Synthesis, spectral investigation and biological interphase of drug-based cytotoxic square pyramidal coordination compounds

Article abstract of DOI:10.1002/aoc.2841

We have synthesized ciprofloxacin-based metal complexes of bipyridine derivatives [Cu(CFL)(Aⁿ)Cl].2H₂O (where CFL=ciprofloxacin and A=bipyridines e.g. A¹=4-(4-fluorophenyl)-6-p-tolyl-2,2′- bipyridine, A⁶=4-(4-(b

Full text of DOI:10.1002/aoc.2841

Full Paper
Received: 26 November 2011
Revised: 11 February 2012
Accepted: 20 February 2012
Published online in Wiley Online Library: 12 April 2012
(wileyonlinelibrary.com) DOI 10.1002/aoc.2841
Synthesis, spectral investigation and biological
interphase of drug-based cytotoxic square
pyramidal coordination compounds
Mohan N. Patela*, Bhupesh S. Bhatta, Promise A. Dosia,
Narasimhacharya V. R. L. Amaravadyb and Hetal V. Movaliyab
We have synthesized ciprofloxacin-based metal complexes of bipyridine derivatives [Cu(CFL)(An)Cl].2H2O (where CFL=ciprofloxacin
and A = bipyridines e.g. A1 = 4-(4-fluorophenyl)-6-p-tolyl-2,2′-bipyridine, A6 = 4-(4-(benzyloxy)phenyl)-6-(4-bromophenyl)-2,2′-
bipyridine, etc.). The ligands and complexes were characterized using analytical (C, H, N elemental analysis, TGA and magnetic
measurement) and spectroscopic methods (1H and 13C NMR, FT-IR, fast atom bombardment mass and reflectance spectroscopy).
The products were evaluated by screening for DNA interaction activity on herring sperm DNA and studies suggest intercalative
mode of DNA binding. The antimicrobial activity was determined in terms of minimum inhibitory concentration. Superoxide
dismutase mimic studies were performed using the NADH/PMS/NBT system. The brine shrimp bioassay was also carried out to
study the in vitro cytotoxic properties of the synthesized metal complexes. Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: cytotoxicity; brine shrimp assay; metallonucleases; spectral characterization
Bayer AG (Wuppertal, Germany). Cupric chloride dihydrate,
p-bromoacetophenone, p-methylacetophenone, m-chloroben-
Introduction
Substantial progress has been made during the past few decades
to develop metal-based small molecules as DNA footprinting as
well as therapeutic agents capable of binding and cleaving
DNA under physiological conditions.[1,2] Metal complexes, in this
context, with their coordination environments and versatile
physicochemical properties offer scope for designing and devel-
oping highly sensitive diagnostic agents for medicinal applications,
as exemplified by chemotherapeutic agents such as cisplatin and
bleomycins.[3] Among the metal complexes so far investigated,
those containing polypyridyl ligands have attracted much attention
by virtue of their propensity to bind nucleic acid under physiologi-
cal conditions.[4,5]
Synthetic nucleases have important potential applications,
ranging from the synthesis of custom-designed artificial restriction
enzymes to the development of new antitumor agents.[6,7] Sigman
and co-workers developed the first chemical nuclease [Cu(phen)2]
that effectively cleaves B-DNA in the minor groove in the presence
of a reducing agent and also exhibits antiviral activity, which
inhibits proviral DNA synthesis.[8] In continuation of earlier work,[9]
our experimental studies provide information regarding nuclease
behaviour of synthesized metal complexes. Various biological
parameters (DNA interaction behaviour, cytotoxic assessment)
were evaluated for known efficient nuclease behaviour.
zaldehyde, m-bromobenzaldehyde, p-fluorobenzaldehyde, p-
benzyloxybenzaldehyde, acetic acid, ethylenediaminetetraacetic
acid (EDTA) and herring sperm DNA (HS DNA) were purchased from
Sigma Chemical Co. (India). Ethidium bromide and Luria broth (LB)
were purchased from Himedia (India). All reactions were performed
in air and chemicals were used as received.
Instrumentation
FT-IR Spectroscopy
FT-IR spectra were recorded on a Shimadzu FTIR-8400S spec-
trometer (Shimadzu Scientific Instruments, Japan). Spectroscopy
IR grade of KBr (S D Fine Chem Ltd, India) were ground with
1.0 wt% of the sample to be analysed. The FT-IR sample chamber
was flushed continuously with N2 prior to data acquisition in the
range 4000–400 cmꢀ1
.
NMR Analysis
All 1H and 13C NMR were performed in CDCl3 and recorded on a
Bruker Avance 400 spectrometer (Germany). 1H NMR spectra
*
Correspondence to: Mohan N. Patel, Department of Chemistry, Sardar Patel
University, Vallabh Vidyanagar-388 120, Gujarat, India. E-mail: jeenen@gmail.
com
Experimental
a Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388
Materials
120, Gujarat, India
All the chemicals and solvents were of reagent grade and used
b BRD School of BioSciences, Sardar Patel University, Vallabh Vidyanagar-388
as purchased. Ciprofloxacin hydrochloride was purchased from
120, Gujarat, India
Appl. Organometal. Chem. 2012, 26, 217–224
Copyright © 2012 John Wiley & Sons, Ltd.

M. N. Patel et al.
were collected at 400 MHz using a 8300 Hz spectral width, a
relaxation delay of 1.0 s, a pulse width of 90ꢁ (12.50 ms), 65 k data
points, and CDCl3 (7.27 ppm) as an internal reference. 13C NMR
spectra were collected at 100 MHz using a 24 000 Hz spectral
width, a relaxation delay of 2.0 s, 65 k data points, a pulse width
of 30ꢁ (7.5 ms), and CDCl3 (77.23 ppm) as the internal reference.
50 ꢃ 2%, m.p. 149ꢁC. Anal. Calc. for C23H17FN2 (340.39):
1
C, 81.16; H, 5.03; N, 8.23%. Found: C, 81.29; H, 5.17; N, 8.04%. H
NMR (CDCl3, 400 MHz): d (ppm) 8.600–8.592 (complex, 1H, H3′),
8.412 (d, 1H, H6’, J = 8 Hz), 8.172–8.162 (complex, 2H, H3,4′), 7.840
(d, 2H, H2′′′,6′′′, J = 8 Hz), 7.796–7.787 (complex, 1H, H5), 7.675 (d, 2H,
H2′′, 6′′, J = 8 Hz), 7.536 (t, 1H, H5′, J = 5.6Hz), 7.442 (d, 2H, H3′′′,5′′′
,
J = 8 Hz),7.394 (t, 2H, H3′′,5′′, J = 6.4Hz), 2.321 (s, 3H, Me). 13C NMR
(CDCl3, 100MHz): d (ppm) 166.47 (C4′′), 152.61 (C2), 151.62 (C2′),
150.23 (C6), 149.22 (C4), 148.86 (C6′), 146.01 (C4′), 143.33 (C1′′),
139.75 (C1′′′), 135.26 (C4′′′), 131.27 (C2′′,6′′), 129.30 (C3′′′,5′′′), 126.77
(C2′′′,6′′′), 125.09 (C3′′,5′′), 124.10 (C5′), 122.63 (C3′), 115.43 (C3),
114.81 (C5), 20.48 (Me).
Fast Atomic Bombardment Mass Spectroscopy (FAB MS)
FAB MS was performed on a Jeol SX 102/Da-600 (Jeol Ltd, India)
mass spectrophotometer/data system using argon/xenon (6 kV,
10 mA) as the FAB gas and m-nitrobenzyl alcohol as the matrix.
The accelerating voltage was 10 kV and spectra were recorded
at room temperature.
4-(4-Benzyloxy)phenyl)-6-p-tolyl-2,2′-bipyridine (A2)
Reflectance Spectroscopy
A similar procedure was followed using 3-(4-(benzyloxy)phenyl)-
1-p-tolylprop-2-en-1-one as the enone. Yield 55 ꢃ 2%, m.p.
151ꢁC. Anal. Calc. for C30H24N2O (428.52): C, 84.08; H, 5.65; N,
6.54%. Found: C, 83.91; H, 5.76; N, 6.70%. 1H NMR (CDCl3,
400 MHz): d (ppm) 8.836 (complex, 3H, H3,3′,6′), 8.275 (dd, 2H, H2′′
′,6′′′, J = 6.4 and 0.8 Hz), 8.050 (complex, 2H, H5,4′), 7.902–7.884
(complex, 2H, H2′′,6′′), 7.626 (dd, 2H, H3′′′,5′′′, J = 3.6 and 1.2 Hz),
Reflectance spectra were recorded on a PerkinElmer Lambda 19
UV/VIS/NIR spectrophotometer (USA) using a slit width of 5.0 nm,
smooth bandwidth of 8.0 nm, scan speed of 240.0 nm minꢀ1, and
data interval of 1.0 nm.
UV–Visible Spectroscopy
Electronic spectra were recorded on a Shimadzu UV-160A UV–
visible spectrophotometer (Shimadzu Scientific Instruments,
Japan). Thermal DNA denaturation was performed using an
Agilent 8453 UV-Visible spectrophotometer (CA, USA).
7.528–7.474 (complex, 6H, H5′,Bz2,3,4,5,6), 7.117 (d, 2H, H3′′,5′′
,
J = 6.4 Hz), 5.343 (s, 2H, OCH2), 2.262 (s, 3H, Me). 13C NMR (CDCl3,
100 MHz): d (ppm) 159.55 (C4′′), 157.73 (C2), 155.96 (C2′), 153.04
(C6), 151.70 (C4), 148.02 (C6′), 137.63 (C1′′′), 136.39 (C4′), 135.24
(CBz1), 133.45 (C1′′), 129.29 (C4′′′), 128.01 (C2′′,6′′), 127.21 (C3′′′,5′′′),
126.63 (CBz3,5), 124.77 (CBz4), 124.28 (C2′′′,6′′′), 123.88 (CBz2,6),
122.90 (C5′), 121.56 (C3′), 115.22 (C3), 114.41 (C5), 113.44 (C3′′,5′′),
69.77 (OCH2), 21.54 (Me).
Thermogravimetric Analysis (TGA)
Thermal stabilities of compounds under N2 were examined
using a 5000/2960 TGA instrument (New Castle, DE, USA). Samples
(5–10 mg) were loaded in alumina pans and ramped to 800ꢁC while
heating at 10ꢁC minꢀ1. The N2 or air flow rate was 60ml minꢀ1
.
4-(3-Chlorophenyl)-6-p-tolyl-2,2′-bipyridine (A3)
Elemental Analysis
A similar procedure was followed using 3-(3-chlorophenyl)-1-p-
tolylprop-2-en-1-one as the enone. Yield 51 ꢃ 2%, m.p. 141ꢁC. Anal.
Calc. for C23H17ClN2 (356.85): C, 77.41; H, 4.80; N, 7.85%. Found:
C, 77.57; H, 4.93; N, 7.95%.1H NMR (CDCl3, 400 MHz): d (ppm)
8.786–8.712 (complex, 3H, H3,3′,6′), 8.219 (d, 2H, H2′′′,6′′′, J = 8 Hz),
7.985–7.946 (complex, 2H, H5,4′), 7.856 (s, 1H, H2′′), 7.779 (dd, 1H,
H6′′, J = 4 and 1.6 Hz), 7.590–7.563 (complex, 2H, H4′′,5′′), 7.455
(dd, 2H, H3′′′,5′′′, J = 8 and 1.6Hz), 7.406 (dt, 1H, H5′, J = 7.6 and
1.2 Hz), 2.303 (s, 3H, Me). 13C NMR (CDCl3, 100 MHz): d (ppm)
157.22 (C2), 155.83 (C2′), 151.93 (C6), 148.80 (C4), 145.63 (C6′), 142.84
(C1′′), 140.32 (C1′′′), 138.86 (C4′), 134.99 (C3′′), 132.04 (C4′′′), 131.43 (C4′
′), 130.46 (C5′′), 129.34 (C3′′′,5′′′), 128.24 (C2′′), 127.01 (C6′′), 125.10 (C2′′
′,6′′′), 124.61 (C5′), 122.27 (C3′), 119.32 (C3), 118.33 (C5), 23.22 (Me).
C, H and N elemental analyses were performed with a model 240
PerkinElmer elemental analyser.
Metal Estimation
Metal contents of the compounds were analysed gravimetrically
and volumetrically[10] after decomposing the organic matter with
an acid mixture (HClO4, H2SO4, and HNO3; 1:1.5:2.5).
Magnetic Measurement
Magnetic moments were measured by Gouy’s method using
mercury tetrathiocyanatocobaltate(II) as the calibrant (wg = 16.44
ꢂ 10ꢀ6 cgs units at 20ꢁC), with a Citizen balance (India).
4-(3-Bromophenyl)-6-p-tolyl-2,2′-bipyridine (A4)
Synthesis of Ligands
Ligands A1–8 were prepared by reacting the appropriate enone
with a respective pyridinium iodide salt.[11] The following
procedure is for a typical preparation of ligands, along with their
1H NMR and 13C NMR assignments as well as analytical data.
A similar procedure was followed using 3-(3-bromophenyl)-1-p-
tolylprop-2-en-1-one as the enone. Yield 53 ꢃ 2%, m.p. 143ꢁC.
Anal. Calc. for C23H17BrN2 (401.30): C, 68.84; H, 4.27; N, 6.98%.
Found: C, 68.69; H, 4.16; N, 7.14%.1H NMR (CDCl3, 400MHz): d
(ppm) 8.788–8.705 (complex, 3H, H3,3′,6′), 8.272 (d, 2H, H2′′′,6′′′
,
4-(4-Fluorophenyl)-6-p-tolyl-2,2′-bipyridine (A1)
J = 8 Hz), 7.985–7.947 (complex, 2H, H5,4′), 7.859 (s, 1H, H2′′), 7.776
(dd, 1H, H6′′, J = 5.6 and 1.6 Hz), 7.591–7.566 (complex, 2H, H4′′,5′′),
7.463 (dd, 2H, H3′′′,5′′′, J = 5.2 and 1.2Hz), 7.406 (dt, 1H, H5′, J = 6.8
and 1.2 Hz), 2.541 (s, 3H, Me). 13C NMR (CDCl3, 100 MHz): d
(ppm) 159.73 (C2), 155.29 (C2′), 151.49 (C6), 148.40 (C4), 145.93
(C6′), 143.87 (C1′′), 142.59 (C1′′′), 138.40 (C4′), 135.88 (C4′′′), 133.32
(C2′′), 131.83 (C4′′), 130.37 (C5′′), 129.40 (C3′′′,5′′′), 128.35 (C2′′′,6′′′),
127.57 (C6′′), 126.98 (C3′′), 125.10 (C5′), 123.65 (C3′), 119.51 (C3),
118.88 (C5), 22.64 (Me).
An excess of ammonium acetate (approximately 10 equiv.) was
added to a mixture of 3-(4-fluorophenyl)-1-p-tolylprop-2-en-1-
one (1.5 mmol) and 1-[2-oxo-2-(pyridyl)ethyl]pyridinium iodide
(1.5 mmol) in methanol (20 ml) in a 100 ml round-bottom flask.
After refluxing with stirring for 5–7 h in air, 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
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Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 217–224

Synthesis and biological activity of cytotoxic compounds
6-(4-Bromophenyl)-4-(4-fluorophenyl)-2,2′-bipyridine (A5)
4-(3-Bromophenyl)-6-(4-bromophenyl)-2,2′-bipyridine (A8)
A similar procedure was followed using 1-(4-bromophenyl)-3-(4-
fluorophenyl)prop-2-en-1-one as the enone. Yield 53 ꢃ 2%, m.p.
148ꢁC. Anal. Calc. for C22H14BrFN2 (405.26): C, 65.20; H, 3.48;
N, 6.91%. Found: C, 65.05; H, 3.33; N, 6.77%.1H NMR (CDCl3,
400 MHz): d (ppm) 8.609–8.597 (complex, 1H, H3′), 8.438
(d, 1H, H6’, J = 8 Hz), 8.183–8.160 (complex, 2H, H3,4’), 7.862
(d, 2H, H2′′′,6′′′, J = 8 Hz), 7.799–7.788 (complex, 1H, H5), 7.699 (d,
A similar procedure was followed using 3-(3-bromophenyl)-1-(4-
bromophenyl)prop-2-en-1-one as the enone. Yield 50 ꢃ 2%, m.
p. 150ꢁC. Anal. Calc. for C22H14Br2N2 (466.17): C, 56.68; H, 3.03;
N, 6.01%. Found: C, 56.57; H, 2.90; N, 6.16%. 1H NMR (CDCl3,
400 MHz): d (ppm) 8.793–8.712 (complex, 3H, H3,3′,6′), 8.278
(d, 2H, H2′′′,6′′′, J = 8 Hz), 7.992–7.952 (complex, 2H, H5,4′), 7.868
(s, 1H, H2′′), 7.788 (dd, 1H, H6′′, J = 5.6 and 1.6 Hz), 7.607–7.571
(complex, 2H, H4′′,5′′), 7.504 (dd, 2H, H3′′′,5′′′, J = 4.2 and 0.8 Hz),
7.418 (dt, 1H, H5′, J = 6.8 and 1.2 Hz). 13C NMR (CDCl3, 100 MHz):
d (ppm) 163.43 (C2), 157.61 (C2′), 153.90 (C6), 149.04 (C4), 147.56
(C6′), 144.19 (C1′′), 141.97 (C1′′′), 138.48 (C4′), 136.72 (C3′′′,5′′′),
131.21 (C2′′), 129.95 (C4′′), 128.04 (C5′′), 127.48 (C2′′′,6′′′), 126.73 (C6′′),
124.87 (C3′′), 124.60 (C4′′′), 123.24 (C5′), 122.84 (C3′), 119.82 (C3),
117.69 (C5).
2H, H2′′, 6′′, J = 8 Hz), 7.606 (t, 1H, H5′, J = 4.8 Hz), 7.571 (d, 2H, H3′′′,5′′′
,
J = 8 Hz), 7.399 (t, 2H, H3′′,5′′, J = 5.6 Hz). 13C NMR (CDCl3, 100MHz):
d (ppm) 169.22 (C4′′), 157.65 (C2), 154.99 (C2′), 151.66 (C6), 150.84
00
(C4), 149.04 (C6′), 146.98 (C4′), 145.46 (C1′′′), 142.10 (C1 ), 135.65 (C3′′
′,5′′′), 133.03 (C2′′,6′′), 129.52 (C2′′′,6′′′), 125.84 (C4′′′), 124.48 (C3′′,5′′),
124.02 (C5′), 123.89 (C3′), 119.46 (C3), 115.76 (C5).
4-(4-(Benzyloxy)phenyl)-6-(4-bromophenyl)-2,2′-bipyridine (A6)
A similar procedure was followed using 3-(4-(benzyloxy)phenyl)-
1-(4-bromophenyl)prop-2-en-1-one as the enone. Yield 49 ꢃ 2%,
m.p. 155ꢁC. Anal. Calc. for C29H21BrN2O (493.39): C, 70.59;
H, 4.29; N, 5.68%. Found: C, 70.71; H, 4.44; N, 5.53%. 1H NMR
(CDCl3, 400 MHz): d (ppm) 8.849 (complex, 3H, H3,3′,6′), 8.281
(dd, 2H, H2′′′,6′′′, J = 11.6 and 1.2 Hz), 8.059 (complex, 2H, H5,4′),
7.911–7.894 (complex, 2H, H2′′,6′′), 7.635 (dd, 2H, H3′′′,5′′′, J = 4 and
1.6 Hz), 7.566–7.479 (complex, 6H, H5′,Bz2,3,4,5,6), 7.214 (d, 2H, H3′
′,5′′, J = 6.4 Hz), 5.402 (s, 2H, OCH2). 13C NMR (CDCl3, 100 MHz): d
(ppm) 160.22 (C4′′), 157.83 (C2), 155.18 (C2′), 154.64 (C6), 152.36
(C4), 146.67 (C6′), 138.55 (C1′′′), 137.69 (C4′), 136.23 (CBz1), 135.94
(C1′′), 128.74 (C3′′′,5′′′), 128.06 (C2′′,6′′), 127.56 (CBz3,5), 126.91 (CBz4),
125.99 (C2′′′,6′′′), 125.57 (CBz2,6), 124.52 (C5′), 122.55 (C3′), 119.82
(C3), 116.58 (C5), 115.79 (C4′′′), 114.34 (C3′′,5′′), 70.94 (OCH2).
Synthesis of Metal Complexes
A methanolic solution (10 ml) of CuCl2.2H2O (1.5 mmol) was added
to a methanolic solution (20 ml) of bipyridines (1.5 mmol), followed
by addition of a previously prepared solution (10 ml) of ciprofloxacin
(1.5 mmol) in methanol containing CH3ONa (1.5 mmol). The pH
was adjusted to ~6.8 using 2% CH3ONa. The resulting solution
was refluxed for 2 h in air, on a water bath, followed by concen-
trating it to half of its volume. A fine amorphous product of
green colour was obtained, which was washed with ether–
hexane and dried in vacuum desiccators. The physicochemical
data (yields, formula weight) are listed in Table 1.
Synthesized Complexes at Biological Interphase
6-(4-Bromophenyl)-4-(3-chlorophenyl)-2,2′-bipyridine (A7)
In Vitro Antimicrobial Behaviour
A similar procedure was followed using 1-(4-bromophenyl)-3-(3-
chlorophenyl)prop-2-en-1-one as the enone. Yield 52 ꢃ 2%, m.p.
146ꢁC. Anal. Calc. for C22H14BrClN2 (421.72): C, 62.66; H, 3.35;
N, 6.64%. Found: C, 62.80; H, 3.22; N, 6.76%.1H NMR (CDCl3,
400 MHz): d (ppm) 8.811–8.733 (complex, 3H, H3,3′,6′), 8.298
(d, 2H, H2′′′,6′′′, J = 8 Hz), 8.112–7.978 (complex, 2H, H5,4′), 7.870
(s, 1H, H2′′), 7.798 (dd, 1H, H6′′, J = 5.6 and 1.6 Hz), 7.622–7.591
(complex, 2H, H4′′,5′′), 7.557 (dd, 2H, H3′′′,5′′′, J = 5.2 and 1.2 Hz),
7.442 (dt, 1H, H5′, J = 7.2 and 1.2 Hz). 13C NMR (CDCl3, 100 MHz):
d (ppm) 162.26 (C2), 159.92 (C2′), 156.85 (C6), 151.29 (C4), 147.58
(C6′), 144.82 (C1′′), 141.47 (C1′′′), 140.49 (C4′), 139.94 (C3′′), 136.95
(C3′′′,5′′′), 134.66 (C4′′), 130.82 (C5′′), 129.70 (C2′′), 128.09 (C2′′′,6′′′),
127.78 (C6′′), 125.89 (C4′′′), 124.36 (C5′), 122.50 (C3′), 118.02 (C3),
117.98 (C5).
In vitro antimicrobial tests were performed against three Gram-
negative (Escherichia coli, Pseudomonas aeruginosa, Serratia
marcescens) and two Gram-positive (Bacillus subtilis, Staphylococcus
aureus) bacteria using twofold serial dilution technique, in terms
of minimum inhibitory concentration (MIC).[12] An appropriate
amount of complexes and sodium salt of ciprofloxacin were
dissolved in DMSO and water, respectively to make their final
concentration 2000mM.
Pre-cultures of bacteria were grown by inoculating the bacterial
strains in 25ml of sterile LB in Erlenmeyer flasks followed by
shaking overnight at 37 ꢃ 1ꢁC until appropriate growth was
achieved. Sterilized stock solutions of compounds were added to
Corning tubes having an appropriate volume of sterile 2% LB,
which results in the desired concentration of the compound (mM).
Table 1. Physical parameters of compounds
Complex empirical
formula
Elemental analysis % found(required)
m.p.
(ꢁC)
Yield
(%)
Formula weight
meff
(BM)
(g molꢀ1
)
C
H
N
M
C40H38ClF2CuN5O5
59.81 (59.62)
62.99 (63.15)
58.60 (58.43)
55.52 (55.43)
53.64 (53.80)
57.86 (57.63)
52.66 (52.80)
50.39 (50.28)
4.56 (4.75)
4.84 (5.07)
4.55 (4.66)
4.57 (4.42)
3.78 (4.05)
4.26 (4.42)
4.20 (3.98)
3.99 (3.79)
8.90 (8.69)
7.60 (7.83)
8.71 (8.52)
7.93 (8.08)
8.19 (8.04)
7.52 (7.30)
7.76 (7.89)
7.67 (7.52)
7.97 (7.89)
7.22 (7.11)
7.66 (7.73)
7.26 (7.33)
7.39 (7.30)
6.74 (6.63)
7.06 (7.16)
6.72 (6.82)
238
220
241
244
238
251
252
260
62.49
61.24
62.93
61.86
64.22
63.66
61.43
60.19
805.76
893.89
822.22
866.66
870.63
958.76
887.08
931.53
1.86
1.88
1.80
1.91
1.93
1.91
1.82
1.84
C
C
C
C
C
C
C
47H45ClFCuN5O6
40H38Cl2FCuN5O5
40H38BrClFCuN5O5
39H35BrClF2CuN5O5
46H42BrClFCuN5O6
39H35BrCl2FCuN5O5
39H35Br2ClFCuN5O5
Appl. Organometal. Chem. 2012, 26, 217–224
Copyright © 2012 John Wiley & Sons, Ltd.
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M. N. Patel et al.
All the implicated Corning tubes were further autoclaved and
brought to room temperature using laminar air flow. Each of the
strains (10 ml) from pre-cultured bacteria were added to Corning
tubes having a previously prepared dilution range of the test
compound and were kept shaking at 37ꢁC for 24 h.
in terms of (ꢀ/ꢀ0)1/3 versus concentration ratio ([complex]⁄[DNA]),
where ꢀ is the viscosity of DNA solution in the presence of complex
and ꢀ0 is the viscosity of the solution of DNA alone.
Thermal DNA Denaturation Studies
MIC is the lowest concentration which inhibits the growth of
microorganism, judged by lack of turbidity (clear solution) in the
tube. If the dilution inhibits growth, a whole experimental proce-
dure was repeated with the next dilution, i.e. half the concentration
of test compound compared with the earlier one. This procedure
was repeated until faint turbidity of the inoculum itself was
observed and the said concentration was termed the MIC. All these
operations were carefully performed under aseptic conditions.
DNA melting experiments were carried out by monitoring the
absorption intensity of herring sperm DNA (100 mM) at 260 nm
in the range 25–100ꢁC in 0.5ꢁC minꢀ1 increments, both in the
absence and presence of the copper(II) complexes. The melting
temperature (Tm) of DNA was determined as the midpoint of
the optically detected transition curves. The ΔTm value was
defined as the difference between Tm of the free DNA and Tm
of the bound DNA.
DNA Binding Study by Absorption Titration
DNA Cleavage Study by Gel Electrophoresis
The ability of Cu(II) complexes to bind HS DNA herring sperm
DNA was measured via DNA-mediated hypochromicity and hyper-
chromicity of the Cu(II) complex using UV–visible absorbance spec-
tra. Hypochromism and bathochromism from the intercalation mode
of binding is due to strong stacking interactions between the
aromatic chromophore and the DNA base pair.[13] Selection of an
appropriate absorption peak was done by performing spectropho-
tometric wavelength scans of Cu(II) complexes. The concentration
of HS DNA was determined by measuring absorbance at 260nm
Gel electrophoresis of plasmid DNA (pUC19 DNA) was carried
out in a TAE buffer (0.04 M Tris–acetate, pH 8, 0.001 M EDTA).
The 15 ml reaction mixture contains 100 mg mlꢀ1 plasmid DNA
in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and 200 mM complex.
Reactions were allowed to proceed for 24 h at 37ꢁC. All reactions
were quenched by addition of 5 ml loading buffer (0.25% bromo-
phenol blue, 40% sucrose, 0.25% xylene cyanol, and 200 mM
EDTA). The aliquots were loaded directly on to 1% agarose gel
and electrophoresed at 50 V in 1ꢂ TAE buffer. Gel was stained
with 0.5 mg mlꢀ1 ethidium bromide and was photographed on
a UV illuminator. After electrophoresis, the proportion of
DNA in each fraction was estimated quantitatively from the
intensity of the bands using AlphaDigiDoc™ RT version V.4.0.0
PC-Image software.
ꢀ1
and using 1.28 x 104 M cmꢀ1 as the molar extinction coefficient
value.[14] HS DNA was added to both the cells (cell containing com-
plex and the reference cell), followed by incubation for 10 min at
room temperature. DNA-dependent spectral changes in the peak
of test compounds were calculated. This was done specifically to
enable direct comparison between the assays, which was required
to interpret the results obtained. The intrinsic binding constant, Kb,
was determined by making it subject to the following equation:
Brine Shrimp Assay
Brine shrimp (Artemia cysts) eggs were hatched in a shallow
rectangular plastic dish (22 ꢂ 32 cm), filled with artificial sea
water, prepared from commercial salt mixture and double distilled
water. An unequal partition was made in the plastic dish with the
help of a perforated device.[15] Approximately 50 mg eggs were
sprinkled into the large compartment, opened to ordinary light.
After 2 days, nauplii were collected using a pipette from the
lighted side.
A sample of the test compound was prepared by dissolving
10 mg of each compound in 10 ml DMSO. From these stock
solutions, solution was transferred to 18 vials to make final
concentrations of 2, 4, 8, 12, 16 and 20 mg mlꢀ1 (three sets for
each dilution were used for each test sample and the mean of
three sets were taken) and three vials were kept as a control
having the same amount of DMSO only. When the nauplii were
ready, 1 ml sea water and 10 nauplii were added to each vial
and the volume was adjusted with sea water to 2.5 ml per vial.
After 24 h the number of survivors was counted.[15] Data were
analysed by simple logit method to determine the LC50 values,
in which log of concentration of samples was plotted against
percentage of mortality of nauplii.[16]
½DNAꢄ=ðea ꢀ efÞ ¼ ½DNAꢄ=ðeb ꢀ efÞ þ 1=Kbðeb ꢀ efÞ
where [DNA] is the concentration of DNA in base pairs, ea is the
apparent extinction coefficient, obtained by calculating Aobs.
/
[Cu(II)complex], ef corresponds to the extinction coefficient of
the complex in its free form, and eb 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/(ea ꢀ ef) and a y-intercept of 1/Kb(eb ꢀ ef). The Kb was
determined from the ratio of the slope to intercept.
DNA Binding Study by Hydrodynamic Volume Measurement
An Ubbelohde viscometer maintained at a constant temperature
of 27 ꢃ 0.1ꢁC in a thermostatic jacket was used to measure the
flow time of DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH
7.2) with an accuracy of 0.01 s and a precision of 0.1 s. The DNA
sample used, with an approximate average length of 200 base
pairs, was prepared by sonicating to minimize the complexities
arising from its flexibility.
Flow time for buffer alone was measured and was termed t0.
By adding a DNA sample of calculated amount, flow time was
measured. The solution of complex of known concentration was
added to achieved the ratio of lowest [complex]/[DNA]. After
mixing and incubating it for 10 min, flow time was measured.
Similarly the flow time for the successive ratio was measured. Flow
time for each case was measured in triplicate and an average flow
time was calculated. The viscosity values were calculated using the
equation ꢀ = (t ꢀ t0), where t0 is the flow time of buffer alone and
t is the flow time for buffer containing DNA. Data are represented
Superoxide Dismutase (SOD) Mimic Behaviour
A non-enzymatic system made up of 30 mM phenazime methosul-
phate (PMS), 79 mM reduced nicotinamide adenine dinucleotide
(NADH), 75 mM nitro blue tetrazolium (NBT) and phosphate buffer
ꢀ
•
(pH = 7.8) was used to produce superoxide anion (O2 ). The
ꢀ
•
scavenging rate of O2 was determined by monitoring the
reduction in rate of transformation of NBT to monoformazan
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Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 217–224

Synthesis and biological activity of cytotoxic compounds
dye under the influence of 0.25–5.0 mM concentration of test com-
pound. The reactions were monitored at 560 nm with a UV–visible
spectrophotometer and the rate of absorption change was deter-
mined. The percent inhibition (ꢀ) of NBT reduction was calculated
using following equation:
Characterization
The ligands were characterized using 1H and 13C NMR spectroscopy
and C,H,N elemental analysis. Several analytical techniques, such as
FAB MS, IR, reflectance spectroscopy, C,H,N elemental analysis and
magnetic measurement, were used to evaluate the structure of the
complexes. Elemental analysis data are in good agreement with the
proposed structures. Table 1 lists the physicochemical parameters.
ꢀðpercent inhibition of NBT reductionÞ ¼ ð1 ꢀ k′=kÞ ꢂ 100
IR Spectroscopy
where k′ and k present the slopes of the straight line of absorbance
values as a function of time in the presence and absence of SOD
mimic or a model compound, respectively. IC50 value of the
complex was determined by plotting the graph of percentage
inhibition of NBT reduction against increase in concentration of
the complex. The concentration of complex causing 50% inhibition
of NBT reduction is reported as IC50.
IR spectra of the Cu(II) complexes show major changes as compared
to the free ligands (supporting information, Supplementary 1).
The absorption bands observed in the case of ciprofloxacin at
1624 and 1340 cmꢀ1 are assigned to n(COO)asy and n(COO)sy
respectively. In complexes these bands are observed at
1552–1575 and 1362–1375 cmꢀ1. The frequency separation (Δn=
nCOOassy ꢀ nCOOsym) in investigated complexes is ~200 cmꢀ1
,
suggesting the unidentate nature of the carboxylato group.[17,18]
The sharp band in quinolone at ~3023 cmꢀ1 is due to hydrogen
bonding,[19] which contributes to ionic resonance structures and
peaks observed to the free hydroxyl stretching vibration. This band
vanishes completely in the spectra of the complexes, indicating
deprotonation of carboxylic proton. The n(C=O) stretching
vibration band in ciprofloxacin appears at 1708 cmꢀ1, while in the
complexes these bands shift to 1613–1627cmꢀ1, suggesting that
coordination occurs through the pyridone oxygen atom.[20] These
data are further supported by n(MꢀO),[21] which appear at
~515 cmꢀ1. The band at ~535cmꢀ1 observed for complexes
suggests the N,N-donating nature of bipyridines.[22]
Results and Discussion
Synthesis
The bipyridine derivatives (A1–8) were prepared by reacting the
appropriate enones with pyridinium salt. The product obtained
from aldol condensation (enone) was allowed to react with previ-
ously prepared pyridinium salts via attack on the active methylene
group of pyridinium salt, subsequently undergo ring closure
reaction following formation of bipyridines. Metal complexes were
prepared in two steps by the template reaction of CuCl2.2H2O and
bipyridines in a 1:1 ratio, followed by reaction with deprotonated
ciprofloxacin solution in equal ratio. These result in stable square
pyramidal metal complexes having three coordinate and two
covalent bonds. The reaction sequences are outlined in Scheme 1.
Electronic and Magnetic Behaviour
Visible reflectance spectrum of the copper(II) complexes, i.e. d9
system, were recorded. The complexes exhibit only a broad lmax
Scheme 1. Reaction scheme for synthesis of ligand and metal complex
Appl. Organometal. Chem. 2012, 26, 217–224
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

M. N. Patel et al.
at ~645 nm attributed to d–d transitions for Cu(II) atoms in a
distorted square pyramidal environment.[23]
E. coli, P. aeruginosa and S. aureus than some copper(II) complexes
reported by Efthimiadou et al.[28]
The magnetic moment measurement for any geometry in
copper(II) complexes generally results in 1.8 BM, which is very
close to the spin-only value, i.e. 1.73 BM. The observed values
in our case are very close to the spin-only values (Table 1)
expected for s = ½ system (1.73 BM.), leading to the conclusion
that the metal centre in synthesized complexes possesses one
unpaired electron.[24]
DNA Interaction Studies by Absorbance Titration
A representative absorption titration curve is shown in Supplemen-
tary 3 (supporting information). The changes observed in the UV
spectra of the complexes after mixing with DNA, i.e. hypochromism
and bathochromism (~4 nm) indicate that the interaction of
complexes with DNA takes place by direct formation of a new
complex with double-helical DNA.[29,30] The extent of the binding
strength of complexes was determined quantitatively by calculating
the intrinsic binding constants Kb of the complexes by monitoring
the change in absorbance at various concentration of DNA. From
the plot of [DNA]/(ea ꢀ ef) versus [DNA], (supporting information,
inset Supplementary 3) the Kb value of complexes were determined
FAB Mass Spectra
The FAB mass spectrum of the representative complex, [Cu(CFL)
(A1)Cl] (supporting information, Supplementary 2) shows peaks at
136, 137, 154, 289 and 307 m/z due to the matrix. The molecular
ion peaks observed at m/z = 768 and 770 in spectra are assigned
to (M) and (M+2) of complex molecule with an intensity ratio of
1:3, indicating presence of chlorine atom. Loss of chlorine gives a
fragment ion peak at m/z = 733, confirming that chlorine atom is
covalently attached to the metal ion.
and were found to be in the range 1.10–1.77 ꢂ 104
M
ꢀ1 (Table 3).
Comparing the intrinsic binding constant of complexes wꢀit1h
those of DNA-intercalative [Ru(bpy)2ppd]+2 (1.18 ꢂ 104
M
)
complex,[31] we can deduce that all of the complex binding is
moderated to DNA by intercalation. The Kb value of complexes is
greater than Co(II) complexes of terpyridines reported by Indumathy
et al.[32] and Cu(II) complexes of type [Cu(phen)2Cl2],[33] while
comparable to ruthenium complexes reported by Tan et al.[34]
These spectral characteristics obviously suggest that the complexes
in our paper interact with DNA most likely through a mode that
Biological Evaluation
In Vitro Antimicrobial Screening
The MIC is defined as the lowest concentration of antimicrobial
agent showing complete inhibition of growth of microorganisms.
The results concerning in vitro antibacterial activity (MIC) are
presented in Table 2. The compounds show better antimicrobial
activity against the five microorganisms than CuCl2.2H2O. Complex
5 has better antibacterial activity than the ciprofloxacin against
tested microorganisms. Complexes 1 and 8 have better antibacte-
rial activity than ciprofloxacin against tested microorganisms,
except of 8 against E. coli, while all the other complexes are moder-
ate to less bacteriostatic.
The increase in antimicrobial activity of metal complexes can
be the result of chelate effects, the nature of the ligands, the
nature of the ion neutralizing the complex, and nuclearity of the
metal centre in the compounds.[25] All complexes show higher
antibacterial activity against S. aureus than ciprofloxacin–
copper(II) complexes reported by Drevensek et al.[26] All com-
plexes show higher antibacterial activity than fluoroquinolone
drug-based metal complexes, [Mg(R-oflo)(S-oflo)(H2O)2].2H2O
and [Mg(S-oflo)2(H2O)2].2H2O against P. aeruginosa.[27] Com-
plexes 1, 5, and 8 have higher antibacterial activity against
Table 3. Binding constants, IC50, ΔTm and LC50 values of compounds
a
a
Complex
Binding
ΔTm
IC50
(mM)
LC50
(mM)
constꢀan1t
(ꢁC)
Kb (M
)
Ciprofloxacin
2.6 ꢂ 103
1.3 ꢂ 104
1.2 ꢂ 104
1.2 ꢂ 104
1.1 ꢂ 104
1.8 ꢂ 104
1.4 ꢂ 104
1.4 ꢂ 104
1.4 ꢂ 104
-
164 (ꢃ2.08) 214 (ꢃ1.15)
1.2 (ꢃ0.03) 6.2 (ꢃ0.16)
1
2
3
4
5
6
7
8
4.8
4.2
5.1
5.0
5.6
5.2
5.4
5.3
1.3 (ꢃ0.04) 13.4 (ꢃ0.19)
1.3 (ꢃ0.02)
1.4 (ꢃ0.05)
0.6 (ꢃ0.01)
9.0 (ꢃ0.13)
9.2 (ꢃ0.17)
6.0 (ꢃ0.15)
0.9 (ꢃ0.02) 12.5 (ꢃ0.18)
1.0 (ꢃ0.01)
1.0 (ꢃ0.03)
9.0 (ꢃ0.16)
9.8 (ꢃ0.11)
aMean of at least three determinations.
Table 2. MICa data of compounds (mM)
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
2404
E. coli
CuCl2.2H2O
2698
2815
2756
3402
Ciprofloxacin
1.6 (ꢃ0.06)
0.8 (ꢃ0.05)
1.4 (ꢃ0.04)
1.4 (ꢃ0.01)
1.3 (ꢃ0.06)
0.5 (ꢃ0.03)
1.1 (ꢃ0.08)
1.1 (ꢃ0.07)
0.7 (ꢃ0.02)
1.1 (ꢃ0.08)
1.1 (ꢃ0.04)
1.6 (ꢃ0.07)
1.2 (ꢃ0.03)
1.4 (ꢃ0.07)
0.4 (ꢃ0.01)
1.2 (ꢃ0.08)
1.0 (ꢃ0.06)
0.8 (ꢃ0.02)
1.6 (ꢃ0.08)
1.0 (ꢃ0.06)
1.5 (ꢃ0.03)
1.6 (ꢃ0.09)
1.5 (ꢃ0.04)
0.7 (ꢃ0.04)
1.0 (ꢃ0.06)
1.2 (ꢃ0.06)
0.9 (ꢃ0.03)
1.4 (ꢃ0.07)
1.3 (ꢃ0.09)
1.7 (ꢃ0.06)
1.7 (ꢃ0.05)
1.6 (ꢃ0.03)
0.7 (ꢃ0.02)
0.9 (ꢃ0.04)
1.1 (ꢃ0.05)
1.0 (ꢃ0.03)
1.4 (ꢃ0.07)
1.2 (ꢃ0.03)
1.8 (ꢃ0.05)
1.6 (ꢃ0.04)
1.7 (ꢃ0.07)
0.6 (ꢃ0.02)
1.1 (ꢃ0.02)
1.2 (ꢃ0.01)
1.6 (ꢃ0.04)
[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)
aMean of at least three determinations.
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Appl. Organometal. Chem. 2012, 26, 217–224

Synthesis and biological activity of cytotoxic compounds
involves a stacking interaction between the aromatic chromophore
and the base pairs of DNA.
Thermal DNA Denaturation Study
The binding of metal complexes to herring sperm DNA was studied
by measuring changes in the melting temperature (Tm) of DNA that
characterizes the transition from a double-stranded nucleic acid to
a single-stranded one. The temperature at which half of a DNA
sample has melted is known as the melting temperature and is
strongly related to the stability of the double-helical structure.
A change in Tm may be observed when the complexes interact
with DNA.
The melting curves for herring sperm DNA in the absence and
presence of the complexes are illustrated in Supplementary 4
(supporting information) and Table 3. The Tm value for the free
herring sperm DNA is 82.8ꢁC. A small change in the DNA melting
temperature (ΔTm) on addition of all the complexes to herring
sperm DNA was observed. The ΔTm values (4.2–5.6ꢁC) suggest
that the complexes may interact with DNA by intercalatiion.[35,36]
Figure 2. Bar diagram for interaction of pUC19 DNA (100 mg mlꢀ1) with
series of copper(II) complexes (200 mM): 1, DNA control; 2, CuCl2.2H2O +
pUC19; 3, ciprofloxacin + pUC19; 4, 1 + pUC19; 5, 2 + pUC19; 6, 3 + pUC19;
7, 4 + pUC19; 8, 5 + pUC19; 9, 6 + pUC19; 10, 7 + pUC19; 10, 8 + pUC19
in binding affinity of the complexes to DNA and the structural
dissimilarities of ligands. The data show higher cleavage ability
of metal complexes than of metal salt and ciprofloxacin.
DNA Binding Study by Viscosity Measurement
Viscometric titration experiments, regarded as a reliable tool in
the absence of crystallographic data, were also carried out to
explore the propensity of DNA binding by the complexes.[37]
A
Cytotoxicity
significant increase in DNA viscosity on addition of any external
species can result only in intercalation, as intercalation leads to
separation of DNA bases and hence to increases in the DNA
effective size.[38] A plot of (ꢀ/ꢀ0)1/3 versus [complex]/[DNA] gives
a measure of the viscosity changes (Fig. 1). A marginal increase in
the relative viscosity was observed on addition of the present
complexes to DNA solution, suggesting the mainly intercalation
nature of the complexes.
Brine shrimp lethality bioassay is a recent development in the
assay of bioactive compounds, which indicates cytotoxicity as well
as a wide range of pharmacological activities (such as anticancer,
antiviral, insecticidal, pesticidal, AIDS) of the compounds.[15,39] All
the synthesized compounds were screened for their cytotoxicity
(brine shrimp bioassay) using the protocol of Meyer et al.[15] From
the data recorded in Table 3, it is evident that compounds 1 and
5 displayed potent cytotoxic activity against Artemia cysts, while
the other compounds were moderately active in this assay.
Gel Electrophoresis: Photo Quantization Technique
SOD-Like Activity: Decomposition of Reactive Oxygen Species
When plasmid DNA was subjected to electrophoresis after
interaction, upon illumination of gel (supporting information,
Supplementary 5) the fastest migration was observed for super-
coiled (SC) form I, whereas the slowest moving was open circular
(OC) form II and the intermediate moving form was the linear (L)
form III generated on cleavage of OC. Figure 2 shows a bar
diagram for the data of plasmid cleavage (supporting informa-
tion, Supplementary 6). The different DNA cleavage efficiency
of the complexes, metal salt and drugs is due to the difference
The NADH/PMS/NBT system was used to generate the superoxide
radical artificially in order to check SOD-like behaviour of the
complexes. The percentage inhibition of formazan formation at
various concentrations of complexes as a function of time was
determined by measuring the absorbance at 560 nm and plotting
to obtain a straight line obeying the equation Y = mX+ C (support-
ing information, Supplementary 7); with an increase in concentra-
tion of the tested complexes a decrease in slope (m) was observed.
Percentage inhibition of the reduction rate of NBT was plotted
against the concentration of the complex (supporting information,
Supplementary 8).
Compounds exhibit SOD-like activity at biological pH, with their
IC50 values ranging from 0.60 to 1.35 mM (Table 3), which is higher
than Cu(II) chloride (0.45 mM). These higher values may be due to
the strong ligand field created by the ligands, which opposes the
interaction of the copper(II) complexes with the superoxide radical.
The IC50 values of our compounds are better than complexes of
type Cu(stz)py3Cl (1.31), Cu(Hstz)2(MeOH)Cl2 (2.51) and Cu(Hstz)2
(EtOH)Cl2 (5.17) reported by Cassanova et al.[40]
Conclusions
Reflectance spectroscopy suggests that ciprofloxacin-based
copper(II) complexes of bipyridine derivatives possess distorted
square pyramidal geometry. The binding properties of copper
Figure 1. Effect on relative viscosity of DNA under the influence of in-
creasing amount of compounds at 27 ꢃ 0.1ꢁC in 5 mM Tris–HCl buffer
(pH 7.2) as a medium
Appl. Organometal. Chem. 2012, 26, 217–224
Copyright © 2012 John Wiley & Sons, Ltd.
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M. N. Patel et al.
[12] M. Alexious, I. Tsivikas, C. Dendreinou-Samara, A. A. Pantazaki,
P. Trikalitis, N. Lalioti, D. A. Kyriakidis, D. P. Kessissoglou, J. Inorg.
Biochem. 2003, 93, 256.
(II) complexes to herring sperm DNA have been studied by
viscosity measurement, absorption titration and thermal DNA
denaturation. The hypochromism and bathochromism (~4 nm)
observed in absorption titration is consistent with the intercalation
mode of metal complex interaction with DNA. The increase in the
relative viscosity of DNA in the presence of metal complexes
suggests a classical intercalation binding mode. The increase in
melting temperature of DNA in the presence of compounds by
~5ꢁC also supports this conclusion. The cleavage of plasmid
pUC19 DNA by compounds as observed in gel electrophoresis
study suggests sufficient interaction of compounds with DNA.
The SOD mimic study suggests that copper(II) complexes can
effectively scavenge superoxide anion radicals at their vacant
coordination side. Cytoxicity assessment shows a potent lethality
(LC50) of metal complexes against brine shrimp.
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The authors thank the Head, Department of Chemistry, and Head,
BRD School of Biosciences, Sardar Patel University, India, for
making it convenient to work in the laboratory, and UGC for
providing financial support under the scheme ’UGC Research
Fellowship in Science for Meritorious Students’.
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