Synthesis, characterization, antibacterial activity and DNA interaction studies of drug-based mixed ligand copper(II) complexes with terpyridines

Article abstract of DOI:10.1007/s00044-011-9799-6

Aseries of ternary copper(II) complexes have been derived using ciprofloxacin and four terpyridine derivatives. Complexes were characterized using metal estimation, IR spectroscopy, FAB-mass spectrometry and reflectance spectra. Compounds were screened fo

Full text of DOI:10.1007/s00044-011-9799-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2723–2733
DOI 10.1007/s00044-011-9799-6
ORIGINAL RESEARCH
Synthesis, characterization, antibacterial activity and DNA
interaction studies of drug-based mixed ligand copper(II)
complexes with terpyridines
•
Mohan N. Patel Promise A. Dosi
•
Bhupesh S. Bhatt
Received: 23 May 2011 / Accepted: 13 September 2011 / Published online: 5 October 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract A series of ternary copper(II) complexes have been
derived using ciprofloxacin and four terpyridine derivatives.
Complexes were characterized using metal estimation, IR
spectroscopy, FAB-mass spectrometry and reflectance spectra.
Compounds were screened for their in vitro antimicrobial
activity against gram(?ve) and gram(-ve) bacterial species.
Binding behaviour of the complexes towards DNA were
determined using UV–Vis absorption titration, viscometric and
DNA thermal denaturation experiment, where as the cleavage
efficacy of the complexes towards pUC19 DNA was deter-
mined by gel electrophoresis in the presence of ethidium bro-
mide. The catalytic activity of the copper(II) complexes
towards the superoxide anion (O•2-) dismutation was assayed
by their ability to inhibit the reduction of nitroblue tetrazolium.
as DNA. The constituents of proteins and nucleic acids
offer adequate sites for metal binding in a specific way and
it can be related to the pharmacological properties of the
complexes. As a result, coordination compounds relevantly
affect cellular processes such as cell division and gene
expression, as well as others like carcinogenicity and
antitumor chemistry (Singh et al., 2005). The factors that
lead the binding modes show that the most important factor
is the molecular shape (Hong et al., 2004).
The interaction of DNA with transition metal complexes
has got intensive attention in the last few years in order to
develop new novel nonradioactive probes of DNA structure
(Erkkila et al., 1999), new therapeutic agents that cleave
DNA (Ren and Chaires 1999; Barton and Raphael 1984;
Chaires et al., 1982) and DNA-mediated electron transfer
reactions (Barton and Raphael 1985). These complexes give
an opportunity to discover the effects of central metal atom,
ligands and coordination geometries on the binding event.
As to the different ligands, it is possible to change the mode
of interaction of the complex with nucleic acids that makes
easy individual applications (Shields and Barton 1995;
Krotz et al., 1993; Sitlani et al., 1992). The application of
octahedral complexes has allowed the targeting of specific
DNA sites by matching the shape, symmetry and func-
tionality of the metal complex to that of the DNA target
(Selvi et al., 2005). Because of the unusual binding prop-
erties and general photo-activity, these coordination com-
pounds will be available candidates as DNA secondary
structure probe, photocleavers and antitumor drugs (Chan
and Wong 1995; Liang et al., 2004; Wang et al., 2004).
The role of copper compounds as pharmaceutical drugs
in the treatment of numerous chronic diseases is well-
established. Numerous copper compounds are able to act as
antioxidant (Naso et al., 2009), antimicrobial (Klayman
et al., 1983), antiparasitic (Agrawal and Santorelli 1978),
Keywords Drug-based complexes of Cu(II) ꢀ FAB-MS ꢀ
Thermal denaturation ꢀ IC50
Introduction
The study of the interaction between DNA with inorganic
compounds is of primary scientific interest, since DNA is
the main target molecule for most anticancer and antibac-
terial therapies according to cell biology. The development
of metal complexes as drugs has been facilitated by the
extensive knowledge of the inorganic chemists in the
coordination and redox properties of metal ions (Sherman
and Lippard 1987). Metal centers being positively charged
are favored to bind with negatively charged molecules such
M. N. Patel (&) ꢀ P. A. Dosi ꢀ B. S. Bhatt
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar 388 120, Gujarat, India
e-mail: jeenen@gmail.com
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Med Chem Res (2012) 21:2723–2733
anti-inflammatory, anticonvulsant (Jones et al., 1965) and
antitumoral agents (Anthroline et al., 1977; Urquiola et al.,
2008). Copper complexes with ligands containing nitro-
genated aromatic rings have deserved a great interest since
the complex of terpyridines proved its ability to break DNA
chains (Zelenko et al., 1998, Morehouse et al., 2000, Bhirud
and Srivastava 1991). The chelation of copper(II) 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
p-electrons over the whole ligand enhances the penetration
of complexes into lipid membranes and blocking of the
metal binding sites in the enzymes of microorganisms
(Chohan et al., 2005). The complexes may also disturb the
respiration process of the cell and thus block the synthesis
of proteins, which restricts further growth of the organism
and thus results in the overall gain in the biological potency.
Herein, we represent the DNA interaction and biological
properties of Cu(II) complexes with the second-generation
quinolone ciprofloxacin and tridentate ligands.
10 mA) as the FAB gas, Massachusetts (USA). The accel-
erating voltage was 10 kV and spectra were recorded at room
temperature. The metal contents of the complexes were
analyzed by Na2H2EDTA titration (Vogel 1978) after
decomposing the organic matter with a mixture of HClO4,
H2SO4 and HNO3 (1:1.5:2.5). At room temperature magnetic
measurement for the complexes was made using Gouy’s
method on Citizen Balance, (India). The Gouy tube was
calibrated using mercury(II)tetrathiocyanatocobaltate(II) as
the calibrant (vg = 16.44 9 10-6 cgs units at 20ꢁC). Photo
quantization of the gel after electrophoresis was done using
AlphaDigiDocTM RT. Version V.4.0.0 PC–Image software,
California (USA).
Synthesis of ligands
40-(4-Benzyloxyphenyl)-2,20:60,200-terpyridine [L1]
An aqueous solution of NaOH (10 mL, 1.5 mmol) was
added to a stirred solution of two moles of 2-acetylpyridine
(l0 mmol) and one mole of 4-benzyloxybezaldehyde
(10 mmol) in ethanol (20 mL) than added ammonia solu-
tion (10 mL). Stirring the solution for 4 h at room tem-
perature, the yellow precipitate was isolated by filtration
and recrystallized from methanol. Yield: 32.4% m.p.:
160–162ꢁC, Anal. Calc. for C28H21N3O (415.49 g/mol):
Calc. (%): C, 80.94; H, 5.09; N, 10.11 Found (%): C,
81.17; H, 4.82; N, 10.37. 1H NMR (CDCl3, 400 MHz)
Experiment
Reagent
All the reagents, chemicals and solvents used were of
analytical grade; double distilled water was used through-
out. Ciprofloxacin hydrochloride was generously supplied
on demand by Bayer AG (Wuppertal, Germany). Cupric
chloride dihydrate was purchased from E. Merck Ltd.
(India). Pyridine, 2-acetyl pyridine, p-benzyloxy benzal-
dehyde, m-benzyloxy benzaldehyde, p-methyl benzalde-
hyde and p-methoxy benzaldehyde were purchased from
Loba Chemie PVT. LTD. (India). Ethidium bromide and
Luria Broth were purchased from Himedia (India). Nico-
tinamide adenine dinucleotide reduced (NADH), NBT and
phenazin methosulphate (PMS) were purchased from Loba
Chemie PVT. LTD. (India). Acetic acid and Na2H2EDTA
were purchased from Sd Fine Chemicals (India). CT DNA
was purchased from Sigma Chemical (India).
0
0
00
d/ppm: 8.79, (s, 2H, H3 ,5 ); 8.76, (dd, 2H, H3,3 ); 8.69,
00
00
(d, 2H, H6,6 ); 7.91, (dt, 2H, H4,4 ); 7.55, (d, 2H, HPh2,6);
7.48–7.37, (complex, 7H); 7.15, (d, 2H, HPh3,5); 5.17,
(s, 2H, CH2), 13C NMR (CDCl3, 100 MHz) d/ppm: 160.58,
0
(CPh4); 156.15, (C2 ,6 ); 155.57, (C2,2 ); 149.8, (C4 );
0
00
0
00
148.94, (C6,6 ); 137.27, (CBz1); 137.13, (C4,4 ); 130.75,
00
(CPh1); 128.86, (CBz3,5); 128.64, (CPh2,6); 127.58, (CBz4);
00
127.13, (CBz2,6); 123.73, (C5,5 ); 121.58, (C3,3 ); 118.12,
00
0
(C3 ,5 ); 114.64, (CPh3,5); 70.7, (CH2).
0
40-(3-Benzyloxyphenyl)-2,20:60,200-terpyridine [L2]
Similar procedure was followed by taking 3-benzyloxy-
bezaldehyde. Yield: 30% m.p.:146–148ꢁC, Anal. Calc. for:
C28H21N3O (415.49 g/mol): Calc. (%): C, 80.94; H, 5.09;
N, 10.11 Found (%): C, 80.76; H, 5.28; N, 10.35. 1H NMR
Physical measurement
00
Elemental analysis (carbon, hydrogen and nitrogen) were
performed on a model Perkin-Elmer 240 elemental analyzer.
The reflectance spectra were recorded on a UV-160A UV–
Vis spectrophotometer, Shimadzu (Japan). Infrared spectra
were recorded on a FT-IR Shimadzu spectrophotometer as
KBr pellets in the range 4000–400 cm-1. The 1H NMR and
13C NMR were recorded on a Bruker Avance (400 MHz).
FAB-mass spectra were recorded on Jeol SX 102/Da–600
mass spectrometer/Data system using Argon/Xenon (6 kV,
(CDCl3, 400 MHz) d/ppm: 8.76, (d, 2H, H3,3 ), 8.72, (s,
0
0
00
2H, H3 ,5 ), 8.67, (d, 2H, H6,6 ), 7.49–7.34, (complex, 11H),
7.28, (d, 1H, HPh6), 7.16, (d, 1H, HPh4), 5.17, (s, 2H, CH2),
13C NMR (CDCl3, 100 MHz) d/ppm: 157.63, (CPh3),
0
0
00
0
00
156.13, (C2 ,6 ), 155.27, (C2,2 ), 151.7, (C4 ), 149.1, (C6,6 ),
00
148.28, (CPh1), 136.86, (C4,4 ), 136.75, (CBz1), 130.26,
(CPh5), 129.03, (CBz3,5), 127.56, (CBz4), 127.08, (CBz2,6),
00
123.7, (C5,5 ), 121.34, (C3,3 ), 119.6, (CPh6), 118.06,
00
0
(C3 ,5 ), 114.76, (CPh2), 113.17, (CPh4), 70.75, (CH2).
0
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Med Chem Res (2012) 21:2723–2733
2725
40-(4-Methoxyphenyl)-2,20:60,200-terpyridine [L3]
00
(d, 2H, H3,3 ), 8.721, (d, 2H, H6,6 ), 7.924, (dd, 2H, H4,4 ),
00
00
00
7.869, (d, 2H, Hph2,6), 7.404, (dd, 2H, H5,5 ), 7.344, (d, 2H,
ph3,5), 2.453, (s, 3H, CH3), 13C NMR (CDCl3, 100 MHz)
Similar procedure was followed by taking 4-methoxybez-
aldehyde. Yield: 34.31% m.p.: 158ꢁC, Anal. Calc. for:
C22H17N3O (339.39 g/mol): Calc. (%): C, 77.86; H, 5.05;
N, 12.38 Found (%): C, 77.65; H, 4.78; N, 12.53. 1H NMR
(CDCl3, 400 MHz) d/ppm: 8.795–8.76, (complex, 4H,
H
0
0
00
0
d/ppm: 156.26, (C2 ,6 ), 155.75, (C2,2 ), 150.18, (C4 ),
00
148.98, (C6,6 ), 139.1, (Cph1), 136.92, (C4,4 ), 135.45,
00
00
(Cph4), 129.66, (Cph3,5), 127.15, (Cph2,6), 123.75, (C5,5 ),
00
121.42, (C3,3 ), 118.7, (C3 ,5 ), 21.25, (CH3).
0
0
0
0
00
H3,3 ,5 ,3 ), 8.71, (d, 2H, H6,6 ), 7.940–7.913, (complex,
00
00
4H), 7.396, (d, 2H, Hph3,5), 7.055, (dd, 2H, H5,5 ), 3.90,
(s, 3H, OCH3), 13C NMR (CDCl3, 100 MHz) d/ppm:
Synthesis of complexes
0
160.57, (Cph4), 156.15, (C2 ,6 ), 155.59, (C2,2 ), 149.81,
0
00
[Cu(cpf)(L1)Cl][I]
0
00
(C4 ), 148.88, (C6,6 ), 137.08, (C4,4 ), 130.63, (Cph1),
00
00
128.55, (Cph2,6), 123.81, (C5,5 ), 121.48, (C3,3 ), 118.41,
00
0
(C3 ,5 ), 114.35, (Cph3,5), 55.38, (OCH3).
0
Cupric chloride dihydrate (1.5 mmol), ciprofloxacin
(1.5 mmol) and 40-(4-benzyloxyphenyl)-2,20:60,200-terpyri-
dine (1.5 mmol) were added in methanol (20 mL) and pH
was adjusted to 6.8 by 10% sodium methoxide solution.
The resulting solution was refluxed for 2 h on a water bath,
followed by concentrating it to half of its volume. A fine
amorphous product of green colour obtained which was
washed with ether/hexane and dried in vacuum desiccators.
The proposed reaction is shown in Scheme 1.
40-(4-Tolyl)-2,20:60,200-terpyridine [L4]
Similar procedure was followed by taking 4-methylbezal-
dehyde. Yield: 1.26 g, 39% m.p.:151–152ꢁC, Anal. Calc.
for C22H17N3 (323.39 g/mol): Calc. (%): C, 81.71; H, 5.30;
N, 12.99 Found (%): C, 81.96; H, 5.13; N, 13.12. 1H NMR
0
0
(CDCl3, 400 MHz) d/ppm: 8.798, (s, 2H, H3 ,5 ), 8.776,
Scheme 1 Reaction scheme for
formation of complex
[Cu(cpf)(L1)Cl]
123

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Med Chem Res (2012) 21:2723–2733
Yield: 62.1%, m.p.: 228ꢁC, leff: 1.86 B.M. Anal. Calc.
compounds. The final volume of bacterial culture was
1 mL and cultures were incubated at 37ꢁC for 2 h. The
100 lL bacterial culture from above was taken and spread
over prepared agar plate. These were incubated for 24 h at
37ꢁC and the visual colonies were calculated in order to
check bactericidal activity of metal complexes, yielding
30–250 colonies.
for: C45H38ClCuFN6O4 (844.82): C, 63.98; H, 4.53; N,
9.95; Cu, 7.52. Found: C, 63.84; H, 4.42; N, 10.06; Cu,
7.44%.
[Cu(cpf)(L2)Cl][II]
It was prepared using 40-(3-benzyloxyphenyl)-2,20:60,200-
terpyridine (1.5 mmol). Yield: 65.6%, m.p.: 218ꢁC, leff:
1.93 B.M. Anal. Calc. for: C45H38ClCuFN6O4 (844.82): C,
63.98; H, 4.53; N, 9.95; Cu, 7.52. Found: C, 64.10; H, 4.44;
N, 10.08; Cu, 7.65%.
DNA interaction activity
Hydrodynamic measurements
Hydrodynamic volume change was measured using
Ubbelohde viscometer immersed in a thermostatic bath
maintained at 27 ( 0.1)ꢁC. Flow times were measured
with a digital stopwatch, each sample was measured three
times, and an average flow time was calculated. Mixing of
test compound and DNA was done by bubbling air to it.
Data are presented as (g/g0)1/3 versus [complex]/[DNA],
where g is the viscosity of DNA in the presence of complex
and g0 is the viscosity of DNA alone. Viscosity values were
calculated from the observed flow time of DNA–containing
solutions (t) corrected for that of the buffer alone (t0),
g = (t-t0) (Basili et al., 2007).
[Cu(cpf)(L3)Cl][III]
It was prepared using 40-(4-methoxyphenyl)-2,20:60,200-ter-
pyridine (1.5 mmol). Yield: 65.6%, m.p.: 230ꢁC, leff: 1.88
B.M. Anal. Calc. for: C39H34ClCuFN6O4 (768.72): C,
60.93; H, 4.46; N, 10.93; Cu, 8.27. Found: C, 61.02; H,
4.59; N, 10.80; Cu, 8.14%.
[Cu(cpf)(L4)Cl][IV]
It was prepared using 40-(4-tolyl)-2,20:60,200-terpyridine
(1.5 mmol). Yield: 61.0%, m.p.: 236ꢁC, leff: 1.82 B.M.
Anal. Calc. for: C39H34ClCuFN6O3 (752.72): C, 62.23; H,
4.55; N, 11.16; Cu, 8.44. Found: C, 62.34; H, 4.66; N,
11.01; Cu, 8.38%.
Absorption titration
The ability of Cu(II) complexes to bind DNA was mea-
sured via DNA-mediated hypochromicity and hyper-
chromicity of the ligand or Cu(II) complex UV–Vis
absorbance spectra (Reichmann et al., 1954; Trommel and
Marzilli 2001; Mudasir and Inoue 1999; Jin and Yang
1997). A solution of CT DNA in 5 mM Tris-HCl buffer
(pH 7.2) gave A260/A280 ratio of 1.9. UV absorbance
indicated that DNA was sufficiently free of protein. So, no
further effort was made on purifying the commercially
obtained DNA. The concentrated stock solution of CT
DNA was prepared, and the concentration of DNA in
nucleotide phosphate was determined by UV absorbance at
260 nm. The molar absorption coefficient of CT DNA was
taken as 6600 mol-1 cm-1 L-1 (Cohen and Eisenberg
1969). Spectroscopic titrations were carried out at room
temperature to determine the binding affinity between
DNA and the metal complex. The solutions of varying
concentration of DNA (50–150 lM) and metal complexes
(15 lM) are prepared along with blank sample of buffered
DNA solution. After the solutions had been mixed for
10 min, the absorption spectra were recorded. The titration
processes were repeated until there was no change in the
spectra for four titrations at least, indicating that binding
saturation had been achieved. The intrinsic binding con-
stant, Kb was determined using equation below (Wolfe
et al., 1987).
Antibacterial activity
Here in, we performed antimicrobial activity by two fold
serial dilution technique in liquid media containing
0.5–3,400 lM variation of test compound concentration
(Alexious et al., 2003), against two gram(?ve) organisms
(Bacillus subtilis and Staphylococcus aureus) and three
gram(-ve) organisms (Escherichia coli, Pseudomonas
aeruginosa and Serratia marcescens). A preculture of
bacteria were grown in LB overnight at the optimal tem-
perature (37ꢁC) for each species. Minimum inhibition
concentration of complexes, ciprofloxacin and metal salt
was determined by their influence on growth of the
microorganism at various concentrations. The lowest con-
centration that inhibited bacterial growth was considered as
the MIC value. All the equipment and culture media used
were sterile.
In addition, minimum inhibitory concentration in terms
of colony forming unit was carried out by colony count. All
the prepared complexes were screened for their activity
against three gram(-ve) and two gram(?ve) bacteria. The
inoculums were prepared by diluting an overnight culture,
grown in LB than culture was diluted 106 times. Bacteria
were exposed to concentrations of 0.25–1.75 lg/mL of
123

Med Chem Res (2012) 21:2723–2733
2727
À
Á
À
Á
À
Á
buffer at pH 7.8, were used for assaying SOD-like activity of
the investigated complexes. The constant flux of superoxide
anion was photogenerated by this system and spectropho-
tometrically detected by monitoring the formation of
monoformazan which absorbs at 560 nm. The NBT reduc-
tion rate was measured in the presence and the absence of the
investigated complex for 3 min. The IC50 (the concentration
of SOD or the complex which causes 50% inhibition of NBT
reduction) of the complex was calculated.
½DNAꢁ= ea ꢂ ef ¼ ½DNAꢁ= eb ꢂ ef þ 1=Kb eb ꢂ ef
where, [DNA] is the concentration of CT-DNA in terms of
nucleotide phosphate [NP], the apparent absorption coef-
ficient ef, ea and eb correspond to the extinction coefficient
of the free complex, the extinction coefficient for each
addition of CT-DNA to the complex and the extinction
coefficient for the complex in the fully bound form,
respectively, and Kb is the ratio of the slope to the
y intercept.
DNA thermal denaturation
Result and discussion
DNA melting experiments were carried out by monitoring
the absorption intensity of CT DNA (100 lM) at 260 nm in
the temperature range from 35 to 90ꢁC with temperature
increments of 0.5ꢁC, both in the absence and presence of
the copper(II) complex (20 lM). Measurements were per-
formed with an Agilent 8453 UV–Vis spectrophotometer.
The melting temperature (Tm) of DNA was determined
as the midpoint of the optically detected transition curves.
The DTm value was defined as the difference between Tm of
the free DNA and Tm of the bound DNA.
Reflectance spectra and magnetism
The magnetic moments of the copper(II) complexes lie in the
1.82–1.93 B.M. range. The observed magnetic moments of
all the complexes correspond to typical high-spin octahedral
complexes. However, the values are slightly higher than the
expected spin-only values due to spin–orbit-coupling con-
tribution (Cotton and Wilkinson, 1988). Copper(II) com-
plexes with octahedral geometry are mock to demonstrate
the absorption spectra in visible region and show one broad
band at about 660 nm (Figgis and Lewis, 1960). Herein our
case, the broad band obtained at kmax = 645 nm points
toward distorted octahedral geometry.
DNA cleavage study
Gel electrophoresis of pUC19 DNA was carried out in TAE
buffer (0.04 M Tris–Acetate, pH 8, 0.001 M EDTA). 15 lL
reaction mixture containing plasmid DNA in TE buffer
(10 mM Tris, 1 mM EDTA, pH 8.0) 300 lg/mL and
200 lM complex. Reactions were allowed to proceed for 3 h
at 37ꢁC. The reactions were satiated by addition of 3 lL
loading buffer (0.25% bromophenol blue, 40% sucrose,
0.25% xylene cyanole and 200 mM EDTA). The aliquots
were loaded directly on to 1% agarose gel and electropho-
resed at 50 V in 19 TAE buffer. Gel was stained with
0.5 lg/mL ethidium bromide and was photographed on a
UV illuminator. The percentage of each form of DNA was
quantities. The degree of DNA cleavage activity was
expressed in terms of the percentage of cleavage of the SC-
DNA according to the following equation (Yang et al., 2000)
Mass spectrometry
FAB-mass spectra of all complexes were obtained using
m-nitro benzyl alcohol as matrix. A mass spectrum of com-
plex [Cu(cpf)(L1)Cl] [1] is shown in Fig. 1. Peaks at 136,
137, 154, 289 and 307 m/z values are of matrix. Peaks at 843
and 845 m/z are assigned to (M) and (M ? 2) molecular ion
peak of complex 1. It can be clearly seen in Fig. 1 that the
peak at 513,515; and 415,417 m/z for the fragments of
complex having single Cl atom. Loss of chlorine atom gave a
fragment ion peak at m/z = 808, which also confirm that
chlorine atom attached to metal ion with covalent bond. The
other fragment corresponds to peaks at 691, 478, 428 and
331 m/z value. The corresponding mass fragments are shown
in supplementary materials.
% DNA clevage
ð% of SC ꢂ DNAÞ control ꢂ ð% of SC ꢂ DNAÞ sample
IR spectra
¼
ð% of SC ꢂ DNAÞ control
ꢃ 100
Ring mode vibrations of the ligands are affected by coor-
dination to Cu(II), 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 substituents of ligand. The prominent IR spectral
data of the complexes are shown in Table 1.
Determination of SOD-like activity
The photoreduction of nitroblue tetrazolium (NBT) was
taken as indicator of O•2- production (Ponti et al., 1978).
Solutions containing the complex (0.5–3.0 lM), NBT
(75 lM), NADH (79 lM) and PMS (30 lM) in phosphate
The complete elimination of broad band at 3010 cm-1
due to –OH stretching of ciprofloxacin carboxylic group
123

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Med Chem Res (2012) 21:2723–2733
Fig. 1 FAB-mass spectrum of
complex 1, that is
[Cu(cpf)(L1)Cl], obtained using
m-nitro benzyl alcohol
Table 1 IR spectra data (cm-1
)
Compounds
m(C=O) pyridone
m(COO)as
m(COO)s
Dm
m(M–N)
m(M–O)
Ciprofloxacin
1708
1617
1620
1622
1621
1624
1564
1574
1582
1586
1340
1363
1372
1376
1384
284
201
202
206
202
–
537
542
539
540
–
505
503
510
512
[Cu(cpf)(L1)Cl] (I)
[Cu(cpf)(L2)Cl] (II)
[Cu(cpf)(L3)Cl] (III)
[Cu(cpf)(L4)Cl] (IV)
can be ascribed to presence of covalent bond between
copper and –COO- of ciprofloxacin. The shifting of m(C=O)
minimum inhibitory concentration (MIC) against bacterial
strain such as E. coli, P. aeruginosa, S. marcescens,
S. aureus and B. subtilis. Table 2 comprises the antibac-
terial activity data from which following conclusion can be
drawn:
stretching vibration band from 1708 to 1617–1622 cm-1
,
confirms ciprofloxacin as coordinating unit in complexes
and the carbonyl oxygen of pyridine ring as coordination
site (Chohan et al., 2005). The characteristic band of car-
bonyl group, i.e. m(COO) asymmetric and symmetric
vibrations, observed as strong absorption band at 1624 and
1340 cm-1 in ciprofloxacin, also shift to 1564–1586 and
1363–1384 cm-1, respectively, in metal complexes. The
difference Dm = mas(COO)-ms(COO) is greater than
200 cm-1, suggests the monodentate coordination behav-
iour of carboxylato group (Chohan et al., 2005; Nakamoto,
1986) of the ciprofloxacin. The band at 537–542 cm-1 are
due to the N–Cu stretch and another at 503–512 cm-1 are
due to the O–Cu stretch (Freedman, 1961). In the investi-
gated ligands, the m(C=N) band of terpyridine derivatives is
observed at about 1584 cm-1. This band shift to higher
frequency at *1626 cm-1 in complexes suggests the tri-
dentate N–N coordination of the ligands (Patel, 1974).
1. S. aureus: All the complexes are more bacteriostatic
compare to ciprofloxacin.
2. B. subtilis: Except complexes IV all are more potent
than ciprofloxacin.
3. S. marcescens: All the complexes are more bacterio-
static compare to ciprofloxacin.
4. P. aeruginosa: Complexes I, II and III exhibit good
activity compare to ciprofloxacin.
5. E. coli: Complexes I, II and III has rather more
potency than ciprofloxacin.
From the data of Table 2, one can conclude that the
complex I, II and III is more active. The compounds show
quite similar antibacterial activity than the Cu(II) com-
plexes synthesized and reported by Eleni et al. against
E. coli, P. aeruginosa and S. aureus (Efthimiadou et al.,
2007a, b).
The good antimicrobial activity is observed may be
studied under following five principles (Dendrinou-Samara
et al., 2001; Russell 1991).
Antibacterial activity
In vitro antimicrobial screening
I
The chelate effect, i.e. ligands that are bound to metal
ions in a bidentate fashion, such as the quinolones
and phenanthroline, bipyridine or bipyridylamine show
Synthesized complexes, ciprofloxacin and metal salt were
checked for their in vitro antibacterial activity in terms of
123

Med Chem Res (2012) 21:2723–2733
2729
Table 2 MIC data of the compounds (lM)
S.aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
CuCl2ꢀ2H2O
Ciprofloxacin
2698.00
1.6
2815.00
1.1
2756.00
1.6
2404.00
1.4
3402.00
1.4
[Cu(cpf)(L1)Cl] (I)
[Cu(cpf)(L2)Cl] (II)
[Cu(cpf)(L3)Cl] (III)
[Cu(cpf)(L4)Cl] (IV)
0.9
0.9
0.5
0.5
0.8
1.2
0.9
0.7
0.6
1.0
1.1
1.0
1.0
0.8
1.3
1.5
1.8
1.8
1.6
1.6
higher antimicrobial efficiency towards complexes with
N-donor ligands.
II Nature of the ligands.
III The total charge of the complex; generally the
antimicrobial efficiency decreases in the order cat-
ionic [ neutral [ anionic complex.
IV The nature of the ion neutralizing the ionic complex
V
The nuclearity of the metal center in the complex;
dinuclear centers are usually more active than mono-
nuclear ones.
Thus, first two factors may be considered for increase in
the activity, i.e. chelate effect.
The study regarding bactericidal activity in terms of
CFU/mL of complexes against three gram(-ve) and two
gram(?ve) microorganisms, which revealed decrease in
number of colonies with increasing the concentration of
compounds. The 1.75 lg/mL was the maximum concen-
tration for all the complexes, which gave lesser number of
colonies. Colonies counted in this technique was 30–250.
The results are shown in Fig. 2 for all the complexes
against S. aureus.
Fig. 2 Relationship between concentration and bactericidal activity
of all complexes against S. aureus
ambiguous and the most critical tests of binding modes in
solution (Patel et al., 2009). The possibility of bending the
helix with larger ring systems and the steric restrictions on
intercalation is supported by viscosity study. A classical
intercalation model demands that the DNA helix lengthens
as base pairs are separated to accommodate the complex,
leading to the increase of DNA viscosity. In contrast,
decrease in DNA viscosity is noted in the partial or non-
classical intercalator, because it may bend DNA helix,
thereby decreasing its effective length and therefore the
viscosity decreases like in ciprofloxacin shown in Fig. 3.
For all the complexes, as increasing the amounts of com-
plexes, the viscosity of DNA increases steadily suggesting
classical intercalative mode of binding.
Metal complex DNA interaction
Hydrodynamic measurement
The geometry 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 interca-
lator, e.g. ethidium bromide; the positively charged poly-
cyclic aromatic compound, which binds to DNA by
inserting itself between the base pairs. Intercalators intro-
duce strong structural perturbations in DNA. The axial
flexibility of DNA in the sense of dynamic flux of changes
in axial and helical parameters allows the compound to
stack between adjacent DNA base pair. The complex is
thought to be stabilized by p-p* stacking interactions
between the compound and DNA bases.
UV–Vis absorption spectra
Optical photophysical probes generally provide neces-
sary, but insufficient clues to support a binding mode.
Hydrodynamic measurements such as viscosity which is
sensitive to length changes are regarded as the least
It is a general observation that the binding of intercalative
molecules to DNA is accompanied by a red shift and
hypochromism in the absorption spectra. The extent
of spectral change is related to the strength of binding
123

2730
Med Chem Res (2012) 21:2723–2733
Fig. 4 Electronic absorption titration curve of [Cu(cpf)(L1)Cl] in the
absence and in the presence of increasing amount of DNA;
50–150 lM in 5 mM Tris-HCl buffer (pH 7.2), [complex] = 15 lM,
[DNA] = 50–150 lM with incubation period of 30 min. at 37ꢁC,
Inset Plot of [DNA]/(ea– ef) versus [DNA]
Fig. 3 Effect on relative viscosity of DNA under the influence of
increasing amount of complexes at 27 0.1ꢁC in 5 mM Tris-HCl
buffer (pH 7.2) as a medium
Table 3 Binding constant (Kb) and IC50 values of copper(II)
complexes
(Long and Barton, 1990; Le Pecq and Paoletti, 1967; Li
et al., 2008; Hirohama et al., 2005). The absorption spectra
of the complex in absence and presence of DNA is illus-
trated in Fig. 4. In order to compare quantitatively the
binding strength of the complexes, the intrinsic binding
constant Kb was obtained by monitoring the changes in
absorbance for complexes with increasing concentration of
DNA. From the Kb value (Table 3) and red shift, it is clear
that complexes bind via intercalation mode. Kb value of all
complexes is found to be in range of 1.05 9 104–
1.30 9 104 M-1. The quinolone Cu(II) complexes with
quite similar Kb value have been reported. (Efthimiadou
et al., 2007a, b). These spectral characteristics are consis-
tent with a mode of interaction that involves a stacking
interaction between the complex and the base pairs of
DNA, which means that the complexes can intercalate into
the double helix structure of DNA.
Complexes
Kb (M-1
)
IC50
[Cu(cpf)(L1)Cl] (I)
[Cu(cpf)(L2)Cl] (II)
[Cu(cpf)(L3)Cl] (III)
[Cu(cpf)(L4)Cl] (IV)
3.01 9 104
2.33 9 104
1.75 9 104
1.57 9 104
0.65
0.78
1.05
1.25
DNA melting experiment
The melting temperature (Tm) of double-stranded DNA
changes with different binding modes. In general, the
melting temperature increases when metal complexes bind
to DNA by intercalation, as intercalation of the complexes
into DNA base pairs causes stabilization of base stacking
and hence raises the melting temperature of the double-
stranded DNA. DNA melting experiments are useful in
establishing the extent of intercalation (Patel et al., 2009).
A large change in the Tm of DNA is indicative of a strong
interaction with DNA. The effect of complexes on the
melting temperature of CT DNA in buffer is shown in
Fig. 5. At the melting temperature, the double helix
denatures into single-stranded DNA. The thermal
Fig. 5 Melting curves of CT DNA in the absence and the presence of
complexes 1–5
denaturation experiment carried out for CT DNA in the
absence of complex gave a Tm 74.2 1ꢁC under our
experimental conditions. The observed melting tempera-
tures in the presence of complexes were 79.3 1ꢁC,
78.8 1ꢁC and 78.2 1ꢁC and 77.9 1ꢁC for com-
plexes I, II, III and IV, respectively. The DTm values of the
123

Med Chem Res (2012) 21:2723–2733
2731
DNA were increases in presence of the complexes and
comparable to those observed for classical intercalation
and suggest that the complex has a moderate DNA-binding
affinity.
Table 4 Gel electrophoresis data
Compounds
%SC
%OC
%LC
% Cleavage
DNA Control
DNA ?Metal salt
DNA ?CPFH
DNA ?I
75
72
67
17
18
20
27
25
28
33
50
51
50
58
–
–
4.00
10.66
77.33
76.00
73.33
64.00
–
Gel electrophoresis
33
31
30
15
DNA ?II
There has been considerable interest in DNA cleavage
reactions that are activated by transition metal complex
(Hertzberg and Dervan 1982; Kumar et al., 2008). The
ability of the complexes to interact double stranded DNA
was examined by agarose gel electrophoresis. The principle
of this method is that molecules migrate in the gel as a
function of their mass, charge and shape, with supercoiled
DNA migrating faster than open circular molecules of the
same mass and charge. When circular plasmid DNA is
subject to electrophoresis, relatively fast migration is
generally observed for the intact supercoiled form. When
scission occurs on one strand (nicking), the supercoil (SC)
relaxes to generate a slower-moving, open-circular (OC)
form. When both strands are cleaved, a linear form (L) is
generated, which migrates between SC form and OC form
DNA. Figure 6 illustrates the gel electrophoretic separa-
tions that show cleavage of pUC19 DNA induced by the
complexes under aerobic conditions. Results clearly show
that the relative binding efficiency of complexes to DNA is
much higher than the binding efficacy of metal salt or
ciprofloxacin (Table 4). The similar behaviour of Cu(II)
complexes with plasmid DNA is shown by compounds of
type [Cu(Hpr-norf)(phen)Cl], [Cu(Hpr-norf)(bipy)Cl],
[Cu(erx)(phen)Cl], [Cu(erx)(bipy)Cl] reported by Katsaros
et al. (2008).
DNA ?III
DNA ?IV
Fig. 7 Plot of absorbance (Abs560) as a function of time (t) for
different concentration complex 1 ranging from 0.5–3 lM
The complexes show the different extent of superoxide
scavenging ability. The higher IC50 can be accredited to the
vacant coordination site facilitating the binding of super-
oxide anion, electrons of aromatic ligands that stabilize
Cu–O2- interaction and not only to the partial dissociation
of complex in solution (Roberts and Robinson 1985).
Difference in electron negativity on terpyridine can also be
taken under consideration for difference in affinity of
complexes towards reactive specie.
SOD-like activity
The measured IC50 values for SOD-like activity at bio-
logical pH ranges from 0.65 to 1.25 lM (Table 3). Fig-
ure 7 represents plot of absorbance values (Abs560) against
time (t) with varying concentration of complex I from 0.5
to 3 lM. Figure 8 illustrate that chromophore requires to
yield 50% inhibition of the reduction of NBT (IC50) value
of complexes are higher than exhibited by the copper salt.
Structure–activity-relationship
The SAR studies revealed that, the fluorine atom and the
1-alkyl, 1,4-dihydro-4-oxo-quinoline-3-carboxylic acid
skeleton of fluoroquinolones is responsible for potency
represented in binding with DNA. Different substituents on
the neutral tridentate ligands are also responsible for
increase and decrease in biological activities. The result
concerning biological aspects of metal complexes show
that electron-withdrawing substituent on the intercalative
ligand can improve the DNA interaction affinity, free
radical scavenging activity and antibacterial activity of
the original complex, whereas the case is reversed for
Fig. 6 Photogenic view of interaction of pUC19 DNA (300 lg/mL)
with series of copper(II) complexes (200 lM): Lane 1 DNA control;
Lane 2, CuCl2ꢀ2H2O; Lane 3 ciprofloxacin; Lane 4 [Cu(cpf)(L1)Cl];
Lane
5
[Cu(cpf)(L2)Cl]; Lane
6
[Cu(L3)(cpf)Cl]; Lane
7
[Cu(cpf)(L4)Cl]
123

2732
Med Chem Res (2012) 21:2723–2733
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