S → Cu(II), and consistent with TDDFT results. A four-line EPR pattern originating from the interaction of the unpaired electron with the central^63/65Cu nucleus (I = 3/2, natural abundances:⁶³Cu, 69.17%;⁶⁵Cu, 30.83%) with the isotropic coupling constant (A_iso) values of 80 ± 1.5 G at RT for all these complexes suggests monomeric nature in solution. The redox behavior of these compounds show either nearly reversible or quasi-reversible Cu(II)/Cu(I) couple with redox potentials within the range ?0.08 to ?0.20 V versus Ag/AgCl. Some of these compounds show strong intercalative DNA binding and its complete cleavage. 1–3 exhibit remarkable cytotoxicity against C6 glioma cell line and human cervical cancer HeLa cell line. IC₅₀values of 2 and 3 for the cervical cancer HeLa cell line reveal that they exhibit higher cytotoxicity than many reported Cu(II) compounds.

Full text of DOI:10.1016/j.ica.2016.10.045

Accepted Manuscript
Research paper
Spectroscopic, electrochemical and DNA binding studies of some monomeric
copper(II) complexes containing N S(thiolate)Cu core and N S(disulfide)Cu
2
4
core
Manjuri K. Koley, Om Prakash Chouhan, Sumit Biswas, Joseph Fernandes,
Arnab Banerjee, Anjan Chattopadhyay, Babu Varghese, Periakaruppan T.
Manoharan, Aditya P. Koley
PII:
S0020-1693(16)30784-8
http://dx.doi.org/10.1016/j.ica.2016.10.045
ICA 17338
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
6 June 2016
25 October 2016
28 October 2016
Please cite this article as: M.K. Koley, O. Prakash Chouhan, S. Biswas, J. Fernandes, A. Banerjee, A. Chattopadhyay,
B. Varghese, P.T. Manoharan, A.P. Koley, Spectroscopic, electrochemical and DNA binding studies of some
monomeric copper(II) complexes containing N S(thiolate)Cu core and N S(disulfide)Cu core, Inorganica Chimica
2
4
Acta (2016), doi: http://dx.doi.org/10.1016/j.ica.2016.10.045
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Spectroscopic, electrochemical and DNA binding studies of some monomeric
copper(II) complexes containing N S(thiolate)Cu core and N S(disulfide)Cu core
2
4
a,
b
b
b
b
Manjuri K. Koley *, Om Prakash Chouhan , Sumit Biswas , Joseph Fernandes , Arnab Banerjee ,
c
d
e,
c,
Anjan Chattopadhyay , Babu Varghese , Periakaruppan T. Manoharan * and Aditya P. Koley *
a
Department of Chemical Engineering, Birla Institute of Technology and Science-Pilani, K.K. Birla
Goa Campus, Goa 403726, India
b
Department of Biological Sciences, Birla Institute of Technology and Science-Pilani, K.K. Birla
Goa Campus, Goa 403 726, India
c
Department of Chemistry, Birla Institute of Technology and Science-Pilani, K.K. Birla Goa
Campus, Goa 403 726, India
d
Sophisticated Analytical Instrument Facility, Indian Institute of Technology-Madras, Chennai 600
036, India
e
Department of Chemistry, Indian Institute of Technology-Madras, Chennai 600 036, India
_________________________________________________________________________________
Abstract
We report the synthesis, characterization and spectroscopic results of the Cu(II) complexes
[
[
(
Cu(pabt)(OH
2
)](ClO
4
) (1), [Cu(pabt)(Imz)](ClO
4
) (2), [Cu(pabt)(N-MeImz)](ClO ) (3),
4
Cu(pabt)Cl] (4), [Cu(pma)Cl] (5), [Cu(pdta)Cl]Cl (6) and [Cu(reduced-pdta)Cl]Cl (7), (Hpabt = N-
2-mercaptophenyl)-2'-pyridylmethylenimine, Hpma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta
=
2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide, reduced-pdta = 2,2'-di(pyridyl-2-
methylamine)diphenyl disulfide, Imz = imidazole, N-MeImz = N-methylimidazole). Electronic
spectra of all these compounds display strong LMCT bands in the visible region mainly associated
with S → Cu(II), and consistent with TDDFT results. A four-line EPR pattern originating from the
6
3/65
interaction of the unpaired electron with the central
Cu nucleus (I = 3/2, natural abundances:
6
3
65
Cu, 69.17%; Cu, 30.83%) with the isotropic coupling constant (Aiso) values of 80±1.5 G at RT for
all these complexes suggests monomeric nature in solution. The redox behavior of these compounds
show either nearly reversible or quasi-reversible Cu(II)/Cu(I) couple with redox potentials within the

2
range −0.08 to −0.20 V versus Ag/AgCl. Some of these compounds show strong intercalative DNA
binding and its complete cleavage. 1-3 exhibit remarkable cytotoxicity against C6 glioma cell line
and human cervical cancer HeLa cell line. IC50 values of 2 and 3 for the cervical cancer HeLa cell
line reveal that they exhibit higher cytotoxicity than many reported Cu(II) compounds.
_______________________________
Keywords: Cu(II) complex; EPR and electronic spectra; Cyclic voltammetry; DNA binding;
Cytotoxicity
_______________________________
*
Corresponding authors. Address: Department of Chemistry, Birla Institute of Technology and
Science, Pilani-K. K. Birla Goa Campus, Goa 403 726, India. Fax: +91 832 255 7033.
Email: manjuri@goa.bits-pilani.ac.in (M. K. Koley); ptm@iitm.ac.in (P.T. Manoharan);
koleyap@yahoo.co.in (A.P. Koley)
1. Introduction
Copper is one among the essential bio-relevant elements and it is present in the active sites of many
metalloenzymes and metalloproteins [1-3] that are involved in electron transfer, oxygen transport
and oxygenation reactions. Based on a number of spectroscopic [4-7] and metal substitution [8,9]
3 4 2 2
studies three basic structural models CuN S, CuN S, and CuN S have been proposed for a number
of copper electron transfer proteins [4]. Such structures are not usually known in the coordination
chemistry of copper. Due to the interest in the spectra of both “blue” and non-blue copper proteins
[
10, 11], a considerable number of studies involving the charge transfer spectra of copper(II) species
with biological significance have been performed, particularly those species containing sulfur and
imidazole ligands [12-20]. Recently, researchers are in search for copper(II) complexes that possess
biologically accessible redox potentials and show high nucleobase affinity, because they are
considered to be potential reagents for cleavage of DNA and may serve as new anticancer drugs.
Thus, a considerable number of Cu(II) complexes have been investigated for DNA interaction and
cleavage study [21-27] as well as antitumor agents [28-30]. Literature shows that mixed ligand
complexes having heterocyclic base adducts like imidazole show higher binding constant as

3
compared with parent complexes [31]. Our present study stems from our interest in designing,
synthesizing and developing the chemistry of biocompatible Cu(II) complexes with selected N,S
donor ligands which will possess suitable redox potentials, and will be capable of effectively binding
and cleaving DNA. We have synthesized five stable four-coordinate Cu(II) complexes
[
[
Cu(pabt)(OH
2
)](ClO
4
) (1), [Cu(pabt)(Imz)](ClO
4
) (2), [Cu(pabt)(N-MeImz)](ClO ) (3),
4
Cu(pabt)Cl] (4), [Cu(pma)Cl] (5) [where, Hpabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine,
−
Hpma = N-(2-pyridylmethyl)-2-mercaptoaniline, both of which act as tridentate NNS ligand [32-
34]] and two six-coordinate complexes [Cu(pdta)Cl]Cl (6) and [Cu(reduced-pdta)Cl]Cl (7), [where,
pdta = 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide [35,36] and reduced-pdta = 2,2'-
di(pyridyl-2-methylamine)diphenyl disulfide [37]. It is to be noted here that the synthesis and
characterization for compound 4 was initially reported by Lindoy and Livingstone [34] and that for
compounds 5 and 7 were initially reported by Kumbhat and coworkers [37]. These studies involved
spectroscopic studies like IR and UV-Vis and conductivity results. Here, we present the synthesis,
spectral (UV-visible, IR, and EPR) as well as electrochemical properties of seven compounds
1
including those three reported. H NMR spectra of compounds 2 and 3 provide valuable information
about the structures of these two compounds in solution. Some of these complexes are found to bind
to DNA effectively and cleaving it. Based on the DNA binding property, we have also studied the
cytotoxicity and antibacterial activity of some of these compounds. We also explored the reactivity
and catechol oxidase activity of a few selected compounds. Apart from these theoretical studies are
performed for three compounds in order to get information about their geometries. The ground state
geometries of the compounds 1-3 that are optimized at the B3LYP/LANL2DZ level of density
functional theory (DFT) suggest that all of them possess distorted square planar geometry. We are
also reporting the crystal structure of Cu(N-MeImz)
synthesis of 3 as well as in the biological activity for comparison. To the best of our knowledge,
crystal structure of Cu(N-MeImz) (ClO (8) is not reported in literature.
4 4 2
(ClO ) (8) [38] which we have used in the
4
4 2
)

4
2. Experimental
2.1. Materials
Pyridine-2-carboxaldehyde, o-aminobenzenethiol, imidazole, N-methyl imidazole, 3,5-di-tert-butyl
catechol, CuCl ⋅2H O and Cu(ClO ⋅6H O were obtained from Aldrich. Acetonitrile was obtained
2
2
4
)
2
2
from Sigma, N,N-dimethylformamide (DMF, GR), absolute ethanol, methanol (GR), DMSO and
dichloromethane (GR) were obtained from Merck and methanol (spectrasol) was obtained from
BDH. Calf thymus (ct) DNA was purchased from Sigma-Aldrich (USA).
2.2. Preparation of 2-(2-Pyridyl)benzothiazoline. This was prepared following a reported method
o
[
32]. Yield 70%; mp 94 C. Anal. Calc. for C12
H
10
2
N S: C, 67.26; H, 4.70; N, 13.07. Found: C, 67.01;
H, 4.68, N, 13.11%.
Care should be taken in the synthesis and recrystallization of this compound because it easily gets
oxidized in solution at elevated temperature in presence of air and produces the corresponding 2-(2-
pyridyl)benzothiazole.
2.3. Preparation of 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide. 2,2'-dithiodianiline was
prepared from o-aminobenzenethiol by following a procedure reported by Livingstone et al. [35].
Then 2, 2'-dithiodianiline (12.4 g, 0.05 mol) was dissolved in about 75 ml absolute ethanol by
stirring at room temperature. To this 10.7 g (0.10 mol) of pyridine-2-carboxaldehyde was slowly
added drop wise with constant stirring and stirring was continued for 1 h at RT when a light yellow
compound separated. This was filtered, washed thoroughly with ethanol and air dried. This was
o
recrystallized from ethanol. Yield 80%, mp 140 C. Anal. Calc. for C24
H
18
N
4
2
S : C, 67.58; H, 4.25; N,
13.13. Found: C, 67.31; H, 4.21, N, 12.91%.
2.4. Preparation of the complexes
The complexes were synthesized as described below. All the synthesized complexes were
recrystallized from a solution of the individual complex in acetonitrile-toluene (2 : 1) at −19 C in
o

5
dark to avoid any decomposition. However, suitable single crystals of any of these compounds were
not grown despite of numerous attempts in different ways.
2 4 4 2 2
2.4.1. [Cu(pabt)(OH )](ClO ) (1). Cu(ClO ) ⋅6H O (1.11 g, 0.003 mol) in cold methanol (15 mL, ∼5
°C) was slowly added to a cold solution of 2-(2-Pyridyl)benzothiazoline (0.645 g, 0.003 mol)
dissolved in 35 mL of methanol during 20 min with stirring below room temperature (∼5 °C). A dark
purple compound separated out from the purple solution. This was filtered, washed with methanol,
and air-dried. Yield ~65%. Anal. Calc. for C12
H
11
N
2
5
O SClCu: C, 36.55; H, 2.81; N, 7.10. Found: C,
+
+
3
5.98, H, 2.75, N, 7.21%. ESI−MS in CH
3 2
CN: m/z 277.1 [Cu(pabt) + H] = [(M−H O) + H] ; m/z
+
4
88.6 [Cu(pabt)(2-(2-pyridyl)benzothiazole)] ; m/z 489.6 [Cu(pabt)(2-(2-pyridyl)benzothiazole) +
+
+
H] ; m/z 490.5 [Cu(pabt)(2-(2-pyridyl)benzothiazole) + 2H] .
2.4.2. [Cu(pabt)(Imz)](ClO
4
) (2). Imidazole (0.136 g, 0.002 mol) in methanol (10 mL) was slowly
o
added to a cold solution of Cu(ClO
4
)
2
⋅6H
2
O (0.741 g, 0.002 mol) in methanol (10 mL, ~ 5 C) during
o
3
0 min with stirring below room temperature (~ 5 C). Then this reaction mixture was slowly added
o
to a cold solution of 2-(2-pyridyl)benzothiazoline (0.43 g, 0.002 mol) in methanol (30 mL, ~ 5 C)
o
during 30 min with stirring at ~ 5 C. A dark purple, almost black, compound separated out from the
purple solution. The solid was filtered, washed well with methanol and dried in vacuo. Anal. Calc.
13 4 4
for C15H N O SClCu: C, 40.55; H, 2.95; N, 12.61. Found: C, 41.01; H, 2.87; N, 13.01%. ESI−MS
+
+
3
in CH CN: m/z 277.1 [Cu(pabt) + H] = [{M−(Imz)} + H] ; m/z 488.6 [Cu(pabt)(2-(2-
+
+
pyridyl)benzothiazole)] ; m/z 489.4 [Cu(pabt)(2-(2-pyridyl)benzothiazole) + H] ; m/z 490.5
+
[
Cu(pabt)(2-(2-pyridyl)benzothiazole) + 2H] .
2.4.3. [Cu(pabt)(N-MeImz)](ClO
4 4 4 2
) (3). Cu(N-MeImz) (ClO ) was synthesized following a
published method [38] and we have characterized it by X-ray crystal structure (vide infra). Solid
Cu(N-MeImz)
4
(ClO
4
)
2
(1.181 g, 0.002 mol) was added to a cold solution of 2-(2-pyridyl)-
o
benzothiazoline (0.43 g, 0.002 mol) in methanol (50 mL) with stirring at ~ 5 C. Then the solution
was slowly brought to room temperature and stirring was continued for an hour. A dark purple,

6
almost black, compound separated out from the violet solution. The solid was filtered, washed well
with methanol followed by water and finally again with methanol, and dried in vacuo. Anal. Calc. for
SClCu: C, 41.93; H, 3.30; N, 12.22. Found: C, 42.01; H, 3.34; N, 12.30%. ESI−MS in
16 15 4 4
C H N O
+
+
+
3
CH CN: m/z 276.1 [Cu(pabt)] = [{M−(N-MeImz)} ]; m/z 277.0 [Cu(pabt) + H] = {M−(N-MeImz)}
+
+
+
+
H] ; m/z 359.6 [Cu(pabt)(N-MeImz) + H] = [M + H] ; m/z 488.5 [Cu(pabt)(2-(2-
+
+
pyridyl)benzothiazole)] ; m/z 489.6 [Cu(pabt)(2-(2-pyridyl)benzothiazole) + H] ; m/z 490.7
+
[
Cu(pabt)(2-(2-pyridyl)benzothiazole) + 2H] .
2.4.4. [Cu(pabt)Cl] (4). To a cold solution of 2-(2-pyridyl)benzothiazoline (0.43 g, 0.002 mol) in
o
methanol (30 mL), a cold solution of CuCl
was rapidly added with constant stirring. The solution turned violet and a red-purple compound
separated. This was filtered and washed with methanol and air-dried. Yield ~80%. Anal. Calc. for
SClCu: C, 46.15; H, 2.90; N, 8.97. Found: C, 45.98, H, 2.87, N, 8.91%. ESI−MS in CH CN:
2 2
⋅2H O (0.341 g, 0.002 mol) in methanol (25 mL, ~ 5 C)
C
12
H
9
N
2
3
+
+
+
+
m/z 276.1 [Cu(pabt)] = [(M−Cl)] ; m/z 277.1 [Cu(pabt) + H] = [(M−Cl) + H] ; m/z 315.9
+
+
+
[
Cu(pabt)Cl + 4H] = [(M+4H) ]; m/z 488.6 [Cu(pabt)(2-(2-pyridyl)benzothiazole)] ; m/z 489.5
+
+
[
Cu(pabt)(2-(2-pyridyl)benzothiazole) + H] ; m/z 551.7 [{Cu(2-(2-pyridyl)benzothiazole)}
2
] .
.4.5. [Cu(pma)Cl] (5). This compound was prepared by modifying the method described by Corbin
(0.204 g, 0.0015 mol) in dry methanol (15 mL) was added to a
2
et al. [33]. A solution of ZnCl
2
solution of 2-(2-pyridyl)benzothiazoline (0.215 g, 0.001 mol) in dry methanol (35 mL) with constant
stirring at room temperature. Then this solution was carefully treated with sodium borohydride
(
2
0.0378 g, 0.001 mol) over a period of 20 min and then H S gas was purged through this solution for
30 min to precipitate ZnS. This was filtered off and the filtrate containing the reduced ligand was
o
collected and neutralized with bubbling hydrogen chloride gas. This solution was cooled to ~ 5 C.
Addition of CuCl ⋅2H O (0.170 g, 0.001 mol) in methanol (10 mL) to this cold solution resulted in a
red-violet solution and a dark, almost black compound separated. This was filtered and the dark
2
2

7
compound was washed with methanol and air-dried. Yield ~60%. Anal. Calc. for C12
H
11
N
2
SClCu: C,
4
5.82; H, 3.53; N, 8.91. Found: C, 45.98, H, 3.58, N, 8.89%.
.4.6. [Cu(pdta)Cl]Cl (6). A solution of CuCl ⋅2H O (0.341 g, 0.002 mol) in methanol (25 mL) was
2
2
2
slowly added to a solution of 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide (0.86 g, 0.002
mol) in methanol (50 mL) with constant stirring at room temperature. The solution turned red-violet
and a red-purple compound separated. This was filtered and washed with methanol and air-dried.
Yield ~85%. Anal. Calc. for C24
N, 9.91%. ESI−MS in CH
H
18
N
4
S
2
Cl
2
Cu: C, 51.38; H, 3.23; N, 9.99. Found: C, 51.98, H, 3.28,
+
3
CN: m/z 213.2 [(2-(2-pyridyl)benzothiazole) + H] ; m/z 276.0 [Cu(2-(2-
+
+
+
pyridyl)benzothiazole) + H] or [Cu(pabt)] ; m/z 277.1 [Cu(2-(2-pyridyl)benzothiazole) + 2H] or
+
+
[
Cu(pabt) + H] ; m/z 311.1 [Cu(2-(2-pyridyl)benzothiazole)Cl] ; m/z 488.5 [Cu(pabt)(2-(2-
+
+
+
pyridyl)benzothiazole)] ; m/z 489.5 [Cu(pdta)] ; m/z 490.6 [Cu(pdta) + H] ; m/z 523.6 [Cu{(2-(2-
+
+
pyridyl)benzothiazole}
.4.7. [Cu(reduced-pdta)Cl]Cl (7). A solution of ZnCl
mL) was added to a solution of 0.001 mol of 2,2'-di(pyridyl-2-methyleneimine)diphenyl disulfide
0.43 g, 0.001 mol) in dry methanol (35 mL) with constant stirring at room temperature. Then this
solution was carefully treated with sodium borohydride (0.0378 g, 0.001 mol) over a period of 20
min and then H S gas was purged through this solution for 30 min to precipitate ZnS. This was
filtered off and the filtrate was collected and neutralized with bubbling hydrogen chloride gas. Slow
addition of CuCl ⋅2H O (0.170 g, 0.001 mol) in methanol (10 mL) to this resulted in a red-violet
2
Cl] ; m/z 525.5 [Cu(pdta)Cl] .
2
2
(0.204 g, 0.0015 mol) in dry methanol (15
(
2
2
2
solution and a dark, almost black compound separated. This was filtered and the purple filtrate was
collected in a beaker and placed in a refrigerator for slow evaporation when a dark crystalline
compound separated. This was collected by filtration and washed with methanol and air-dried. Yield
~50%. Anal. Calc. for C24
22 4 2 2
H N S Cl Cu: C, 51.02; H, 3.92; N, 9.91. Found: C, 50.98, H, 3.86, N,
9.88%.

8
3
. Methods for biological activity
.1. Assay for antibacterial activity
3
The antibacterial activities of all the copper compounds were determined using disc diffusion
method. The Gram negative bacterium Escherichia coli (E.coli) was used as the standard bacterial
strain. A single colony from a fresh plate was transferred to 5 ml of Luria-Bertani (LB) broth and
allowed to grow at 37°C with shaking at 120 rpm till the culture reached an OD600 of 0.8. This
bacterial culture was swabbed on to LB agar media culture plates using sterile cotton swabs thrice to
ensure that the entire plate surface is covered with bacterial cells. Inoculated plates were kept for 2
minutes in laminar air flow before the disc was applied. Sterile Whatman filter paper (number 3)
discs of 5 mm diameter were prepared and sterilized at 121°C temperature and 15 psi before use. All
the copper samples were dissolved in DMSO so as to achieve a final concentration of 100 µg/mL and
sterilized individually by passing through a 0.22 micron membrane filter. The sterilized discs were
then dipped in the sterilized sample solutions and allowed to air dry in laminar air flow for 2 minutes
on a sterile surface. The air dried discs were applied to the LB agar culture plate surface (which was
previously swabbed by bacterial culture) ensuring complete contact of the disc on agar surface. The
plates were incubated at 37 °C overnight (approximately 16 hours) and the growth inhibition zones
(
clear zone) around each disc were measured.
3.2. DNA binding studies
Since DNA cleavage was observed (vide infra) for most of the compounds under study, we wanted
to figure out the kind of binding to DNA. Thus, we have chosen 1 and 2 as representative compounds
for the DNA binding studies. These studies were carried out for compounds 1 and 2 in aqueous
medium by monitoring the overall electronic spectral change after addition of plasmid DNA
o
(
dissolved in Tris buffer, pH 7) as described below. A cold (∼10 C) aqueous solution (1.2 mL) of
−4
−1
compound 2 [0.92 × 10 mol L ] was treated with 10 µL of cold DNA solution (140.5 ng/µL) in

9
Tris buffer, allowed to stand to reach equilibrium and the electronic spectrum was recorded. This
titration was repeated ten times, by adding 10 µL of cold DNA solution each time, until there is no
further change observed in the electronic spectrum indicating the reaction reached a steady state after
addition of a total of 100 µL of cold DNA solution amounting to 10.8 µg/mL DNA in the reaction
o
−4
−1
mixture. In the case of compound 1, 1 mL of cold (∼10 C) aqueous solution [1.51 × 10 mol L ]
was directly treated with 83.4 µL of cold DNA solution and the electronic spectra were recorded
with time. The spectrum became steady within 40 min and no further change was observed for
another 25 min.
In addition, in order to get the binding constant for the compounds 1-3, electronic spectra of the
compounds with incremental amounts of calf thymus DNA (ct-DNA) have been recorded using a
Jasco V-570 UV/VIS/NIR spectrophotometer. The absorption titration experiments were performed
by keeping the complex concentration constant and by varying the concentration of ct-DNA from 0
to 40 µM in 10 mM Tris–HCl buffer (pH 7.43).
3.3. Nucleic acid degradation activity
Since DNA binding and its cleavage studies being one of the major objective for the present work,
we have carried out DNA cleavage studies for all seven compounds as well as another reported
4 4 2
compound Cu(N-MeImz) (ClO ) (8) as a reference. For DNA degradation assay, E. coli plasmid
DNA purified by Qiagen plasmid isolation kit was subjected to treatment with each copper
compound. The quantity of plasmid DNA was estimated to be 250 µg/µL. All the samples were
dissolved in DMSO and sterilized as mentioned in the previous section. 5 µL (1250 µg) of the
purified plasmid DNA was mixed with sample so that each reaction mixture contained 100 µg of the
sample. The total reaction volume was 10 µL (made up with DNA loading dye). All the vials
containing the reaction mixture were kept at room temperature for 12 h. The samples were run in a
1
% agarose gel at 60 V for 2 h to achieve complete separation of nucleic acid bands.

10
3.4. Cytotoxicity
3.4.1. Cell culture and treatment
The C6 glioma cell line and the cervical cancer HeLa cell line were maintained in DMEM
(
Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS). On
4
achieving 80% confluency, the cells were seeded into 96 well plate with a density of 4 × 10 per well
and treated with different concentrations of the compounds 1-3 dissolved in DMSO (10 µM, 20 µM,
5
0 µM, 100 µM) in triplicates, further incubating for two different incubation times, 24 h and 48 h,
respectively. Each set of experiment was further repeated three times.
.4.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
3
To see the impact of the three compounds on biological system, MTT assay was performed. In this
assay, cell viability was determined by the reduction of yellow MTT into purple formazan product by
the enzymatic activity of mitochondrial dehydrogenase in the live cells based on the method of
Mabley et al. [39]. C6 glioma cells and HeLa cells were plated on 96 well plates at a density of 4 ×
4
1
0 per well and treated with 1, 2, and 3 as described earlier. After stipulated time period, media was
removed and fresh media with MTT (final conc. 500 µg/µl) added. After 4h MTT solution was
removed carefully and 100 µL of DMSO was added to solubilize the purple formazan crystals.
Absorbance of the colored solution was measured at a test wavelength of 570 nm with a reference at
wavelength of 620 nm. The absorbance obtained from treated cells was expressed as the percentage
of absorbance obtained from untreated cells.
3.5. Detection of hydrogen peroxide in the catalytic reaction of catechol oxidase activity
The formation of H
2
O
2
during the catalytic reaction of catechol oxidation by the compounds 1 and 2
−
was detected iodometrically by assaying I
3
that was formed from the reaction of KI with the
−
reaction mixture using a spectrophotometer since I
3
has a characteristic absorption band at 353 nm
−1
−1
(
ε = 26000 M cm ) in water. The oxidation reaction of 3,5-DTBC by complex 1 or 2 in methanol

11
was carried out as described in the kinetic experiment for catechol oxidase activity (vide infra).
When the formation of 3,5-DTBQ reached a desired value at 401 nm after 1 h, the reaction mixture
−3
was acidified by adding an equal volume of cold H
2
SO
4
(20 × 10 M) to quench the reaction. Then
the quinone was extracted from the reaction mixture with CH
2
Cl until the organic layer was
2
colorless. Then, 1 mL of a KI solution (10% aqueous) was added to the pink aqueous layer, divided
into two parts, and spectra were recorded separately with both parts, one part without and the other
part with a catalytic amount of ammonium molybdate (3% solution) to accelerate the formation of
−
I
3
. Blank experiment was performed under identical condition in the absence of the catalyst, but
−
only very minor formation of I
3
was observed as compared to that formed in presence of 1 and 2.
3.6. Physical measurements
Elemental analyses (for C, H, and N) were performed on a Perkin-Elmer model 2400 series II
CHNS/O analyzer. Mass spectra were recorded on a Varian Inc, USA Liquid Chromatograph Mass
Spectrometer Model 410 Prostar Binary LC with 500 MS IT PDA Detectors operating in ESI mode.
Infrared and far-infrared spectra were measured with a Jasco IR report-100 and a Shimadzu IR
Affinity - 1 FT-IR and a Bruker IFS 66V FT-IR spectrometer using KBr and polyethylene pellet,
respectively. Electronic absorption spectra were recorded on a CARY 5E UV-Vis-NIR
spectrophotometer (Varian) and a Jasco V-570 UV/VIS/NIR spectrophotometer using a pair of
matched quartz cell of path length of 1 cm. Diffuse reflectance spectral measurements were done
with JASCO-ISN470 attachment with solid sample holder JASCO SSH-506. Electron paramagnetic
resonance spectra were recorded on a Varian E-112 X/Q-band EPR spectrometer and a JEOL, Japan
Model: JES - FA200 ESR spectrometer with X and Q band. Instrumental parameters: modulation
frequency = 100 kHz, modulation amplitude = 1 G, microwave power = 20 mW.
Diphenylpicrylhydrazyl (DPPH) was used as an internal field marker. Room temperature solution
EPR spectra were recorded using an aqueous cell and frozen glass spectra were recorded in liquid

12
nitrogen using a quartz dewer. Proton NMR spectra were recorded using a Bruker AVANCE III 500
MHz (AV 500) multi nuclei solution NMR Spectrometer. Conductivity measurement was done using
a Mettler Toledo dual conductivity/pH meter model SevenMulti equipped with Inlab 730 and Inlab
413 electrodes. Electrochemical measurements were done with the help of a Bioanalytical system
CV-27 electrochemical analyzer and a BAS model X-Y recorder at 298K under dinitrogen. A
standard three electrode cell consisting of a platinum working electrode, a platinum auxiliary
electrode and a Ag/AgCl reference electrode was used. Tetrabutylammonium hexafluorophosphate
(
4 6 4 4
[N(n-Bu) ]PF ) or tetrabutylammonium perchlorate ([N(n-Bu) ]ClO ) was used as supporting
electrolyte.
3
.7. Computational details
In our present work, computational studies have been performed at the density functional theory
DFT) [40] and time-dependent density functional theory (TD-DFT) [41] level using Gaussian 09 suite
(
of program [42]. Becke’s [43] three parameter exchange function (B3) with Lee-Yang-Parr
correlation [44] function (LYP) have been employed using LANL2DZ basis sets for the geometry
optimization calculations. The estimation of the peak positions of these complexes has been done at
the Td-B3LYP/LANL2DZ level. Calculations with solvent (methanol) have been carried out using
the Polarizable Continuum Model (PCM) [45, 46] in G09. The relevant molecular orbitals are
analyzed through the visualization software Chemcraft.
3
.8. X-ray crystallography for Cu(N-MeImz)
Purple crystals were obtained from the slow evaporation of an aqueous solution of Cu(N-
MeImz) (ClO [38] at room temperature. A crystal with approximate size of 0.35 × 0.35 × 0.30 mm
was mounted on a Bruker Axs Kappa Apex2 diffractometer equipped with graphite-monochromated
Mo Kα, λ = 0.71073 Å] radiation. The unit cell parameters were determined by the method of
4 4 2
(ClO )
4
4 2
)
[

13
difference vectors using reflections scanned from three different zones of the reciprocal lattice. The
°
intensity data were measured using ω and φ scan with frame width of 0.5 . The frames integration
and data reduction were performed using Bruker SAINT-Plus (Version 7.06a) software [47]. The
multi-scan absorption corrections were applied to the data using SADABS software. The crystal is
indexed in monoclinic system with space group P2 /n and lattice parameters a = 8.0844(6) Å, b =
1
ο
ο
ο
1
0.4731(6) Å, c = 14.3954(10) Å, α = 90.00 , β = 93.519(2) , γ = 90.00 . SIR-92 program [48] was
used for solving the structure. Structure was refined using SHELXL-97 program [49]. Successive
difference fourier map showed the positions of all hydrogen atoms. However, the hydrogen positions
were geometrically fixed and refined through riding model. The full matrix structure refinement was
2
2
2
2
2
2
carried out through minimization of the function∑(w(F
o
− F
c
)) , where w = [σ (F
o
) + (0.0667P) +
−1
2
2
2
2
0
.7873P] and P = (F
o
+ 2F
c
)/3, F
o
is the measured intensity (i.e., intensity observed) and F
c
is
the intensity calculated. The final residual factors were R = 0.0387 and wR = 0.1214. The largest
3
difference map peak was 0.640 e/Å .
4. Results and discussion
We have studied the reactions of Cu(ClO
4
)
2
⋅6H
2
O or CuCl
2
⋅2H
2
O and Cu(N-MeImz)
4
(ClO
4
)
2
with 2-
(
2-pyridyl)-benzothiazoline in methanol to prepare the complexes of the corresponding Schiff base
−
N-(2-mercaptophenyl)-2'-pyridylmethylenimine with NNS coordination [32-34] and also the
reactions of CuCl ⋅2H O with another Schiff base 2,2'-di(pyridyl-2-methyleneimine)diphenyl
2
2
disulfide (pdta) [35,36] and its reduced form 2,2'-di(pyridyl-2-methylamine)diphenyl disulfide [37]
with NNSNN coordination [36], and also with N-(2-pyridylmethyl)-2-mercaptoaniline (Hpma)
[
32,33] derived from the reduction of N-(2-mercaptophenyl)-2'-pyridylmethylenimine (Hpabt) as
shown in Fig. 1.

14
Fig.1. Structures of the ligands.
The complexes are readily soluble in methanol (blue purple solution), acetonitrile (red purple
solution) and in DMF (dark purple to violet solution), but are insoluble in dichloromethane except
the compounds [Cu(pabt)Cl] (4) and Cu(pma)Cl] (5) which are found to be moderately soluble in
dichloromethane producing a bright blue solution in each case. Conductivity measurements with
fresh solutions of the compounds 1 - 3 show these are 1 : 1 electrolyte while 4 and 5 are
nonelectrolyte in nature. On the other hand, conductivity measurements for 6 and 7 in DMF show Λ
−1
2
−1
−1
2
−1
=
76.6 Ohm cm mol for 6 and Λ = 88.5 Ohm cm mol for 7, respectively, indicating that
they are 1 : 1 electrolyte in this solvent [50]. However, on standing, the molar conductivity increases
−1
2
−1
−1
2
−1
−1
2
−1
from 76.6 Ohm cm mol to 162.48 Ohm cm mol for 6 and from 88.5 Ohm cm mol to
−1
2
−1
−
1
70.91 Ohm cm mol for 7. This strongly suggests that one of the two Cl ions is coordinated to
2
+
the Cu ion resulting octahedral complex in each case. On standing, the conductivity of these
solutions changes with time and become steady after several hours when it shows 2 : 1 electrolyte
−
[
50] indicating that the coordinated Cl is gradually replaced by DMF. However, this change is not

15
observed in acetonitrile for both 6 and 7, and both of them are found to be 1 : 1 electrolyte in
CH CN. But when the CH CN solution of 6 is reacted with solid imidazole (1 : 1 mmol ratio), the
conductivity immediately increased and became steady within 20 min and the conductivity value
3
3
−1
2
calculated based on the initial concentration was found to be changed from 125 to 220 Ohm cm
−1
−
mol for 6. The isolated product (6A) (vide infra) confirms the substitution of the coordinated Cl
ion by imidazole and making the final species 2 : 1 electrolyte in CH
in the positive-ion mode) for complexes 1–4 and for complex 6 have been recorded in acetonitrile
3
CN [50]. The ESI-MS spectra
(
and the results are presented in Fig. S1(Supplementary material). The complexes 1−4 showed a peak
+
at m/z 277.1 corresponding to [Cu(pabt) + H] while complex 6 displayed peaks at m/z 489.5
+
+
corresponding to [Cu(pdta)] , at m/z 490.6 corresponding to [Cu(pdta) + H] and at m/z 525.5
+
corresponding to [Cu(pdta)Cl] . Based on the above observations, elemental analyses, conductivity
results and X-band RT solution EPR results (vide infra) the proposed structures of 1-7 are shown in
Fig. 2.
Fig. 2. Proposed structures of the compounds 1-7 in solution.

16
1
.1. H NMR results
4
Though it is very difficult to get well resolved NMR spectra for paramagnetic mononuclear Cu(II)
compounds due to unfavorable electronic relaxation time and only few mononuclear complexes are
1
reported to have H NMR spectra with considerable broadness [51-53], we were keen to get some
1
information from H NMR for at least a few of our compounds in the absence of any crystal structure
since single crystals for any of the compounds under study could not be grown in spite of trying
many different ways. So we have chosen compounds 2 and 3 which are very similar except the
1
imidazole N−H proton in 2 is replaced by N–CH
compounds were recorded in d
broadening [52,53] in both cases are found to be remarkably similar except that the broad peak at
1.552 ppm observed for 2 is absent in the spectrum of 3, and the latter contains a peak at 4.261 ppm
which is absent in the case of 2. All other peaks appear to be very similar for both these compounds
Fig. 3). This broad peak at 11.552 ppm observed for 2 can be assigned to NH proton [54] which is
not observed in 3, on the other hand the signal at 4.261 ppm (corresponds to three protons) observed
for 3 but not for 2 can be assigned to N-CH protons [54]. The broad peak at 14.059 ppm observed
3
group in 3. The H NMR spectra for these two
6
-DMSO and shown in Fig. 3. The spectra which show significant
1
(
3
for 2 or at 14.023 ppm observed in the spectrum of 3 is appearing for 2′−H of the imidazole group
1
[
55]. Though signals for all the protons did not show up in the H NMR spectra of 2 and 3, the
number of signals observed has provided valuable information about the structures of these two
compounds in solution.

17
1
Fig. 3. H NMR results of compounds 2 and 3 in d -DMSO at RT.
6
4.2. Electronic structures and spectral properties
Compounds 1-5 produce intense purple to violet color in methanol, acetonitrile and DMF while
compounds 4 and 5 produce blue color in dichloromethane. All these solutions exhibit optical spectra
at room temperature inclusive of an intense absorption in the UV region between 210 and 390 nm

18
*
involving principally intraligand π → π transitions and another strong, relatively intense but broad
band centered around 530 -573 nm in the visible region which is absent in the absorption spectrum of
any of the free ligands is arising due to S→Cu(II) charge transfer transitions [10,11,16]. This band in
the visible region involves both the ligand and the metal center and exhibits a significant
solvatochromic shift [11, 56, 57], characteristic of a large dipole moment change between the ground
and the excited state, and indicative of charge transfer (CT) character [56]. A blue shift with
increasing solvent polarity is observed (Fig. 4, panels B, C), indicating a reduction in the dipole
moment upon electronic excitation [11, 56]. Electronic spectra of 1-3 in methanol are found to be
very similar (Fig. 4, panel A) and show a strong and broad band centered around 540 nm. Electronic
spectra for 4 [34] and 5 [37] in CH
broad band in the visible region (λmax at 573 nm). This λmax, for example, is found to be shifted to
34 nm for 5 in methanol, and the blue color in CH Cl is changed to purple in CH OH (and also in
CH CN or DMF). The λmax for 1 in CH OH is at 541 nm whereas the same in water is found to be at
78 nm causing a blue shift of 63 nm. Similarly, λmax for 2 in CH OH is at 540 nm whereas in water
it is found to be at 480 nm causing a blue shift of 60 nm (Fig. 4, panel C).
2 2
Cl are found to be very similar (Fig. 4, panel B) and exhibit a
5
2
2
3
3
3
4
3

19
Fig. 4. Electronic spectra of the compounds. Panel A. A quantitative comparison of the electronic
−4
−1
−4
spectra of 1-3 in methanol. Black curve a for 1 [1.89 × 10 mol L ], red curve b for 2 [2.17 × 10
−1
−4
−1
mol L ] and blue curve c for 3 [1.84 × 10 mol L ], respectively. Panel B. Blue curve a. 4 in
−
4
−1
−4
−1
CH
CH
in water [2.19 × 10 mol L ]. Panel D. Blue curve a. 6 in CH CN [0.8 × 10 mol L ]. black
2
Cl
2 2 2
[6.1 × 10 mol L ]. black curve b: 5 in CH Cl [2.1 × 10 mol L ], purple curve c: 5 in
−
4
−1
−4
−1
3
OH [3.2 × 10 mol L ]. Panel C. black curve a: 2 in CH OH [2.17 × 10 mol L ], red curve b:
3
−4
−1
−4
−1
2
3
−4
−1
−4
3 3
curve b: 7 in CH CN [0.6 × 10 mol L ]. Inset of panel D. Blue curve a. 6 in CH CN [7.0 × 10
−1
−4
−1
3
mol L ]. black curve b: 7 in CH CN [2.6 × 10 mol L ].
To check the difference between the solution spectrum with that of the solid, we have recorded the
diffused reflectance spectra with the powder samples of 1 and 2. Both of them have strong and broad
absorbance in the visible as well as in the UV region as shown in Fig. S2 (Supplementary material).
It should be pointed out in this context that the overall spectral features for all these complexes are
comparable in the visible region and these are very similar to that reported in literature [16] for a
2
−
pseudo-tetrahedral Cu(II) complex with a dianionic tetradentate N
2
S
2
ligand. The ligand field bands
for our complexes under study are not seen due to the broad tail of the LMCT band masking these
bands. This can be resolved only by deconvolution [32] of the strong and broad band in the visible
and near-IR region (Fig. S3 of Supplementary material) that clearly suggests a number of electronic
transitions in this region.
Compound 6 is found to be highly soluble in acetonitrile producing a purple to violet solution (λmax
at 547 nm) while 7 which is moderately soluble in acetonitrile produces a red-purple solution (λmax at
545 nm) and both these solutions exhibit very similar electronic spectra (Fig. 4, panel D). It is to be
noted here that the electronic spectra exhibited by these six-coordinate compounds 6 and 7 in the

20
visible region are found to be very similar to those obtained for 1-5 in the same solvent. This
observation along with the intense purple color for compounds 6 and 7 in acetonitrile (also in
methanol and DMF) strongly suggest equatorial coordination of the sulfur ligand (one of the two
sulfur atoms of the disulfide) in these complexes also, resulting in a tetragonal elongated octahedral
complex. It should be mentioned here that normally in such cases the axially coordinated ligands in
general do not show any charge transfer (CT) absorption [11]. The hexacoordinated Cu(II) thiolato
complexes with sulfur ligands in the equatorial positions reported by Bhardwaj and coworkers [16]
exhibited an intense band centered around 600 nm that was resulted from the good overlap between
2
2
the equatorially bound thiolate and the d
x
- orbital on Cu(II) and was assigned as σ(thiolate) →
y
Cu(II) LMCT transition [16]. The equatorial S coordination for the pentadentate ligand used in our
study has been established in the crystal structure of chloro[bis[2-(2-pyridylmethyl)imino]phenyl]-
disulfide]nickel(II)perchlorate reported by Warner et al. [36] where the ligand is found to act as a
pentadentate NNSNN donor with an equatorial S (from the disulfide) coordination to Ni(II).
4
.3. Computational results
We have carried out theoretical studies to get an understanding of the ground state geometries of the
compounds 1-3 that are optimized at the B3LYP/LANL2DZ level of density functional theory (DFT)
Fig. 5, and Table S1 of Supplementary Materials). All of them possess distorted square planar
(
geometry as revealed from the bond angles and dihedral angles (Table 1, and Fig. S4 of
Supplementary material) and the extent of distortion is found to be more in the cases of 2 and 3 as
compared to 1. It is to be noted here that the RT solution EPR spectra for 1 and 2 in methanol also
showed that these are more distorted toward tetrahedral in this solvent (vide infra). The calculated
peak positions obtained from the TDDFT results are presented in Table 1. The experimentally
observed positions of the peaks are almost reproduced by the TDDFT results (Table 1) with slight
deviations. Interestingly, all these peaks are found to be dominated by configurations where the

21
electrons from the inner beta orbitals are excited to the first unoccupied beta spin orbital which is
2
2
x y
antibonding in nature and has the characteristic of dx -y orbital on Cu with p /p orbitals on the sulfur
and the nitrogens (Fig. 6). Among different inner beta orbitals from where this excitation originates,
the one having strong bonding nature between the copper d orbital and the p orbital of sulfur has a
good contribution in all the studied complexes. In other words, the Cu−S bond weakens due to the
electronic transfer to the vacant beta spin orbital where the electronic cloud is delocalized on copper
2
2
d
x -y orbital and on p orbitals of sulfur, nitrogens and oxygen.
Table 1. Theoretically predicted peak positions and the oscillator strength of the electronic transitions
corresponding to these peaks, and the bond angles and dihedral angles obtained from the optimized
geometry of the compounds 1-3 (without and with solvent).
Complex
Peak position
Oscillator strength of
the corresponding
transition
Bond angles and
dihedral angles
(without solvent)
Bond angles and
dihedral angles (in
methanol)
(
nm)
a
b
b
b
a
b
b
b
0
0
1
516 , 535
0.025 , 0.014
N2-Cu-S,170 ;
N2-Cu-S, 170 ;
0
0
N1-Cu-O, 180 ;
N1-Cu-O, 177 ;
0
0
C7-N2-Cu-S, 180 ; C7-N2-Cu-S, 180 ;
0
0
N2-Cu-S-C5, 0 ;
N2-Cu-S-C5, 0 ;
0
0
C7-N2-Cu-O, 0 .
C7-N2-Cu-O, 0 .
a
a
0
0
2
3
493 , 503
0.009 , 0.023
N2-Cu-S, 168 ;
N1-Cu-N4, 176 ;
N2-Cu-S, 168 ;
N1-Cu-N4, 180 ;
0
0
0
0
C7-N2-Cu-S, 164 ; C7-N2-Cu-S, 177 ;
0
0
N2-Cu-S-C5, 14 ;
N2-Cu-S-C5, 2 ;
C7-N2-Cu-N4, 0 .
0
0
C7-N2-Cu-N4, 5 .
a
a
0
0
495 , 505
0.016 , 0.026
N2-Cu-S, 168 ;
N1-Cu-N4, 176 ;
N2-Cu-S, 168 ;
N1-Cu-N4, 177 ;
0
0
0
0
C7-N2-Cu-S, 162 ; C7-N2-Cu-S, 168 ;
0
0
N2-Cu-S-C5, 15 ;
N2-Cu-S-C5, 11 ;
0
0
C7-N2-Cu-N4, 6 .
C7-N2-Cu-N4, 4 .
a
b
without solvent, in methanol

22
Fig. 5. Ground state optimized structures of the complexes 1, 2, and 3 without and with solvent,
respectively. The numbers between the atoms are respective bond distances in Å.

23
Fig. 6. Orbitals involved in the electronic excitations corresponding to the dominant configurations
for compounds 1, 2, and 3, respectively.
4.4. Reactivity of the complexes
For chemical reactivity we have chosen compounds 2 and 6 to react with imidazole, because 2, if it
reacts at all, will undergo only addition reaction to form either a 5-coordinate or a 6-coordinate
−
product while compound 6 should undergo only Cl substitution reaction. Thus interpretation of the
spectral data is expected to be simple and easy to derive kinetics parameters. Reactions of these two
complexes in CH CN at RT with imidazole have been studied by monitoring the electronic spectral
3
change in the visible region as shown in Fig. 7. Imidazole readily reacts with 2 as evident from the
electronic spectral change. When 2-fold solid imidazole is added to the acetonitrile solution of 2
having the spectrum shown in Fig. 7, curve a of panel A, the intensity of the peak increases
immediately as shown in curve b, and becomes steady within 5 min and no further change is
observed up to 20 min (curve c). The λmax is found to be shifted from 533 nm to 540 nm upon

24
3
reaction with imidazole. On the other hand, 6 reacts with imidazole in CH CN and the λmax at 546
nm (curve a, Fig. 7 panel B) immediately blue shifts with increased intensity (curve b, Fig. 7 panel
B) and then slowly increases and becomes steady within 20 min when the λmax is found to be at 542
nm (and the absorbance is increased from c.a. 0.94 to 1.47) (curve e, Fig. 7 panel B).
Fig. 7. Electronic spectral changes for the reactions of the compounds. Panel A. Reaction of 2 with
−4
−1
imidazole. Black curve a. 2 in CH
3
CN [2.17 × 10 mol L ], red curve b: within 5 min after
addition of 2-fold excess of solid imidazole, and blue curve c: after 20 min. Panel B. Reaction of 6
−4
−1
with imidazole. Black curve a. 6 in CH
adding 2-fold excess of solid imidazole, curve d: recorded after 15 min, and curve e: recorded after
0 min. Curves d and e are identical indicating the steady state reached within 15 min. Panel C. Plot
3
CN [5.50 × 10 mol L ], blue curve b: immediately after
2
of absorbance change at 546 nm with time for the spectral data presented in panel B. The solid line
represents the fitting of the experimental points (circles) using the model
.
Based on the electronic spectral change shown in Fig. 7 panel A for the reaction of 2 with imidazole,
and the final product isolated from this reaction (vide infra), it is concluded that imidazole undergoes
3 4
addition reaction forming a six-coordinate octahedral complex [Cu(pabt)(Imz) ](ClO ) which has a
strong absorbance at 540 nm and exhibit a weak broad ligand field band [16] in the 700-800 nm
region (shown in inset of Fig. 7, panel A) apart from other charge transfer bands in the UV region.
2
2
The λmax at 533 nm which is assigned as σS(1) → Cu(II) d
x y
- [10,11 16, 58] is found to be shifted
to 540 nm with increased intensity. The higher intensity of this band in the reaction product is most

25
2
2
likely due to a better overlap between the Cu(II) d
x
-
y
and the equatorially bound thiolate donor
16]. This reaction product was easily synthesized from the reaction of 2 in CH CN with imidazole
1 : 2) by stirring the solution at RT for 30 min, and the volume was then reduced in vacuum to get a
[
(
3
solid. This was filtered and the dark compound was washed with methanol and air dried. The
elemental analysis of the isolated product was found to be consistent with the formulation of
[
Cu(pabt)(Imz)
3
](ClO ) (2A).
4
Unlike the reaction of 2 with imidazole that resulted in an abrupt change in the spectrum due to the
formation of a six-coordinate complex owing to addition of the incoming ligand, 6 shows only a
gradual change of its λmax at 546 nm which is blue shifted to 542 nm with enhanced intensity (Fig. 7
curve e of panel B) upon completion of the reaction within 20 min due to the slow substitution of the
−
Cl by the incoming imidazole ligand in the sixth coordination site, and this is supported by the
increase in conductivity of 6 in CH
mentioned resulting a 2 : 1 electrolyte. Isolation of this reaction product followed by purification was
found to be consistent with the formulation [Cu(pdta)(Imz)]Cl (6A). A plot of absorbance change at
46 nm with time for the spectral data presented in panel B of Fig. 7 clearly shows an exponential
rise to maximum (Fig. 7, panel C) and this was fitted using the single exponential equation (1)
1)
3
CN after addition of solid imidazole (1 : 1 mmol) already
2
5
(
−1
This fitting yielded the kinetic parameters: c = 0.938± 0.013, a = 0.526± 0.015 and k (min ) =
0.324± 0.024 for this ligand substitution reaction.
4.5. DNA binding studies
DNA binding studies were carried out for compounds 1 and 2 in aqueous medium by monitoring the
overall electronic spectral change (Fig. S5, Supplementary material) after addition of plasmid DNA
dissolved in Tris buffer, pH 7. The final spectra (Fig. 8) clearly suggests that both the compounds

26
strongly bind with the DNA [24, 29, 30]. Compound 1 exhibits hypochromism of ~43% of the π→π*
absorption band at 333 nm and compound 2 exhibits hypochromism of ~51% of the π→π*
absorption band at 332 nm.
−4
−1
Fig. 8. Panel A. Electronic spectra (a) compound 1 in water [1.51 × 10 mol L ], (b) compound 1 in
−4
−1
water [1.51 × 10 mol L ] + 10.8 µg/mL plasmid DNA, and (c) plasmid DNA in water [10.8
−4
−1
µg/mL]. Panel B. Electronic spectra (a) compound 2 in water [0.92 × 10 mol L ], (b) compound 2
−4
−1
in water [0.92 × 10 mol L ] + 10.8 µg/mL plasmid DNA, and (c) plasmid DNA in water [10.8
µg/mL].
In order to get the binding constant for the compounds 1-3, absorption titration experiments were
performed with incremental amounts of calf thymus DNA (ct-DNA) in 10 mM Tris–HCl buffer (pH
7.43) and by keeping the individual complex concentration constant. The binding of complexes to
the ct-DNA exhibits hypochromism (Fig. S5, Supplementary material) with a minor red shift of the
band at around 332 nm indicating intercalative binding, resulting in strong stacking between the
DNA base and aromatic chromophore [59].
b
The binding constant K has been calculated from the kinetic data obtained using the following
equation (2).

27
(2)
where, [DNA] is the concentration of ct-DNA used, and ε
extinction coefficients for the complex i.e., Abs/[complex] in the presence of DNA, in the
absence of DNA and to fully bound DNA, respectively. A plot of [DNA]/(ε − ε ) versus
DNA] yields a slope = 1/(ε − ε ), and the intercept = 1/K (ε − ε ), respectively. The binding
constant K is calculated from the ratio of the slope to the intercept.
a f b
, ε and ε correspond to apparent
a
f
[
b
f
b
b
f
b
5
−1
5
−1
5
The binding constant K
b
is found to be 1.47 × 10 M for 1, 2.03 × 10 M for 2, and 2.09 × 10
M for 3, respectively. The K values are comparable to that reported for a classical intercalator
such as ethidium bromide (K
The higher K values for 2 and 3 as compared to 1 suggest that 2 and 3 possessing the imidazole or
−1
b
5
−1
b
= 1.40 × 10 M ) in 25 mM Tris-HCl buffer (pH 7.33) [60].
b
N-methyl imidazole group interact with DNA more strongly than complex 1. This is consistent with
the observation of complete cleavage of the DNA by 2 and 3, but not 1 (vide infra). Similar
observations of enhanced cleavage activity of copper(II) complexes showing stronger DNA-binding
affinity resulting from a partially intercalating co-ligand dipyrido-[3,2-d:2′,3′-f]-quinoxaline (dpq)
are reported in literature [61-63].
4.6. Catechol oxidase activity
We have chosen 1 and 2 as representative compounds, both being 4-coordinate, to explore the
catechol oxidase activity, to see the influence of imidazole present in 2 as the fourth ligand in this
activity as compared to the water molecule present as the fourth ligand in 1. The catalytic activity of
−4
−1
compounds 1 and 2 were performed by treating 1 × 10 mol L complex solutions in methanol with
−2
−1
1
× 10 mol L of 3,5-di-tert-butyl catechol (3,5-DTBC) as model substrate in methanol at RT in
presence of air. Fig. 9 shows the spectral change for 1 and 2, respectively. The reaction was

28
monitored for ∼24 h after. Initially, in the absence of 3,5-DTBC, complexes 1 and 2 show very low
absorbance at ∼401 nm (bottom curve i). Upon addition of 3,5-DTBC the spectra recorded
immediately shows increment of the absorbance at 401 nm. Since it is well established [64] that 3,5-
di-tert-butyl benzoquinone (3,5-DTBQ) shows band maxima at 401 nm in pure methanol, the
experiment unequivocally proves oxidation of 3,5-DTBC to 3,5-DTBQ catalyzed by the Cu(II)
complexes. The chemical kinetics of 3,5-DTBC oxidation was determined by monitoring the
increase of the absorbance at 401 nm for the formation of the product 3,5-DTBQ, and to get an idea
about the number of intermediate species involved in this oxidation process, the plot of absorbance
change at 401 nm with time (figure 9, panel C) was fitted using the model (3)
(3)
It is also evident from this plot that compound 2 was found to be more efficient in the catalytic
oxidation of 3,5-DTBC than compound 1 under identical experimental conditions.
−4
−1
Fig. 9. The catalytic activity of compounds 1 and 2 were performed by treating 1 × 10 mol L
−2
−1
complex solutions with 1 × 10 mol L of 3,5-di-tert-butyl catechol (3,5-DTBC) as model substrate
in methanol at RT. Panel A and B show the electronic spectral change for the formation of 3,5-
DTBQ from the catalytic oxidation of 3,5-DTBC with compounds 1 and 2, respectively, in methanol
at RT. Panel C shows the plot of absorbance change at 401 nm with time showing the relative rate of
formation of 3,5-DTBQ. The solid line represents the fitting of the experimental points (circles)
using the model
.

29
3
Table 2. Kinetic parameters for the reaction of compounds 1 and 2 with 3,5-DTBC in CH OH at RT.
Data for the plot of absorbance change at λmax = 401 nm with time for the formation of 3,5-DTBQ in
both the cases were fitted using the equation (3).
Complex 1
Complex 2
Constant c
0.233±0.004
0.564±0.014
0.015±0.001
2.477±0.021
0.0012±0.00003
0.322±0.007
0.284±0.011
0.037±0.003
2.958±0.011
0.0022±0.00003
a
1
−
1
k
1
(min )
a
2
−
1
k
2
(min )
______________________________________________________________________________
It is to be noted here that there are recent reports of mononuclear Cu(II) [65] and Zn(II) [64]
complexes apart from dinuclear complexes [66-69] that show catechol oxidase activity. During the
catalytic oxidation of 3,5-DTBC by mononuclear Zn(II) complexes, Guha et al. [64] have detected
spectroscopically two catalyst-substrate adducts intermediates for only the mononuclear Zn(II)
catalysts but not for the dinuclear Zn(II) catalysts. Gajewska et al. [65] have reported the catechol
2 2 2
oxidation reaction of [Cu(bdtbpza) (tmeda)(H O) ] (where, bdtbpza = bis(3,5-di-t-butylpyrazol-1-
yl)acetate and tmeda = tetramethylethylenediamine) with 3,5-DTBC and have proposed a mechanism
which includes an equilibrium between two intermediates, one of them being a radical species as
suggested from the weak EPR signal at g = 1.9807 that was detected during the reaction of the said
2 2
complex with 3,5-DTBC in CH Cl . Thus the observation of two exponent fit for the kinetic data in
our cases (figure 9, panel C) of 1 and 2 with the reaction of 3,5-DTBC in methanol to form 3,5-
DTBQ is not surprising and proceeds through two intermediates, one major and another minor, to
form the final product 3,5-DTBQ. 2 is much efficient than 1 in the oxidation process as obvious from
the kinetic curves (Fig. 9, panel C) as well as from the kinetic parameters presented in Table 2.
We have also carried out the kinetics experiment to determine the catalytic activity for these two
compounds as described below.
The kinetics of the 3,5-DTBC oxidation catalyzed by the compounds 1 and 2 was studied by
monitoring the increase in the product 3,5-DTBQ which has a characteristic absorption band maxima