Synthesis, crystal structures, DNA binding and cleavage activity of water soluble mono and dinuclear copper(II) complexes with tridentate ligands

Article abstract of DOI:10.1016/j.ica.2014.01.010

Water soluble mono and dinuclear copper(II) complexes of novel tridentate ligands have been synthesized and characterized by spectroscopic methods and single crystal X-ray diffraction analysis. X-ray crystallographic studies reveal that the structure of m

Full text of DOI:10.1016/j.ica.2014.01.010

Inorganica Chimica Acta 413 (2014) 174–186
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Synthesis, crystal structures, DNA binding and cleavage activity of water
soluble mono and dinuclear copper(II) complexes with tridentate ligands
1
⇑
Madur Pragathi , Katreddi Hussain Reddy
Department of Chemistry, Sri Krishnadevaraya University, Anantapur 515003, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2013
Received in revised form 16 January 2014
Accepted 16 January 2014
Available online 24 January 2014
Water soluble mono and dinuclear copper(II) complexes of novel tridentate ligands have been synthe-
sized and characterized by spectroscopic methods and single crystal X-ray diffraction analysis. X-ray
crystallographic studies reveal that the structure of mononuclear copper(II) complex with 2-[1-(methyl-
amino-ethylimino)-ethyl]-phenol [Cu(HAPMEN)(H2O)2]NO3 has distorted square pyramidal structure
whereas the dinuclear complex with 2-[1-(proylamino-ethylimino)-methyl]-phenol [Cu(SAPEN)]2(ClO4)2
has square planar geometry. Electrochemical behavior of these complexes was investigated by cyclic vol-
tammetric studies. The complexes show quasi reversible cyclic voltammetric responses for the Cu(II)/
Cu(I) couple. The binding properties of these complexes with calf-thymus DNA have been investigated
by using absorption spectrophotometry. Mono nuclear complexes show unusually high binding affinity
possible due to presence the labile aqua ligands. Cleavage activities of these complexes have been inves-
tigated on double stranded pBR 322 plasmid DNA by gel electrophoresis experiments under different
conditions. Dinuclear complexes exhibit higher nuclease activity when compared with those of mononu-
clear complexes. The studies indicate that the cleavage of DNA takes place by oxidative mechanism in the
presence of oxidant (H2O2).
Keywords:
Water soluble mono and dinuclear
copper(II) complexes
Tridentate
Absorption spectrophotometry
Nuclease activity
Gel electrophoresis
Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction
have been shifted to non-platinum based agents in order to find
different metal complexes [10–17]. Among these copper(II) com-
DNA is a significant cellular receptor, many chemicals bring to
bear their antitumor effects by binding to DNA and by this means
changes the replication of DNA and inhibits the growth of the tu-
mor cells, which is the basis of designing new and more efficient
antitumor drugs. During the last decade several transition metal
complexes have been used as tools for understanding DNA struc-
ture, as agents for mediation of DNA cleavage or as chemothera-
peutic agents. Metal complexes offer an opportunity to explore
the effects of the effects of central metal atom, the ligands and
the coordination geometries on the binding event. Moreover, their
activity depends on the mode and affinity of their binding ability to
the DNA strands [1–3]. A number of metal chelates have been used
as agents for mediation of strand scission of duplex DNA, probes of
DNA structure in solution and chemotherapeutic agents [4–6].
Platinum-based complexes had been in primary focus of research
on chemotherapy agents [7–9]. The interests in this field recently
plexes appear to be very promising agents for anticancer therapy
having effective cytotoxic activities [18–21]. Copper is known
essential metal. It is widely distributed in living cells and body. It
is of interest to investigate DNA binding and cleavage activity of
metal complexes of essential metals and thus attracted much
attention.
Tridentate Schiff-bases are very efficient in coordinating with
metal ions which prefer square-planar or square-pyramidal geom-
etry. Such complexes are also important as models for copper pro-
teins containing active metal sites [22–26]. A large number of
mono and polynuclear complexes of imino-Schiff bases have been
reported [27–31], but there are a few reports available in the liter-
ature on DNA binding and cleavage activities of such complexes
[32].
In the light of the above and in continuation of our ongoing re-
search on DNA binding and cleavage activities of transition metal
complexes [32–37], herein we report the synthesis, crystal struc-
tures, DNA binding and cleavage activities of water soluble mono
and dinuclear copper(II) complexes derived from tridentate Schiff
base ligands.
⇑
Corresponding author. Tel.: +91 8554224347.
E-mail address: khussainreddy@yahoo.co.in (K. Hussain Reddy).
1
Address: Department of Chemistry, Government Degree College (Men), Anantapur
515001, Andhra Pradesh, India.
http://dx.doi.org/10.1016/j.ica.2014.01.010
0020-1693/Ó 2014 Elsevier B.V. All rights reserved.

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
175
30 min. A green coloured crystalline product was obtained which
was filtered and washed with a minimum volume of methanol.
Yield: 52% (0.881 g); Decomposition temperature: 274–276 °C.
Anal. Calc. for C10H17CuN3O6: C, 35.42; H, 5.06; N, 12.42. Found:
C, 35.45; H, 5.09; N, 12.48%.
2. Experimental
2.1. Materials and methods
The reagents used in preparation of ligands were of reagent
grade (Aldrich) and were used without further purification. Metal
salts used in the synthesis of complexes were of reagent grade
(Merck). Solvents used in the present study were distilled before
use. Calf thymus DNA and plasmid pBR 322 were purchased from
Genie Bio Labs, Bangalore, India. All other chemicals were of AR
grade and used without further purification.
Caution! Although no problems were encountered in this work,
perchlorate salts containing organic ligands are potentially explo-
sive. They should be prepared in small quantities and handled with
care.
2.1.3. Synthesis of mononuclear copper(II) complex (2) 2-[1-(proylamino-
ethylimino)-methyl]-phenol [Cu(SAPEN)(H2O)2]NO3
To a methanolic solution (50 mL) of 5 mmol (1.2 g) cop-
per(II)nitrate trihydrate,
a methanolic solution (10 mL) of
5 mmol/1.025 g SAPEN was added and stirred magnetically for
30 min. A green coloured product was obtained which was filtered
and washed with a minimum volume of methanol. Yield: 59%
(1.123 g); Decomposition temperature: 268–270 °C. Anal. Calc. for
C12H21CuN3O6: C, 39.29; H, 5.77; N, 11.45. Found: C, 39.22; H,
5.79; N, 11.43%.
2.1.1. Synthesis of ligands
5 mmol of 2-hydroxyacetophenone/salicylaldehyde (0.61/
0.52 mL) dissolved in 30 mL of methanol were added to a metha-
nolic solution (30 mL) of 5 mmol N-methylethylenediamine/N-
propylethylenediamine (0.44/0.62 mL). The contents were refluxed
over water bath for 1 h. The contents were then cooled and filtered.
On evaporation of the solvent, 2-[1-(methylamino-ethylimino)-
methyl]-phenol (SAMENH)/2-[1-(propylamino-ethylimino)-methyl]-
2.1.4. Synthesis of synthesis of mononuclear copper(II) complex (3)
2-[1-(methylamino-ethylimino)-ethyl]-phenol
[Cu(HAPMEN)(H2O)2]NO3
To a methanolic solution (50 mL) of 5 mmol copper(II) nitrate
trihydrate, a methanolic solution (10 mL) of 5 mmol/0.955 g HAP-
MEN was added and stirred magnetically for 30 min. A green col-
oured product was obtained which was filtered and washed with
a minimum volume of methanol. Dark green single crystals suit-
able for X-ray analysis were grown by dissolving the product in
methanol and slow dispersion of n-hexane. Yield: 49% (0.865 g);
phenol
SAPENH/2-[1-(Methylamino-ethylimino)-ethyl]-phenol
(HAPMENH)/2-[1-(propylamino-ethylimino)-ethyl]-phenol (HAP-
PENH) (Fig. 1) were yielded as reddish yellow coloured viscous
liquids. The FT-IR spectra of ligands show vibrations at 3501–
Decomposition temperature: 278–280 °C. Anal. Calc. for C11H19
CuN3O6: C, 37.45; H, 5.43; N, 11.91. Found: C, 37.49; H, 5.42; N,
11.96%.
-
3510 cmꢀ1 and 1615–1662 cmꢀ1 corresponding to
m(NH) of sec-
ondary amine and azomethine
m(C@N) groups, respectively. The
ligands were subsequently used in the preparation of metal com-
plexes. 1H NMR: SAPENH: d(8.0) (singlet 1H), d(7.45–6.76) (multi-
plet 4H), d(5.0) (singlet 1H), d(3.67) (triplet 2H), d(3.67–2.91)
(multiplet 4H), d(2.0) (multiplet 1H), d(2.55–2.15) (multiplet 4H)
and d(0.96) (triplet of triplet 3H) assigned to ACH@, aromatic ring
protons, –OH, –CH2–, –NH– and –NH–CH3, respectively. HAP-
MENH: d(7.15–6.78) (multiplet 4H), d(5.56) (singlet 1H), d(3.52–
3.01) (multiplet 4H), d(2.38) (multiplet 1H), d(1.52) (singlet 3H)
and d(1.2) (doublet 3H) assigned to aromatic ring protons, –OH,
–CH2–, –NH–, –CH3 and –NH–CH3 respectively. For each complex
the ligand is prepared separately and completely used for synthesis
of one complex.
2.1.5. Synthesis of mononuclear copper(II) complex (4) 2-[1-(propylamino-
ethylimino)-ethyl]-phenol [Cu(HAPPEN)(H2O)2]NO3
To a methanolic solution (50 mL) of 5 mmol (1.2 g) cop-
per(II)nitrate trihydrate,
a methanolic solution (10 mL) of
5 mmol/1.095 g HAPPEN was added and stirred magnetically for
30 min. A green coloured product was obtained which was filtered
and washed with a minimum volume of methanol. Yield: 52%;
decomposition temperature: 265–267 °C (D). Anal. Calc. for C13H23
-
CuN3O6: C, 40.99; H, 6.09; N, 11.03. Found: C, 40.92; H, 6.01; N,
11.07%.
2.1.6. Synthesis of dinuclear copper(II) complex (5) 2-[1-(methylamino-
ethylimino)-methyl]-phenol [Cu(SAMEN)]2(ClO4)2
2.1.2. Synthesis of mononuclear copper(II) complex (1) 2-[1-(methylamino-
ethylimino)-methyl]-phenol [Cu(SAMEN)(H2O)2]NO3
To a methanolic solution (50 mL) of 5 mmol (1.853 g) Cu(ClO4)2ꢁ
6H2O, a methanolic solution (10 mL) of 5 mmol/0.885 g SAMEN
was added and stirred magnetically for 30 min. Triethylamine
(5 mmol, 0.85 mL) was added drop wise to the contents with con-
stant stirring. A green coloured product was obtained which was
filtered and washed with a minimum volume of methanol. Yield:
59% (2.006 g); decomposition temperature: 255–257 °C. Anal. Calc.
for C20H26Cl2Cu2N4O10: C, 35.41; H, 3.94; N, 8.23. Found: C, 35.27;
H, 3.82; N, 7.97%.
To a methanolic solution (50 mL) of 5 mmol (1.2 g) cop-
per(II)nitrate trihydrate,
a methanolic solution (10 mL) of
5 mmol/0.885 g SAMEN was added and stirred magnetically for
R1
N
H
R2
N
2.1.7. Synthesis of dinuclear copper(II) complex (6) 2-[1-(proylamino-
ethylimino)-methyl]-phenol [Cu(SAPEN)]2(ClO4)2
OH
To a methanolic solution (50 mL) of 5 mmol Cu(ClO4)2ꢁ6H2O, a
methanolic solution (10 mL) of 5 mmol/1.025 g SAPEN was added
and stirred magnetically for 30 min. Triethylamine (5 mmol,
0.85 mL) was added drop wise to the contents with constant stir-
ring. A green coloured product was obtained which was filtered
and washed with a minimum volume of methanol. Dark green col-
oured hexagonal single crystals suitable for X-ray analysis were
grown by dissolving the product in acetonitrile solution and slow
dispersion of n-hexane. Yield: 54% (1.98 g); decomposition tem-
Ligand
R1
R2
SAMENH
SAPENH
HAPMENH
HAPPENH
H
H
CH3
CH3
CH3
C3H7
CH3
C3H7
Fig. 1. Structure of tridentate ligands.

176
M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
perature: 242–244 °C. Anal. Calc. for C24H34Cl2Cu2N4O10: C, 39.14;
H, 4.65; N, 7.61. Found: C, 39.26; H, 4.58; N, 7.66%.
mined using 25 reflections and then the intensity data of a given
set of reflections are collected automatically by the computer. An
IBM compatible PC/AT 486 is attached to micro VAX facilitating
the data transfer onto a DOS floppy of 5.2500 or 3.500. Maximum X-
ray power is 40 mA ꢂ 50 kV.
2.1.8. Synthesis of dinuclear copper(II) complex (7) 2-[1-(methylamino-
ethylimino)-ethyl]-phenol [Cu(HAPMEN)]2(ClO4)2
To a methanolic solution (50 mL) of 5 mmol (1.853 g) Cu(ClO4)2-
ꢁ6H2O, a methanolic solution (10 mL) of 5 mmol/0.955 g HAPMEN
was added and stirred magnetically for 30 min. Triethylamine
(5 mmol, 0.85 mL) was added drop wise to the contents with con-
stant stirring. A green coloured product was obtained which was
filtered and washed with a minimum volume of methanol. Yield:
49% (1.734 g); decomposition temperature: 270–272 °C. Anal. Calc.
for C22H30Cl2Cu2N4O10: C, 37.28; H, 4.27; N, 7.91. Found: C, 37.46;
H, 4.23; N, 7.75%.
The data collected was reduced using SAINT program [38]. The
trial structure was obtained by direct method [39] using
SHELXS-86, which revealed the position of all non-hydrogen atoms
and refined by full-matrix least squares on F2
(SHELXS-97) [40] and
graphic tool was DIAMOND for windows [41]. All non-hydrogen atoms
were refined anisotropically, while the hydrogen atoms were treated
with a mixture of independent and constrained refinements.
2.4. DNA binding experiments
2.1.9. Synthesis of dinuclear copper(II) complex (8) 2-[1-(propylamino-
ethylimino)-ethyl]-phenol [Cu(HAPPEN)]2(ClO4)2
The interaction of the complexes with DNA was carried out in
tris-buffer. Solution of calf thymus-DNA (CT-DNA) in (0.5 mM
NaCl/5 mM Tris–HCl; pH 7.0) buffer gave absorbance ratio at 260
and 280 nm of 1.89 indicating that the DNA was sufficiently free
of proteins. The DNA concentration per nucleotide was determined
by absorption coefficient (6600 dm3 molꢀ1 cmꢀ1) at 260 nm. Stock
solutions stored at 4 °C were used after no more than four days.
The electronic spectra of metal complexes in aqueous solutions
were monitored in the absence and presence of CT-DNA. Absorp-
tion titrations were performed by maintaining the metal complex
concentration 20 ꢂ 10ꢀ6 M and varying the nucleic acid concentra-
tion (0–26.4 ꢂ 10ꢀ6 M). The ratio of r = [complex]/[DNA] values
vary from 6.51 to 0.72. Absorption spectra were recorded after
each successive addition of DNA solution.
To a methanolic solution (50 mL) of 5 mmol (1.85 g) Cu(ClO4)2ꢁ
6H2O, a methanolic solution (10 mL) of 5 mmol/1.095 g HAPPEN
was added and stirred magnetically for 30 min. Triethylamine
(5 mmol, 0.85 mL) was added drop wise to the contents with con-
stant stirring. A green coloured product was obtained which was
filtered and washed with a minimum volume of methanol. Yield:
52% (1.986 g); decomposition temperature: 185–187 °C. Anal. Calc.
for C26H38Cl2Cu2N4O10: C, 40.83; H, 4.97; N, 7.34. Found: C, 40.89;
H, 4.87; N, 7.45%.
2.2. Physical measurements
Infrared spectra in KBr disc were recorded in the range
4000–400 cmꢀ1 with a Perkin-Elmer spectrum 100 spectrometer.
Electronic spectra were recorded in N,N-dimethylformamide with
a Perkin-Elmer UV Lamda-50 spectrophotometer. Elemental analy-
ses were carried out on a Heraeus Vario EL III Carlo Erba 1108
instrument. Magnetic measurements of all the complexes at
298 K in the present study are obtained on a Faraday’s magnetic
susceptibility balance (Sherwood Scientific, Cambridge, UK). High
purity copper sulfate pentahydrate was used as a standard. The
conductance measurements at 298 2 K in water were carried
out on CM model 162 Conductivity cell (ELICO). ESR spectra were
recorded in solid state and in solution state at 298 K and at liquid
nitrogen temperature (L.N.T) on Varian E-112 spectrometer with
100 kHz field modulation. Temperature dependent magnetic sus-
ceptibility measurements were carried out with a Lakeshore VSM
7410 magnetometer, at SAIF, IIT-M, Chennai, under an applied
magnetic field of 5000 Oe. Magnetic data were corrected for dia-
magnetic contributions estimated from Pascal tables and for the
sample holder contributions of a sample holder. Cyclic voltamme-
try was performed with a CH Instruments 660C Electrochemical
Analyzer and a conventional time electrode, Ag/AgCl reference
electrode, glassy carbon working electrode and platinum counter
electrode. Nitrogen was used as purge gas and all solutions were
prepared in water containing 0.1 M concentration in tetrabutylam-
monium hexafloro phosphate (TBAPF6).
2.5. DNA cleavage experiments
The extent of cleavage of DNA mono and dinuclear copper(II)
complexes was monitored using agarose gel electrophoresis with
pBR 322 DNA. After incubation for 30 min at 37 °C, the samples
were added to the loading buffer containing 0.25% bromophenol
blue + 0.25% xylene cyanol + 30% glycerol, and solutions were
loaded on 0.8% agarose gel containing 100 lg of ethidium bromide.
Electrophoresis was performed at 75 V in TBE buffer until the bro-
mophenol blue reached to 3/4 of the gel. Bands were visualized by
UV Transilluminator and photographed. The efficiency of DNA
cleavage was measured by determining the ability of the complex
to form open circular (OC) or nicked circular (NC) DNA from its
supercoiled (SC) form. The reactions were carried out under differ-
ent conditions.
3. Results and discussion
Reactions of tridentate ligands with Cu(NO3)ꢁ3H2O yielded in
the formation of mononuclear complexes (complexes 1–4)
whereas the same ligands in presence of triethylamine react with
Cu(ClO4)2ꢁ6H2O yielding in the formation of dinuclear copper(II)
complexes (complexes 5–8). All the complexes are dark green col-
oured and freely soluble in water and many organic solvents. Molar
2.3. X-ray crystallography
conductivity values (58–69
X
ꢀ1 cm2 molꢀ1 for complexes 1–4 and
124–178
X
ꢀ1 cm2 molꢀ1 for complexes 5–8) suggest that mononu-
Crystal data were collected by using the Enraf Nonius
CAD4-MV31 single crystal X-ray diffractometer, Indian Institute
of Technology-Madras, Chennai. Enraf Nonius CAD4-MV31 single
crystal X-ray diffractometer is a fully automated four circle instru-
ment controlled by a computer. It consists of an FR 590 generator, a
goniometer, CAD4F interface and a microVAX3100 equipped with a
printer and plotter. The detector is a scintillation counter. A single
crystal is mounted on a thin glass fiber fixed on the goniometer
head. The unit cell dimensions and orientation matrix are deter-
clear complexes are 1:1 electrolytic, whereas the dinuclear metal
complexes are 1:2 electrolytic [42]. Magnetic susceptibility mea-
surements reveal that complexes 1–4 (leff = 1.78–1.90 BM) are
monomeric and there are no metal–metal interactions [43]. The
magnetic moment values of complexes 5–8 are found to be in
the range of 1.14–1.26 BM which are sub-normal to spin only va-
lue. The data reveal that these complexes are dimeric and there
are strong metal–metal interactions between the two copper
centers.

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
177
Table 1
Spin Hamiltanian and orbital reduction parameters for the copper(II) complexes.
Complex
In solid state at 77 K
In solution state at 77 K
g
g\
gav
G
g
g\
gav
G
k
K
K\
A
k
(cmꢀ1
)
A\ (cmꢀ1
)
Aav (cmꢀ1
)
g /A
k
a2
k
k
k
k
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
Complex 6
Complex 7
Complex 8
2.046
2.153
2.207
2.213
2.066
2.079
2.070
2.080
2.011
2.040
2.021
2.036
2.049
2.056
2.057
2.054
2.023
2.077
2.040
2.070
2.055
2.064
2.062
2.061
4.752
4.007
4.117
4.011
1.344
1.428
1.247
1.506
2.144
2.180
2.197
2.216
2.204
2.127
2.198
2.126
2.030
2.043
2.050
2.055
2.098
2.059
2.082
2.054
2.068
2.088
2.099
2.109
2.133
2.081
2.120
2.078
5.046
4.373
4.098
4.005
2.107
2.199
2.496
2.392
283
365
408
441
541
331
494
319
1.020
1.001
0.991
0.988
0.871
0.874
0.901
0.890
0.908
0.959
0.980
0.989
1.429
1.179
1.147
1.151
0.0105
0.0103
0.0098
0.0096
0.0134
0.0072
0.0131
0.0065
0.0023
0.0023
0.0017
0.0014
0.0062
0.0051
0.0060
0.0031
0.0050
0.0049
0.0044
0.0041
0.0086
0.0058
0.0084
0.0043
204
211
224
230
164
295
167
327
0.097
0.052
0.018
0.010
0.523
0.391
0.635
0.366
Fig. 2. (A) X-band powder ESR spectra of [Cu(HAPMEN(H2O)2]NO3 at 300 K, (B) at LNT, (C) in DMF solution at 300 K and (D) at LNT in DMF solution.
+
H2O
O
OH2
-
Cu
NO3
C
N
HN
R2
R1
Where R1 = H/CH3 and R2 = CH3/C3H7
Fig. 3. Suggested structure for mononuclear copper(II) complexes 1–4.
3.1. Spectral characterization
The electronic spectral data of copper(II) complexes are re-
corded in water and in dimethylformamide (DMF). In aqueous
solutions electronic spectra of complexes 1–4 show medium
bands in the region 27244–27984 cmꢀ1 are due to metal to
ligand charge transfer transition (MLCT). Bands observed in the
region 12484–12285 cmꢀ1 followed by a band in the regions
16130–16548 cmꢀ1 are attributed due to d-d transitions suggest
square pyramidal geometry around Cu(II) atom [43–45]. In DMF
the absorption bands are shifted to higher wavelengths (batho-
Where R1 = H/CH3 and R2 = CH3/ C3H7
Fig. 4. Suggested structure for dinuclear copper(II) complexes 5–8.
complexes 5–8 show medium intensity bands in the region
27548–28818 cmꢀ1 is due to metal to ligand charge transfer
transition (MLCT). Whereas one broad band observed in the
region 16447–16722 cmꢀ1 (k = 598–608 nm) is assigned to d–d
transition. In DMF the absorption bands are shifted to higher
chromic shift,
Dk = 9–14 nm) suggesting the involvement of sol-
vent molecules in coordination. In aqueous solution the dinuclear

178
M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
Fig. 5. ORTEP view of complex 3.
Fig. 6. Close packing diagram of complex 3.
wavelengths suggesting the involvement of solvent molecules in
coordination. Bathochromic shift of absorption bands in d–d tran-
and 1584–1637 cmꢀ1 corresponding to
m(NH) and
m
(C@N), respec-
ꢀ
tively. When ClO4 ions are considered, m3 mode bands in the re-
gion 1077–1115 cmꢀ1 are slightly broadened, but the m4 bands in
the region 624–626 cmꢀ1 is the devoid of any splitting and is consis-
tent with the IR normal modes for a Td symmetry, suggesting that
these anions [ClO4ꢀ] are not coordinated to the metal atom [47].
sition region is
Dk = 6–13 nm. The electronic spectra of these com-
plexes display weak d–d bands in the low intensity 16233–
16393 cmꢀ1 region are assigned to 2Eg ? 2T2g electronic transition
in favor of octahedral structure in DMF medium. Six coordination
for copper(II) is facilitated by axial coordination of DMF solvent
molecules.
3.1.1. ESR spectral studies
This band is shifted to lower wave number in IR spectra of all
the complexes suggesting the participation of azomethine nitrogen
atom in coordination with metal atom.
The spin Hamiltonian and orbital reduction parameters of com-
plexes 1–8 are shown in Table 1. Typical X-band ESR spectra of
[Cu(HAPMEN)(H2O)2]NO3 (complex 3) are shown in Fig. 2. The g
k
The FT-IR spectra of ligands show vibrations at 3501–
and g\ values are computed from the spectrum using (TCNE) free
radical as ‘g’ marker. From the observed values of complexes 1–4
at 300 K and 77 K in solid state spectrum, it is clear that
3510 cmꢀ1 and 1615–1662 cmꢀ1 corresponding to
m(NH) of sec-
ondary amine and azomethine
m(C@N) groups respectively. The
FT-IR spectra of complexes 1–4 show vibrations in the regions
g > g\ > 2.00 which suggest the fact that the unpaired electron lies
k
3416–3454, 3100–3200 and 1610–1635 cmꢀ1 corresponding to
predominantly in the dx2
orbital [48] characteristic of square
ꢀy2
m
(NH) of secondary amine,
m
(OH) of coordinated water molecule
pyramidal geometry in copper(II) complexes [45]. For ESR spectra
at 77 and 300 K, the ‘G’ values are found to be more than 4, which
suggest that there are no interactions between copper(II) ions in
the solid state or in solution state. The solution EPR spectra show
four-hyperfine signals at 300 K, which were not observed in DMF
at 77 K. The spin–orbit coupling constant, k value of the complexes
and azomethine (C@N) groups respectively. The bands shifted to
m
lower wave number in IR spectra of all the complexes suggest
the participation of azomethine nitrogen atom in coordination
with metal atom. The sharp bands observed in the region
1360–1338 cmꢀ1 are due to the non-coordinated nitrate ions
present outside the coordination sphere [46]. FT-IR spectra of
complexes 5–8 display vibrations in the regions 3413–3436 cmꢀ1
calculated using the relations, gav = 1/3[g + 2 g\] and gav
=
k
2(1 ꢀ 2k/10Dq), is less than the free Cu(II) ion (ꢀ832 cmꢀ1) which

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
179
Table 2
Crystal data and structure refinement for [Cu(HAPMEN)(H2O)2]NO3 and [Cu(SAPEN)]2(ClO4)2.
[Cu(HAPMEN)(H2O)2]NO3
[Cu(SAPEN)]2(ClO4)2
Empirical formula
Formula weight
T (K)
C11H19CuN3O6
352.83
293(2)
C24H34Cl2Cu2N4O10
736.53
293(2)
Wavelength (Mo K
Crystal system
Space group
a) (Å)
0.71073
monoclinic
P21/n
0.71073
monoclinic
P21/c
Unit cell dimensions
a (Å)
b (Å)
c (Å)
7.874(5)
20.908(4)
9.119(2)
107.324(5)
1433.2(10)
4
1.635
1.555
11.9790(5)
14.7940(5)
17.0060(6)
102.0400(10)
2947.46(19)
4
1.660
1.685
1512
b (°)
V (Å3)
Z
Calculated density,
Absorption coefficient
F(000)
q
(Mg mꢀ3
(mmꢀ1
)
l
)
732
Crystal size (mm)
h Range for data collection (°)
Limiting indices
0.30 ꢂ 0.20 ꢂ 0.20
0.20 ꢂ 0.20 ꢂ 0.15
2.53–24.99
2.22–25.00
ꢀ9 6 h 6 9, ꢀ23 6 k 6 24, ꢀ10 6 l 6 10
13991
ꢀ14 6 h 6 14, ꢀ17 6 k 6 17, ꢀ20 6 l 6 20
26437
Reflections collected
Unique reflections (Rint
Completeness to h (%)
Absorption correction
Maximum and minimum transmission
Refinement method
)
2513 (0.0236)
24.99–99.8
semi-empirical from equivalents
0.7512 and 0.6462
full-matrix least-squares on F2
2513/8/212
5184 (0.0304)
25.00–100.0
semi-empirical from equivalents
0.7942 and 0.7190
full-matrix least-squares on F2
5184/70/426
Data/restraints/parameters
Goodness-of-fit (GOF) on F2
1.181
1.039
Final R indices [I > 2
R indices (all data)
r
(I)]
R1 = 0.0465a, wR2 = 0.1061b,c
R1 = 0.0572a, wR2 = 0.1362b,c
0.685 and ꢀ0.400
R1 = 0.0281, wR2 = 0.0694
R1 = 0.0372, wR2 = 0.0755
0.621 and ꢀ0.289
Largest difference in peak and hole (e Aꢀ3
)
P
P
a
b
c
R1
=
(|FO| ꢀ |FC|)/ |FO|.
P
P
wR2 = { [w(FO ꢀ FC2)2]/ [w(FO2)2]}1/2
.
2
w = 1/[
r
2(FO2)+(aP)2 + bP] with P = [Fo2 + 2Fc2]/3, a = 0.0612 and b = 0.24.
Table 3
Hydrogen bond geometry for [Cu(HAPMEN)(H2O)2]NO3 [Å and °].
D–Hꢁ ꢁ ꢁA
d(D–H)
d(Hꢁ ꢁ ꢁA)
d(Dꢁ ꢁ ꢁA)
\(DHA)
O(2)-H(2B)...O(3)#1
O(2)-H(2A)...O(4)#2
O(2)-H(2A)...O(4)#2
O(2)-H(2A)...O(6)#2
O(2)-H(2A)...N(3)#2
O(3)-H(3A)...O(4)
0.875(19)
0.875(19)
0.895(19)
0.895(19)
0.895(19)
0.880(19)
0.890(2)
2.12(4)
2.54(5)
1.92(3)
2.47(5)
2.54(3)
1.95(3)
1.98(3)
2.871(5)
3.169(5)
2.799(6)
3.162(8)
3.384(6)
2.812(6)
2.808(5)
144(6)
129(5)
165(5)
135(5)
157(6)
165(5)
154(6)
O(3)-H(3B)...O(1)#1
Symmetry transformations used to generate equivalent atoms: #1 ꢀx + 2, ꢀy,
ꢀz + 3; #2 x + 1, y, z.
supports the covalent nature of M–L bond in the complexes [49].
The greater value of g compared to g\ proposes a distorted square
k
pyramidal structure and rules out the possibility of a trigonal bipy-
ramidal structure which is expected to have g\ > g [50,51].
k
Increasing steric hindrance caused by bulky ligands results in the
lowest A and highest g values, which in turn reflects in square
k
k
pyramidal distortion of copper(II) geometries. As the bulkiness in-
creases, an increased distortion in square pyramidal structure is
observed which is reflected in the values of g /A [52]. Also, the ob-
k
k
served g values of less than 2.3 provide evidence for the covalent
k
character of bonding between Cu(II) ion and the ligand [53]. The
observed K > K\ relation for mononuclear copper(II) complexes
k
indicates the presence of out-of-plane
p-bonding [50]. The in-
plane bonding parameter a2 values are calculated using the rela-
Fig. 7. ORTEP view of the complex 6. Ellipsoids drawn at 50% probability level.
tion
a
2 = ꢀ(A /0.036) + (g ꢀ 2.0023) + 3/7 (g\ ꢀ 2.0023) + 0.04.
k
k
For ESR spectra of complexes 5–8 at 77 and 300 K, the ‘G’ values
are found to be less than 4 suggesting metal–metal interactions be-
tween copper(II) ions in the solid state which further supports the
for dinuclear copper(II) complexes indicates the presence of in-
plane
p-bonding [50]. Increasing steric hindrance caused by bulky
dinuclear nature of the complexes. The observed K < K\ relation
ligands results in the lowest A and highest g values.
k
k
k

180
M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
Table 4
view is shown in Fig. 5 together with the numbering scheme in the
metal coordination sphere. The molecule contains a five coordi-
nated Cu(II) atom, while the basal plane is occupied by three donor
atoms O(1), N(1), N(2) of the tridentate ligand together with two
oxygen atoms O(2) and O(3) of the coordinated water molecules.
The axial Cu(1)–O(3) bond distance of 2.417(4) Å is larger than
the basal Cu(1)–O(1) and Cu(1)–O(2) bond distances of 1.878(3)
and 2.006(3) Å, respectively. This indicates distorted square-pyra-
midal geometry of mononuclear complex [54,55]. Close packing
diagram of [Cu(HAPMEN)(H2O)2]NO3 is shown in Fig. 6. Geometric
details describing the intermolecular O(3)–H(3A)ꢁ ꢁ ꢁO(4) hydrogen
bond in the complex: H(3A)ꢁ ꢁ ꢁ1.95(3) Å, O(3)ꢁ ꢁ ꢁO(4) = 2.812(6) Å
with angle at H(2B) = 165(5). Intermolecular interactions operating
in the crystal structure of [Cu(HAPMEN)(H2O)2]NO3: O(2)–
H(2B)ꢁ ꢁ ꢁO(3)#1 = 2.14(4) Å; O(2)–O(3) = 2.871(5) Å with angle at
H(2B) = 144(6) for symmetry operation #1 ꢀx + 2, ꢀy, ꢀz + 3;
Selected bond lengths (Å) and bond angles (°) for [Cu(HAPMEN)(H2O)2]NO3.
Bond lengths (Å)
Bond angles (°)
N(1)–Cu(1)
N(1)–H(1)
N(2)–Cu(1)
N(3)–O(5)
N(3)–O(6)
N(3)–O(4)
O(1)–Cu(1)
O(2)–Cu(1)
O(2)–H(2B)
O(2)–H(2A)
O(3)–Cu(1)
O(3)–H(3A)
O(3)–H(3B)
1.999(4)
0.97(2)
C(11)–N(1)–Cu(1)
C(10)–N(1)–Cu(1)
C(7)–N(2)–Cu(1)
C(9)–N(2)–Cu(1)
C(1)–O(1)–Cu(1)
Cu(1)–O(2)–H(2B)
Cu(1)–O(2)–H(2A)
Cu(1)–O(3)–H(3A)
Cu(1)–O(3)–H(3B)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
O(1)–Cu(1)–O(2)
N(2)–Cu(1)–O(2)
N(1)–Cu(1)–O(2)
O(1)–Cu(1)–O(3)
N(2)–Cu(1)–O(3)
N(1)–Cu(1)–O(3)
O(2)–Cu(1)–O(3)
122.0(4)
103.3(3)
127.7(3)
111.0(3)
127.0(3)
118(4)
121(4)
118(4)
115(4)
93.25(13)
175.89(18)
84.74(15)
88.27(13)
169.88(15)
93.09(15)
88.71(13)
105.44(14)
95.27(18)
84.59(16)
1.955(3)
1.178(6)
1.183(6)
1.241(6)
1.878(3)
2.006(3)
0.875(19)
0.895(19)
2.417(4)
0.880(19)
0.89(2)
O(3)–H(3B)ꢁ ꢁ ꢁO(1)#1 = 1.98(4) Å;
O(3)–O(1) = 2.808(5) Å
with
angle at H(2B) = 154(6) for symmetry operation #1 ꢀx + 2, ꢀy,
ꢀz + 3
and
O(2)–H(2A)ꢁ ꢁ ꢁO(4)#2 = 2.54(5) Å;
O(2)ꢁ ꢁ ꢁO(4) =
3.168(5) Å with angle at H(2)A = 129(5)° for #2 x + 1, y, z. Hydrogen
bonding data are listed in Table 3. Selected bond lengths and bond
angles are summarized in Table 4.
Based on physico-chemical and spectral data, structures of
mono and dinuclear complexes are given in Figs. 3 and 4.
3.2. Description of crystal structures
3.2.2. Description of crystal structure of [Cu(SAPEN)]2(ClO4)2
(complex 6)
3.2.1. Description of crystal structure of [Cu(HAPMEN)(H2O)2]NO3
(complex 3)
The complex crystallizes in monoclinic, space group P21/n. Crys-
tal data and structure refinements are shown in Table 2. The ORTEP
The X-ray crystallographic analysis reveals that complex 6 is a
binuclear entity with the formula C24H34Cl2Cu2N4O10. The complex
crystallizes in monoclinic, space group P21/c. Crystal data and
structure refinements are shown in Table 2. The structure is shown
Fig. 8. Close packing diagram of complex 6.

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
181
Fig. 9. ZFC magnetization curve of complex 6.
Table 6
Table 5
Selected bond lengths (Å) and bond angles (°) for [Cu(SAPEN)]2 2ClO4.
Hydrogen bonds for [Cu(SAPEN)]2 2ClO4 (Å and °).
D–Hꢁ ꢁ ꢁA
d(D–H)
d(Hꢁ ꢁ ꢁA)
d(Dꢁ ꢁ ꢁA)
\(DHA)
Bond lengths (Å)
Bond angles (°)
N(1)–H(1A)ꢁ ꢁ ꢁO(60)#1
N(1)–H(1A)ꢁ ꢁ ꢁO(6)#
N(3)–H(3A)ꢁ ꢁ ꢁO(3)
N(3)–H(3A)ꢁ ꢁ ꢁO(40)
0.835(17)
0.835(17)
0.825(17)
0.825(17)
2.26(3)
2.28(2)
2.233(19)
2.34(3)
3.082(18)
3.084(6)
3.037(6)
3.03(2)
170(3)
162(2)
165(3)
141(3)
N(1)–Cu(1)
N(2)–Cu(1)
N(3)–Cu(2)
N(4)–Cu(2)
O(1)–Cu(1)
O(1)–Cu(2)
O(2)–Cu(2)
O(2)–Cu(1)
Cu(1)–Cu(2)
2.004(2)
1.931(2)
2.005(2)
1.923(2)
1.9299(17)
1.9420(16)
1.9435(17)
1.9655(16)
2.9278(4)
C(10)–N(1)–Cu(1)
Cu(1)–N(1)–H(1A)
C(7)–N(2)–Cu(1)
C(8)–N(2)–Cu(1)
C(21)–N(3)–Cu(2)
C(22)–N(3)–Cu(2)
Cu(2)–N(3)–H(3A)
C(19)–N(4)–Cu(2)
C(20)–N(4)–Cu(2)
C(1)–O(1)–Cu(1)
C(1)–O(1)–Cu(2)
Cu(1)–O(1)–Cu(2)
C(13)–O(2)–Cu(2)
C(13)–O(2)–Cu(1)
Cu(2)–O(2)–Cu(1)
O(1)–Cu(1)–N(2)
O(1)–Cu(1)–O(2)
N(2)–Cu(1)–O(2)
O(1)–Cu(1)–N(1)
N(2)–Cu(1)–N(1)
O(2)–Cu(1)–N(1)
O(1)–Cu(1)–Cu(2)
N(2)–Cu(1)–Cu(2)
O(2)–Cu(1)–Cu(2)
N(1)–Cu(1)–Cu(2)
N(4)–Cu(2)–O(1)
N(4)–Cu(2)–O(2)
O(1)–Cu(2)–O(2)
N(4)–Cu(2)–N(3)
O(1)–Cu(2)–N(3)
O(2)–Cu(2)–N(3)
N(4)–Cu(2)-Cu(1)
O(1)–Cu(2)–Cu(1)
O(2)–Cu(2)–Cu(1)
N(3)–Cu(2)–Cu(1)
117.52(16)
107.9(19)
126.6(2)
112.55(18)
104.63(16)
118.66(16)
106.6(19)
126.62(18)
111.97(16)
127.55(16)
134.09(16)
98.25(7)
128.10(15)
134.88(16)
97.00(7)
90.88(8)
78.04(7)
168.91(8)
163.58(9)
85.82(9)
104.87(8)
41.03(5)
128.42(7)
41.21(5)
133.79(7)
167.01(8)
92.33(8)
78.28(7)
86.31(9)
104.20(8)
172.15(8)
132.85(7)
40.72(5)
Symmetry transformations used to generate equivalent atoms: #1 x + 1, y, z.
Dinuclear cation of the complex comprises two [Cu(SAPEN)]
subunits, which are interconnected through two oxygen bridges
afforded by the oxygen atoms of the ligands. The copper center
has square-planar geometry with the coordination of two nitrogen
atoms and one oxygen atom from the tridentate ligand while the
oxygen atoms of the ligands forming bridges in between the two
Cu atoms. In the cation [(CuSAPEN)]22+, the average bond lengths
in the square planar coordination sphere are Cu–N(amino)
2.0045 Å, Cu–N(imino) 1.927 Å, Cu–O(ligand) 1.9367 Å, and Cu–
O(axial) 1.9537 Å, respectively. The axial Cu–O bonds are longer
than those of equatorial oxygen atoms of the ligands. The distance
between the two copper atoms is 2.9278 Å and the Cu–O–Cu bridg-
ing angles are 98.25(7)° and 97(7)°. The sums of the bond angles
around the bridging oxygen atoms are 359.89° and 359.98°, indi-
cating that these bonds are essentially planar. Selected bond
lengths and selected bond angles are presented in Table 5. Hydro-
gen bonding data are listed in Table 6.
There are some other reports in the literature [56,57], in which
similar type of crystal structures derived from same type of ligands
are found. In both the reports dinuclear copper(II) complexes were
found, but the difference is that the Cu(II) centres are five-coordi-
nated and have square-pyramidal geometry. Moreover in both the
reports the bridging oxygen atoms are afforded by H2O ligands. In
present study we report four coordinated complexes with square
planar geometry around the Cu(II) moieties and the bridging oxy-
gen atoms are from phenolic moieties.
41.78(5)
137.30(6)
in Fig. 7 together with the numbering scheme in the metal coordi-
nation sphere. Close packing diagram of the complex is shown in
Fig. 8.
The single crystal X-ray diffraction analysis confirms the struc-
tures suggested in Figs. 3 and 4 based on physico-chemical and
spectral analysis.

182
M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
Table 9
Electronic absorption data upon addition of CT-DNA to the complexes.
Complex
kmax (nm)
D
k
H (%)
Kb (Mꢀ1
)
Free
bound
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
Complex 6
Complex 7
Complex 8
366
332
348
341
368
352
346
341
351
330
344
342
361
349
344
342
15
02
04
01
07
03
02
01
ꢀ16.07
08.21
9.91 ꢂ 106
9.25 ꢂ 106
8.68 ꢂ 106
8.36 ꢂ 106
2.24 ꢂ 105
1.25 ꢂ 105
1.68 ꢂ 105
1.15 ꢂ 105
ꢀ09.64
06.23
11.23
18.21
ꢀ12.25
16.36
The structural data for compound 6 indicate that the bridging
oxygen atoms of phenolic moieties connected in an apical position
to both the copper ions. As a consequence, there is no overlap with
the magnetic orbitals of the metal centres and the exchange inter-
action through this bridging ligand is expected to be very weak.
Fig. 10. Cyclic voltammetric profiles of complex 4 at different scan rates 25, 50 and
75 mV sꢀ1
.
3.3. Magnetic properties
The temperature dependent molar magnetic susceptibilities, vM
for complexes 3 and 6 were measured in the temperature range
20–300 K. At 300 K the magnetic moment value for complex 3 is
in good agreement with the value expected. For complex 6 the va-
3.4. Electrochemical studies
Redox behavior of the copper(II) complexes has been investi-
gated by cyclic voltammetry using 0.1 M tetrabutylammonium-
hexaflourophosphate (TBAHEP) as supporting electrolyte. The
cyclic voltammetric profile of [Cu(HAPPEN)(H2O)2]NO3 is given in
Fig. 10. The electrochemical data of copper(II) complexes are pre-
sented in Table 7.
lue of vM at 300 K is 0.99 emu which is subnormal to spin only va-
lue. This value increases (vM ꢂ T value decreases) slightly as the
temperature is lowered, and below 100 K it increases very rapidly.
This is a characteristic magnetic behavior of an antiferromagneti-
cally coupled dimer. The ZFC magnetization curve of complex 6
as a function of temperature is shown in Fig. 9. To estimate the
magnitude of antiferromagnetic coupling the magnetic susceptibil-
Repeated scans at various scan rates suggest that the presence
of stable redox species in solution. E1/2 values of 1–4 complexes ob-
served at potential range of 0.122–0.397 V versus Ag/AgCl [58–60].
These values vary in the range of 0.322–0.397 V versus. Ag/AgCl for
dinuclear complexes. It may be inferred that all the Cu (II) com-
plexes undergo reduction to their respective Cu(I) complexes.
The non-equivalent current in cathodic and anodic peaks (ic/
ity data were fitted to the Bleaney Bowers equation
v
MT = 2Ng2b2/
k[3 + exp(ꢀJ/kT)] for two interacting copper(II) ions (S = ½) with
the Hamiltonian in the form H = ꢀJS1S2. The magnetic data fitting
with the above equation affords the parameters J = ꢀ35.05 cmꢀ1
with g = 2.10.
ia = 1.192–1.801 for 1–4 and 0.326–0.812 for 5–8 at 100 mV sꢀ1
)
Table 7
Cyclic voltammetric data of complexes.
Go
747
a
b
Complex
CV cathodic (Epc
)
CV anodic (Epa
)
D
Ep
E1/2
ꢀic/ia (mV)
Log Kc (V)
D
Complex 1
Complex 2
Complex 3
Complex 4
0.268
0.292
0.526
0.446
0.446
0.265
258
154
550
285
0.397
0.369
0.171
0.122
1.268
1.192
1.658
1.801
0.130
0.218
0.061
0.117
1251
350
ꢀ0.104
ꢀ0.020
672
Complex 5
Complex 6
Complex 7
Complex 8
0.168
0.146
0.218
0.107
0.626
0.582
0.546
0.537
458
436
328
285
0.397
0.364
0.382
0.322
0.326
0.452
0.658
0.812
0.073
0.077
0.102
0.117
420
442
588
676
a
LogKc = 0.434 ZF/RT
D
Ep.
b
D
G
o = ꢀ2.303 RT logKc.
Table 8
Details of electronic spectral titrations of DNA binding activity of complexes.
S. No.
Addition of DNA (
lL)
Total volume of DNA (
l
L)
Total volume of solution (
lL)
Conc. of complex (
l
M)
Conc. of DNA (
lM)
r = [complex]/[DNA]
1
2
3
4
5
6
7
8
9
10
0
0
2000
2010
2020
2030
2040
2050
2060
2070
2080
2090
20
19.9
0
10
10
10
10
10
10
10
10
10
10
20
30
40
50
60
70
80
90
3.0547
6.0792
9.0738
12.0392
14.9756
17.8835
20.7632
23.6153
26.4401
6.51
3.25
19.801
19.704
19.607
19.512
19.417
19.323
19.231
19.138
2.171
1.628
1.303
1.086
0.931
0.844
0.724

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
183
cept complexes 1, 3 and 7 showed an increase in absorbance
(Hyperchromism). The three complexes showed a decrease in
intensity exhibiting hypochromism. It is evident from the table,
that all the complexes bind with DNA with high affinities and,
the estimated binding constants are in the range 8–9 ꢂ 106 Mꢀ1,-
whereas the dinuclear complexes 5–8 show bathochromic shift
(kmax: 1–7 nm) and binding constants as in the range of 1.15–
2.24 ꢂ 105 Mꢀ1. The change in absorbance values with increasing
amounts of CT-DNA was used to evaluate the intrinsic binding con-
stant Kb, for the complexes. Typical absorption spectra of [Cu(HAP-
MEN)]2(ClO4)2 in presence and in absence of DNA are shown in
Fig. 11. The binding constants (Kb) for DNA interaction of the
complexes have been calculated by using the relation [DNA]/
(
ea
ꢀ
e
f) = [DNA]/(eb
ꢀ
e
f)+1/Kb(eb
ꢀ
ef) where [DNA] is the molar
concentration of DNA, ea
,
eb and ef are apparent extinction coeffi-
cient (Aabs/[M]), the extinction coefficient for the complex in fully
bound form and the extinction coefficient for free metal ion
respectively.
Binding affinity of metal complexes decreases with increasing
molecular weight; this may be due to the steric hindrance of bulky
moieties present in the organic ligands. Also the binding constants
of the dinuclear complexes are less than those of mononuclear
copper(II) complexes. This may be due to the bulky nature of dinu-
clear complexes. Also the presence of good leaving groups (H2O
molecules) in mononuclear complexes causes the higher bind-
ing affinity towards DNA. Since the binding constants are high
105–106 Mꢀ1), the complexes may be regarded as efficient
intercalaters of DNA.
Fig. 11. Absorption spectra of complex 7 in the absence and in the presence of
increasing concentration of CT-DNA; top most spectrum is recorded in the absence
of DNA and below spectra on addition of 10
f) vs. [DNA] is shown in the inset.
ll DNA each time; A plot of [DNA]/
(
ea ꢀ e
indicate quasi-reversible behavior [61]. The difference
DEp in all
the complexes exceeds the Nerstian requirement 59/n mV
(n = number of electrons involved in oxidation reduction) which
suggests quasi-reversible character associated with a considerable
reorganization of the coordination sphere during electron transfer
[62]. The complexes have large separation between anodic and
cathodic peaks indicating quasi-reversible character. The E1/2 val-
ues of copper complexes are inversely related to the size of the
complex.
3.6. DNA cleavage activities
Nuclease activity of mono and dinuclear complexes derived
from tridentate Schiff base ligands has been studied by agarose
gel electrophoresis using pBR 322 plasmid DNA in Tris–HCl/NaCl
(50 mM/5 mM) buffer (pH-7) in the presence and absence of
H2O2 after 30 min incubation period at 37 °C. Concentration effect
has been studied with the complex [Cu(SAPEN)]2(ClO4)2 (complex
3.5. DNA binding studies
The interaction of metal complexes with calf-thymus DNA was
monitored by UV–visible spectroscopy. The absorption spectra of
complexes in aqueous solutions were compared in the absence
and in the presence of CT-DNA. Absorption spectra were recorded
in the range of 250–500 nm. The details of electronic spectral titra-
tions of DNA binding activity of complexes are given in Table 8.
Electronic absorption spectral data upon addition of CT-DNA and
binding constants of these complexes are given in the Table 9. In
the presence of increasing amounts of CT-DNA, the UV–Visible
absorption spectra of mononuclear complexes 1–4 show batho-
chromic shift (kmax: 1–15 nm). The spectra of all the complexes ex-
6) from 100 to 250 lM. In presence of H2O2 all the super coiled
DNA (form 1) is changed into nicked form (form II), which was fur-
ther cleaved into linear form i.e., form III. From the experiments it
is known that at higher concentrations, the complex exhibited sig-
nificant DNA cleavage activity even in the absence of an oxidant,
which may be due to catalytic hydrolysis [36]. Fig. 12 (a) and (b)
show the cleavage activity of mononuclear copper(II) complexes.
In presence of H2O2 the complexes cleave DNA more effectively
(even lanes except lane 2), which may be due to the reaction of hy-
Fig. 12. Agarose gel (0.8%) showing results of electrophoresis of 1
l
l of pBR 322 Plasmid DNA; 4
ll of Tris–HCl/NaCl (50 mM/5 mM) buffer (pH-7); 2 ll of complex in
DMF(1 ꢂ 10ꢀ3 M); 11
ll of sterilized water; 2
ll of H2O2 (total volume 20 l) were added, respectively, incubated at 37 °C (30 min); (a) Mononuclear complexes: Lane 1: DNA
l
control; Lane 2: DNA control + H2O2; Lane 3: complex 1 + DNA; Lane 4: complex 1 + DNA + H2O2; Lane 5: complex 1 + DNA + EDTA; Lane 6: complex 1 + DNA + DTT; Lane 7:
complex 3 + DNA; Lane 8: complex 3 + DNA + H2O2; Lane 9: complex 3 + DNA + EDTA; Lane 10: complex 3 + DNA + DTT; (b) Lane 1: DNA control; Lane 2: DNA control + H2O2;
Lane 3: complex 2 + DNA; Lane 4: complex 2 + DNA + H2O2; Lane 5: complex 2 + DNA + EDTA; Lane 6: complex 2 + DNA + DTT; Lane 7: complex 4 + DNA; Lane 8: complex
4 + DNA + H2O2; Lane 9: complex 4 + DNA + EDTA; Lane 10: complex 4 + DNA + DTT;

184
M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
droxyl radical with DNA. The percentage of the three forms of DNA
is presented in Tables 10 and 11. From the data it is clear that
percentage of cleavage activity of mononuclear copper complexes
follows the order [Cu(HAPMEN)(H2O)2]NO3 > [Cu(HAPPEN)(H2O)2]
NO3 > [Cu(SAMEN)(H2O)2]NO3 > [Cu(SAPEN)(H2O)2]NO3. Fig. 13(a)
and (b) show the cleavage activity of dinuclear copper(II) com-
plexes. These complexes also cleave DNA more effectively in pres-
ence of H2O2 the complexes cleave DNA more effectively.
Quantification of the gel afforded data of three forms is presented
in Tables 12 and 13. From the data it is clear that percentage of
cleavage activity of dinuclear copper complexes follows the order
[Cu(HAPPEN)]2(ClO4)2 > [Cu(HAPMEN)]2(ClO4)2 > [Cu(SAPEN)]2
(ClO4)2 > [Cu(SAMEN)]2(ClO4)2. Further more it is clear that dinu-
clear complexes exhibit higher nuclease activity when compared
with those of mononuclear complexes. Figs. 12 and 13 and Tables
10, 11 and 13 reveal that dinuclear complexes cleave DNA more
effectively as the nicked form (form II) was further cleaved into
linear form (form III).
Nuclease activity of complexes was also investigated in pres-
ence of chelating agent EDTA (1 ꢂ 10ꢀ6 M) and reducing agent
DTT (1 ꢂ 10ꢀ6 M). In the absence of H2O2 the complexes cleaved
supercoiled DNA (Form 1) into nicked DNA (Form II). From Figs. 12
and 13 it is evident that copper complexes cleave DNA more effec-
tively in the presence of oxidant which may be due to hydroxyl rad-
ical (OH) reaction with DNA [63]. Chelating agent EDTA diminishes
the nuclease activity of copper complexes, whereas in presence of
reducing agent (DTT) the cleavage activity of copper complexes is
enhanced. This may be due to formation of copper(I) complex by cat-
alytic reduction. In presence of chelating agent EDTA the cleavage
activity of the complexes was considerably reduced.
Table 10
Selected SC pBR322 DNA cleavage data of copper complexes in Fig. 11(a).
Lane No
Reaction condition
Percentage of
Form I
Form II
Form III
1
2
3
4
5
6
7
8
9
10
DNA
DNA + H2O2 (10
94.12
91.33
76.25
22.92
58.32
51.44
52.13
21.79
28.30
37.26
5.88
8.67
ND
ND
ND
1.43
ND
ND
ND
1.99
ND
ND
l
M)
DNA + complex 1 (62.5
DNA + complex 1 (62.5
DNA + complex 1 (62.5
DNA + complex 1 (62.5
DNA + complex 3 (62.5
DNA + complex 3 (62.5
DNA + complex 3 (62.5
DNA + complex 3 (62.5
l
l
l
l
l
l
l
l
M)
23.75
75.65
41.68
49.56
47.87
76.22
74.70
62.74
M) + DNA + H2O2 (10 lM)
M) + EDTA (10
M) + DTT (10
M)
M) + H2O2 (10
M) + EDTA(10
l
M)
lM)
lM)
lM)
M) + DTT(10 lM)
Table 11
Selected SC pBR322 DNA cleavage data of copper complexes in Fig. 11(b).
Lane No
Reaction condition
Percentage of
Form I
Form II
Form III
1
2
3
4
5
6
7
8
9
10
DNA
DNA + H2O2 (10
96.15
91.64
74.29
48.14
52.63
44.46
50.92
22.36
50.46
29.43
3.85
8.36
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
l
M)
DNA + complex 2 (62.5
DNA + complex 2 (62.5
DNA + complex 2 (62.5
DNA + complex 2 (62.5
DNA + complex 4 (62.5
DNA + complex 4 (62.5
DNA + complex 4 (62.5
DNA + complex 4 (62.5
l
l
l
l
l
l
l
l
M)
25.71
51.90
47.36
55.54
49.07
78.63
49.54
70.56
M) + DNA + H2O2 (10 lM)
M) + EDTA (10
M) + DTT (10
M)
M) + H2O2 (10
M) + EDTA (10
l
M)
lM)
l
M)
M)
M)
l
l
M) + DTT (10
Fig. 13. Agarose gel (0.8%) showing results of electrophoresis of 1
l
l of pBR 322 Plasmid DNA; 4
l
l of Tris–HCl/NaCl (50 mM/5 mM) buffer (pH-7); 2
l
l of complex in
DMF(1 ꢂ 10ꢀ3 M); 11
ll of sterilized water; 2
ll of H2O2 (total volume 20 ll) were added, respectively, incubated at 37 °C (30 min); (a) Dinuclear complexes: Lane 1: DNA
control; Lane 2: DNA control + H2O2; Lane 3: complex 5 + DNA; Lane 4: complex 5 + DNA + H2O2; Lane 5: complex 5 + DNA + EDTA; Lane 6: complex 5 + DNA + DTT; Lane 7:
complex 7 + DNA; Lane 8: complex 7 + DNA + H2O2; Lane 9: complex 7 + DNA + EDTA; Lane 10: complex 7 + DNA + DTT; (b) Lane 1: DNA control; Lane 2: DNA control + H2O2;
Lane 3: complex 6 + DNA; Lane 4: complex 6 + DNA + H2O2; Lane 5: complex 6 + DNA + EDTA; Lane 6: complex 6 + DNA + DTT; Lane 7: complex 7 + DNA; Lane 8: complex
7 + DNA + H2O2; Lane 9: complex 7 + DNA + EDTA; Lane 10: complex 7 + DNA + DTT;

M. Pragathi, K. Hussain Reddy / Inorganica Chimica Acta 413 (2014) 174–186
185
Table 12
Selected SC pBR322 DNA cleavage data of copper complexes in Fig. 12(a).
Lane No
Reaction condition
Percentage of
Form I
Form II
Form III
1
2
3
4
5
6
7
8
9
10
DNA
DNA + H2O2 (10
96.32
92.66
53.82
20.96
63.33
55.20
50.48
24.04
80.77
44.23
3.68
7.34
ND
ND
ND
35.21
ND
ND
ND
45.19
ND
16.44
l
M)
DNA + complex 5 (62.5
DNA + complex 5 (62.5
DNA + complex 5 (62.5
DNA + complex 5 (62.5
DNA + complex 7 (62.5
DNA + complex 7 (62.5
DNA + complex 7 (62.5
DNA + complex 7 (62.5
l
l
l
l
l
l
l
l
M)
46.18
43.83
36.68
44.80
49.52
30.76
19.23
39.33
M) + DNA + H2O2 (10
M) + EDTA (10
M) + DTT (10
M)
M) + H2O2 (10
M) + EDTA (10
lM)
l
M)
lM)
l
M)
M)
M)
l
l
M) + DTT (10
Table 13
Selected SC pBR322 DNA cleavage data of copper complexes in Fig. 12 (b).
Lane No
Reaction condition
Percentage of
Form I
Form II
Form III
2
3
4
5
6
7
8
9
DNA
DNA + H2O2 (10
97.33
94.35
64.92
21.66
49.47
28.11
51.04
23.70
49.98
25.25
2.67
5.65
ND
ND
ND
42.42
ND
47.91
ND
56.30
2.11
48.42
l
M)
DNA + complex 6 (62.5
DNA + complex 6 (62.5
DNA + complex 6 (62.5
DNA + complex 6 (62.5
DNA + complex 8 (62.5
DNA + complex 8 (62.5
DNA + complex 8 (62.5
DNA + complex 8 (62.5
l
l
l
l
l
l
l
l
M)
35.08
35.92
50.53
21.06
48.96
20.00
47.91
26.32
M) + DNA + H2O2 (10
M) + EDTA (10
M) + DTT (10
M)
M) + H2O2 (10
M) + EDTA (10
lM)
l
M)
lM)
l
M)
M)
M)
10
11
l
l
M) + DTT (10
(Sanction No. SR/S1/IC-37/2007) for financial support and to SAIF,
IIT-Bombay for providing ESR spectral data. We are also thankful
to SAIF, IIT-Madras for providing X-ray crystallographic data and
VSM measurements. The authors also thank UGC and DST for pro-
viding equipment facility under SAP and FIST programs
respectively.
4. Conclusions
Water soluble mono and dinuclear copper(II) complexes of no-
vel tridentate ligands have been synthesized and characterized.
Physico-chemical and spectral studies reveal that mono nuclear
complexes have square pyramidal structure, whereas the dinuclear
complexes have square planar geometry. X-ray diffraction studies
confirm the suggested structures. Mononuclear complexes show
higher binding affinity towards CT-DNA when compared with
dinuclear complexes possibly due to the presence of labile aqua
ligands. In presence of H2O2 the complexes cleave DNA more
effectively which may be due to the reaction of hydroxyl radical
with DNA. All the complexes cleave DNA via oxidative path. Dinu-
clear complexes exhibit higher nuclease activity when compared
with those of mononuclear complexes Nuclease activity is
enhanced with higher nuclearity of the copper complexes. The
highlights of the present investigations are: (i) The structures of
the complexes are determined by single crystal X-ray diffraction
studies, (ii) Both mono and dinuclear complexes are synthesized
with given tridentate ligand. (iii) Solubility of present complexes
is an attractive feature to develop chemotherapeutic drug. (iv)
DNA binding and cleavage activities of the complexes are investi-
gated. Mononuclear complexes bind DNA more strongly than cor-
responding dinuclear complexes possibly due to the labile aqua
ligands indicating that the former complexes may bind DNA bases
via substitution. Dinuclear complexes exhibit higher nuclease
activity when compared with those of mononuclear complexes.
The present observation is consistent with the idea that as nuclear-
ity complex increases, DNA cleavage activity increases.
Appendix A. Supplementary material
CCDC 892541 and 892618 contains the supplementary crystal-
lographic data for 3 and 6. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2014.01.010.
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