Synthesis and crystal structure of a series of pyrazolone based Schiff base ligands and DNA binding studies of their copper complexes

Article abstract of DOI:10.1016/j.molstruc.2012.01.006

PMP (5-methyl-4-(4-methyl-benzoyl)-2-phenyl-2,4-dihydro-pyrazol-3-one), PTPMP (5-methyl-4-(4-methyl-benzoyl)-2-p-tolyl-2,4-dihydro-pyrazol-3-one) and MCPMP (2-(3-Chloro-phenyl)-5-methyl-4-(4-methyl-benzoyl))-2,4-dihydro-pyrazol-3- one) were synthesized and used for the synthesis of Schiff base ligands. Schiff base ligands were characterized by FT-IR, ¹H NMR, Mass and single crystal X-ray analysis. Cu(II) complexes of synthesized ligands were prepared and characterized by elemental analysis, FT-IR, TGA-DTA, UV-Visible, ESI mass and ESR spectroscopy. On the basis of analytical and spectroscopic techniques, distorted octahedral geometry of the complexes was proposed. The interaction of Cu(II) complexes with CT-DNA was investigated by Absorption titration, Viscosity and fluorescence spectroscopy. Results suggest that the synthesized complexes bind to DNA via an intercalative mode and can quench the fluorescence intensity of EB bound to DNA.

Full text of DOI:10.1016/j.molstruc.2012.01.006

Journal of Molecular Structure 1013 (2012) 86–94
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis and crystal structure of a series of pyrazolone based Schiff base
ligands and DNA binding studies of their copper complexes
a
a
b
R.N. Jadeja a, , Sanjay Parihar , Komal Vyas , Vivek K. Gupta
⇑
a Department of Chemistry, Faculty of Science, The M.S. University of Baroda, Vadodara 390 002, India
b Department of Physics, University of Jammu, Jammu Tawi 180 006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
PMP (5-methyl-4-(4-methyl-benzoyl)-2-phenyl-2,4-dihydro-pyrazol-3-one), PTPMP (5-methyl-4-(4-
methyl-benzoyl)-2-p-tolyl-2,4-dihydro-pyrazol-3-one) and MCPMP (2-(3-Chloro-phenyl)-5-methyl-4-
(4-methyl-benzoyl))-2,4-dihydro-pyrazol-3-one) were synthesized and used for the synthesis of Schiff
base ligands. Schiff base ligands were characterized by FT-IR, 1H NMR, Mass and single crystal X-ray anal-
ysis. Cu(II) complexes of synthesized ligands were prepared and characterized by elemental analysis, FT-
IR, TGA–DTA, UV–Visible, ESI mass and ESR spectroscopy. On the basis of analytical and spectroscopic
techniques, distorted octahedral geometry of the complexes was proposed. The interaction of Cu(II) com-
plexes with CT-DNA was investigated by Absorption titration, Viscosity and fluorescence spectroscopy.
Results suggest that the synthesized complexes bind to DNA via an intercalative mode and can quench
the fluorescence intensity of EB bound to DNA.
Received 22 November 2011
Accepted 6 January 2012
Available online 28 January 2012
Keywords:
Schiff base
Pyrazolone
Crystal structure
DNA binding
Cu(II) complex
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
and analytical applications [11,12]. Investigations on copper com-
plexes to probe nucleic acids are becoming more prominent in the
Pyrazolone and its derivatives form an important class of com-
pounds and have attracted considerable scientific and applied
interest. Pyrazolones especially, Acylpyrazolones are an interesting
class of b-diketone compounds which are widely used as solvent
extractions of metal ions, laser working materials and NMR shift-
reagents [1–3]. The 4-acyl-pyrazolone derivatives are broadly used
in many fields, especially in biological, clinical and analytical appli-
cations [4–7].
The interest in the coordination chemistry of pyrazolones has
increased greatly in the last decade [8]. Due to the presence of
two oxygen donor atoms and facile keto–enol tautomerism, they
easily coordinate with metal ions after deprotonation of the enolic
hydrogen and provide stable metal complexes with six-membered
chelate rings. Pyrazolones are used in analytical chemistry for the
determination and isolation of almost all metal ions due to their
high extracting ability, intense color of the complex extracts and
low solubility of the complex in some solvents [9].
research area of bioinorganic chemistry [13–15]. Studies pertain-
ing to DNA cleavage by synthetic reagents are of considerable
interest because of their utility tools in molecular biology. This
has resulted in the development of both sequence specific DNA
cleavers [16] and DNA foot printing agents [17].
In our previous work, a series of Schiff base of 4-acylpyrazolone
derivatives and their transition metal complexes have been re-
ported [18,19]. In this paper, our group has focused much on syn-
thesizing new Schiff bases of 4-acyl pyrazolone derivatives (see
Scheme 1), studying on their crystal structures, synthesis of their
transition metal complexes and also studying their DNA binding
activities. In this present paper we are reporting the binding stud-
ies of Cu(II) complexes with CT-DNA by UV–Visible, fluorescence
spectroscopy and viscosity measurements.
2. Experimental
The well-known Cu(II) ion forms a series of coordination com-
pounds with well defined structures. It plays an important role in
the numerous biological processes that involve electron transfer
reactions or the activation of some anti-tumor substances [10]. In
addition, 4-acyl pyrazolones can form a variety of Schiff bases
and are reported to be superior reagents in biological, clinical
2.1. Materials and physical measurements
All reagents and solvents were purchased from commercial
sources and were further purified by the standard methods, if nec-
essary [20]. Pyrazolones were obtained from Nutan Dye Chem. Sa-
chin, Surat. Copper acetate and naphthylamine were purchased
from Loba Chem., Mumbai. Disodium salt of calf thymus DNA
(highly polymerized), purchased from Sigma, was stored at 4 °C
and used as received. The stock solution of DNA was prepared by
⇑
Corresponding author. Tel.: +91 265 2795552.
E-mail address: rajendra_jadeja@yahoo.com (R.N. Jadeja).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2012.01.006

R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
87
H3C
H3C
Cl
O
H3 C
O
N
O
N
Ca(OH)2/ Dioxane
reflux
2M HCl
Ca-complex
N
O
N
CH3
Cl /H
H/ CH 3
Cl /H
H/ CH3
1-Aminonaphtalene
Ethanol/ reflux
H3C
H3C
N
N
O
N
H /Cl
H/ CH3
Scheme 1. Synthetic route for the synthesis of Schiff base ligands.
dissolving appropriate amount of DNA in H2O and stored at 4 °C.
The stock solution was used after no more than 2 days. The ratio
of the absorbance at 260 and 280 nm (A260/A280) was checked to
be ꢀ1.86, indicating that the DNA is sufficiently free from protein.
The concentration of DNA was determined spectrophotometrically
at 260 nm after 1:100 dilutions using the known molar extinction
coefficient value of 6700 Mꢁ1 cmꢁ1. Ethidium bromide (EB) was
obtained from Hi-media laboratories Pvt. Ltd., Mumbai. Double
distilled water was used for preparing all the solutions.
SII EXSTAR6000 TG/DTA 6300. The experiments were performed
in N2 at a heating rate of 10 °C minꢁ1 in the temperature range
25–750 °C using a platinum pan. Purity of the ligands and their
complexes was evaluated by thin layer chromatography. Gravi-
metric and volumetric analysis were performed for the determina-
tion of copper after decomposition of complexes in nitric acid.
2.2. General
Infrared (IR) spectra were recorded on a Perkin Elmer Fourier
transform IR (FT-IR) spectrum RX 1 spectrometer as KBr pellets.
1H NMR spectra of ligands were recorded with AV 400 MHz Bruker
FT-NMR instruments. Mass spectra of the ligands were recorded on
Trace GC ultra DSQ II. Elemental analysis of C, H and N were deter-
mined using a Perkin Elmer series-II 2400 elemental analyzer.
X-ray intensity data were collected on Bruker CCD area-detector
2.2.1. Synthesis of PMP, PTPMP and MCPMP
PMP, PTPMP and MCPMP were synthesized according to meth-
od reported previously [21].
2.2.2. Synthesis of Schiff base ligands
PMP (10 mmol) was dissolved in minimum amount of absolute
ethanol. To this solution,
a solution of 1-aminonaphthalene
diffractometer equipped with graphite monochromated Mo K
a
(10 mmol) in 20 ml was added drop wise. The reaction mixture
was refluxed for 12 h. After cooling a microcrystalline yellow com-
pound was separated. The compound was filtered and washed
with water and then with ethanol and dried in vacuum. The suit-
able crystals for single crystal X-ray study were grown in
acetonitrile.
radiation (k = 0.71073 Å). Electronic spectra were recorded on a
Perkin Elmer Lambda 35 UV–Vis spectrometer. Fluorescence spec-
tra were recorded on JASCO FP-6300 spectrofluorometer. ESR spec-
tra of complexes were recorded on X-band instrument at ESR
laboratory, SAIF, IIT, Bombay, at room temperature and liquid
nitrogen temperature. ESI-mass spectra were recorded on Waters
Q-ToF micromass. A simultaneous TG/DTA was carried out on a
Ligand L1: Yield: 3.5 g, 84%, m.p: 184 °C, 1H NMR (CDCl3,
400 MHz, TMS): d 1.67(s, 3H), 2.33(s, 3H), 6.85(d, 1H), 7.12(d,

88
R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
Table 1
Analytical data of Cu(II) complexes.
Compound
Molecular formula
Color
Yield (%)
Elemental analysis
%C
%H
%N
% Cu
[Cu(L1)2(H2O)2]
[Cu(L2)2(H2O)2]
[Cu(L3)2(H2O)2]
C60H56CuN6O4
C58H52CuN6O4
C56H46Cl2CuN6O4
Brown
Brown
Dark brown
67
60
64
71.97 (72.12)
71.82 (72.52)
66.23 (67.16)
5.15 (5.19)
5.38 (5.46)
4.16 (4.63)
9.12 (9.01)
8.78 (8.75)
8.39 (8.39)
6.8 (6.81)
6.58 (6.62)
6.33 (6.35)
Table 2
Summary of crystallographic data for the ligand L1, L2 and L3.
The UV–Visible experiments were conducted by adding solution
of DNA at different concentrations (0–350 M) to the samples con-
taining 3 mM copper complexes. Absorption spectra were recorded
l
Compound
L1
L2
L3
in visible region within the range 570–800 nm.
Crystal color
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
Yellow
Yellow
C29H25N3O
431.52
Triclinic
P-1
7.0212(5)
11.2828(6)
15.8177(10)
71.306(5)
80.636(5)
84.692(5)
1170.04(13)
2
1.225
0.075
456
399
0.0601
0.1256
0.956
Yellow
From the absorption data, the intrinsic binding constant Kb was
C
28H23N3O
C28H22N3OCl
451.94
Triclinic
P-1
7.0658(12)
10.8912(18)
15.837(3)
70.928(3)
81.102(3)
86.555(3)
1137.9(3)
2
1.319
0.194
472
386
0.0521
0.1369
1.001
417.49
Orthorhombic
Pbca
15.518(3)
15.158(3)
18.110(4)
90
determined from a plot of [DNA]/(ea
equation:
ꢁ
ef) versus [DNA] using the
½DNAꢂ=ðea
ꢁ
ef Þ ¼ ½DNAꢂ=ðeb
ꢁ
ef Þ þ ½Kbðeb
ꢁ
ef Þꢂꢁ1
b (Å)
c (Å)
where [DNA] is the concentration of DNA in base pairs. The appar-
a
(°)
ent absorption coefficients ea, ef, and eb correspond to Aobsd/[M]
b (°)
90
90
(where M = metal = Cu+2), to the extinction coefficient for the free
Cu(II) complex and to the extinction coefficient for the Cu(II) com-
plex in the fully bound form, respectively.
C
(°)
V (Å3)
4260.0(15)
8
1.302
0.080
1760
Z
qcalcd. (g cmꢁ3
)
Viscosity experiments were carried out by using an Ostwald’s
capillary viscometer, immersed in a thermostated water bath with
the temperature setting at 30 0.1 °C for 15 min. DNA samples
with an approximate average length of 200 base pairs were pre-
pared by sonication in order to minimize complexities arising from
DNA flexibility [22]. The concentration changes of the Cu(II) com-
plexes was realized by adding different volume of Cu(II) complex
stock solution. Flow time was measured with a digital stopwatch,
and each sample was measured triply, and an average flow time
Abs. coeff.,
l
(cmꢁ1
)
F(000)
No. of parameters refined
Final R
wR(F2)
Goodness-of-fit
381
0.0489
0.1132
1.004
2H), 7.15(m, 5H), 7.20(m, 1H), 7.43(m, 2H), 7.54(m, 1H), 7.61(m,
1H), 7.64(d, 1H), 7.84(d, 1H), 8.0(m, 2H), 8.26(d, 1H), 13.3(s, 1H);
IR spectra (KBr, cmꢁ1): 2923, 1589, 1485, 1266, 898, 768; EI-MS
m/z: 417.08 M+ (calculated = 417.18).
was obtained. Data were presented as (
tio of Cu(II) complex to DNA. Where is the viscosity of DNA in the
g/g0)
1/3 versus the mole ra-
g
presence of complex and g0 is the viscosity of DNA alone.
Ligand L2: Yield: 3.4 g, 79%, m.p: 161 °C, 1H NMR (CDCl3,
400 MHz, TMS): d 1.66(s, 3H), 2.33(s, 3H), 2.38(s, 3H), 6.84(d,
1H), 7.16(m, 5H), 7.28(t, 2H), 7.56(t, 1H), 7.62(m, 2H), 7.73(d,
1H), 7.84(d, 1H), 7.93(d, 2H), 8.26(d, 1H), 13.31(s, 1H); IR spectra
(KBr, cmꢁ1): 2919, 1592, 1486, 1208, 945, 781; EI-MS m/z:
431.1 M+ (calculated = 431.19).
3. Result and discussion
The synthesized ligands were characterized by FT-IR, 1H NMR,
Mass spectra and single crystal X-ray analysis. All spectral data
are agreed with the Schiff base ligand structures. The Cu(II) com-
plexes having the general composition [Cu(L)2(H2O)2] have been
synthesized by a general procedure based on the mixing of an
aqueous solution of copper acetate with an ethanolic solution of
the ligand in 1:2 M ratio and isolation of final precipitate by filtra-
tion. The complexes are stable to air and moisture without any
kind of decomposition also after several months. The complexes
are insoluble in water, but soluble in DMF and DMSO. All the com-
plexes were characterized by elemental analysis, FT-IR, Mass spec-
trometry, TGA–DTA, ESR and UV–Visible spectroscopic techniques.
Analytical data of the synthesized complexes were given in Table 1.
Ligand L3: Yield: 3.2 g, 71%, m.p: 146 °C, 1H NMR (CDCl3,
400 MHz, TMS): d 1.66(s, 3H), 2.33(s, 3H), 6.86(d, 1H), 7.14(m,
5H), 7.18(m, 1H), 7.36(t, 1H), 7.57(m, 1H), 7.64(m, 2H), 7.85(d,
1H), 8.05(m, 1H), 8.18(t, 1H), 8.24(d, 1H), 13.19(s, 1H); IR spectra
(KBr, cmꢁ1): 2922, 1585, 1480, 1268, 1209, 967, 778; EI-MS m/z:
451.1 M+ (calculated = 451.14).
2.2.3. Synthesis of Cu(II) complexes
All the Cu(II) complexes of Schiff bases were prepared by the
following method. The metal salt (2 mmol) was dissolved in mini-
mum amount of water and the solution was added to a hot etha-
nolic solution of the corresponding Schiff base (4 mmol). After
the complete addition small amount of Na-acetate was added
and the reaction mixture was refluxed for 4 h. A crystalline solid
was formed which was filtered, washed with hot distilled water
and then from ethanol and dried under vacuum.
3.1. Crystal structure
The crystallographic data for ligands were summarized in Ta-
ble 2. X-ray intensity data were collected on Bruker CCD area-
detector diffractometer equipped with graphite monochromated
Mo Ka radiation (k = 0.71073 Å). ORTEP views of the ligands were
2.3. DNA binding experiment
given in Fig. 1. The structure was solved by direct methods using
SHELXS97 [23]. All non-hydrogen atoms of the molecule were lo-
cated in the best E-map. Full-matrix least-squares refinement
was carried out using SHELXL97. All hydrogen atoms were in-
cluded as idealized atoms riding on the respective carbon atoms
with CAH bond lengths appropriate to the carbon atom hybridiza-
tion. Atomic scattering factors were taken from International
Fluorescence quenching experiments were conducted by add-
ing solutions of copper complexes at different concentrations (0–
20 lM) to the samples containing 40 lM EB and 50 lM DNA. All
the samples were excited at 340 nm and emission was recorded
at 550–650 nm.

R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
89
Fig. 1. ORTEP views of ligands L1, L2 and L3.
Tables for X-ray Crystallography (1992, Vol. C, Tables 4.2.6.8 and
6.1.1.4). Single crystal data reveals that all the Schiff base ligands
are present in amine-one [12] form in the solid state.
13.31 and 13.19 d (for ANHA) showing all the ligands exist in
the amine-one form in the solution state.
Examination of non bonded contacts reveals intramolecular
hydrogen bonding in ligands. Hydrogen bonding data for one of
the ligand are summarized in Table 3. The packing of molecules
3.3. IR spectral studies
FT-IR spectra of ligands exhibits the bands within the range
1580–1590 cmꢁ1 which can be assign to
tC@N (cyclic). All the com-
in the unit cell is further stabilized by
p
–
p
stacking interactions
in L2 (Fig. 2). Summary of
Table 4.
p–p stacking interaction is given in
plexes shows absorption in 1560–1570 cmꢁ1 shifted to lower
wavelength which is due to the interaction with metal ion. The
complexes show the absorption within 3420–3450 cmꢁ1 which
can be assigned to the coordinated water molecules. The com-
plexes show bands within the range 585–595 cmꢁ1 and 470–
490 cmꢁ1 due to the tMAN and tMAO.
3.2. 1H NMR of the ligands
The synthesized ligands were characterized by the 1H NMR
spectra in CDCl3. All the spectra are in good agreement with the
proposed structure of the ligands. In the spectra two, three and
two singlet appeared for the methyl proton of L1, L2 and L3 respec-
tively. Due to the aryl protons of naphthalene and two phenyl rings
doublets, overlapped doublet and multiplet were observed in the
region 6.8–8.3 ppm d. The ligands are showing singlet at 13.3,
3.4. Mass spectral studies
The mass spectra of Schiff base ligands were in good agreement
with the proposed structures. Schiff base L1 shows molecular ion
peak at m/z = 417.08 with a relative intensity near to 70% which

90
R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
Table 3
Table 5
Selected bond lengths and bond angles in the ligands.
Geometry of
centroids; CgIꢃ ꢃ ꢃP, the perpendicular distance of the centroid of one ring from the
plane of the other. is the dihedral angle between the planes of rings I and J; b is the
angle between normal to the centroid of ring I and the line joining ring centroids; is
p–p interactions. CgIꢃ ꢃ ꢃCgJ represents the distance between the ring
Ligand
L1
Ligand
L2
Ligand
L3
a
D
the displacement of the centroid of rings J relative to the intersection point of the
normal to the centroid of ring I and the least-squares plane of ring J. Cg4 denotes the
centroid of the C27AC32 ring.
Bond distances (Å) with esd’s in parentheses
N1AN2
N1AC5
N1AC6
N2AC3
C3AC4
C3AC12
C5AO1
C4AC13
N3AC13
N3AC22
1.406(2)
1.375(3)
1.414(2)
1.306(3)
1.439(3)
1.490(3)
1.254(2)
1.389(3)
1.339(3)
1.426(3)
1.404(2)
1.374(3)
1.411(3)
1.308(3)
1.445(3)
1.491(3)
1.252(3)
1.394(3)
1.345(3)
1.427(3)
1.401(2)
1.382(3)
1.413(2)
1.311(2)
1.436(3)
1.498(2)
1.247(2)
1.406(3)
1.329(2)
1.425(3)
CgI
4
CgJ
4ii
CgIꢃ ꢃ ꢃCgJ (Å)
3.634(2)
CgIꢃ ꢃ ꢃP (Å)
3.444
a
(°)
b (°)
D
(Å)
0.03
18.64
1.15
Symmetry code: (i) 1 ꢁ x, 1 ꢁ y, 1 ꢁ z.
Bond angles (°) with esd’s in parentheses
N2AN1AC5
N2AN1AC6
C5AN1AC6
N1AN2AC3
C3AC4AC5
N1AC5AO1
C4AC5AO1
C13AN3AC22
111.7(1)
118.4(1)
129.9(2)
106.7(2)
104.9(2)
126.3(2)
128.8(2)
127.6(2)
111.8(2)
118.7(2)
129.2(2)
106.7(2)
105.0(2)
125.6(2)
129.3(2)
127.3(2)
112.1(2)
119.5(2)
128.4(2)
106.3(2)
105.4(2)
126.1(2)
129.4(2)
128.5(2)
Fig. 3. ESR spectrum of complex [Cu(L2)2(H2O)2] at liquid nitrogen temperature.
Table 6
Thermal analysis data of Cu(II) complexes.
Complexes
TGA
range
(°C)
DTA DTG Mass loss (%) Assignments
obs. (calcd.)
[Cu(L1)2(H2O)2] 200–350 264 391 3.85 (3.86)
Loss of two coordinated
water molecules
350–600
600–800
44.65 (44.66) Loss of one L1 ligand
8.53
Remaining CuO residue
[Cu(L2)2(H2O)2] 200–350 271 326 3.93 (3.90)
Loss of two coordinated
water molecules
350–600
600–800
45.2 (45.25)
(8.63)
Loss of one L2 ligand
Remaining CuO residue
[Cu(L3)2(H2O)2] 200–350 280 379 3.70 (3.70)
Loss of two coordinated
water molecules
350–600
600–800
44.98 (44.98) Loss of one L3 ligand
(8.18)
Remaining CuO residue
Fig. 2.
p–p
stacking interaction in Ligand L2, different colors showing different
layers of molecule.
is equal to its molecular mass. The other peaks appeared in the
mass spectrum (abundance range 1–100%) are attributed to the
fragmentation of ligand molecule obtained from the rupture of dif-
ferent bonds inside the molecule. ESI-Mass spectra of complexes
[Cu(L2)2(H2O)2] and [Cu(L3)2(H2O)2] were recorded. Complex
[Cu(L2)2(H2O)2] shows molecular ion peak m/z = 925.6 (M++1),
926.6(M++2) and base peak at m/z = 432.3. The base peak is attrib-
uted to the ligand L2 (M++1). Complex [Cu(L3)2(H2O)2] shows
molecular ion peak m/z = 967.5 (M++2) and base peak at m/
z = 452.2. The base peak is attributed to the ligand L3 (M++1). Mass
spectra of both the complexes are well agreed with the proposed
structures of the complexes. Fragmentation of weakly coordinated
water molecule/ion in ESI mass spectra is not unlikely. To confirm
the presence of weakly coordinating H2O molecules in the com-
plexes, we carried out thermogravimetric analyses of complexes.
Table 4
Hydrogen–bonding geometry in ligand L2 (e.s.d.’s in parentheses). Cg1, Cg2, Cg3 and
Cg4 denote the centroid of the N1AC5, C6AC11, C15AC20 and C27AC32 rings
respectively.
DAHꢃ ꢃ ꢃA
DAH (Å)
Hꢃ ꢃ ꢃA (Å)
Dꢃ ꢃ ꢃA (Å)
DAHꢃ ꢃ ꢃA (°)
N22AH22ꢃ ꢃ ꢃO3
1.04(3)
0.97(2)
1.04(3)
0.89(4)
0.93(4)
1.81(3)
2.88(2)
2.53(3)
3.11(5)
2.87(5)
2.701(3)
3.534(3)
3.521(3)
3.761(5)
3.690(6)
142(2)
125(2)
159(2)
132(4)
147(4)
C24AH24ꢃ ꢃ ꢃCg1i
C29AH29ꢃ ꢃ ꢃCg2ii
C21AH213ꢃ ꢃ ꢃCg3iii
C12AH121ꢃ ꢃ ꢃCg4iv
Symmetry code: (i) ꢁ1 + x, y, z, (ii) 2 ꢁ x, 1 ꢁ y, 1 ꢁ z, (iii) 1 ꢁ x, 2 ꢁ y, ꢁz, (iv) 1 + x,
ꢁ1 + y, z.

R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
91
3.5. Electronic spectral studies
Electronic spectra of all the complexes were recorded in DMF.
For octahedral Cu(II) complexes, the expected transition is
2B1g ? 2A1g with respective absorption at 500–700 nm. Due to
Jahn–Teller (J–T) distortions, Cu(II) complexes give a broad absorp-
tion between 600 and 700 nm. All the complexes under this study
exhibit a broad band in the region of 590–680 nm. Absorption coef-
ficients were found to be 863, 832 and 859 (in nm) for the com-
plexes [Cu(L1)2(H2O)2], [Cu(L2)2(H2O)2] and [Cu(L3)2(H2O)2]
respectively.
3.6. Thermogravimetric analysis
The synthesized copper complexes were found to be air stable
and have higher thermal stability. The thermal study was carried
out using the thermogravimetric technique with a heating rate of
10 °C minꢁ1. The experimental results (Table 5) revealed that the
degradation occurred in multiple stages. Up to 200 °C mass loss
is not observed indicating the absence of lattice water molecule
in the complexes. The complexes slowly started to decompose
within the range of 200–350 °C, corresponds to the loss of coordi-
nated water molecules. In the temperature range 350–600 °C deg-
radation due to pyrolysis of the ligand molecule is observed. In the
temperature range 600–800 °C the mass loss is due to the pyrolysis
of another ligand molecule. The final product of the thermal
decomposition CuO was determined by elemental analysis.
Fig. 4. Effect of increasing amounts of complex 1(A), complex 2(B) and complex
3(C) on the relative viscosities of DNA at 30.0 0.1 °C. [DNA] = 1 mM, [Complex]/
[DNA] = 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, respectively.
Table 7
ESR spectral data of the Cu(II) complexes.
Complex
gll
g?
gav
All
A
?
Aav
[Cu(L1)2(H2O)2]
[Cu(L2)2(H2O)2]
[Cu(L3)2(H2O)2]
2.307
2.319
2.319
2.336
2.371
2.343
2.326
2.354
2.335
140
77.5
150
11.6
28
8.3
38.9
7.16
44.4
Fig. 5. Electronic spectra of complexes (A) = [Cu(L1)2(H2O)2], (B) = [Cu(L2)2(H2O)2] and (C) = [Cu(L3)2(H2O)2] in DMF in the absence and presence of plasmid DNA. Arrow shows
the absorbance changes upon increasing DNA concentrations.

92
R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
3.7. ESR spectral studies
of BZPT-Cu > MCPT-Cu > PTPT-Cu, may depend on its affinity to
DNA. The results obtained from viscosity measurements substanti-
ate those obtained from spectroscopic studies (see Table 7).
The full range (3200–2000 G) X-band ESR spectra for the copper
complexes (frozen liquid state and room temperature solid state)
were recorded. The ESR spectrum of the complex 2 in liquid nitro-
gen temperature is shown in Fig. 3. The ESR spectra of all the com-
plexes shows typical four line pattern which suggest that single
copper is present in the molecule, i.e. it is mononuclear. The g-val-
ues and A-values were computed from the spectra using TCNE free-
radical as g marker. ESR spectral data are included in Table 6.
3.8.2. Absorption spectroscopy
The binding of the complex to DNA has been characterized clas-
sically through absorption titration. A complex bound to DNA
through intercalation is characterized by the change in absorbance
(hypochromism) and red-shift in wavelength, due to the intercala-
tive mode involving a stacking interaction between the aromatic
chromophore and the DNA base pairs. The electronic absorption
spectra of all the complexes exhibited broad absorption bands in
3.8. DNA binding studies
the region 590–630 nm, typical for transitions between the p-elec-
3.8.1. Viscosity studies
tronic energy levels of the pyrazolone skeleton. The electronic
spectra of the complexes in the presence and absence of DNA were
monitored at a wavelength range of 570–800 nm, as shown in
Fig. 5. Upon the increasing of concentration of CT DNA (0–
In order to clarify the interactions between the compounds and
DNA, viscosity measurements were carried out. Hydrodynamic
measurements that are sensitive to length change (i.e. viscosity
and sedimentation) are regarded as the least ambiguous and the
most critical tests of binding in solution in the absence of crystal-
lographic structural data [24]. The effects of all the synthesized
complexes on the viscosity of DNA at 30 0.1 °C are shown in
Fig. 4. Viscosity experimental results clearly show that all the com-
plexes can intercalate between adjacent DNA base pairs, causing an
extension in the helix, and thus increase the viscosity of DNA. The
complexes can intercalate strongly, leading to a greater increase in
viscosity of the DNA with an increasing concentration of com-
plexes. The increased degree of viscosity which follows the order
350 lM), a considerable drop in the absorptivity (hypochromicity)
at about 623 nm was observed for all three complexes. The hypo-
chromicity suggests that the complexes may bind to DNA by inter-
calation mode, due to a strong interaction between the electronic
states of the intercalating chromophore and those of the DNA
bases.
To further illustrate the DNA binding strength, the intrinsic
binding constant Kb was determined by the standard given equa-
tion for all the complexes (Fig. 6), which were found to be
5.0 ꢄ 104 Mꢁ1
(BZPT-Cu),
1.0 ꢄ 104 Mꢁ1
(PTPT-Cu)
and
Fig. 6. Plots of [DNA]/(
ea ꢁ e
f) versus [DNA] for the titration of DNA with the complexes (A) = [Cu(L1)2(H2O)2], (B) = [Cu(L2)2(H2O)2] and (C) = [Cu(L3)2(H2O)2].

R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
93
Fig. 7. Emission spectra of EB bound to DNA in the absence and presence of complexes (A) = [Cu(L1)2(H2O)2], (B) = [Cu(L2)2(H2O)2] and (C) = [Cu(L3)2(H2O)2]. [EB] = 40
lM,
[DNA] = 50 lM, [Complex] = 0, 5, 10, 15, 20, lM, respectively; kex = 340 nm. The arrow shows the intensity changes on increasing the complex concentration.
2.5 ꢄ 104 Mꢁ1 (MCPT-Cu). The binding constants of these com-
plexes were lower in comparison to those observed for typical clas-
sical intercalators (ethidium–DNA, 1.4 ꢄ 106 Mꢁ1) [25]. Our results
are consistent with earlier reports on preferential binding to DNA
in the Cu complexes [26,27]. These results suggest an intimate
association of the compounds with DNA and it is also likely that
compounds bind to the helix via intercalation mode.
H/ CH3
CH3
Cl/ H
OH2
Cu
CH3
N
N
N
O
3.8.3. Fluorescence spectroscopy
To understand the interaction pattern of the complex with DNA
more clearly, fluorometric competitive binding experiment was
carried out using ethidium bromide (EB) as a probe that shows
no apparent emission intensity in buffer solution because of sol-
vent quenching. However, EB emits intense fluorescent light in
the presence of DNA due to its strong intercalation between the
adjacent DNA base pairs. A competitive binding of small molecular
to CT DNA could result in the displacement of EB or quenching of
the bound EB by the compound decreasing its emission intensity.
It was previously reported that the enhanced fluorescence could
be quenched by the addition of another molecules [28,29]. The
emission spectra of EB bound to DNA in the absence and in the
presence of the complexes are given in Fig. 7. The emission band
between 640–645 nm of the DNA–EB system decreased in inten-
sity with the increasing concentration of the complexes. It has
been reported that small organic molecules interact with DNA by
intercalation when the concentration ratio of them to DNA (CM/
CDNA) is less than 100 and the fluorescence intensity of EB–DNA
system decreases by 50% [30,31]. Figure shows the quenching ex-
tent has reached up to 50% when Ccomplex/CDNA = 2.5. The changes
observed here are often characteristic of intercalation. Moreover,
N
OH2
O
N
N
CH3
Cl/ H
CH3
H/ CH3
Fig. 8. Proposed structure for the Cu(II) complexes.
this phenomenon indicates that the complexes compete with EB
in binding to DNA. This type of results may be due to the complex
interacts with DNA through intercalative binding, so releasing
some free EB from DNA–EB complex, [11] which is consistent with
the above absorption spectral results.

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R.N. Jadeja et al. / Journal of Molecular Structure 1013 (2012) 86–94
4. Conclusion
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SCCDC-817985, 854557 and 817983 contain the supplementary
crystallographic data for the ligands (L1, L2 and L3). This data can be
obtained free of charge at www.ccdc.cam.ac.uk/conts/retrie-
vel.html or from the Cambridge Crystallographic Data Center, 12
Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223/336 033.
email: deposit@ccdc.ac.uk. Supplementary data associated with
this article can be found, in the online version, at doi:10.1016/
j.molstruc.2012.01.006.