Spectroscopic study of DNA hydrolysis, DNA intercalative, and electrostatic interaction activity exerted by drug based coordination compounds

Article abstract of DOI:10.1002/zaac.201100307

Our work emphasized on synthesizing and characterizing neutral mononuclear copper(II) complexes with second generation fluoroquinolone drug ciprofloxacin (CFL) and some bipyridine derivatives (Aⁿ) of type [Cu(CFL)(A ⁿ)Cl]·2H₂

Full text of DOI:10.1002/zaac.201100307

ARTICLE
DOI: 10.1002/zaac.201100307
Spectroscopic Study of DNA Hydrolysis, DNA Intercalative, and
Electrostatic Interaction Activity Exerted by Drug Based Coordination
Compounds
Mohan N. Patel,*[a] Bhupesh S. Bhatt,[a] and Promise A. Dosi[a]
Keywords: Hydrolytic cleavage; DNA cleavage; DNA degradation; Bactericidal activity; Copper
Abstract. Our work emphasized on synthesizing and characterizing
tration, and gel electrophoresis experiments. Also superoxide dismu-
neutral mononuclear copper(II) complexes with second generation tase (SOD)-like activity (IC50 values) and antibacterial activity of
fluoroquinolone drug ciprofloxacin (CFL) and some bipyridine deriva-
tives (An) of type [Cu(CFL)(An)Cl]·2H2O. The DNA binding free en-
ergies were evaluated by studying the effect of salt concentrations on
DNA binding. DNA interactions were investigated by using DNA
melting temperature studies, viscosity measurements, absorption ti-
metal complexes were studied. To validate the proper mechanistic
pathway for plasmid DNA cleavage, gel electrophoresis experiments
were carried out in presence of radical scavenging agents. The bacteri-
cidal activity of metal complexes was evaluated in terms of colony
forming unit.
toxicity, carcinogenicity, anti-tumor, etc. Copper is found in a
variety of enzymes, including the copper atoms of cytochrome-
c oxidase and the enzyme superoxide dismutase (SOD, con-
taining copper and zinc), and is the central metal ion in the
oxygen-carrying pigment hemocyanin. In accumulation to its
enzymatic roles, copper is used for biological electron trans-
port. Due to extensive bioavailability and less toxicity of cop-
per in comparison with other metal ions, it is the metal of
choice for the latest research in bioinorganic and medicinal
chemistry. Since the discovery of bis(phen)copper(I) complex
as the first copper-based “chemical nuclease”,[9] many
attempts have been made to study copper(II) complexes of
phenanthrolines/bipyridines/bipyridylamines in combination
with the quinolone family drugs nalidixic acid,[10] cinox-
acin,[11] ciprofloxacin,[12] N-propyl-norfloxacin,[13] oflox-
acin,[14] as antitumor, antibacterial, antimicrobial,[15] DNA in-
tercalating,[16] artificial nucleases,[17] and SOD mimic
agents.[18] Copper can be extremely useful in mounting the
new agents that can serve mankind by their action against se-
vere diseases.
Ciprofloxacin, the second generation fluoroquinolone drug,
is a broad-spectrum antibiotic that is active against Gram(+ve)
and Gram(–ve) microorganisms. It functions by suppressing
cell growth by inhibiting one step of the multi-step activity of
the enzyme DNA gyrase, which can introduce super-coils into
closed-circular DNA using the free energy of ATP hydrolysis.
Quinolone-induced DNA damage was first reported in
1986.[19–21] Ciprofloxacin has 12 FDA approved human uses
and other veterinary uses. Since the evolving problem of drug
resistance to microorganisms, the latest researches are going
on finding applicability of coordination effect to enhance the
biological potency of ligands or drug molecules.[22,23]
Introduction
DNA-interacting molecules are of importance in medicine
and the understanding of their interaction with specific se-
quences is the fundamental key in any attempt to control gene
expression through drug binding.[1–3] Deoxyribonucleic acid
(DNA) is the primary target molecule for most anticancer and
antiviral therapies according to cell biology.[4] Transition metal
complexes can interact non-covalently with DNA in various
binding modes e.g. intercalation, groove binding, and external
electrostatic binding. Investigations of the interactions of DNA
with transition metal complexes are performed to design new
types of the pharmaceutical molecules, to elucidate the mecha-
nisms involved in the site specific recognition of DNA and to
determine the principles governing the recognition.[5]
Metal ions are often classed as toxic or non-toxic, however
their biological activity depends very much on speciation and
it is now widely accepted that, with carefully controlled coor-
dination chemistry, even toxic metals can exhibit therapeutic
properties.[6] Among all the mechanism of detoxification, che-
lation by various ligands or proteins like metallothioneins or
phytochelatins provides greater resistance to the toxic effects
of the heavy metals.[7] Metal ions and coordination compounds
are known to affect the cellular processes dramatically,[8] not
only by affecting the natural processes like cell division, gene
expression, etc, but also by some non-natural processes like
* Prof. Dr. M. N. Patel
E-Mail: jeenen@gmail.com
[a] Department of Chemistry
Sardar Patel University,
Vallabh Vidyanagar
388120 Gujarat, India
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201100307 or from the au-
thor.
The thermodynamic profile of DNA binding is needed for
the proper understanding of interactions between DNA and
152
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2012, 638, (1), 152–162

DNA Hydrolysis, DNA Intercalative, and Electrostatic Interaction Activity
binding moieties. Our experimental studies provide data of ge- behavior of the carboxylato group[26–28] of ciprofloxacin.
neral interest and use in addressing the larger issues concerned These data are further supported by ν(M–O) bands,[29] which
with developing appropriate thermodynamic descriptions of appear at 509–520 cm–1 for complexes.
DNA binding free energy. Researching DNA binding model of
The ν(C=N) band of bipyridine derivatives is usually ob-
the fluoroquinolones and their transition metal complexes can served at about 1414 cm–1. It is shifted to higher frequency at
bring many innovations in the antitumor, antiviral, antibacte- approx. 1459 cm–1 in the complexes, which suggests the biden-
rial, and antiaging fields of medicine. In continuation to our tate N–N coordination of the ligand.[26,30,31] The ν(M–N) band
earlier work,[24] in this paper, mixed ligand complexes of CuII at 532–544 cm–1 is assigned to NǞM bonding of com-
with ciprofloxacin and neutral N,N-donor bidentate ligands plexes.[26,32] So the IR spectroscopic study suggests coordina-
were synthesized in order to evaluate their DNA binding prop- tion of ciprofloxacin from pyridone oxygen and carboxylic hy-
erties. Bactericidal activity against Gram(+ve) and Gram(–ve) droxyl group, whereas coordination of bipyridines occurs
bacteria were assayed in terms of colony forming unit.
through two nitrogen atoms.
FAB-Mass Spectrometry
Results and Discussion
Figure 1 represents the FAB-mass spectrum of complex
[Cu(CFL)(A1)Cl]·2H2O (1) obtained using m-nitro benzyl
alcohol as matrix. Peaks at 136, 137, 154, 289, and 307 m/z
value are due to usage of matrix. The molecular ion peak is
observed at m/z = 772. The M+2 peak at 774 m/z value with
1/3 intensity to molecular ion peak demonstrate Cl– ion attach-
ment with metal ion. Furthermore, the peak at 737 m/z can be
ascribed for removal of covalently bonded Cl– ion. The most
intense peak was observed at m/z = 342. Figure 1 shows peaks
at 737, 442, 405, 342, and 330 m/z values. The m/z values
correspond to peaks for particular fragments are duly high-
lighted in fragmentation pattern of complex 1 (Figure S1, Sup-
porting Information)).
Characterization of Metal Complexes
The elemental analysis is in good concurrence with the pro-
posed 1:1:1, metal:CFL:An formulation.
IR Spectroscopy
Ring mode vibrations of the ligands are affected by coordi-
nation to CuII, corresponding to shift in energy for bands in
the 1700–500 cm–1 region of IR spectra. In the IR spectra of
complexes, these bands are also slightly sensitive to the sub-
stituent of ligands. The prominent IR spectroscopic data of the
complexes are shown in Table 1. The broad band in quinolone
at approx. 3010 cm–1 is due to hydrogen bonding, which con-
tributes to ionic resonance structure and the observed peak was
of free hydroxyl stretching vibration. The complete elimination
of this broad band can be ascribed to presence of covalent
Reflectance Spectroscopy and Magnetic Behavior
Complexes of copper(II) with different coordination num-
bond between copper and –COO– of ciprofloxacin. The bers result in different arrangements. The electronic absorption
ν(C=O) stretching vibration band appears at 1708 cm–1 for cip- bands of copper(II) complexes resulting from the d–d transi-
rofloxacin, whereas for the complexes it appears at 1619– tions are referred as ligand field or crystal field bands because
1627 cm–1. This shift towards lower energy suggests that coor- their energies shift correspond to the positions of ligands in
dination occurs through the carbonyl oxygen of pyridine the spectrochemical series. The copper(II) complexes exhibit
ring.[25] The strong absorption bands at 1624 cm–1 and broad bands at ambient temperature in the region 16000–
1340 cm–1 in ciprofloxacin could be assigned for ν(COO) 18000 cm–1. These bands are characteristic of CuII d–d transi-
asymmetric and symmetric vibrations respectively, whereas in tion in tetragonal field, in which the CuII atom is in a distorted
the metal complexes, these bands are observed at 1561–1581 square-pyramidal coordination environment,[10,33] which is the
and 1355–1378 cm–1. The difference
νsym(CO2) is very much informative in determining the coordi-
Δ
=
νasym(CO2)– only possible arrangement for the metal complexes.
The magnetic moments measurement for any arrangement
nation mode of the ligands. If the difference is greater than in copper(II) complexes generally results in 1.8 BM, which is
200 cm–1, it would point towards monodentate coordination very close to spin-only value i.e. 1.73 BM. The observed val-
Table 1. Characteristic absorptions bands /cm–1 of IR spectra of complexes 1–8 and CFLH.
ν(C=O) pyridone
ν(COO)asy
ν(COO)sy
Δν
ν(M–N)
ν(M–O)
CFLH
1708
1620
1626
1627
1623
1619
1622
1624
1618
1624
1576
1581
1579
1561
1569
1577
1575
1580
1340
1372
1373
1361
1355
1362
1378
1364
1370
284
204
208
212
206
207
199
211
210
–
–
1
2
3
4
5
6
7
8
544
540
537
541
539
532
536
545
515
520
509
519
518
510
516
507
Z. Anorg. Allg. Chem. 2012, 152–162
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153

M. N. Patel, B. S. Bhatt, P. A. Dosi
ARTICLE
Figure 1. FAB-mass spectra of [Cu(CFL)(A1)Cl]·2H2O at the accelerating voltage was 10 kV.
ues in our case are very close to the spin-only values expected
for s = ½ system (1.73 BM), which lead a path to conclude
that central metal atoms in synthesized complexes have one
unpaired electron responsible for s = ½ system.[34]
Thermogravimetric Analysis
Thermogravimetric analysis was carried out at a heating rate
of 10 K per minute in the range of 20–800 °C in a nitrogen
atmosphere. The characteristic thermogravimetric curve (mass
loss in % to temperature in °C) shows three distinct mass
losses.[35] Mass lost (approximately 4%) occurring during first
decomposition step between 50–120 °C corresponds to loosely
bind two molecules of crystallization water. The mass loss (ap-
prox. 42%) during second step between 180 to 300 °C corre-
sponds to decomposition of neutral bidentate ligand, in which
two coordination bonds are involved. The third step (approx.
41%) corresponds to decomposition of CFL, in which one co-
ordination bond and one covalent bond are involved leaving
behind the CuO (approx. 9%) as residue. So these three de-
composition steps show ternary nature of the complexes.
The structure of complex 1, assigned according to above
analytical facts is shown in Figure 2.
Figure 2. Proposed structure of complex 1.
be governed in certain degree by the facility of coordination
at the metal atom as well as electronic nature of the ligands.
The biomolecular interaction with metal complexes can be the
cause for inhibition of biological synthesis and this prevent
the organisms from reproducing.[28] The copper chelation with
ligand reduces polarity of metal ion by overlap of the ligand
orbital and partial sharing of the positive charge of the metal
ion with the donor groups. Increase in delocalization of π elec-
trons over the whole ligand enhances the penetration of com-
plexes into lipid membranes and blocking of the metal binding
sites in the enzymes of microorganisms. The complexes may
Antibacterial Activity
The antibacterial efficiencies of the CuCl2·2H2O, ligands, also disturb the respiration process of the cell and thus block
standard drugs, and the complexes were tested against two the synthesis of proteins, which restricts further growth of the
Gram(+ve) Staphylococcus aureus, Bacillus subtilis, and three organism and thus results in the overall gain in the biological
Gram(–ve) Serratia marcescens, Escherichia coli, and Pseu- potency. It has been proposed that quinolone in solution form
domonas aeruginosa microorganisms in terms of MIC in μM. the stable bonds with metal ion.[36] Our data also supported
The minimum inhibitory concentration (MIC) is defined as the the fact that metal ion is required for tight binding of DNA
lowest concentration of antimicrobial agent showing complete with quinolone,[37] since synthesized metal complexes or che-
inhibition of growth of microorganisms. The results concern- lates (metal bounded quinolone) show better antimicrobial ac-
ing in vitro antibacterial activity (MIC) are presented in tivity than free quinolone.
Table 2. The antimicrobial activity of all the complexes against
In addition, our study regarding bactericidal activity in terms
the five microorganisms is much higher than that of of CFU per mL of the metal complexes against same microor-
CuCl2·2H2O and free ligands. The inhibition activity seems to ganisms reveal decrease in number of colonies with increasing
154
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DNA Hydrolysis, DNA Intercalative, and Electrostatic Interaction Activity
Table 2. MIC in terms of μM.
Gram positive
Compounds
Gram negative
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl22H2O
Ciprofloxacin
2698.0
1.6
2815.0
1.1
2756.0
1.6
2404.0
1.4
3402.0
1.4
Gatifloxacin
5.1
4.0
2.9
1.0
2.9
Norfloxacin
2.5
2.5
4.1
3.8
2.8
Enrofloxacin
1.9
3.9
1.7
1.4
1.4
Pefloxacin
2.1
2.4
5.1
5.7
2.7
Levofloxacin
1.7
2.2
1.7
1.7
1.0
Sparfloxacin
1.3
2.0
1.5
1.5
1.3
Ofloxacin
1.9
1.4
1.7
2.2
1.4
A1
194.0
130.0
194.0
255.0
184.0
194.0
167.0
194.0
0.7
1.6
1.7
0.8
2.1
169.0
250.0
169.0
278.0
139.0
169.0
145.0
169.0
0.5
0.4
0.3
1.8
2.3
272.0
506.0
272.0
275.0
272.0
286.0
272.0
244.0
1.8
0.8
0.7
0.8
2.1
255.0
154.0
255.0
267.0
255.0
250.0
255.0
255.0
1.5
1.8
1.3
1.5
2.1
278.0
129.0
278.0
268.0
278.0
506.0
278.0
278.0
1.5
1.6
1.4
1.5
2.0
A2
A3
A4
A5
A6
A7
A8
[Cu(CFL)(A1)Cl]·2H2O (1)
[Cu(CFL)(A2)Cl]·2H2O (2)
[Cu(CFL)(A3)Cl]·2H2O (3)
[Cu(CFL)(A4)Cl]·2H2O (4)
[Cu(CFL)(A5)Cl]·2H2O (5)
[Cu(CFL)(A6)Cl]·2H2O (6)
[Cu(CFL)(A7)Cl]·2H2O (7)
[Cu(CFL)(A8)Cl]·2H2O (8)
1.5
0.7
1.6
1.8
0.3
0.6
1.4
0.3
1.0
0.5
0.5
1.2
1.7
1.6
1.6
the concentration of compounds. The results are shown in Fig- hyperchromic effect on the absorption spectra of DNA bases
ure 3 for all the complexes against S. aureus. The maximum (λmax = 260 nm). In order to identify this transition process,
the melting temperature Tm, which is defined as the tempera-
ture, where half of the total base pairs are unbounded, is usu-
ally introduced. The binding of metal complexes to the double
stranded DNA usually stabilizes the duplex structure to some
extent depending on the strength of interaction with nucleic
acid.[38] The binding should lead to an increase in the melting
temperature (Tm) of DNA as compared to DNA itself.
concentration reveals bactericidal activity for all the complexes
is 1.75 μM. The number of colonies counted in this technique
was 30–250 CFU per mL.
According to the literature the interaction of metallointercal-
ators generally results in a considerable increase in melting
temperature (Tm). DNA (100 μM) melting experiments reveal
Tm of Herring sperm DNA 82.8Ϯ1 °C in the absence of the
complex (Figure 4). With addition of complexes 1–8, Tm in-
Figure 3. Relationship between concentration and bactericidal activity
of all complexes against S. aureus.
Thermal Denaturation Studies
Thermal behaviors of DNA in the presence of complexes
can give insight of their conformational changes when tem-
perature is raised, and offer information about the interaction
strength of complexes with DNA. It is well known that when
the temperature of the solution increases, the double stranded
Figure 4. Melting curves of herring sperm DNA in the absence and
DNA gradually dissociates to single strands and generates a presence of complexes 1–8.
Z. Anorg. Allg. Chem. 2012, 152–162
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155

M. N. Patel, B. S. Bhatt, P. A. Dosi
ARTICLE
creased to 88.2Ϯ1 °C, 86.5Ϯ1 °C, 88.5Ϯ1 °C, 86.7Ϯ1 °C,
All copper(II) complexes exhibit an intense absorption band
86.9Ϯ1 °C, 87.5Ϯ1 °C, 87.9Ϯ1 °C and 88.0Ϯ1 °C, respec- around 274 nm, which is attributed to intraligand n–π* or π–
tively. The increased Tm (3.7–5.7 °C) value of the DNA after π* transition of intecalative ligands. The increasing concentra-
addition of the complexes is comparable to that observed for tion of herring sperm DNA results in the obvious tendency of
classical intercalators.[39]
hypochromism with a bathochromic (approx. 4 nm) shift. To
compare the binding strength of complexes quantitatively, the
intrinsic binding constants Kb of the complexes were deter-
mined by monitoring the changes of absorbance at 274 nm
with increasing concentration of herring sperm DNA (Fig-
ure 6). From the plot of [DNA]/(εa–εf) versus [DNA], the Kb
value of all the complexes were found to be 1.14ϫ104 to
1.67ϫ104 M–1 (Table S1, Supporting Information). For each
set of compounds, the spectral changes for the individual com-
pounds differ only slightly from each other, which indicates
that their interactions with DNA were similar and the com-
plexes have comparable binding constant. The observed ba-
thochromicity and hypochromicity suggest an intimate associa-
tion of the complexes with DNA and also that the complexes
may bind to the helix by intercalation. These values are com-
parable to that observed for [Ru(phen)(phi)]2+ (2.72ϫ104)[42]
and [Cu(erx)2(H2O)] (3.12ϫ104),[43] but smaller than those
observed for [Ru(bpy)(dppz)]2+ (Ͼ106).[44]
Viscosity Measurements
The arrangement of a supercoiled molecule may be altered
by any factor, which affects the intrinsic twisting of the DNA
helix. One important factor is the presence of an intercalator,
e.g. ethidium bromide, the positively charged polycyclic aro-
matic compound, which binds to DNA by inserting itself be-
tween the base pairs. Intercalators introduce strong structural
perturbations in DNA. The axial flexibility of DNA in the
sense of dynamic flux of changes in axial and helical param-
eters allows the compound to stack between adjacent DNA
base pair. The complex is thought to be stabilized by π–π
stacking interactions between the compound and DNA bases.
The possibility of bending the helix with larger ring systems
and the steric restrictions on intercalation is supported by vis-
cosity study. A classical intercalation model demands that the
DNA helix lengthens as base pairs are separated to accommo-
date the complex, leading to the increase of DNA viscosity. In
contrast, decrease in DNA viscosity is noted in the partial or
non-classical intercalator like in ciprofloxacin, because it may
bend (or kink) DNA helix, thereby decreasing its effective
length. For all the complexes, as increasing the amounts of
complexes, the viscosity of DNA increase steadily (Figure 5).
Similar mechanism was suggested for the complexes
[Ru(tpy)(pta)]2+ and [Ru(tpy)(ptp)]2+ reported by Chao et
al..[40]
Figure 6. Electronic absorption spectra of [Cu(CFL)(A1)Cl]·2H2O
(10 μM) in phosphate buffer(Na2HPO4 /NaH2PO4, pH 7.2) in the ab-
sence and presence of increasing amount of DNA (50–150 μM). (Inset:
Plot of [DNA]/(εa–εf) versus [DNA]. Arrow shows the absorbance
change upon increasing DNA concentrations).
Figure 5. Viscometric curve for complexes showing classical intercal-
ative mode of binding.
Salt-dependence of Binding Constants
The typical plots of [DNA] versus [DNA]/(εa–εf) for deter-
mination of binding constant (Kb) of complex 1 to herring
sperm DNA at various concentrations of NaCl based on Equa-
tion (1) (see Experimental Section) was used (Figure S2, Sup-
Absorption Titration
It is a general observation that the binding of intercalative porting Information). The detailed results are collected in
molecules to DNA is accompanied by a red shift and hypoch- Table 3. The reported binding constants are obtained over the
romism in the absorption spectra due to the strong stacking concentration range 0.005 to 0.1 M NaCl, in order to apply
interaction between aromatic chromophore of the complex and polyelectrolyte theory to the calculation of the non-electro-
the base pairs of DNA. The extent of spectral change is related static binding constants and separate the binding free energy
to the strength of binding. The spectra for intercalators are change into its electrostatic and nonelectrostatic contributions.
more perturbed than those for groove binders.[41]
The salt concentrations of 0.005–0.1 M were selected in this
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DNA Hydrolysis, DNA Intercalative, and Electrostatic Interaction Activity
study, because the polyelectrolyte theories are strictly appli- and the remaining terms are constants for double stranded
cable to salt concentrations of lower than 0.1 M. It has been DNA in B form, i.e. ξ = 4.2 and δ = 0.56. Results of the
reported that the dependence of Kb on salt concentration be- calculations are summarized in Table 4 along with the percen-
comes non linear at higher concentrations of salt.[45] The plot tage of Kto contribution to the total binding constants (Kb) at
of ln[Na+] against lnKb for the binding of complex 1 to DNA various concentrations of Na+. The Kto can be taken as a mea-
is given in Figure 7. It is clear from the plots that the binding sure how large the non-electrostatic forces stabilize the ligand–
constant decreases with increasing salt concentration. This is DNA interaction. In contrast to the Kb values, which are salt-
due to the stoichiometric amount of ion release that follows dependent, the magnitude of Kto is constant throughout the
the binding of charged ligand, i.e. copper(II) complex,[46] sug- concentration of NaCl employed with the average value of
gesting that electrostatic interaction is involved in the DNA- 1.18ϫ103 M–1 bp. This is in agreement with the expectation
binding event.
for the salt-independency of this parameter. Although the val-
ues of Kto are constant throughout the concentrations of salt,
the percentage of Kto contributions to the Kb increases signifi-
cantly and reach a maximum of 30.6% at [Na+] = 0.1 M. It
can be expected that at higher concentrations of salt, e.g. at
physiological condition (Na+ ≈ 0.2 M), the non-electrostatic
forces would play a major role in the DNA binding of the
copper(II) complex. The value of 23.1% for the non-electro-
static binding constant found in the DNA binding of complex
1 at NaCl = 0.05 M is considered to be high even when it is
compared with those of other proven intercalators such as ethi-
dium (12.3%) at the same ionic strength.[47]
Table 3. Equilibrium binding constant (Kb) for the binding of complex
1 to herring sperm DNA in 0.2 M phosphate buffer (pH 7.2) at 25 °C
and various concentrations of NaCl.
[Na+] /M
Kb/103 /M–1 bp
Kto/103 /M–1 bp
Kto/Kb /%
0.005
0.025
0.050
0.075
0.100
12.0
7.1
5.2
4.1
3.6
1.2
1.3
1.2
1.1
1.1
10.0
18.3
23.1
26.8
30.6
Average = 6.4Ϯ0.33 (103)
Table 4. Thermodynamic parameters for the binding of copper(II)
complexes to herring sperm DNA at 0.005 m NaCl.
DNA
Binders
Kb/103 /
M–1 bp
Kto/103 (%Kto/
Kb) /M–1 bp
ΔGto (%
ΔGto/ΔGo)
o
ΔGo
SK
ΔGpe
–18.3
(86.3)
–17.2
(83.1)
–18.7
(87.4)
–17.9
(86.1)
–18.8
(88.7)
–18.8
(88.3)
–19.2
(89.3)
–18.9
(88.3)
–27.2
(84.5)
1
5.2
4.3
5.8
4.5
5.4
5.6
6.0
5.8
494
–21.2 0.399 –2.9 1.2 (23.1)
–20.7 0.474 –3.5 0.9 (20.9)
–21.4 0.360 –2.7 1.8 (31.0)
–20.8 0.394 –2.9 1.2 (26.7)
–21.2 0.323 –2.4 1.8 (33.3)
–21.3 0.342 –2.5 1.8 (32.1)
–21.5 0.318 –2.3 2.1 (35.0)
–21.4 0.345 –2.5 1.8 (31.0)
–32.2 0.75 –5.0 61.0 (12.3)
2
3
4
5
6
7
8
EtBra)
a) Taken from reference.[48]
Figure 7. Salt dependence of binding constant (Kb) for the binding of
complex 1 (10 μM) to herring sperm DNA.
By further analysis is also possible to dissect the binding
free energy change (ΔGo) for the binding of complex 1 to
Using the slope of linear fitting of Figure 7, we may calcu- DNA into its electrostatic (ΔGpeo) and non-electrostatic (ΔGto)
late non-electrostatic binding constant (Kto) at various concen- contributions at a given concentration of NaCl.[44] Table 4
trations of NaCl ([Na+]) according to the following polyelec- summarizes the results of energetics calculation for the binding
trolyte theory:[46]
lnKb = lnKto + Zξ–1 {ln(γϮδ)} + Zψ ln[Na+],
of metal complexes to DNA in 0.05 M NaCl as well as proven
organic intercalator, EtBr,[46,48] for the purpose of comparison.
The total binding free energy changes listed in Table 4 were
calculated based on the standard Gibbs relation:
where Zψ is estimated from the slope of the regression line in
Figure 7. Z is partial charge on the binding ligand involved in
the DNA interaction as predicted by polyelectrolyte theory, ψ
ΔGo = –RT ln[Kb]
is the fraction of counterions associated with each DNA phos- where R is the gas constant and T is the temperature in Kelvin.
phate unit (ψ = 0.88 for double-stranded B-form DNA), γϮ is The salt dependence of the binding constant is defined as the
the mean activity coefficient at cation concentration of Na+, slope, SK.
Z. Anorg. Allg. Chem. 2012, 152–162
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
157

M. N. Patel, B. S. Bhatt, P. A. Dosi
ARTICLE
The SK value can be used to calculate the polyelectrolyte
contribution of the free energy change (ΔGpeo) to the overall
free energy change (ΔGo) at a given NaCl concentration by
the relation:[46,48]
ΔGpe = (SK) RT ln[Na+]
o
The difference between the Gibbs free energy change (ΔGo)
o
and ΔGpe is defined as the non-electrostatic free energy
change:
ΔGto = ΔGo – ΔGo
The quantity ΔGto corresponds to the portion of the binding
free energy change, which is independent of salt concentra-
tions and contains a minimal contribution from polyelectrolyte
effects such as coupled ion release.
Figure 8. Photogenic view of cleavage of pUC19 DNA: Lane 1, DNA
control; Lane 2, pUC19 DNA + CuCl2·2H2O; Lane 3, pUC19 DNA +
Ciprofloxacin; Lane 4, pUC19 DNA + [Cu(CFL)(A1)Cl]·2H2O; Lane
5, pUC19 DNA + [Cu(CFL)(A2)Cl]·2H2O; Lane 6, pUC19 DNA +
pe
[Cu(CFL)(A3)Cl]·2H2O; Lane 7, pUC19 DNA
+
[Cu(CFL)(A4)
Cl]·2H2O; Lane 8, pUC19 DNA + [Cu(CFL)(A5)Cl]·2H2O; Lane 9,
pUC19 DNA + [Cu(CFL)(A6)Cl]·2H2O; Lane 10, pUC19 DNA +
[Cu(CFL)(A7)Cl]·2H2O; Lane 11, pUC19 DNA + [Cu(CFL)(A8)
Cl]·2H2O.
DNA Cleavage Studies
spontaneously dismutes to O2 and hydrogen peroxide (H2O2)
quite rapidly (approx. 105 M–1s–1 at pH 7). SOD is biologically
necessary because superoxide reacts even faster with certain
targets such as the NO radical, which makes peroxynitrite.
The system used as a source of superoxide radical generator
was NBT/NADH/PMS system in order to check SOD like ac-
tivity of the synthesized complexes. Copper is known to give
good SOD activity, although their structure are totally unre-
lated with native enzyme.[53] The IC50 value (chromophore re-
quire to yield 50% inhibition of the reduction of NBT) of the
present complexes are lower than the value exhibit by the cop-
per salt (Figure S5, Supporting Information). The copper(II)
complexes showed SOD-like activity, which was evaluated by
the scavenger concentration which causes 50% inhibition in
the detector formation i.e. IC50. Compounds exhibit SOD-like
activity at biological pH with their IC50 values ranging from
0.6 to 1.3 μM. The superoxide scavenging data (Table S3, Sup-
porting Information) suggest that all the complexes are active
compared to the complexes of type [Cu(sulfathiazole)(py)3Cl]
(1.31–5.17 μM) reported by Casanova et al..[54] The lower
IC50 can only be accredited to the vacant coordination, which
facilitates the binding of superoxide anion, electrons of aro-
There has been considerable interest in DNA cleavage reac-
tions that are activated by transition metal complexes.[49,50]
Figure 8 illustrates the gel electrophoretic separations showing
the cleavage of pUC19 DNA induced by the complexes under
aerobic conditions.[51] When circular plasmid DNA is con-
ducted by electrophoresis, the fastest migration is observed for
the supercoiled form (SC). If one strand is cleaved, the su-
percoiled will relax to produce a slower-moving open circular
form (OC). If both strands are cleaved, a linear form (L) is
generated that migrates in between. This clearly shows that the
relative binding efficacy of the complexes to DNA is much
higher than the binding efficacy of metal salt itself or cipro-
floxacin (Table S2, Supporting Information). The different
DNA-cleavage efficiency of the complexes was due to the dif-
ferent binding affinity of the complexes to DNA, which was
observed in other cases. The involvement of reactive oxygen
species (hydroxyl and singlet oxygen) in the nuclease mecha-
nism can be inferred by monitoring of the quenching of the
DNA cleavage in the presence of radical scavengers in solu-
tion. Complexes 1–8 do not show inhibition of DNA cleavage
in the presence of scavengers of hydroxyl radicals (DMSO)
(Figure S3, Supporting Information) and singlet oxygen (so-
dium azide) (Figure S4, Supporting Information). This indi-
cates that the cleavage of DNA probably follows a hydrolytic
cleavage mechanism. Hydrolytic DNA cleavage involves
cleavage of phosphoester bond to generate fragments those
could be subsequently relegated. Such DNA hydrolysis by
copper(II) complexes have been reported earlier.[52]
·–
matic ligands that stabilize Cu–O2 interaction and not only
to the partial dissociation of complex in solution. Copper(II)
·–
complexes mimic the SOD enzyme and bind with O2 at its
vacant coordinating site resulting in formation of octahedral
arrangement (Figure S6, Supporting Information). The interac-
·–
tion mechanism could involve a direct attack of the O2 ion
on the free axial position of the CuII ion passing through a six-
coordinate adduct, which is highly unstable due to the Jahn–
Teller effect.[55] As a consequence of this interaction, the cen-
tral metal ion CuII undergoes rapid reduction to CuI. It is as-
SOD: SOD-like Activity
·–
sumed that electron transfer between the central metal and O2
occurs by direct binding.[56]
Superoxide is one of the main reactive oxygen species
(ROS) in the cell and as such, super oxide dismutase (SOD)
serves a key antioxidant role. SOD outcompetes damaging re-
actions of superoxide, thus protecting the cell from superoxide
toxicity. The reaction of superoxide with non-radicals is spin
forbidden in biological systems; this means, its main reactions
Conclusions
The spectroscopic data suggests square pyramidal arrange-
are with itself (dismutation) or with another biological radical ment for all the complexes. The CuII complexes mimic the
such as nitric oxide (NO). The superoxide anion radical (O2
158 www.zaac.wiley-vch.de
·–
)
SOD enzyme and mechanism suggests transformation of
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2012, 152–162

DNA Hydrolysis, DNA Intercalative, and Electrostatic Interaction Activity
pyridyl)-3-(4-chlorophenyl)propen-1-one (1.5 mmol) and 1-[2-oxo-2-
square pyramidal – octahedral arrangement, while scavenging
superoxide radicals. The effect of CuII coordination in enhanc-
ing the antimicrobial properties is clearly visualized by MIC
data as the resulting complexes are more bacteriostatic even
than standard tested drugs. Additionally biocidal activity
shows decrease in number of colonies as compound concentra-
tion increases. The viscometry data are in good agreement with
bathochromicity and hypochromicity observed in UV/Vis ab-
sorption curve and suggests classical intercalation for all the
(pyridyl)ethyl]pyridinium iodide (1.5 mmol) in methanol (20 mL). Af-
ter heating to reflux for 5–7 h, the reaction mixture was allowed to
cool, which led to the formation of greenish yellow needles of pure
product. The solid was filtered off, washed with cold methanol, and
dried under vacuum. Yield 51.3%, m.p. 140 °C, Anal. C22H15ClN2
(342.82): calcd. C 77.87; H 4.41; N 8.17%; found: C 77.58; H 4.55;
1
N 7.99%. H NMR (CDCl3, 400 MHz): δ = 8.754–8.710 (complex, 2
H, H3Ј,6Ј), 8.659 (s, 1 H, H3), 8.220 (d, 2 H, H2ЈЈЈ,6ЈЈЈ), 7.960 (s, 1
H, H5), 7.910 (t, 1 H, H4Ј), 7.789 (d, 2 H, H2ЈЈЈ, 6ЈЈ), 7.578–7.474
complexes. The above DNA interaction data are well sup- (complex, 5 H, H3ЈЈ,5ЈЈ,3ЈЈЈ,4ЈЈЈ,5ЈЈЈ), 7.386 (t, 1 H, H5Ј) ppm. 13C
NMR (CDCl3, 100 MHz): δ = 157.43 (C2), 155.86 (C2Ј), 150.22 (C6),
149.09 (C4), 147.22 (C6’), 139.28 (C1Љ), 138.25 (C1ЈЈЈ), 137.08 (C4’),
135.30 (C4ЈЈЈ), 129.30 (C2ЈЈЈ,6ЈЈЈ), 128.80 (C2ЈЈ,6ЈЈ), 128.55
(C3ЈЈЈ,5ЈЈЈ), 127.10 (C3ЈЈ,5ЈЈ), 124.08 (C4ЈЈ), 121.78 (C5Ј), 118.40
(C3Ј), 117.50 (C3), 102.75 (C5) ppm.
ported by thermal denaturation study. The gel electrophoresis
experiments validate again sufficient interaction between DNA
and CuII complexes and gel electrophoresis in presence of radi-
cal scavenging agents suggest the hydrolytic cleavage mecha-
nism for plasmid DNA. The thermodynamic profile of com-
plex–DNA interaction is illustrated as binding free energy by
absorption data. The effect of increasing salt concentration is
clearly visualized and suggests electrostatic interaction exis-
tence in presence of electrolytic material.
6-Phenyl-4-p-tolyl-2,2Ј-bipyridine (A2): A similar procedure was fol-
lowed by taking 1-(2-pyridyl)-3-(4-tolyl)propen-1-one as enone. Yield
56.4%, m.p. 121 °C. Anal. C23H18N2 (322.40): calcd. C 85.68; H 5.63;
N 8.69%; found: C 85.83; H 5.51; N 8.77%. 1H NMR (CDCl3,
400 MHz): δ = 8.776–8.723 (complex, 3 H, H3,3Ј,6Ј), 8.232 (dd, 2 H,
H2ЈЈЈ,6ЈЈЈ), 8.020 (s, 1 H, H5), 7.934 (t, 1 H, H4Ј), 7.787 (d, 2 H,
H2ЈЈ,6ЈЈ), 7.560 (t, 2 H, H3ЈЈЈ,5ЈЈЈ), 7.485 (t, 1 H, H4ЈЈЈ), 7.404 (t, 1
H, H5Ј), 7.358 (d, 2 H, H3ЈЈ,5ЈЈ), 2.446 (s, 3 H, CH3) ppm. 13C NMR
(CDCl3, 100 MHz): δ = 157.20 (C2), 156.05 (C2Ј), 155.47 (C6),
150.27 (C4), 148.41 (C6Ј), 139.52 (C1ЈЈ), 139.18 (C1ЈЈЈ), 137.56
(C4Ј), 135.63 (C4ЈЈ), 129.80 (C3ЈЈ,5ЈЈ), 129.12 (C3ЈЈЈ,5ЈЈЈ), 128.77
(C2ЈЈЈ,6ЈЈЈ), 127.12 (C2ЈЈ,6ЈЈ,4ЈЈЈ), 123.88 (C5Ј), 121.85 (C3Ј), 118.47
(C3), 117.53 (C5), 21.29 (CH3) ppm.
Experimental Section
Materials and Methods: All the chemicals and solvent used were of
analytical grade. Ciprofloxacin hydrochloride was purchased from
Bayer AG, Wuppertal, Germany. Pyridine, 2-acetyl pyridine, p-chloro
benzaldehyde, m-chloro benzaldehyde, p-bromo benzaldehyde, m-
bromo benzaldehyde, p-fluoro benzaldehyde, p-methyl benzaldehyde,
p-methoxy benzaldehyde, and p-benzyloxy benzaldehyde were pur-
chased from Loba Chemie Pvt. Ltd., India. Ethidium bromide and Lu-
ria Broth were purchased from Himedia, India. Acetic acid and EDTA
were purchased from Sd fine Chemicals, India. Herring sperm DNA
was purchased from Sigma Chemical Co., India.
4-(4-Bromophenyl)-6-phenyl-2,2Ј-bipyridine (A3): A similar pro-
cedure was followed by taking 1-(2-pyridyl)-3-(4-bromophenyl)pro-
pen-1-one as enone. Yield 51.7%, m.p. 146 °C. Anal. C22H15BrN2
(387.27): calcd. C 68.23; H 3.90; N 7.23%; found: C 68.36; H 4.05;
N 7.08%. 1H NMR (CDCl3, 400 MHz): δ = 8.882 (s, 1 H, H3), 8.828–
8.806 (complex, 2 H, H3Ј,6Ј), 8.201 (dd, 2 H, H2ЈЈЈ,6ЈЈЈ), 8.091 (t, 1
H, H4Ј), 8.009 (d, 1 H, H5), 7.807 (d, 2 H, H2ЈЈ,6ЈЈ), 7.683 (d, 2 H,
H3ЈЈ,5ЈЈ), 7.578–7.482 (complex, 4 H, H5Ј,3ЈЈЈ,4ЈЈЈ,5ЈЈЈ) ppm. 13C
NMR (CDCl3, 100 MHz): δ = 157.83 (C2), 154.42 (C2Ј) 149.48 (C6),
146.61 (C4), 143.55 (C6Ј), 139.75 (C1ЈЈ), 138.85 (C1ЈЈЈ), 137.00
(C4Ј), 132.36 (C2ЈЈЈ,6ЈЈЈ), 129.48 (C4ЈЈЈ), 128.94 (C2ЈЈ,6ЈЈ), 128.87
(C3ЈЈЈ,5ЈЈЈ), 127.11 (C3ЈЈ,5ЈЈ), 124.52 (C4ЈЈ), 123.87 (C5Ј), 122.92
(C3Ј), 118.92 (C3), 118.26 (C5) ppm.
Instrumentation: IR spectra were recorded with a FT-IR Shimadzu
spectrophotometer with sample prepared as KBr pellets in the range
4000–400 cm–1. C, H, and N elemental analyses were performed with
a model Perkin–Elmer 240 elemental analyzer. The metal contents of
the complexes were analyzed by EDTA titration[57] after decomposing
the organic matter with a mixture of HClO4, H2SO4, and HNO3
(1:1.5:2.5). MIC study was carried out by means of laminar air flow
cabinet, Toshiba, Delhi, India, Thermogravimetric analyses was ob-
1
tained with a model 5000/2960 SDTA, TA instrument (USA). The H
NMR and 13C NMR were recorded with a Bruker Avance (400 MHz).
The electronic spectra were recorded with a UV-160A UV/Vis. spec-
trophotometer, Shimadzu (Japan). The magnetic moments were mea-
sured by Gouy’s method using mercury tetrathiocyanatocobaltate(II)
as the calibrant (χg = 16.44ϫ10–6 cgs units at 20 °C), Citizen Balance.
The diamagnetic correction was made using Pascal’s constant. The
FAB mass spectra were recorded with a Jeol SX 120/Da-600 mass
spectrometer/ data system using argon/xenon (6 kV, 10 mA) as the
FAB gas. The accelerating voltage was 10 kV and spectra were re-
corded at room temperature.
4-(4-Methoxyphenyl)-6-phenyl-2,2Ј-bipyridine (A4): A similar pro-
cedure was followed by taking 1-(2-pyridyl)-3-(4-methoxyphenyl)pro-
pen-1-one as enone. Yield 48.5%, m.p. 107 °C. Anal. C23H18N2O
(338.40): calcd. C 81.63; H 5.36; N 8.28%; found: C 81.50; H 5.49;
1
N 8.40%. H NMR (CDCl3, 400 MHz): δ = 8.797–8.747 (complex, 3
H, H3,3Ј,6Ј), 8.217 (dd, 2 H, H2ЈЈЈ,6ЈЈЈ), 8.000–7.978 (complex, 2 H,
H5,4Ј), 7.860 (d, 2 H, H2ЈЈ,6ЈЈ), 7.555 (t, 2 H, H3ЈЈЈ,5ЈЈЈ), 7.502–7.441
(complex, 2 H, H5Ј,4ЈЈЈ), 7.071 (d, 2 H, H3ЈЈ,5ЈЈ), 3.907 (s, 3 H,
OCH3) ppm. 13C NMR (CDCl3, 100 MHz): δ = 160.78 (C4’’), 157.58
(C2), 157.12 (C2Ј), 153.48 (C6), 150.16 (C4), 146.73 (C6Ј), 139.22
(C1ЈЈЈ), 137.22 (C4Ј), 130.32 (C1ЈЈ), 129.25 (C4ЈЈЈ), 128.81 (C2ЈЈ,6ЈЈ),
128.62 (C3ЈЈЈ,5ЈЈЈ), 127.14 (C2ЈЈЈ,6ЈЈЈ), 124.35 (C5Ј), 122.84 (C3Ј),
118.62 (C3), 117.93 (C5), 114.57 (C3ЈЈ,5ЈЈ), 55.43 (OCH3) ppm.
Synthesis of Ligands
Ligands A1–A8 were prepared by reaction of the appropriate enone
with pyridinium salt.[58] The following procedure is for typical prepa-
4-(4-(Benzyloxy)phenyl)-6-phenyl-2,2Ј-bipyridine (A5): A similar
procedure was followed by taking 1-(2-pyridyl)-3-(4-benzyloxy-
phenyl)propen-1-one as enone. Yield 53.3%, m.p. 117 °C. Anal.
C29H22N2O (414.50): calcd. C 84.03; H 5.35; N 6.76%; found: C
1
ration along with their H NMR and 13C NMR spectroscopic assign-
ments and analytical data.
4-(4-Chlorophenyl)-6-phenyl-2,2Ј-bipyridine (A1): An excess of am-
monium acetate (approx. 10 equiv.) was added to a mixture of 1-(2- 84.20; H 5.50; N 6.88%. 1H NMR (CDCl3, 400 MHz): δ = 8.793
Z. Anorg. Allg. Chem. 2012, 152–162
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
159

M. N. Patel, B. S. Bhatt, P. A. Dosi
ARTICLE
(complex, 3 H, H3,3Ј,6Ј), 8.222 (dd, 2 H, H2ЈЈЈ,6ЈЈЈ), 8.006 (complex, A fine amorphous product of green color was obtained, which was
2 H, H5,4Ј), 7.877 (complex, 2 H, H2ЈЈ,6ЈЈ), 7.558 (t, 2 H, H3ЈЈЈ,5ЈЈЈ), washed with diethyl ether/hexane and dried in vacuo desiccator. Yield
7.507–7.375 (complex, 7 H, H5Ј,4ЈЈЈ,Bz2,3,4,5,6), 7.151 (d, 2 H, 67.8%, m.p. 225 °C. Anal. C39H36Cl2CuFN5O5 (808.22): calcd. C
H3ЈЈ,5ЈЈ), 5.172 (s, 2 H, OCH2) ppm. 13C NMR (CDCl3, 100 MHz):
δ = 159.84 (C4ЈЈ), 157.37 (C2), 156.82 (C2Ј), 154.28 (C6), 149.81
(C4), 147.93 (C6Ј), 139.48 (C1ЈЈЈ), 137.29 (C4Ј), 136.76 (CBz1),
134.52 (C1ЈЈ), 129.15 (C2ЈЈ,6ЈЈ), 129.08 (C3ЈЈЈ,5ЈЈЈ), 128.76 (CBz3,5),
128.66 (CBz4), 128.09 (C4ЈЈЈ), 127.52 (C2ЈЈЈ,6ЈЈЈ), 127.09 (CBz2,6),
123.95 (C5’), 122.15 (C3Ј), 118.26 (C3), 116.44 (C5), 115.44
(C3ЈЈ,5ЈЈ), 70.16 (OCH2) ppm.
57.67; H 4.96; N 8.62; Cu 7.82%; found: C 57.85; H 4.85; N 8.70;
Cu 7.85%.
In similar way, complexes 2–8 were prepared with the use of the corre-
sponding ligands. The proposed reaction scheme is shown in
Scheme 1.
4-(3-Chlorophenyl)-6-phenyl-2,2Ј-bipyridine (A6): A similar pro-
cedure was followed by taking 1-(2-pyridyl)-3-(3-chlorophenyl)pro-
pen-1-one as enone. Yield 54.1%, m.p. 117 °C. Anal. C22H15ClN2
(342.82): calcd. C 77.87; H 4.41; N 8.17%; found: C 77.69; H 4.58;
N 8.29. 1H NMR (CDCl3, 400 MHz): δ = 8.780–8.708 (complex, 3
H, H3,3Ј,6Ј), 8.225(d, 2 H, H2ЈЈЈ,6ЈЈЈ), 7.954 (complex, 2 H, H5,4Ј),
7.840 (s, 1 H, H2ЈЈ), 7.760 (dd, 1 H, H6ЈЈ), 7.564 (complex, 2 H,
H4ЈЈ,5ЈЈ), 7.514–7.450 (complex, 3 H, H3ЈЈЈ,4ЈЈЈ,5ЈЈЈ), 7.427 (dt, 1 H,
H5Ј) ppm. 13C NMR (CDCl3, 100 MHz): δ = 157.51 (C2), 155.78
(C2Ј)148.96 (C6), 148.48 (C4), 143.56 (C6Ј), 140.59 (C1ЈЈ), 139.12
(C1ЈЈЈ), 137.62 (C4Ј), 135.16 (C3ЈЈ), 130.39 (C4ЈЈ)129.32 (C5ЈЈ),
129.07 (C2ЈЈ), 128.81 (C2ЈЈЈ,6ЈЈЈ), 127.45 (C4ЈЈЈ), 127.16 (C3ЈЈЈ,5ЈЈЈ),
125.54 (C6ЈЈ), 124.08 (C5Ј), 121.91 (C3Ј), 118.61 (C3), 117.65 (C5)
ppm.
4-(3-Bromophenyl)-6-phenyl-2,2Ј-bipyridine (A7): A similar pro-
cedure was followed by taking 1-(2-pyridyl)-3-(3-bromophenyl)pro-
pen-1-one as enone. Yield 49.7%, m.p. 132 °C. Anal. C22H15BrN2
(387.27): calcd. C 68.23; H 3.90; N 7.23%; found: C 68.40; H 3.75;
Scheme 1. Reaction scheme for ligand (A1) and complex 1.
1
N 7.06%. H NMR (CDCl3, 400 MHz): δ = 8.763–8.714 (complex, 2
[Cu(CFL)(A2)Cl]·2H2O (2): It was prepared using 6-phenyl-4-p-
tolyl-2,2Ј-bipyridine (1.5 mmol). Yield 66.4, m.p. 215 °C. Anal.
C40H39ClCuFN5O5 (788.72): calcd. C 60.68; H 5.47; N 8.84; Cu
8.03%; found: C 60.59; H 5.55; N 8.76; Cu 8.17%.
H, H3Ј,6Ј), 8.650 (s, 1 H, H3), 8.231 (d, 2 H, H2ЈЈЈ,6ЈЈЈ), 7.990 (s, 1
H, H5), 7.960 (s, 1 H, H2ЈЈ), 7.912 (t, 1 H, H4Ј), 7.780 (d, 1 H, H6ЈЈ),
7.620 (d, 1 H, H4ЈЈ), 7.565 (t, 2 H, H3ЈЈЈ,5ЈЈЈ), 7.496 (t, 1 H, H4ЈЈЈ),
7.436–7.376 (complex, 2 H, H5Ј,5ЈЈ) ppm. 13C NMR (CDCl3,
100 MHz): δ = 157.60 (C2), 154.92 (C2Ј), 149.00 (C6), 148.44 (C4),
143.19 (C6’), 140.70 (C1’’), 139.10 (C1ЈЈЈ), 135.76 (C4Ј), 132.08
(C3ЈЈ), 130.68 (C2ЈЈ), 130.25 (C5ЈЈ), 129.40 (C4ЈЈ), 128.83
(C2ЈЈЈ,6ЈЈЈ), 127.13 (C4ЈЈЈ), 126.10 (C3ЈЈЈ,5ЈЈЈ), 124.28 (C6ЈЈ), 123.23
(C5Ј), 122.23 (C3Ј), 118.78 (C3), 117.93 (C5) ppm.
[Cu(CFL)(A3)Cl]·2H2O (3): It was prepared using 4-(4-bro-
mophenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 61.9.2%, m.p.
236 °C. Anal. C39H36BrClCuFN5O5 (852.67): calcd. C 54.68; H 4.71;
N 8.18; Cu 7.42%; found: C 54.51; H 4.82; N 8.31; Cu 7.35%.
[Cu(CFL)(A4)Cl]·2H2O (4): It was prepared using 4-(4-meth-
oxyphenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 65.2%, m.p.
217 °C. Anal. C40H39ClCuFN5O6 (803.80): calcd. C 59.47; H 5.37; N
8.67; Cu 7.87%; found: C 59.55; H 5.49; N 8.60; Cu 7.99%.
4-(4-Fluorophenyl)-6-phenyl-2,2Ј-bipyridine (A8): Similar pro-
cedure was followed by taking 1-(2-pyridyl)-3-(4-fluorophenyl)pro-
pen-1-one as enone. Yield 52.4%, m.p. 126 °C. Anal. C22H15FN2
(326.37): calcd. C 80.96; H 4.63; N 8.58%; found: C 80.77; H 4.52;
N 8.41%. 1H NMR (CDCl3, 400 MHz): δ = 9.092 (s, 1 H, H3), 8.938–
8.899 (complex, 2 H, H3’,6’), 8.266 (t, 1 H, H4Ј), 8.203 (dd, 2 H,
H2ЈЈЈ,6ЈЈЈ), 8.074 (s, 1 H, H5), 8.015 (dd, 2 H, H2ЈЈ,6ЈЈ), 7.700 (t, 1
H, H5Ј), 7.590–7.498 (complex, 3 H, H3ЈЈЈ,4ЈЈЈ,5ЈЈЈ), 7.258 (t, 2 H,
H3ЈЈ,5ЈЈ) ppm. 13C NMR (CDCl3, 100 MHz): δ = 164.79 (C4ЈЈ),
157.46 (C2), 155.39 (C2Ј), 154.93 (C6), 149.43 (C4), 147.86 (C6Ј),
139.17 (C1ЈЈЈ), 138.29 (C1ЈЈ), 134.56 (C4Ј), 129.29 (C2ЈЈ,6ЈЈ), 129.06
(C3ЈЈЈ,5ЈЈЈ), 127.10 (C2ЈЈЈ,6ЈЈЈ), 124.17 (C4ЈЈЈ), 122.22 (C5Ј), 118.63
(C3Ј), 117.84 (C3), 116.19 (C5), 115.98 (C3ЈЈ,5ЈЈ) ppm.
[Cu(CFL)(A5)Cl]·2H2O (5): It was prepared using 4-(4-(benzyl-
oxy)phenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 71.3%, m.p.
206 °C. Anal. C46H43ClCuFN5O6 (879.8): calcd. C 62.74; H 4.89; N
7.96; Cu 7.22%; found: C 62.88; H 4.73; N 7.85; Cu 7.35%.
[Cu(CFL)(A6)Cl]·2H2O (6): It was prepared using 4-(3-chlo-
rophenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 66.7%, m.p.
220 °C. Anal. C39H36Cl2CuFN5O5 (808.22): calcd. C 57.67; H 4.96;
N 8.62; Cu 7.82%; found: C 57.80; H 4.83; N 8.76; Cu 7.67%.
[Cu(CFL)(A7)Cl]·2H2O (7): It was prepared using 4-(3-bro-
mophenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 62.2%, m.p.
232 °C. Anal. C39H36BrClCuFN5O5 (852.67): calcd. C 54.68; H 4.71;
N 8.18; Cu 7.42%; found: C, 54.81; H 4.83; N 8.04; Cu 7.31%.
Synthesis of Complexes
[Cu(CFL)(A1)Cl]·2H2O (1): A methanol solution of CuCl2·2H2O
(1.5 mmol) was added to methanol solution of 4-(4-chlorophenyl)-6-
phenyl-2,2Ј-bipyridine (1.5 mmol), followed by addition of a pre-
viously prepared solution of ciprofloxacin (1.5 mmol) in methanol in
[Cu(CFL)(A8)Cl]·2H2O (8): It was prepared using 4-(4-fluo-
presence of CH3ONa (1.5 mmol). The pH was adjusted at 6.8 using rophenyl)-6-phenyl-2,2Ј-bipyridine (1.5 mmol). Yield 69.5%, m.p.
dilute solution of CH3ONa. The resulting solution was refluxed for 2
h on a water bath, followed by concentrating it to half of its volume.
218 °C. Anal. C39H36ClCuF2N5O5 (791.76): calcd. C 58.86; H 5.07;
N 8.80; Cu 7.99%; found: C 58.67; H 5.25; N 8.93; Cu 8.11%.
160
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Z. Anorg. Allg. Chem. 2012, 152–162

DNA Hydrolysis, DNA Intercalative, and Electrostatic Interaction Activity
Antibacterial Activity: The MIC was determined using two fold serial where [DNA] is the concentration of DNA in terms of nucleotide phos-
dilutions in liquid media, of the compound being tested against control phate [NP], εa, εf, and εb are the apparent, free and bound metal com-
with no active ingredients, taking DMSO as the solvent. All equipment plex extinction coefficients respectively. Kb is the ratio of the slope to
and culture media were sterilized. The incubation conditions comprise the y-intercept.
Luria Broth as media for culturing of bacteria at 37 °C. This culture
was used as a control to examine if the growth of the bacteria tested
Measurements of Salt Dependence in DNA Binding: The salt de-
pendent equilibrium binding constant (Kb) of copper(II) complex to
was normal. In a similar second set, 20 μL of the bacterial solution as
herring sperm DNA was determined by spectrophotometric titration
well as the tested compound at the desired concentration were added.
over the concentration range 0.005 to 0.100 M NaCl. A fixed amount
We monitored bacterial growth by measuring the turbidity of the cul-
of copper(II) complex in 0.2 M phosphate buffer at pH 7.2 and various
ture after 18 h. If a certain concentration of a compound inhibited
concentrations of NaCl was titrated with increasing amounts of DNA
bacterial growth, half the concentration of the compound was tested.
stock solutions. The hypochromicity and bathochromicity due to metal
This procedure was carried on to a concentration for bacteria grown
complex–DNA interaction was monitored by UV-VIS spectrophotom-
normally. The lowest concentration that inhibited bacterial growth was
eter. The Kb values at various NaCl concentrations were calculated on
the basis of Equation (1).
determined as the MIC value.
The bactericidal action of all compounds was also evaluated against
The salt concentration dependence of Kb for the DNA binding of the
same bacterial culture. The inoculum was prepared by diluting an over-
copper(II) complexes was evaluated by plotting lnKb versus ln[Na+] to
night culture grown in Luria Broth to obtain 106 viable bacteria·mL–1
obtain SK value, which is essential for polyelectrolyte analysis. Each
confirmed in each experiment by colony counts. Bacteria were exposed
measured point was the average value of at least three separate mea-
to concentrations of 0.25–1.75 μg·mL–1 of compounds. The final vol-
surements with a relative standard deviation (RSD) normally less than
ume was 1 mL. Cultures were incubated at 37 °C for 2 h. The 100 μL
15%.
bacterial culture from above was taken and spread over previously
prepared agar plate. These were incubated for 24 h at 37 °C and the
visual colonies were calculated in order to check biocidal activity of
metal complexes, yielding 30–250 colonies.
Gel Electrophoresis: Isolation of plasmid DNA from pure culture of
E. coli was carried out by conventional method.[62] The basic principle
employed is “alkali-lysis”, in which at the alkaline pH, both the geno-
mic and plasmid DNA are denatured. On reduction of the pH the plas-
mid DNA molecule being small in size, quickly reanneals itself, while
the large genomic DNA is not. The denatured genomic DNA is sedi-
mented, while the plasmid DNA remains in solution. Subsequently,
this is precipitated.
DNA Melting Temperature: Thermal denaturation studies were per-
formed with a Perkin–Elmer Lambda 35 spectrophotometer equipped
with a Peltier temperature-controlling programmer (Ϯ0.1 °C). The ab-
sorbance at 260 nm was continuously monitored for solutions of
Herring sperm DNA (100 μM) in the absence and the presence of com-
plex (20 μM). The temperature of the solution was increased by
1 °C·min–1.
For the gel electrophoresis experiments, supercoiled pUC19 DNA in
TE buffer was treated with different CuII complexes; and in the pres-
ence of peroxide hydrogen in the reaction solution. The samples were
incubated for 30 min at 37 °C and a loading buffer containing 10 mmol
TE (pH 7.5), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60%
glycerol, and 60 mmol EDTA was added and electrophoresis was per-
formed at 100 V for 2 h in TAE buffer using 1.0% agarose gel contain-
ing 0.5 μg·mL–1 ethidium bromide. Bands were visualized by UV light
and photographed on a capturing system (AlphaDigiDocTM RT. Ver-
sion V.4.1.0 PC-Image software). For mechanistic investigations, ex-
periments were carried out in the presence of radical scavenging
agents, viz., DMSO (OH· radical) and NaN3 (1O2 radical), which were
added to SC DNA prior to the addition of the complex.
Viscosity Measurement: Viscosity measurements were carried out
using an Ubbelodhe viscometer thermostatted at 27.0Ϯ0.1 °C. Flow
time was measured with a digital stopwatch, three times for each sam-
ple and an average flow time was calculated. Data are presented as (η/
η0)1/3 versus binding ratio,[59] where η and η0 are the viscosity of DNA
in the presence and in absence of the complex respectively. Viscosity
values were calculated from the observed flow time of DNA-contain-
ing solutions (t Ͼ100 s) corrected for the flow time of buffer alone
(t0): η = t–t0.
Absorption Titration: A solution of herring sperm DNA in 0.2 M
phosphate buffer (Na2HPO4 / NaH2PO4, pH 7.2) gave A260/A280 ratio
1.82 UV absorbance indicated that DNA was sufficiently free of pro-
tein. So, no further effort was made on purifying the commercially
obtained DNA. The concentration of DNA was determined by absorp-
Determination of SOD-Like Activity: SOD-like activity of the inves-
tigated complexes was assayed using phenazenmethosulfate (PMS) to
photogenerate a reproducible and constant flux of superoxide anion
radicals at pH = 7.2 (phosphate buffer). Reduction of nitroblue tetrazo-
lium to blue formazan was used as an indicator of O2·– production and
followed spectrophotometrically at 560 nm by the addition of PMS
(30 μM) to an aqueous solution of NBT (75 μM), NADH (79 μM),
0.25 to 2.0 μM complex solution and phosphate buffer.[63] The reaction
in blank samples and in the presence of copper complexes was mea-
sured for 5 min. The plot of time t against varying concentration of
complex is drawn.
tion spectroscopy having
260 nm.[60]
ε
value of 12858 mol–1·cm–1·L–1 at
Spectroscopic titrations were carried out at room temperature to deter-
mine the binding affinity between DNA and the metal complex. The
solutions of varying concentration of DNA (50–150 μM) and metal
complexes (10 μM) were prepared along with blank sample of buffered
DNA solution. After the solutions were mixed for 10 min, the absorp-
tion spectra were recorded. The titration processes were repeated until Supporting Information (see footnote on the first page of this article):
there was no change in the spectra for four titrations at least, indicating Mass fragmentation pattern of compound; DNA binding constant and
that binding saturation was achieved. Afterwards, the observed data IC50 value of compounds; DNA cleavage data; plots for salt concen-
were utilized to acquire the intrinsic binding constant, Kb using Equa-
tion (1):[61]
tration effect on DNA binding and % inhibition of NBT reduction;
gel images for gel electrophoresis experiment in presence of radical
scavenging agents; and mechanism for damaging effect of ROS are
[DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/Kb (εb–εf)
Z. Anorg. Allg. Chem. 2012, 152–162
(1) provided.
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
161

M. N. Patel, B. S. Bhatt, P. A. Dosi
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Published Online: November 29, 2011
162
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Z. Anorg. Allg. Chem. 2012, 152–162