Cytotoxic, DNA interaction, SOD mimic, and antimicrobial activities of square pyramidal copper(II) complexes

Article abstract of DOI:10.1002/zaac.201200052

The copper(II) complexes with NS donor ligand and ciprofloxacin were synthesized. The synthesized complexes were characterized by physicochemical parameters like elemental and thermal analysis, electronic, FT-IR, and LC-MS spectroscopy. The complexes were

Full text of DOI:10.1002/zaac.201200052

ARTICLE
DOI: 10.1002/zaac.201200052
Cytotoxic, DNA Interaction, SOD Mimic, and Antimicrobial Activities of
Square Pyramidal Copper(II) Complexes
Mohan N. Patel,*[a] Chintan R. Patel,[a] and Hardik N. Joshi[a]
Keywords: NS donor ligand; Ciprofloxacin; Antibacterial activity; DNA interaction; Cytotoxicity; Copper
Abstract. The copper(II) complexes with NS donor ligand and cipro-
floxacin were synthesized. The synthesized complexes were charac-
drug. Viscosity measurement and absorption titration were employed
to determine the mode of binding of complexes with DNA. DNA
terized by physicochemical parameters like elemental and thermal cleavage activity was carried out by gel electrophoresis experiment
analysis, electronic, FT-IR, and LC-MS spectroscopy. The complexes using supercoiled form of pUC19 DNA. The complexes were screened
were tested for their antibacterial activity against two Gram(+ve) i.e.
Staphylococcus aureus, Bacillus subtilis and three Gram(–ve) i.e., Ser-
for their SOD mimic activity in terms of IC50 value. The complexes
were also checked for their cytotoxicity using brine shrimp assay
ratia marcescens, Pseudomonas aeruginosa, Escherichia coli bacteria method.
in terms of MIC (μM) and the results were compared with the parent
costs. To avoid such limitations, there has been a considerable
1 Introduction
interest in developing synthetic SOD mimics that have low
molecular weight, biological stability, membrane permeability,
nontoxic and low cost.[15,16]
Quinolones are synthetic antibacterial agents widely used
in clinical practice. Ciprofloxacin [1-cyclopropyl-6-fluoro-1,4-
dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid]
is a member of this large family and is used for the treatment
of certain diseases caused by various Gram(-ve) and some
Gram(+ve) positive microorganisms.[1] Fluoroquinolone drugs
act intravenously by inhibiting topoisomerase II (DNA gyrase)
or topoisomerase.[2] It is generally accepted that the quinolone
target the bacterial enzyme gyrase–DNA complex, which is
responsible for the supercoiling of bacterial DNA.[3] Many
transition metal complexes are used as potential probes of nu-
cleic acid structure and for potential application as drugs.[4]
The complexes of vanadium(IV), copper(II), magnesium(II),
uranium(VI), manganese(II), iron(III), cobalt(II), nickel(II),
molybdenum(II), and europium(III) with ciprofloxacin have
been synthesized and explored for their biological activities
because of its biological relevance.[5–12]
In continuation of our previous work,[17] herein we report
ternary CuII complexes with ciprofloxacin and neutral biden-
tate ligands with NS donor atom. In-vitro antimicrobial activity
was performed against two Gram(+ve) and three Gram(–ve)
microorganisms using double dilution method. The synthe-
sized complexes were checked for their DNA interactions
using absorption titration, viscosity measurement, and gel elec-
trophoresis. The SOD mimic behavior of the complexes was
checked under non-enzymatic condition. The synthesized com-
plexes were also checked for their cytotoxic effect using brine
shrimp lethality bioassay.
2 Result and Discussion
Superoxide anion (O2·–) is essential for the biological de- 2.1 Synthesis and Characterization of Complexes
fence system against the invasion of bacteria and viruses, but it
The ternary CuII complexes were prepared by the reaction
is also known for its pathogeneses of many disease processes,
of CuII chloride with ciprofloxacin and different bidentate NS
including inflammatory damage and DNA damage and
donor ligands (A1–A6) in a ratio of 1:1:1 refluxed for 3 h on
aging.[13,14] Use of synthetic SOD mimic for pharmaceutical
water bath maintaining pH = 6.8 of the reaction mixture. A
purposes are problematic due to difficulties associated with the
fine amorphous product obtained was washed with chloroform
systematic infection of protein, i.e. the circulation lifetime, cell
and dried in vacuum. The synthesized complexes were charac-
impermeability, immunogenicity, tissue, antigenicity, and high
terized by FT-IR, UV/Vis spectroscopy, magnetic measure-
ment, thermogravimetric analysis and LC-MS techniques. All
the complexes are insoluble in water, ethanol, methanol,
dichloromethane, chloroform, acetonitrile, hexane, and DMF,
whereas they are soluble in DMSO, so it is difficult to grow
crystals for X-ray diffraction analysis. All the physicochemical
parameters data are in good agreement and suggest the square
pyramidal arrangement around the central metal ion. The pro-
* Prof. Dr. M. N. Patel
E-Mail: jeenen@gmail.com
[a] Department of Chemistry
Sardar Patel University
Vallabh Vidyanagar-388 120
Gujarat, India
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201200052 or from the au-
thor.
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Square Pyramidal Copper(II) Complexes
Scheme 1. General synthesis of ligands and proposed structures.
posed structure and general synthesis of ligands and complexes 2.1.2 IR Spectroscopy
are shown in Scheme 1 and Scheme 2, respectively.
In the IR spectrum of ciprofloxacin the valence vibration of
the carboxylic stretch ν(C=O)carb is found at 1708 cm–1 and
the pyridone stretch ν(C=O)p at 1624 cm–1. The characteriza-
tion of quinolones metal complexes can be achieved by study-
ing the most typical vibrations that are characteristic of the
coordination type of quinolones.
On complexation, these bands appear between 1575–1584
and 1352–1360 cm–1. The frequency of separation Δν =
ν(COO)as – ν(COO)s in investigated complexes is ca. 200 cm–1,
suggesting the unidentate nature for the carboxylato
group.[20,21] A sharp band at 3520 cm–1 due to stretching vi-
bration of free hydroxyl in quinolone moiety is completely dis-
appeared in the spectra of complexes.[22] The band at 1624 cm–1
responsible for ν(C=O)p in ciprofloxacin is observed between
1622–1632 cm–1 in case of complexes.[12,23] These data are
further supported by the weak bands observed at 575–540,
525–490, and 485–440 cm–1, which are assigned to ν(M–O),
ν(M–N),[24,25] and ν(M–S)[26] vibrations, respectively
(Table 2).
Scheme 2. Proposed structures and general synthesis of complexes.
2.1.3 Electronic Spectra and Magnetic Data
A visible emission spectra of the copper(II) complexes i.e.
d9 system were recorded in DMSO. The complexes exhibited
2.1.1 Spectrophotometric Titration
The amount of copper was determined by spectrophotomet- the only broad peak at λmax = ca. 660 nm, which was attributed
ric titration technique. Sample solutions were prepared by de- to d–d transition, in which the CuII atom was in a distorted
composing organic matter of complex with acid mixture and square pyramidal environment.[27,28] The possibility of trigonal
making it to total volume of 20 mL with double distilled water. bipyrimidal arrangement at the central metal atom was ruled
Ten different sets of solutions were prepared by taking a fixed out because a peak of λmax greater than 800 nm along with a
amount of complex solution (2 mL), 2 mL acetate buffer solu- shoulder at ca. 660 nm was not observed in the case of synthe-
tion and varying aliquots of 0.0005 M EDTA and making it to sized complexes.[29,30]
total volume of 10 mL with double distilled water. Absorbance
The magnetic moments measurement for any arrangement
was measured at 745 nm using buffer as a reference. The in copper(II) complexes generally results in the range 1.76–
amount of copper was determined using the plot of absorbance 1.88 BM, which is very close to the spin-only value i.e.
against different volume of EDTA used during the spectropho- 1.73 BM. The observed values in our case were very close to
tometric titration.[18,19] The calculated results from the equiva- the spin-only values (Table 1) for single unpaired electrons and
lent endpoint reveal the metallic content of the complexes confirm the copper in +2 state with d9 configuration
6
3
(Table 1 and Supporting Information).
(t2g eg ).[31,32]
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M. N. Patel, C. R. Patel, H. N. Joshi
ARTICLE
Table 1. Theoretical and found copper content for the synthesized complexes 1–6.
1
2
3
4
5
6
Cu experimental /%
Cu theoretically /%
7.73
7.64
8.09
7.81
7.49
7.5
7.26
7.12
8.01
7.68
7.66
7.54
Table 2. Characteristic absorptions bands of IR spectra of the complexes and CPFH /cm–1.
ν(C=O) pyridone
ν(COO)as
ν(COO)s
Δν
ν(M–O)
ν(M–N)
ν(M–S)
CPFH
1624
1632
1627
1622
1625
1630
1629
1708a
1584
1577
1575
1577
1576
1578
–
–
–
–
–
[Cu(CPF)A1Cl]·2H2O (1)
[Cu(CPF)A2Cl]·2H2O (2)
[Cu(CPF)A3Cl]·2H2O (3)
[Cu(CPF)A4Cl]·2H2O (4)
[Cu(CPF)A5Cl]·2H2O (5)
[Cu(CPF)A6Cl]·2H2O (6)
1356
1360
1352
1354
1355
1358
228
217
223
223
221
220
575
560
545
570
565
540
490
520
515
505
510
525
470
485
450
465
455
440
a) As ν(COOH).
2.1.4 LC-MS Spectra Analysis
due to fragmentation of bidentate ligand and the peak at 247.14
m/z corresponds to loss of piperidine moiety from ciproflox-
acin. The peak at 203.21 m/z is due to the loss of the COOH
group from fragment of ciprofloxacin.
Material 2 (Supporting Information) shows the mass spec-
trum of complex [Cu(CPF)(A1)Cl]·2H2O. The mass spectrum
of complex 1 (Figure 1, mass fragmentation pattern of complex
1) shows molecular ion peak [M+] at 795.18 m/z and [M + 2]
at 797.19 m/z. Peaks at 464.85 m/z and 466.55 m/z are due to
ligand attached with copper and one chlorine atom. The peaks
at 429.40 m/z and 431.33 m/z correspond to the removal of
2.1.5 Thermogravimetric Analysis
TGA data indicate that complex 1 decomposes in three
coordinated chlorine atom from the central metal ion. The peak steps.[33] From the characteristic thermogravimetric curve
at 429.33 m/z is due to ciprofloxacin attached to copper(II) (mass loss in % to temperature in °C), it is clear that loss
including coordinated chlorine atom. The peaks at 365.85 m/z occurring during the first step i.e. 40–120 °C (found ca. 4.5%,
and 367.75 m/z are due to the loss of copper from neutral theoretical 4.33%) corresponds to loosely bonded two mole-
bidentate ligand. The peak at 331.15 m/z corresponds to cipro- cules of water of crystallization. The mass lose (found ca.
floxacin moiety. The peaks at 283.10 m/z and 285.15 m/z are 44%, theoretical 44.13%) during the second step (200–
Figure 1. Proposed mass fragmentation pattern of [Cu(CPF)A1Cl]·2H2O (1).
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© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Square Pyramidal Copper(II) Complexes
Table 3. Antimicrobial activities of CPFH, copper(II) salt, and complexes 1–6 in terms of Minimum Inhibitory Concentration (MIC) /μM.
Gram positive
Gram negative
S.aureus
B.subtilis
S.marcescens
P .aeruginosa
E. coli
CuCl2·2H2O
CPFH
2698.0
1.52
0.72
0.96
0.75
0.80
1.28
1.50
2815.0
1.04
0.20
0.50
0.25
0.40
0.70
0.96
2756.0
1.52
0.40
0.80
0.45
0.48
0.95
1.60
2404.0
1.32
0.82
1.15
0.90
1.10
1.25
1.32
3402.0
1.36
0.80
1.10
0.85
0.94
1.30
1.35
[Cu(CPF)A1Cl]·2H2O (1)
[Cu(CPF)A2Cl]·2H2O (2)
[Cu(CPF)A3Cl]·2H2O (3)
[Cu(CPF)A4Cl]·2H2O (4)
[Cu(CPF)A5Cl]·2H2O (5)
[Cu(CPF)A6Cl]·2H2O (6)
470 °C) corresponds to loss of ciprofloxacin and chlorine. In creasing the concentration of complexes. The results are shown
the third step mass loss (found ca. 44.10%, theoretical in Figure 2 for all the complexes against B. subtilis (see Sup-
44.01%) corresponds to decomposition of neutral bidentate li- porting Information). The number of colonies counted in this
gand (470–835 °C) occurs, leaving behind metal oxide as resi- technique was 30–300. From the minimum inhibitory concen-
due (found ca. 9.70%, theoretical 9.57%) (see Supporting In- tration (MIC) values and colony forming units (CFU), one can
formation).
conclude that complex 1 showed more potency against all the
five microorganisms than other complexes.
2.2 Biological Evaluation
2.2.1 In-vitro Antimicrobial Screening
Synthesized complexes, ciprofloxacin, and metal salt were
checked for their in-vitro antibacterial activity in terms of
minimum inhibitory concentration (MIC) against bacterial
strain such as E. coli, P. aeruginosa, S. aureus, B. Subtilis, and
S. marcescens (Table 3).
Following conclusion is drawn from the antibacterial data:
(1) S. aureus: Complexes 1, 2, 3, and 4 are more active
compared to other complexes and ciprofloxacin.
(2) B. subtilis: All the complexes show better activity com-
pare to ciprofloxacin.
(3) S. marcescens: Except complex 6 all other complexes
are more potent than ciprofloxacin.
(4) P. aeruginosa: Complexes 1, 3 and 4 are more active
than other complexes and ciprofloxacin.
Figure 2. Plot of log(CFU/mL) versus concentration (µg/mL) for all
complexes against B. subtilis.
(5) E. coli: Complexes 1, 2, 3, and 4 are more active com-
pared to other complexes and ciprofloxacin.
The good antimicrobial activity is observed may be studied
under following five principles.[34]
2.2.2 DNA Binding Study by Absorption Titration
DNA can provide three distinctive binding sites for the quin-
(1) The chelate effect, i.e. ligands that are bound to metal olone complexes; namely, groove binding, binding to phos-
ions in a bidentate fashion, such as the quinolones and phenan- phate groups, and intercalation. This behavior is of great im-
throline, bipyridine, or bipyridylamine show higher antimicro- portance with regard to the biological role of fluoroquinolone
bial efficiency towards complexes with nitrogen donor ligands. antibiotics in the human body.[35] When complex interacts with
(2) Nature of the ligands.
DNA by intercalative mode it results in hypochromism and red
(3) The total charge of the complex; generally the antimicro- shift, as intercalative mode involving a strong stacking interac-
bial efficiency decreases in the order cationic Ͼ neutral Ͼ an- tion between the aromatic chromophore and the base pairs of
ionic complex.
(4) The nature of the ion neutralizing the ionic complex.
(5) The nuclearity of the central metal atom in the complex; interaction.[36] It is a general observation that the binding of
dinuclear centers are usually more active than mononuclear intercalative molecules to DNA is accompanied by a red shift
ones.
Thus, first two factors may be considered for increase in the spectral change is related to the strength of binding. The ab-
activity i.e. chelate effect. sorption spectra of the complex in absence and presence of
DNA. The magnitude of the hypochromism and red shift are
commonly found to depend on the strength of the intercalative
and hypochromism in the absorption spectra.[37] The extent of
In addition, our study regarding bactericidal activity in terms DNA is illustrated in Figure 3. In order to compare quantita-
of CFU·mL–1 of above metal complexes against same microor- tively the binding strength of the complexes, the intrinsic bind-
ganisms reveals that, decrease in number of colonies with in- ing constant Kb was obtained by monitoring the changes in
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M. N. Patel, C. R. Patel, H. N. Joshi
ARTICLE
absorbance for complexes with increasing concentration of
DNA. This is lower than Kb value of classical intercalator ethi-
dium bromide but higher than reported CuII complexes[38–41]
and different metal quinolone complexes.[42,43] From the Kb
value and red shift, it is clear that the complexes bind by inter-
calation mode and complex 1 has the highest binding ability
(Table 4).
Figure 4. Effects of increasing amounts of EtBr, CPFH and complexes
on the relative viscosity of herring sperm DNA at 37 Ϯ 0.1 °C.
2.2.4 DNA Cleavage Study by Gel Electrophoresis
There has been considerable interest in DNA cleavage reac-
tions that are activated by transition metal complexes.[45,46]
Agarose gel electrophoresis was used as a base for monitoring
plasmid DNA cleavage reaction. When plasmid DNA is sub-
jected to electrophoresis, relatively fast migration is observed
for the intact supercoil form (Form I). The scission on one
strand (nicking) will relax supercoil to generate a slower-mov-
ing open circular (Form II), and if both strands are cleaved, a
linear (Form III) that migrates between Form I and Form II
will be generated.[47] The data of the cleavage study obtained
from Figure 5 are presented in Table 5, which show that syn-
thesized complexes have better cleavage ability than metal salt
and ciprofloxacin.
Figure 3. Electronic absorption spectra of [Cu(CPF)A1Cl]·2H2O (1)
in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2) in the absence and
presence of increasing amount of DNA. The [Cu] complex = 10 μM;
[DNA] = 0–150 μM. The incubation period is 10 min. at room tem-
perature, Inset: Plot of [DNA]/(εa–εf) versus [DNA]. Arrow shows the
absorbance change upon increasing DNA concentrations.
Table 4. Binding constant (Kb), IC50, and LC50 values of synthesized
complexes.
Kb /M–1
IC50 /μM
LC50 /μM
[Cu(CPF)A1Cl] ·2H2O (1) 3.17ϫ105
[Cu(CPF)A2Cl] ·2H2O (2) 1.36ϫ105
[Cu(CPF)A3Cl] ·2H2O (3) 2.42ϫ105
[Cu(CPF)A4Cl] ·2H2O (4) 2.16ϫ105
[Cu(CPF)A5Cl] ·2H2O (5) 0.88ϫ105
[Cu(CPF)A6Cl] ·2H2O (6) 0.49ϫ105
0.59
0.97
0.68
0.83
1.03
1.11
6.02
10.30
7.96
10.02
16.73
19.28
Figure 5. Photogenic view of cleavage of pUC19 DNA (300 µg/mL)
with series of copper(II) complexes (200 µM) using 1% agarose gel
containing 0.5 µg/mL ethidium bromide. All reactions were incubated
in TE buffer (pH 8) in a final volume of 15 µL, for 24 h. at 37 °C.
Lane 1, DNA control; Lane 2, CuCl2·2H2O; Lane 3, Ciprofloxacin;
Lane 4, [Cu(CPF)A1Cl]·2H2O (1); Lane 5, [Cu(CPF)A2Cl]·2H2O (2);
Lane 6, [Cu(CPF)A3Cl]·2H2O (3); Lane 7, [Cu(CPF)A1Cl]·2H2O (4);
Lane 8, [Cu(CPF)A5Cl]·2H2O (5); Lane 9, [Cu(CPF)A6Cl]·2H2O (6).
2.2.3 DNA Binding Study by Viscosity Measurement
Optical photophysical probes provide necessary, but insuf-
ficient clues to support binding mode. In absence of crystallo-
graphic structural data viscosity measurement, sensitive to
length change is regarded as the least ambiguous and the most
critical tests for determining binding mode in solution state.[44]
A classical intercalation model usually resulted in lengthening
the DNA helix, as base pairs were separated to accommodate
the binding ligand leading to the increase of DNA viscosity.[41]
Intercalation of a molecule into DNA could result in lengthen-
ing, unwinding, and stiffening of the helix and is usually ac-
2.2.5 SOD-like Activity
The system used as a source of superoxide radical generator
companied by increase in solution viscosity.[45] Figure 4 shows in order to check SOD like activity for the synthesized com-
the binding ability of ethidium bromide, complexes, and cipro- plexes was the NBT/NADH/PMS system. The percent inhibi-
floxacin. In our case increase in viscosity was observed, which tion of formazan formation at various concentrations of com-
was less compared to classical intercalator ethidium bromide plexes as a function of time was determined by measuring the
and more compared to ciprofloxacin. Hence, complexes bind absorbance at 560 nm and plotted to have a straight line obey-
to DNA by classical intercalation mode and complex 1 inter- ing equation Y = mX + C (Figure 6); with increase in concen-
acts more strongly compared to the other complexes.
tration of tested complexes deterioration in slope (m) was ob-
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Square Pyramidal Copper(II) Complexes
Table 5. Complex mediated DNA cleavage data by gel electrophoresis.
Lane No.
Compound
Form I
SC
Form II
OC
Form III
L
Cleavage /%
1
2
3
4
5
6
7
8
9
Control
CuCl2·2H2O
Ciprofloxacin
82
76
61
10
20
18
20
24
29
18
24
39
60
51
52
54
49
41
–
–
–
30
29
31
26
27
30
–
7.31
25.60
87.80
73.17
78.04
75.60
70.73
64.63
[Cu(CPF)A1Cl]·2H2O (1)
[Cu(CPF)A2Cl]·2H2O (2)
[Cu(CPF)A3Cl]·2H2O (3)
[Cu(CPF)A4Cl]·2H2O (4)
[Cu(CPF)A5Cl]·2H2O (5)
[Cu(CPF)A6Cl]·2H2O (6)
Figure 6. Plot of absorbance (Abs560) as a function of time (t) to determine % inhibition of formazan formation at various concentrations of
complex 1 (0.5 µm to 3 µm) as function of time and the plot of percentage of inhibiting NBT reduction with an increase in the concentration
of complex 1.
served. Percent inhibition of the reduction of nitro blue tetrazo- bioassay. And among all the complexes, complex 1 shows
lium (NBT) was plotted against the concentration of the com- maximum activity.
plex 1 to obtain IC50 values (Figure 6). Complexes exhibit
SOD-like activity at biological pH with their IC50 values rang-
ing from 0.59 to 1.11 μM. The superoxide scavenging data are
presented in Table 4. The best IC50 value among synthesized
complexes is observed for complex 1. The higher IC50 can
3 Conclusions
only be accredited to the vacant coordination site facilitating
the binding of superoxide anion, electrons of aromatic ligands
that stabilize Cu–O2 interaction and not only to the partial
dissociation of complexes in solution. The mechanism for
scavenging of superoxide radical moves forward via unstable
octahedral adduct under the influence of Jahn-Teller ef-
fect,[48,49] and there is a possibility of rapid inter-conversion
between CuII and CuI via electron transfer between copper and
reactive oxygen radical anion following the principle of elec-
troneutrality.[50]
Data of magnetic behavior and electronic spectral measure-
ment points towards the d9 system with distorted square py-
ramidal arrangement. The electronic absorption data are in
good accordance with viscosity titration curves. The reason
behind increase in potency of drug is due to its coordination
with metal ion. The data from SOD mimic activity suggests
that, the higher activity is due to vacant coordination site at
the central metal ion. The DNA cleavage study of pUC19
shows that all complexes have higher cleavage ability than
metal salt and drug. Such a trend in result shows that the DNA
interaction ability, free radical scavenging ability, antimicrobial
activity, and cytotoxicity of the synthesized complexes can be
controlled by the substituent of the intercalative ligand. It also
–
2.2.6 Cytotoxic Activity - Brine Shrimp Lethality Bioassay
From the data, percent of mortality was calculated and plot- concluded from the results that as the electron withdrawing
ted against log dose (see Supporting Information). LC50 values ability of substituent (F, Cl, Br) on the intercalative ligand in-
of test complexes observed after 24 h are shown in Table 4. creases, the DNA interaction ability, free radical scavenging
From the results, it is found that the complexes show consider- ability and antimicrobial activity increases and vice-versa for
able cytotoxicity in the brine shrimp (Artemia cysts) lethality the electron releasing substituent (CH3, OCH3).
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M. N. Patel, C. R. Patel, H. N. Joshi
ARTICLE
m.p. Ͼ300 °C, μeff: 1.80 B.M. m/z = 795.18. C38H34Cl2CuF2N4O5S
(831.21): calcd. (found) C 54.91 (55.10); H 4.12 (4.01); N 6.74 (6.67);
4. Experimental Section
4.1 Materials and Reagents
S 3.86 (3.92), Cu 7.64 (7.73)%. λmax(solid) = 676 nm, λmax(solution)
656 nm, ε = 94.11 M–1·cm–1 (1.02ϫ10–3M).
=
All solvents, chemicals, and reagents used were of analytical reagent
grade and were used as such; double distilled water was used through-
out. Ciprofloxacin (CPFH) was purchased from Bayer AG (Wuppertal,
Germany). Cupric chloride was purchased from E. Merck (India) Ltd.
Mumbai. Benzaldehyde, 4-fluoro benzaldehyde, 4-chloro benzalde-
[Cu(CPF)A2Cl]·2H2O (2): prepared by using 2-(4-chlorophenyl)-4-
phenyl-6-(thiophen-2-yl)pyridine (A2) (1 mmol). Yield 49%, m.p.
Ͼ300 °C, μeff: 1.84 B.M. m/z = 777.02. C38H35Cl2CuFN4O5S
(813.22): calcd (found) C 56.12 (56.60); H 4.34 (4.45); N 6.89 (7.05);
hyde, 4-bromo benzaldehyde, 4-methyl benzaldehyde, 4-methoxy S 3.94 (4.05), Cu 7.81 (8.09)%. λmax(solid) = 680 nm, λmax(solution)
=
benzaldehyde, and 2-acetyl thiophene were purchased from Spectro-
chem Pvt. Ltd. Mumbai (India). Luria Broth, agarose, ethidium brom-
ide, TAE (tris-acetyl-EDTA), bromophenol blue, and xylene cyanol FF
were purchased from Himedia, India. Nicotinamide adenine dinucleo-
tide reduced (NADH), nitro blue tetrazolium (NBT) and phenazin me-
thosulphate (PMS) were purchased from Loba Chemie Pvt. Ltd. Her-
ring Sperm DNA was purchased from Sigma Chemical Co. (India).
670 nm, ε = 78.27 M–1·cm–1 (5.75ϫ10–4M).
[Cu(CPF)A3Cl]·2H2O (3): prepared by using 2,4-bis(4-chlo-
rophenyl)-6-(thiophen-2-yl)pyridine (A3) (1 mmol). Yield 65%, m.p.
Ͼ300 °C, μeff: 1.76 B.M. m/z = 811.47. C38H34Cl3CuFN4O5S (847.67):
calcd (found) C 53.23 (53.29); H 4.04 (3.90); N 6.61 (6.47); S 3.78
(3.65), Cu 7.50 (7.49)%. λmax(solid) = 651 nm, λmax(solution) = 636 nm,
ε = 100.77 M–1·cm–1 (8.13ϫ10–4M).
[Cu(CPF)A4Cl]·2H2O (4): prepared by using 4-(4-bromophenyl)-2-
(4-chlorophenyl)-6-(thiophen-2-yl)pyridine (A4) (1 mmol). Yield 46%,
m.p. Ͼ300 °C, μeff: 1.78 B.M. m/z = 856.04. C38H34Cl2CuBrFN4O5S
(892.12): calcd (found) C 51.16 (51.20); H 3.84 (3.68); N 6.28 (6.30);
4.2. Physical Measurements
Elemental analyses (C, H, and N) of the synthesized complexes were
performed with a model 240 Perkin-Elmer elemental analyzer, Massa-
chusetts (USA). Metallic content of the complex was determined after
decomposing it under effect of acid mixture and titrating against EDTA
solution volumetrically. Room temperature magnetic measurement for
the complexes was made using Gouy magnetic balance. The Gouy
tube was calibrated using mercury(II)tetrathiocyanatocobaltate(II) as
the calibrant (χg = 16.44ϫ10–6 cgs units at 20 °C). The electronic
spectra were recorded with a UV-160A UV-Vis spectrophotometer,
Shimadzu, Kyoto (Japan). Infrared spectra were recorded with a FT-
IR ABB Bomen MB-3000, (Canada) spectrophotometer as KBr pellets
in the range 4000–400 cm–1. MIC study was carried out by means of
laminar air flow cabinet, Toshiba, Delhi, (India). The thermogram of
complexes was recorded with a Mettler Toledo TGA/DSC 1 thermo-
gravimetric analyzer. The LC-MS spectra were recorded with a
Thermo mass spectrophotometer (USA). Photo quantization of the gel
after electrophoresis was done using AlphaDigiDocTM RT. Version
V.4.0.0 PC–Image software, California (USA).
S 3.59 (3.43), Cu 7.12 (7.26)%. λmax(solid) = 680 nm, λmax(solution)
665 nm, ε = 76.38 M–1·cm–1 (7.72ϫ10–4M).
=
[Cu(CPF)A5Cl]·2H2O (5): prepared by using 2-(4-chlorophenyl)-6-
(thiophen-2-yl)-4-p-tolylpyridine (A5) (1 mmol). Yield 57%, m.p.
Ͼ300 °C, μeff: 1.88 B.M. m/z = 791.16. C39H37Cl2CuFN4O5S
(827.25): calcd (found) C 56.62 (56.35); H 4.51 (4.42); N 6.77 (6.67);
S 3.88 (3.82), Cu 7.68 (8.01)%. λmax(solid) = 685 nm, λmax(solution)
675 nm, = 88.76 M–1·cm–1 (8.33ϫ10–4M).
=
[Cu(CPF)A6Cl]·2H2O (6): prepared by using 2-(4-chlorophenyl)-4-
(4-methoxyphenyl)-6-(thiophen-2-yl)pyridine (A6) (1 mmol) Yield
45%, m.p. Ͼ300 °C, μeff
:
1.77 B.M. m/z
=
807.17.
C39H37Cl2CuFN4O6S (843.25): calcd (found) C 55.55 (55.90); H 4.42
(4.30); N 6.64 (6.60); S 3.80 (3.75), Cu 7.54 (7.66)%. λmax(solid)
665 nm, λmax(solution) = 650 nm, ε = 82.94 M–1·cm–1 (9.40ϫ10–4M).
=
4.5 Biological Screening of Synthesized Complexes
4.3. General Synthesis of Ligands
The ligands were prepared by a modified Krönke pyridine synthesis[51]
using halo pyridinium salt of 2-acetyl thiophene, ammonium acetate,
and different chalcone in methanol. The mixture was reflux for 6–7 h
on a sand bath. The product was obtained by keeping the solution in
an ice bath and purified by crystallization in n-hexane. Ligands were
characterized by elemental analysis, 1H and 13C NMR spectroscopy
(see Supporting Information).
4.5.1 In-vitro Antimicrobial Screening
The antibacterial activity of the complexes was studied against E. coli
(MTCC-433), P. aeruginosa (MTCC-1688), B. subtilis (MTCC-7193),
S. aureus (MTCC-3160) and S. marcescens (MTCC-7103). Screening
was performed by determining the minimum inhibitory concentration
(MIC) using Luria Broth (LB) as a medium. The complexes were dis-
solved in DMSO. Cultured for Gram(+ve) and Gram(–ve) were incu-
bated at 37 °C. Control test with no active ingredient was also per-
formed.[52] MIC was determined using two fold serial dilution in liquid
media containing 0.2–3,500 μM variation of test complex concentra-
tion. A pre-culture of bacteria was grown in LB overnight at the opti-
mal temperature of each species. Bacterial growth was monitored by
measuring the turbidity of the culture after 18 h. If a certain concentra-
tion of a complex inhibits the bacterial growth, half the concentration
of the complex was tested. This procedure was carried on to a concen-
tration that bacteria grow normally. The lowest concentration that in-
hibits the bacterial growth was determined as the MIC value. All
equipment and culture media were sterile.
4.4. General Synthesis of Complexes
A methanol solution of CuCl2·2H2O (1 mmol) was added to a meth-
anol solution of neutral bidentate ligand (An) (1 mmol), followed by
addition of previously prepared methanol solution of ciprofloxacin in
presence of CH3ONa (1 mmol). The pH of reaction mixture was ad-
justed at ca. 6.8 using dilute solution of CH3ONa. The resulting solu-
tion was refluxed for 3 h. on a water bath, followed by concentrating
it to half of its volume. A fine amorphous product obtained was
washed with chloroform and dried in vacuo desiccators.
[Cu(CPF)A1Cl]·2H2O (1): prepared by using 2-(4-chlorophenyl)-4- In addition to MIC, the bactericidal action of all complexes was evalu-
(4-fluorophenyl)-6-(thiophen-2-yl)pyridine (A1) (1 mmol). Yield 53%,
ated against two Gram(+ve) and three Gram(–ve) bacteria in terms of
1230
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© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2012, 1224–1232

Square Pyramidal Copper(II) Complexes
colony forming unit (CFU). The inoculum for CFU of 106 viable bac-
30 μM PMS system containing 75 μM NBT, phosphate buffer (pH =
teria per mL was prepared by diluting an overnight culture grown in 7.8), and 0.25 to 3.0 μM tested complex. The amount of reduced NBT
LB. The bacteria were exposed to various concentrations of com-
plexes. The final volume was 1 mL. Cultures were incubated at 37 °C
for 24 h. The 100 μL bacterial culture from above was taken and
was spectrophotometrically detected by monitoring the concentration
of blue formazan form, which absorbs at 560 nm. The reduction rate
of NBT was measured in presence and absence of test compounds at
spread over previously prepared agar plate and incubated for 24 h at various concentration of complex in the system. All the measurements
37 °C. The visual colonies were calculated in order to check biocidal
activity of metal complexes, yielding 30–300 colonies.
were carried out at room temperature. The percent inhibition (η) of
NBT reduction was calculated using following equation[58]
4.5.2 DNA Binding Study by Absorption Titration
η (percent inhibition of NBT reduction) = (1 – kЈ/k)ϫ100
Hypochromism and bathochromism from intercalation mode of bind-
ing are due to strong stacking interaction between an aromatic chromo-
phore and DNA base pair.[53,54] Selection of an appropriate absorbance
peak was done by performing spectrophotometric wavelength scans of
CuII complexes. After addition of equivalent amount of DNA to refer-
ence cell, both were kept for incubation of 10 min at room temperature
followed by absorption measurement. It was specifically done to en-
able direct comparison between the assays that was required to inter-
pret the results obtained. The intrinsic binding constant, Kb was deter-
mined by making it subject in following equation.[55]
where, kЈ and k present the slopes of the straight line of absorbance
values as a function of time in presence and absence of SOD mimic
or a model complex, respectively. The IC50 value of the complexes
was determined by plotting the graph of percent inhibition of NBT
reduction against increase in concentration of the complex. Concentra-
tion of the complex, which causes 50% inhibition of NBT reduction
is reported as IC50 (μM).
4.5.6 Cytotoxic Activity – Brine Shrimp Lethality Bioassay
Brine shrimp (Artemia cysts) lethality bioassay technique was applied
for the determination of general toxic property of complexes. The in
vitro lethality test was carried out using brine shrimp eggs i.e. Artemia
cysts. Brine shrimp eggs were hatched in a shallow rectangular plastic
dish (22ϫ32 cm), filled with artificial seawater, which was prepared
with commercial salt mixture and double distilled water. An unequal
partition was made in the plastic dish with the help of a perforated
device. Approximately 50 mg of eggs were sprinkled into the large
compartment, which was darkened, while the minor compartment was
opened to ordinary light. After two days nauplii were collected by a
pipette from the lighter side. A stock solution of the test complex was
prepared in DMSO. From this stock solution, solutions were transfer-
red to the vials to make final concentration 2, 4, 6, 8, 10, 12 μM etc.
(three for each dilutions were used for each test sample and LC50 is
the mean of three values) and three vial was kept as control having of
DMSO only. After two days, when the nauplii were ready, 1 mL of
seawater and 10 of nauplii were added to each vial and the volume
was adjusted with seawater to 2.5 mL per vial.[59] After 24 h each vial
was observed using a magnifying glass and the number of survivors
in each vial was counted and noted. Data were analyzed by simple logit
method to determine the LC50 values, in which log of concentration of
samples were plotted against percent of mortality of nauplii.[60]
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb(εb – εf)
where, [DNA] is the concentration of DNA in base pairs, εa the appar-
ent extinction coefficient is obtained by calculating Aobs./[complex], εf
corresponds to the extinction coefficient of the complex in its free
form and εb refers to the extinction coefficient of the complex in the
fully bound form. When each set of data, fitted to the above equation,
gave a straight line with a slope of 1/(εa – εf) and y-intercept of
1/Kb(εb – εf). The Kb value was determined from the ratio of the slope
to intercept.
4.5.3 DNA Binding Study by Viscosity Titration
An ubbelohde viscometer immersed in a thermostatic bath maintained
at 37Ϯ0.1 °C was used to measure the change in hydrodynamic vol-
ume with change in complex concentration. Digital stopwatch with
least count of 0.01 s was engaged for flow times measurement with
accuracy ofϮ0.01 s plot of (η/η0)1/3 vs. [complex]/[DNA] is used to
study the behavior of binding, where η is the viscosity of DNA in
presence of complex and η0 is the viscosity of DNA alone. Viscosity
values were calculated from the observed flow time of DNA-contain-
ing solutions (t) corrected for that of the buffer alone (t0), η =
(t – t0).[56]
Supporting Information (see footnote on the first page of this article):
Material 1: Spectrophotometric titration curves and Table for equiva-
lent endpoint determination of the complexes. Material 2: LC-MS
spectrum of complex [Cu(CPF)A1Cl]·2H2O. Material 3: Thermogram
of complex 1. Material 4: CFU/mL for all complexes against different
microorganisms. Material 5: Effect of complexes on brine shrimp le-
thality bioassay. Material 6: Physicochemical parameters of ligands
A1–A6.
4.5.4 DNA Cleavage Study by Gel Electrophoresis
For the gel electrophoresis experiments, total volume of 15 μL contain
300 μg·mL–1 of pUC19 DNA in TE buffer (10 mM Tris, 1 mM EDTA,
pH 8.0) was treated with different complexes (200 μM) and the mix-
ture was incubated for 24 h at 37 °C in the dark. The samples were
analyzed on the base of charge and size difference on 1% agarose gel
bed consisting 0.5 μg·mL–1 of ethidium bromide at 50 V after quench-
ing the reaction with 5 μL loading buffer (40% sucrose, 0.2% bro-
mophenol blue). The whole bed was immersed in 1 X TAE buffer
(0.04 M Tris-Acetate, pH 8, 0.001 m EDTA). Bands were visualized
by UV light and photographed followed by estimating the intensity of
the bands of DNA using AlphaDigiDocTM RT. Version V.4.1.0 PC-
Image software; gel documentation system. The degree of DNA cleav-
age activity was expressed in terms of the percent of cleavage of the
SC-DNA according to the following equation.[57]
Acknowledgement
The authors thank Head, Department of Chemistry, Sardar Patel Uni-
versity, India for making it convenient to work in laboratory and
U.G.C. for providing financial support under the Scheme “UGC Re-
search Fellowship in Science for Meritorious Students”.
4.5.5 SOD-like Activity
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The NBT/NADH/PMS system was used to study SOD-like behavior
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Received: February 11, 2012
Published Online: June 04, 2012
1232
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© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2012, 1224–1232