DNA interactions and cytotoxic studies of cis-platin analogues of substituted 2,2′-bipyridines

Article abstract of DOI:10.1016/j.saa.2012.05.043

Platinum(II) complexes [Pt(4″-fpbpy)Cl₂] (1), [Pt(4″-mepbpy)Cl₂] (2), [Pt(4″-mpbpy)Cl₂] (3) and [Pt(4″-bopbpy)Cl₂] (4) {where 4″-fpbpy = 4-(4″-fluorophenyl)-6-phenyl-2,2′-bipyridine, 4″-mepbpy = 4-(4″-methylphen

Full text of DOI:10.1016/j.saa.2012.05.043

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA interactions and cytotoxic studies of cis-platin analogues of substituted
2,20-bipyridines
⇑
Mohan N. Patel , Pradhuman A. Parmar, Deepen S. Gandhi, Anshul P. Patidar
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
Increasing the absorbance shows square planar complexes of Pt(II) binding to the DNA via covalent mode.
"
"
"
"
Synthesis of Pt(II) complexes with
bipyridines.
Characterization by IR, UV–visible,
TGA and mass spectrometry.
All Pt(II) complexes have been tested
for their cytotoxic studies.
The interaction of complexes with
DNA has been performed.
a r t i c l e i n f o
a b s t r a c t
Platinum(II) complexes [Pt(400-fpbpy)Cl2] (1), [Pt(400-mepbpy)Cl2] (2), [Pt(400-mpbpy)Cl2] (3) and [Pt(400-
bopbpy)Cl2] (4) {where 400-fpbpy = 4-(400-fluorophenyl)-6-phenyl-2,20-bipyridine, 400-mepbpy = 4-(400-
methylphenyl)-6-phenyl-2,20-bipyridine, 400-mpbpy = 4-(400-methoxyphenyl)-6-phenyl-2,20-bipyridine,
400-bopbpy = 4-(400-benzyloxyphenyl)-6-phenyl-2,20-bipyridine} have been synthesized and character-
ized. The binding strength and binding mode of the complexes with HS DNA (Herring Sperm) have been
investigated by absorption titration and viscosity measurement studies. The results have been revealed
that the complexes bind to DNA by covalent mode with intrinsic binding constant Kb ranging from
6.05 ꢀ 104 Mꢁ1 to 3.48 ꢀ 105 Mꢁ1. The unwinding angle of pUC19 DNA has been evaluated by gel electro-
phoresis assay. The brine shrimp bioassay has been performed to study the in vitro cytotoxic properties of
the synthesized metal complexes.
Article history:
Received 10 January 2012
Received in revised form 28 April 2012
Accepted 17 May 2012
Available online 26 May 2012
Keywords:
Pt(II) complexes
Substituted bipyridines
Spectral characterization
Cytotoxicity
Ó 2012 Elsevier B.V. All rights reserved.
Introduction
Among these, only the 2,30- and the 3,30-bipyridines are found to
be naturally abundant in certain varieties of tobacco [2]. In contrast
Since the discovery of 2,20-bipyridines at the end of the 19th
century, it has been extensively used in the complexation with me-
tal ions due to its robust redox stability and ease of functionaliza-
tion [1]. The 2,20-, 3,30- and 4,40- are symmetrical isomers of
bipyridine, while 2,30-, 2,40- and 3,40- are asymmetrical isomers.
to other monoanionic ligands, such as catechol, which is dianionic;
and derivatives of the acetylacetonate ion, 2,20-bipyridine (bpy) is a
neutral ligand, which can forms charged complexes with metal cat-
ions, and this property has been exploited in the design and syn-
thesis of metal-bipyridine complexes [3]. The use of ligands
containing several bpy subunits to produce multinuclear com-
plexes finds wide attraction. This interest includes the elaboration
of binuclear and polynuclear complexes, which can be involved in
a large range of application, i.e. mimicking of enzyme’s active-site,
⇑
Corresponding author. Tel.: +91 2692 226856x218.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.05.043

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
55
light emitting materials, films of coordination polymers [4], in the
Synthesis of complexes
production of the herbicide and as potential antitumor agents [5].
2,20-Dipyridylamine (dpa) is an aromatic amine in some ways sim-
ilar to bpy, but the central amine unit introduces several differ-
ences: (1) dpa coordinates with formation of six-membered
chelate ring instead of five-membered ring for bpy; (2) the coordi-
nated pyridine rings are not necessarily forced to near coplanarity
as in bpy; (3) the ligand field strength of dpa is greater than ethy-
lenediamine, but closer to that of a single aromatic amine [6]. Tran-
sition metal complexes can be designed to have long lifetimes in
fluid solution, visible light excitation and emission, and versatile
binding modes to biomolecules [7]. The chemistry of platinum
complexes is important from a biological and medicinal point of
view. Some Pt(II) complexes, viz., cis-platin, carboplatin and
oxaliplatin, are extensively used as anticancer drugs in chemother-
apy. Over the last 30 years many other platinum drugs have been
developed in an attempt to improve on cis-platin [8]. The DNA-
damaging drug cis-platin, cis-[PtCl2(NH3)2], has been employed
with great success in the treatment of testicular, ovarian and other
cancers. The proposed principal mechanism of action of platinum
drugs is the formation of platinum–DNA adducts, resulting in cell
death [9].
Synthesis of [Pt(400-fpbpy)Cl2] (1)
The complex was synthesized by heating 1:1 ratio of 400-fpbpy
(0.337 mmol) and K2PtCl4 (0.337 mmol) in water–ethanol system
(50 mL) at 80 °C with a drop of hydrochloric acid (free acid is to
avoid displacement of Clꢁ by OHꢁ) until the solution became color-
less (0.5–5 h) (Scheme 1). Reaction mixture was allowed to cool at
room temperature. The obtained product was washed with hot
water and dried under vacuum. Yield: 81%, mp: >300 °C, mol. wt.
592.35, Anal. calc. for C22H15Cl2FN2Pt (%); C, 44.61; H: 2.55; N,
4.73, Found: C, 44.23; H, 2.62; N, 4.79%, kmax (nm): 326, 363,
433, FAB MS: m/z = 593 [Pt(400-fpbpy)Cl2 + H]+, 557 [Pt(400-
fpbpy)Cl + H]+, 524 [Pt(400-fpbpy) + 2H]+, 326 [400-fpbpy]+, FT–IR
(KBr, cmꢁ1):
d(C–H) 768(s),
m
(C–H) 3069(m),
m
(C@C)1655 (s),
m(C@N) 1492 (s),
m
(M–N)sym 584 (s),
m(M–N)asym 476 (s).
Synthesis of [Pt(400-mepbpy)Cl2] (2)
The complex was synthesized in a manner identical to that de-
scribed for [Pt(400-fpbpy)Cl2], with 400-mepbpy(0.337 mmol) in
place of 400-fpbpy. Yield: 73%, mp: >300 °C, mol. wt. 588.39, Anal.
calc. for C23H18Cl2N2Pt (%): C, 46.95; H, 3.08; N, 4.76, Found: C,
47.12; H, 3.16; N, 4.85%, kmax (nm): 334, 368, 442, FAB MS: m/
z = 588 [Pt(400-mepbpy)Cl2 + H]+, 554 [Pt(400-mepbpy)Cl + H]+, 519
[Pt(400-mepbpy) + 2H]+, 322 [400-mepbpy]+, FT–IR (KBr, cmꢁ1):
Present article describe the synthesis and characterization of
some bipyridines and their Pt(II) complexes. Mode and extent of
interaction of complexes have been determined by viscosity mea-
surements and absorption titration. Gel electrophoresis technique
has been used to determine the unwinding angle of pUC19 DNA.
The experimental studies provide information regarding nuclease
behavior of synthesized metal complexes. Cytotoxic activity has
been carried out to measure the LC50 value of the compounds.
m(C–H) 3063(m),
m
(C@C)1642 (s),
m(C@N) 1488 (s), d(C–H) 762(s),
m(M–N)sym 585 (s),
m
(M–N)asym 481 (s).
Synthesis of [Pt(400-mpbpy)Cl2] (3)
The complex was synthesized in a manner identical to that de-
scribed for [Pt(400-fpbpy)Cl2], with 400-mpbpy (0.337 mmol) in place
of 400-fpbpy. Yield: 78%, mp: >296 °C, mol. wt. 604.39, Anal. Calc. for
C23H18Cl2N2OPt (%); C, 45.71; H, 3.00; N, 4.64, Found: C, 45.56; H,
Experimental
3.12; N, 4.75%, kmax (nm): 338, 371, 445, FAB MS: m/z = 606
[Pt(400-mpbpy)Cl2 + 2H]+, 569 [Pt(400-mpbpy)Cl + H]+, 535 [Pt(400-
Materials and instrumental details
mpbpy) + 2H]+, 338 [400-mpbpy]+; FT–IR (KBr, cmꢁ1):
m(C–H)
3065(m),
N)sym 591 (s),
m
(C@C)1653 (s),
(M–N)asym 483 (s).
m(C@N) 1491 (s), d(C–H) 763(s), m(M–
Potassium tetrachloro platinate(II) was purchased from
Chemport (India). Acetophenone, 4-fluorobenzaldehyde, 4-methyl-
benzaldehyde, 4-methoxybenzaldehyde, 4-benzyloxybenzalde-
hyde and 2-acetylpyridine were purchased from Spectrochem
(Mumbai, India). Agarose, Ethidium Bromide, TAE (Tris-Acetyl-
EDTA), bromophenol blue and xylene cyanol FF were purchased
from Himedia (India). Herring Sperm (HS) DNA was purchased
from Sigma Chemical Co. (India). Culture of pUC19 bacteria (MTCC
47) was purchased from Institute of Microbial Technology (Chandi-
garh, India). C, H and N elemental analyses were performed with a
model 240 Perkin Elmer elemental analyzer. TGA was carried out
using a 5000/2960 SDTA, TA instrument (USA) operating at a heat-
ing rate of 10 °C per minute in the range of 20–800 °C in N2. Infra-
red spectra were recorded on Fourier transform IR (FTIR) Shimadzu
spectrophotometer as KBr pellets in the range 4000–400 cmꢁ1. The
1H and 13C NMR were recorded on a Bruker Avance (400 MHz). The
fast atomic bombardment mass spectra (FAB MS) were recorded on
Jeol SX 102/Da–600 mass spectrophotometer/data system using
Argon/Xenon (6 kV, 10 mA) as the FAB gas. The accelerating volt-
age was 10 kV and spectra were recorded at room temperature.
The electronic spectra were recorded on a UV-160A UV–Vis spec-
trophotometer, Shimadzu (Japan). Photo quantization of the gel
after electrophoresis was carried out on AlphaDigiDoc™ RT. Ver-
sion V.4.0.0 PC-Image software.
m
Synthesis of [Pt(400-bopbpy)Cl2] (4)
The complex was synthesized in a manner identical to that de-
scribed for [Pt(400-fpbpy)Cl2], with 400-bopbpy (0.337 mmol) in
place of 400-fpbpy. Yield: 69%, mol. wt. 680.48, mp: >300 °C Anal.
Calc. for C29H22Cl2N2OPt (%); C, 51.19; H, 3.26; N, 4.12, Found: C,
51.35; H, 3.13; N, 4.24%; kmax (nm): 337, 369, 446, FAB MS: m/
z = 681 [Pt(400-bopbpy)Cl2 + 2H]+, 645 [Pt(400-bopbpy)Cl + H]+, 611
[Pt(400-bopbpy) + 2H]+, 414 [400-bopbpy], FT–IR (KBr, cmꢁ1):
H) 3061(m), (C@C)1644 (s), (C@N) 1496 (s), d(C–H) 761(s),
(M–N)sym 590 (s), (M–N)asym 479 (s).
m(C–
m
m
m
m
Evaluation of binding constants by absorption spectral study
The experiment was performed using HS DNA (
e
= 12858
dm3molꢁ1cmꢁ1) in phosphate buffer solution (pH 7.2). The stock
solutions of the complexes were prepared by dissolving the com-
plexes in DMSO. The prepared solutions were diluted using phos-
phate buffer in such a manner that the concentration of complex
became 5% v/v. The absorption titration was carried out by keeping
the concentration of complex constant (50 lM) and varying the
concentration of DNA. After addition of equivalent amount of
DNA to both the cells and incubated for 10 min at room tempera-
ture. The change in absorbance was recorded after each addition of
DNA aliquot. The intrinsic binding constant Kb was determined
according to the following equation:
Synthesis of ligands
All bidentate ligands were prepared by reacting appropriate en-
one with pyridinium salt by following literature procedure [10].
½DNAꢂ=ðea
ꢁ
ef Þ ¼ ½DNAꢂ=ðeb
ꢁ
ef Þ þ 1=Kbðeb
ꢁ
ef
Þ

56
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
Scheme 1. General scheme for the synthesis of complex via 2,20-bipyridine.
where, [DNA] is the concentration of DNA in base pairs, the appar-
ent absorption coefficient ea ef and eb correspond to Aobs./[Pt], the
Brine shrimp assay
,
extinction coefficient for the free platinum complex for each addi-
tion of DNA and the extinction coefficient for the platinum complex
in the fully bound form, respectively. In plots [DNA]/(ea–ef) versus
[DNA], Kb is given by the ratio of slope to the y intercept.
Brine shrimp (Artemia Cysts) eggs were hatched in a shallow
rectangular plastic dish (22 ꢀ 32 cm), filled with artificial seawater
which was prepared with commercial salt mixture and double dis-
tilled 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 and was opened to or-
dinary light. After two days, nauplii were collected by a pipette
from the lighter side of the compartment. A sample of the test
compound was prepared by dissolving 10 mg of each compound
in 10 mL of DMSO. Solutions were transferred from the stock solu-
tions to 18 vials to make final concentration 2, 4, 8, 12, 16 and
Viscosity measurement
Cannon–Ubbelodhe viscometer maintained at a constant tem-
perature of 27.0 0.1 °C in a thermostatic jacket was used to mea-
sure the relative viscosity of DNA solutions in the presence of
ethidium bromide or platinum(II) complexes. A 3.0 mM stock solu-
tion of each platinum(II) complex was prepared. A 0.40 mM solu-
tion of HS DNA was used. The [Complex]/[DNA] ratio was
maintained in the range of 0–0.2. Flow time was measured with
a digital stopwatch with an accuracy of 0.01 s. The flow time of
each sample was measured three times and an average flow time
20 l
g mLꢁ1 (three sets for each dilutions were used for each test
sample and mean of three sets were taken) and three vials were
kept as control having same amount of DMSO only. When the
shrimp nauplii were ready, 1 mL of seawater and 10 nauplii were
added to each vial and the volume was adjusted with seawater
to 2.5 mL per vial. After 24 h the numbers of survivors were
counted [14]. Data were analyzed to determine the LC50 values
by simple method in which log of concentration of samples were
plotted against percentage of mortality of nauplii [15].
was calculated. Data were represented graphically as (
sus concentration ratio ([Complex]/[DNA]), where is viscosity of
g/g0)
1/3 ver-
g
DNA in the presence of complex and g0 is viscosity of DNA alone
[11]. Viscosity values were calculated from the observed flow time
of DNA–containing solutions (t > 100 s) corrected for the flow time
of buffer alone (t0) i.e.
g
/ t–t0.
Results and discussion
Unwinding of pUC19 DNA
Thermogravimetric analysis and magnetic moments
Unwinding of supercoiled pUC19 DNA was measured by aga-
rose gel electrophoresis mobility shift assay [12]. The unwinding
Thermal analyses of the Pt(II) complexes were carried out in or-
der to ascertain the nature of associated water molecules and the
compositional difference of the complexes. No weight loss occurs
up to 320 °C, indicates the absences of coordinated as well as lat-
tice water molecules. Room temperature magnetic moment value
of the synthesized complexes was found to be zero, suggesting
d8-system with low–spin configuration i.e. dsp2 hybridization,
pointing toward square planar Pt(II) complexes.
angle
U
,
induced per Pt–DNA adduct was calculated by
U
= ꢁ18
r
/rb(c), where is the superhelical density and rb is the
r
bound drug-to-nucleotide ratio, rb(c) is the ration at which the
supercoiled and nicked forms comigrate i.e. the ratio at which
the complete transformation of the supercoiled to relaxed form
of the plasmid is attained. Plasmid were incubated with platinum
complex for 48 h, precipitated by ethanol and redissolved in TAE
buffer (0.04 M Tris-acetate +1 mM EDTA, pH 7.0). An aliquot of
the precipitated sample was subjected to electrophoresis on 1%
agarose gel at room temperature in TAE buffer at constant voltage
of 50 V. The gel was stained with aqueous EB, followed by photo
capturing on UV–transilluminator.
Electronic absorption analyses
In the synthesized Pt(II) complexes of bipyridine, only one d-d
band at 433 to 446 nm is observed, which is assigned for
d2z ! d2x ꢁ y2 transition, where as other two bands are masked by
high intensity charge transfer bands. The two bands observed at
363 to 371 nm and 326 to 338 nm correspond to the charge
transfer transitions. Same pattern was observed in electronic spec-
tra for K2PtCl4 [16], Pt(DPymPz)Cl2 [17] and PtCl2(FFAAPTS),
U
¼ 18r
=rbðcÞ
The superhelical density (r) of the plasmid was determined from
rb(c) values obtained for cis-platin and its known unwinding angle
(U
= 13° for the site-specific 1,2 intrastrand cross-link [13].

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
57
Fig. 1. Absorption spectral traces of complex 1 with increasing amount of Sperm
Herring DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2). [complex] = 50 M,
[DNA] = 0–60 M with incubation period of 15 min at 37 °C. Arrow shows the
absorbance change upon increasing DNA concentrations. Inset: Plots of [DNA]/(ea
f) versus [DNA] for the titration of DNA with platinum(II) complexes.
Fig. 2. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 27 0.1 °C in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2).
l
l
–
e
PtCl2(TMBAAPTS) [18] complexes. All these bands points toward
the low spin complex of d8–system having square planar geometry.
Absorption spectral study
Fig. 3. Unwinding of supercoiled pUC19 plasmid DNA modified by [Pt(400-
fpbpy)Cl2]. Lane 1: Control, unmodified DNA; Lane 2–8, rb = 0.04, 0.05, 0.06, 0.07,
0.075, 0.08, 0.085, respectively.
Absorption titration is employed universally to determine the
binding mode and binding strength of the complexes with DNA.
Transition metal complexes can bind to DNA via covalent (a labile
ligand is replaced with a nitrogen atom of DNA base, such as guan-
ine N7) and/or noncovalent interactions (intercalative, electro-
static and groove binding). Complex bind to DNA through
intercalation usually results in hypochromism (decrease in absor-
bance) and bathochromism (red shift), because intercalative mode
involves a strong stacking interaction between aromatic chromo-
phore and the base pairs of DNA [22]. Electrostatic interaction of
complex with DNA shows lower hypochromicity with no batho-
chromic shift [23].
that of the classical intercalator (ethidium bromide) but are compa-
rable to intercalative [PtCl2(4,7-Me2phen)] complex (6.35 0.2
ꢀ 104 Mꢁ1) [25]. The lower Kb values can be accredited to non–pla-
narity of bipyridines, i.e. phenyl and substituted phenyl at 6- and 4-
positions respectively [26]. The substitution on 6-position of one
pyridine ring of 2,20–bipyridine ligand may cause severe steric
constraints near the metal core, when the complex approaches
the DNA base pairs. The binding constant of complex 4 is low due
to the presence of extended non–planar benzyloxy group.
Complex binds to DNA through covalent binding generally re-
sults in hyperchromism resulting from breakage of secondary
structure of DNA due to the fact that phosphate group can provide
the suitable anchors for coordination with complexes. Presence of
red shift indicates coordination of complex with DNA through N7
position of guanine. The extent of hyperchromism also reveals
the nature of binding affinity [24]. The absorption spectra of
Pt(II)-bipyridine complex with increasing concentration of HS
DNA is shown in (Fig. 1). As the concentration of DNA increases,
hyperchromism is observed in the charge transfer band of each
complex along with the red shift of about 2 nm which suggest
covalent binding of metal complexes with DNA.
Viscosity measurement
Viscosity measurement study was carried out by varying the
concentration of the added metal complexes to confirm the bind-
ing mode of the complexes. The ethidium bromide is good interca-
lator which is used to compare the binding efficiency of complexes
with DNA. A classical intercalation mode results in the lengthening
of the DNA helix, leading to an increase in the DNA viscosity. In
contrast, partial intercalators as well as covalent binders could
bend DNA helix, reduce its effective length and thereby its viscos-
ity [27]. All the Pt(II) complexes decrease relative viscosity of DNA
solution (Fig. 2). So, all the Pt(II) complexes bind to DNA either via
covalent binding or partial intercalation mode. Similarly in absorp-
tion titration experiment, we observed hyperchromism and red
The Kb values were calculated from the perturbation observed in
charge transfer band of the complexes. The values obtained for Pt(II)
complexes 1, 2, 3 and 4 are 1.33 ꢀ 105, 3.23 ꢀ 105, 3.48 ꢀ 105 and
6.05 ꢀ 104, respectively (Table 1). These values are much lower than
Table 1
Absorption titration data upon addition of Herring Sperm DNA.
Complex
kmax (nm)
Hyperchromism Ha (%)
Binding constant Kb (Mꢁ1
)
Free
Bound
D
k
1
2
3
4
291
294
296
295
293
296
298
296
2
2
2
1
24.8
25.2
24.3
20.1
1.33 ꢀ 105
3.23 ꢀ 105
3.48 ꢀ 105
6.05 ꢀ 104
a
H% = 100 ꢀ (Abound–Afree)/Afree
.

58
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
Table 2
Data for determining the unwinding angle of pUC19 DNA under influence of complexes.
Complex
Complex-to-nucleotide ratioa rb (C)
Unwinding angle
U
Reference
1
2
3
4
0.08
0.09
0.075
0.09
–
12
11
13
11
15
This work
This work
This work
This work
[30]
[PtCl2(1,4-DACH)]
a
Complex-to-nucleotide ratio at which supercoiled and nicked forms comigrates.
Table 3
Effect of compounds on brine shrimp lethality bioassay.
Compound
1
Concentration (
l
g mLꢁ1
)
Log(Conc.)
No. of nauplii taken
No. of nauplii dead
% of mortality
LC50
8.46
(l
g mLꢁ1
)
LC50 (lM)
2
4
8
12
16
20
0.30
0.60
0.90
1.08
1.20
1.30
10
10
10
10
10
10
1
2
4
6
8
9
10
20
40
60
80
90
14.28
15.52
19.70
28.14
2
3
4
2
4
8
12
16
20
0.30
0.60
0.90
1.08
1.20
1.30
10
10
10
10
10
10
1
2
3
5
7
9
10
20
30
50
70
90
9.13
2
4
8
12
16
20
0.30
0.60
0.90
1.08
1.20
1.30
10
10
10
10
10
10
1
1
2
3
4
7
10
10
20
30
40
70
11.91
19.15
2
4
8
12
16
20
0.30
0.60
0.90
1.08
1.20
1.30
10
10
10
10
10
10
0
1
1
2
3
5
0
10
10
20
30
50
shift for the Pt(II) complexes. So, Conclusion from the results of
both the studies is that complexes 1, 2, 3 and 4 bind covalently
to DNA.
lethality bioassay. The aim of this method is to provide a front-line
screen that can be backed up by more specific and more expensive
bioassays once the active compounds have been isolated. Brine
shrimp lethality bioassay (BSLA) is a recent development in the as-
say procedure of bioactive compound, which indicates cytotoxicity
as well as a wide range of pharmacological activities (such as anti-
cancer, antiviral, insecticidal, pesticidal, AIDS etc.) of the com-
pounds [14,29]. All the synthesized compounds were screened
for their cytotoxicity (brine shrimp bioassay) using the protocol
of Meyer et al. [14]. From the data recorded in (Table 3), it is evi-
dent that compounds 1 and 2 having less crowded structural units
(–F, –CH3), displayed potent cytotoxic activity against Artemia
Cysts, while the other compounds were moderately active in this
assay. The LC50 values of synthesized complexes ranging from 14
Unwinding of pUC19 DNA
Agarose gel electrophoresis was used as a base for monitoring
plasmid DNA cleavage reaction and determines the unwinding in-
duced in negatively supercoiled pUC19 plasmid by monitoring the
degree of supercoiling (Fig. 3) [12]. The degree of supercoiling de-
crease upon binding of unwinding agents which causes a decrease
in the rate of migration through agarose gel, which makes it possi-
ble to observe and to quantify the mean value of unwinding. Due to
binding of unwinding agents linear form of DNA is not observed
and SC form of DNA is completely converted to OC form. Under
to 28 lM in terms of brine shrimp lethality bioassay, which is com-
the present experimental conditions,
ꢁ0.055 on the basis of the data of cis-platin for which the rb(c)
was determined in this study and = 13° was assumed. Using this
r
was calculated to be
parable to that of cisplatin (cis-diamminedichloroplatinum(II)
complexes reported using cell line (MCF-7, HL-60, EJ, BGC823
and HCT-8 cells) [30,31].
U
approach, the DNA unwinding angles were determined (Table 2).
The highest unwinding angle observed for complex 3 (13°), while
the lowest unwinding angle observed for complexes 2 and 4
(11°). These are similar to those found for [PtCl2(1,4-DACH)]
(15 2°) [28].
Conclusions
From analytical and spectral investigations, we can conclude
square planar nature of all Pt(II) complexes derived from bipyri-
dine derivatives. The measurement of DNA binding affinity of Pt(II)
complexes towards HS DNA using absorption spectral analyses
suggest highest Kb (DNA binding constant) value for complex 3
and lowest Kb value for complex 4. The lowest Kb value for complex
Cytotoxicity
A general bioassay that appears capable of detecting a broad
spectrum of bioactivity present in complex is the brine shrimp

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 97 (2012) 54–59
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)
Declaration of interest
[15] M.R. Islam, S.M.R. Islam, A.S.M. Noman, J.A. Khanam, S.M.M. Ali, S. Alam, M.W.
Lee, Mycobiology 35 (1) (2007) 25–29.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
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Acknowledgments
Authors thank Head, Department of Chemistry Sardar Patel Uni-
versity, India for making it convenient to work in laboratory and
UGC for providing financial assistance of UGC grant 40-95/
2011(SR).
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