Nucleic acid interaction and antibacterial behaviours of a ternary palladium(II) complexes

Article abstract of DOI:10.1016/j.saa.2011.10.077

The bidentate ligands and Pd(II) complexes have been synthesized and characterized using elemental analysis (C, H, N), ¹H NMR, ¹³C NMR, electronic spectra, FT-IR and FAB mass spectroscopy. The binding of palladium complexes with calf thymus DNA (CT DNA) has been explored using absorption titration, DNA melting temperature and viscosity measurements. The cleavage reaction on pUC19 DNA has been monitored by agarose gel electrophoresis. The results suggest that complexes can bind to DNA by intercalative modes and exhibit nuclease activities in which supercoil form is converted to open circular form. The antibacterial activity of ligands and complexes has been performed against three Gram(-ve) and two Gram(+ve) microorganisms and the study indicates that all the complexes show better microbial inhibition activity than ligands and palladium salt.

Full text of DOI:10.1016/j.saa.2011.10.077

Spectrochimica Acta Part A 86 (2012) 508–514
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Nucleic acid interaction and antibacterial behaviours of
a ternary palladium(II) complexes
Mohan N. Patel∗, Promise A. Dosi, Bhupesh S. Bhatt
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar – 388 120 Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The bidentate ligands and Pd(II) complexes have been synthesized and characterized using elemental
analysis (C, H, N), 1H NMR, 13C NMR, electronic spectra, FT-IR and FAB mass spectroscopy. The binding
of palladium complexes with calf thymus DNA (CT DNA) has been explored using absorption titration,
DNA melting temperature and viscosity measurements. The cleavage reaction on pUC19 DNA has been
monitored by agarose gel electrophoresis. The results suggest that complexes can bind to DNA by inter-
calative modes and exhibit nuclease activities in which supercoil form is converted to open circular form.
The antibacterial activity of ligands and complexes has been performed against three Gram(−ve) and
two Gram(+ve) microorganisms and the study indicates that all the complexes show better microbial
inhibition activity than ligands and palladium salt.
Received 3 September 2011
Received in revised form 22 October 2011
Accepted 28 October 2011
Keywords:
Ternary palladium(II) complexes
Absorption titration
Minimal inhibitory concentration (MIC)
Gel electrophoresis
Melting temperature
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
directed to synthesis and evaluation of complexes with biologically
interesting ligands with the aim of widening spectrum of com-
There is a steadily growing interest in investigations of transi-
tion metal complexes other than the traditional platinum-based
compounds for use as chemotherapeutic agents against cancer.
Metal-based drugs research is moving towards the development
of the new agents able to improve effectiveness and reduce the
severe side effects of cis-diamminedichloroplatinum(II) (cisplatin)
and its analogues that are still the most widely used anticancer
therapeutics [1]. The special attention has been turned to platinum
complexes that are structurally different to those agents as well as
to complexes of the other platinum-group metals like ruthenium,
rhodium, palladium and iridium [2].
Among them palladium(II) complexes are very interesting can-
didates for alternative metal-based drugs, since the coordination
geometry and complex forming processes of palladium(II) are very
similar to those of platinum(II). Because of their higher lability
(105-fold) relative to platinum (II) analogues [3], chelating ligands
have been used to impose the cis-coordination of the more labile
ligands (chloride or nitrate) to palladium (II) as a suitable way
to synthesize antitumor compounds. Some palladium complexes
with aromatic N containing ligands, e.g. derivatives of pyridine,
quinoline, pyrazole and 1,10-phenanthroline, have shown very
promising antitumor characteristics [4–6]. As the biological activ-
ity of a complex strongly depends on the nature of the ligands and
on the metal coordination pattern, the recent research has been
plex activity. The combination of metallic and organic activity may
reveal new modes of action [7].
During the last decade, the interaction between transition metal
complexes and DNA has been extensively studied [8–10]. Binding
studies of small molecules to DNA are very important in the devel-
opment of new therapeutic reagents and DNA molecular probes
[11]. The design of small complexes that bind and react with DNA
becomes important as we begin to delineate the expression of
genetic information on a molecular level. A complete understand-
ing of how to target DNA sites with specificity will lead not only to
novel chemotherapeutics but also to a greatly expanded ability for
chemists to probe DNA and to develop highly sensitive diagnostic
agents [12–16]. Transition-metal complexes are being used at the
forefront of many of these efforts. Stable, inert, and water-soluble
complexes containing spectroscopically active metal centre are
extremely valuable in the researches of biological systems. More
recently, such metal complexes have been applied to explore both
structural and functional aspects of nucleic acid chemistry [17–19].
In continuation of our ongoing research work [20], we repre-
sent the complexation of Pd(II) with the bidentate ligands and an
attempt to examine the mode of coordination and biological prop-
erties of resultant complexes.
2. Experiments
2.1. Materials
∗
Sodium tetrachloropalladate(II) was purchased from Chemport
(India). Picolinic acid, 1,2-diaminobenzene, 2-acetyl thiophene,
Corresponding author. Tel.: +91 2692 226856x221.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.077

M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
509
glyoxal and 2-amino thiophenol were purchased from Spec-
trochem (Mumbai, India). Agarose, ethidium bromide (EB), TAE
(Tris-acetyl-EDTA), bromophenol blue and xylene cyanol FF were
purchased from Himedia (India). CT DNA was purchased from
Sigma Chemical Co. (India). Culture of pUC19 bacteria (MTCC 47)
was purchased from Institute of Microbial Technology (Chandigarh,
India).
extract was washed with water (2 × 20 mL) and dried over anhy-
drous CaCl2. Yield: 38%, m.p.: 105 ◦C, chemical formula (mol.wt.):
C
11H16N4 (204.27 g/mol), elemental analysis, calc. (%): C, 64.68;
H, 7.89; N, 27.43, found: C, 64.56; H, 7.77; N, 27.31, 1H NMR:
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
6.032 (s, 2H, H4 ,4 ), 5.087 (s, 2H, H1), 2.403 (6H, methyl5 ,5 ), 2.059
(6H, methyl, 3 ,3 ), 13C NMR: 147.42 (C5 ,5 ), 140.42 (C3 ,3 ), 106.00
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
(C4 ,4 ), 59.31 (CH2) 13.78 (methyl3 ,3 ), 11.18 (methyl5 ,5 ), FT-IR
(KBr, cm−1): ꢀ(C–H)aliphatic, 2924 (m); ꢀ(C C), 1512 (s); ꢀ(C N),
1420 (s).
2.2. Physical measurements
Elemental analysis (C, H, N) were performed with a model Var-
ioMICRO superuser elementar analysensysteme GmbH. Infrared
spectra were recorded on Fourier transform infrared (FTIR) ABB
Bomen MB 3000 spectrophotometer as KBr pellets in the range
4000–400 cm−1. 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 spectropho-
tometer/data system using Argon/Xenon (6 kV, 10 mA) as the FAB
gas. The accelerating voltage was 10 kV and spectra were recorded
at room temperature. The electronic spectra were recorded on
a UV-160A UV–Vis Spectrophotometer, Shimadzu (Japan). Photo
quantization of the gel after electrophoresis was carried out on
AlphaDigiDocTM RT. Version V.4.0.0 PC-Image software.
2.3.3. 2,4-Bis(4-chlorophenyl)-6-(thiophen-2-yl)pyridine [L3]
An excess of ammonium acetate (approximately 10 equiv)
was added to a mixture of 1,3-bis(4-chlorophenyl)prop-2-en-1-
one (2.88 mmol) and 1-(2-oxo-2-(thiophen-2-yl)ethyl)pyridinium
iodide (2.88 mmol) in methanol (20 mL). After refluxing for 5–7 h,
the reaction mixture was allowed to cool which led to the forma-
tion of greenish yellow needles of product. The solid was filtered off,
washed with cold methanol and dried under vacuum and further
recrystallized using hexane. A further crop of the desired prod-
uct was obtained upon refluxing the filtrate for another 5–6 h and
subsequent workup.
Yield: 23%, m.p.: 175 ◦C, chemical formula (mol.wt.):
C
21H13Cl2NS (382.31 g/mol), elemental analysis, calc. (%): C,
65.97; H, 3.43; N, 3.66, found: C, 65.86; H, 3.31; N, 3.54, 1H NMR:
2.3. Synthesis of ligands
ꢀꢀ ꢀꢀ
ꢀꢀꢀ ꢀꢀꢀ
ꢀ
ꢀꢀ ꢀꢀ
8.133 (d, 2H, H1 ), 7.751 (d, 3H, H3
,5 ), 7.678 (d, 2H, H,4 ),
,3
,5
,6
,3,5), 7.179 (t, 1H, H 4 ), 13C
ꢀ
ꢀꢀꢀ ꢀꢀꢀ
ꢀ
7.541–7.462 (complex, 5H, H3 ,2
2.3.1. 2-(2-Pyridyl)benzimidazole [L1]
,6
ꢀ
NMR: 156.28 (C2), 153.03 (C6), 151.26 (C4), 149.13 (C2 ), 145.02
2-Picolinic acid (20.0 mmol) and 1,2-diaminobenzene
(20.0 mmol) were added to 28 g PPA (polyphosphric acid),
and stirred at 160 ◦C for 8 h. The resulting viscous solution was
poured into 500 mL water, producing a solid which was collected
by suction filtration. The solid was suspended in 500 mL of aqueous
0.5 M Na2CO3, and filtered giving a pale yellow powder. Recrystal-
lization from methanol/water. Yield: 43%, m.p.: 219 ◦C, chemical
formula (mol.wt.): C12H9N3 (195.22 g/mol), elemental analysis,
calc. (%): C, 73.83; H, 4.65; N, 21.52, found: C, 73.75; H, 4.54; N,
ꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀ
ꢀꢀ ꢀꢀ
(C2 ), 137.31 (C1 ), 137.09 (C4 ), 135.44 (C5 ), 129.38 (C4 ),
,6
ꢀꢀꢀ ꢀꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀꢀ ꢀꢀꢀ
), 128.40 (C1 ), 128.29 (C2 ), 128.06 (C3),
,6
128.94 (C3
,5
,3
ꢀ
ꢀ
127.98 (C5), 124.89 (C3 ), 116.21 (C4 ), 115.26 (C5), FT-IR (KBr,
cm−1): ꢀ(C–H), 3024 (m); ꢀ(C C), 1585 (s); ꢀ(C N), 1524 (m);
ꢀ(C–Cl), 1040 (s); ꢀ(C–S), 663 (s), ı(C–H), 733 (s).
2.3.4. 2,2ꢀ-Bisbenzimidazole[L4]
Anhydrous ammonia (20 mL) was slowly bubbled into 250 mL
glyoxal that the temperature was maintained between 40 and 50 ◦C.
After 10 h, the solution was filtered and washed with water to give
19.2 g of crude brown product. The crude product was dissolved in
a solution of 4 L water and 4 g NaOH, and on slow cooling; a light
brown, fibrous material was recovered. Yield: 33%, m.p.: 180 ◦C,
chemical formula (mol.wt.): C14H10N4 (234.26 g/mol), elemental
analysis, calc. (%): C, 71.78; H, 4.30; N, 23.92, found (%): C, 71.68; H,
21.43, 1H NMR: 8.213 (d, 2H, H4 ,5 ), 8.169 (d, 1H, H6 ), 7.964 (dt,
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
1H, H4 ), 7.737 (d, 1H, H3 ), 7.699–7.592 (complex, 4H, H 3 ,6 ,1,5 ),
13C NMR: 159.36 (C2 ), 156.91 (C2), 146.76 (C6 ), 137.35 (C2 ),
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
133.22 (C1 ), 130.54 (C4 ), 129.50 (C5 ), 129.09 (C4 ), 128.55 (C3 ),
128.30 (C3 ), 127.41 (C6 ), 117.44 (C5 ), FT-IR (KBr, cm−1): ꢀ(NH),
3399 (s); ꢀ(C–H), 3055 (m); ꢀ(C C), 1545 (s); ꢀ(C N), 1532 (s);
ı(C–H), 741 (s).
ꢀ
ꢀ
ꢀꢀ
4.18; N, 23.81, 1H NMR: 13.543 (NH), 7.760 (d, 2H, H2 ,2 ), 7.567 (d,
ꢀ
ꢀꢀ
2.3.2. Bis(3,5-dimethyl-1H-pyrazol-1-yl)methane [L2]
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
2H, H5 ,5 ), 7.290 (complex, 4H, H3 ,3
). 13C NMR: 144.28 (C1,2),
ꢀꢀ
,4 ,4
Hydrazine sulphate (0.5 mmol) was dissolved in 400 mL 10%
NaOH in a 1 L RB flask, fitted with a separating funnel. The flask
was immersed in an ice bath and cooled. When the temperature
of the mixture reached 15 ◦C, than acetylacetone (0.50 mmol) was
added drop wise with stirring while the temperature is maintained
at about 15 ◦C. The addition required about 30 min and the mix-
ture was stirred for 1 h at 15 ◦C. The contents of the flask were
diluted with 200 mL of water to dissolve precipitated salts, trans-
ferred to a 1 L separating funnel, and shaken with 125 mL ether. The
layers were separated, and the aqueous layer was extracted with
4 × 40 mL ether. The ether extracts were combined, washed once
with saturated NaCl solution, and dried over anhydrous K2CO3. The
ether was removed by distillation and the slightly yellow residue
of crystalline 3,5-dimethylpyrazole obtained by drying at reduced
pressure.
3,5-Dimethylpyrazole (52.1 mmol) was dissolved in 50 mL
DMSO and added 5.84 g finely powdered KOH in it. The suspen-
sion was vigorously stirred for 1 h at 80 ◦C, and CH2Cl2 in 10 mL
DMSO were added dropwise over 30 min. Stirring at 80 ◦C was con-
tinued for an additional 4 h, and the reaction mixture was poured
into 200 mL water and extracted with chloroform (4 × 40 mL). The
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
143.97 (C1 ,1 ), 135.30 (C6 ,6 ), 124.70 (C2 ,2 ), 122.70 (C5 ,5 ), 119.64
(C4 ,4 ), 112.52 (C3 ,3 ), FT-IR (KBr, cm−1): ꢀ(NH), 3364 (s); ꢀ(C–H),
3063 (m); ꢀ(C C), 1566 (s); ꢀ(C N), 1535 (s); ı(C–H), 733 (s).
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
2.3.5. 4,5-Diazafluorene-9-one (L5)
1,10-Phenanthroline (0.025 mmol) and KOH (0.046 mmol) were
added to 200 mL water and a resulting mixture was brought to a
reflux followed by dropwise addition of aqueous solution of potas-
sium permanganate (0.08 mmol) to it. After addition, the solution
was refluxed for 30 min and filtered off to remove MnO2. When the
solution was cooled, crude 4,5-diazafluoren-9-one precipitated as
yellow needles. Crystallization from water gives a desired ketone
in pure form. Yield: 23.02%, m.p.: 212–213 ◦C, chemical formula
(mol.wt.): C11H6N2O (182.18 g/mol), elemental analysis: calc. (%):
C, 72.52; H, 3.32; N, 15.38, found (%): C, 72.49; H, 3.34; N, 15.35.
1H NMR (CDCl3, 400 MHz) ı/ppm: 8.828, (dd, 2H, H3,6); 8.020, (dd,
2H, H1,8); 7.381, (dd, 2H, H2,7), 13C NMR (CDCl3, 100 MHz) ı/ppm:
188.42, (C9); 155.11, (C11,13); 152.38, (C3,6); 131.69, (C10,12); 129.40,
(C1,8); 124.84, (C2,7), FT-IR (KBr, 4000–626 cm−1): ꢀ(C–H), 3032;
ꢀ(C O), 1713, ꢀ(C C), 1535; ꢀ(C N), 1497; ı(C–H), 710 (s).

510
M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
2.3.6. 2-(2-Pyridyl)benzothiazole [L6]
2.4.6. [Pd(L6)Cl2] [6]
2-Picolinic acid (0.66 g, 5.4 mmol) and 2-aminothiophenol
(0.66 g, 5.3 mmol) were added together to PPA (18 g) at 120 ◦C.
The reaction was stirred under N2 for 20 h at 160 ◦C. The mixture
was cooled, added to water (125 mL) and filtered to give a small
quantity of solid and a yellow filtrate. The filtrate was neutralized
slowly with solid Na2CO3 and NaOH, resulting in the formation of
a yellow precipitate. This was recrystallized from methanol to as a
pale solid. Yield: 45% m.p.: 133–134 ◦C, chemical formula (mol.wt.):
Complex 6 was prepared using 2-(2-pyridyl)benzothiazole by
following above method. Yield: 59.2%, m.p.: 244 ◦C, anal. calc. for:
C
12H8Cl2N2SPd (389.60): C, 36.99; H, 2.07; N, 7.19; Pd, 27.32,
found: C, 37.04; H, 2.02; N, 7.11; Pd, 27.26%, FT-IR (KBr, cm−1):
ꢀ(C–H), 3065; ꢀ(C C), 1569; ꢀ(C N), 1540; ꢀ(C–S), 665; ı(C–H),
745; ꢀ(M–N), 537.
2.5. Antibacterial activity
C
12H8N2S (212.27 g/mol), elemental analysis: calc. (%), C, 67.90; H,
3.80; N, 13.20, found (%): C, 67.99; H, 3.69; N, 13.09. 1H NMR (CDCl3,
The complexes were dissolved in DMSO (dimethylsulfoxide)
and tested against five bacterial strains i.e. two Gram(+ve), Staphy-
lococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 7193) and
three Gram(–ve) Serratia marcescens (MTCC 7103), Escherichia
coli (MTCC 433), Pseudomonas aeruginosa (MTCC P09) for their
inhibitory activities. The antimicrobial assays were performed
using the two fold serial dilution method.
The minimal inhibitory concentration of each of the tested com-
pounds was defined as the lowest concentration exhibiting no
visible growth. Luria Broth was employed as the bacterial growth
medium. Serial concentrations of each of the tested compounds
in the range from 15 to 2820 ␮M were prepared for the determi-
nation of MIC and inoculated with five different microorganisms.
After incubation at 37 ◦C for 24 h, the without turbidity solution
was determined as the minimal inhibitory concentration.
ꢀ
ꢀ
ꢀꢀ
400 MHz) ı/ppm: 8.177, (d, 2H, H4 ,5 ); 7.976, (d, 1H, H6 ); 7.876, (dt,
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
1H, H2,7), 7.546 (d, 1H, H3 ), 7.471–7.409 (complex, 3H, H3 ,6 ,5 )
13C
NMR (CDCl3, 100 MHz) ı/ppm: 157.45, (C2 ); 155.21, (C2); 147.99,
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
(C6 ); 136.41, (C1 ); 133.16, (C2 ); 130.39 (C4 ), 129.41, (C5 ), 128.76
ꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
(C4 ), 128.34 (C6 ), 128.19 (C3 ), 126.49 (C3 ), 121.20 (C5 ), FT-IR
(KBr, cm−1): ꢀ(C–H), 3055 (m); ꢀ(C C), 1532 (s); ꢀ(C N), 1507 (s);
ı(C–H), 740 (s); ꢀ(C–S), 662 (s).
2.4. Synthesis of complexes
2.4.1. [Pd(L1)Cl2] [1]
The complexes were prepared by the method of Rund [20]. The
methanolic solution of 2-(2-pyridyl)benzimidazole added in a solu-
tion of 1 equiv of Na2PdCl4 in H2O with 1 drop of conc. HCl until the
water solution was colourless (0.5–5 h) [21]. Evaporated the solu-
tion to half of the volume and kept solution of complex overnight at
room temperature. Filter the solution and wash with chloroform.
The reaction procedure of all the complexes is shown in Scheme 1.
Yield: 53.5%, m.p.: 228 ◦C, anal. calc. for: C12H9Cl2N3Pd (337.09):
C, 42.76; H, 2.69; N, 12.47; Pd, 31.57, found: C, 42.67; H, 2.75; N,
12.38; Pd, 31.45%, FT-IR (KBr, cm−1): ꢀ(NH), 3418; ꢀ(C–H), 3055;
ꢀ(C C), 1589; ꢀ(C N), 1551; ı(C–H), 741 (s); ꢀ(M–N), 532.
2.6. DNA binding experiments
All the experiments involving the interaction of the complexes
with CT DNA were carried out in Tris–HCl buffer (50 mM Tris–HCl,
pH 7.2) at room temperature. A solution of CT DNA in the buffer gave
a ratio of UV absorbance at 260 and 280 nm of about 1.9, indicating
the DNA sufficiently free from protein [22]. The DNA concentration
per nucleotide was determined by absorption spectroscopy using
the molar absorption coefficient of 6600 M−1 cm−1 at 260 nm [23].
2.4.2. [Pd(L2)Cl2] [2]
Complex 2 was prepared using bis(3,5-dimethyl-1H-pyrazol-1-
yl)methane by following above method. Yield: 53.0%, m.p.: 241 ◦C,
anal. calc. for: C11H16Cl2N4Pd (381.60): C, 34.62; H, 4.23; N, 14.68;
Pd, 27.89, found: C, 34.59; H, 4.16; N, 14.74; Pd, 27.96%, FT-IR (KBr,
cm−1): ꢀ(C–H), 2930; ꢀ(C C), 1550; ꢀ(C N), 1460; ꢀ(M–N), 550.
2.6.1. Absorption titration
Absorption titration experiments were performed by maintain-
ing the palladium complex concentration as constant at 20 ␮M,
while varying the concentration of the CT DNA within 50–150 ␮M.
While measuring the absorption spectra, equal quantity of CT DNA
was added to both the complex solution and the reference solution
to eliminate the absorbance of DNA itself.
From the absorption data, the intrinsic binding constant Kb was
determined from a plot of [DNA]/(εa–εf) versus [DNA] using the
following equation:
2.4.3. [Pd(L3)Cl2] [3]
Complex
3 was prepared using 2,4-bis(4-chlorophenyl)-6-
(thiophen-2-yl)pyridine by following above method. Yield: 57.1%,
m.p.: 236 ◦C, anal. calc. for: C21H13Cl4NSPd (559.63): C, 45.07; H,
2.34; N, 2.50; Pd, 19.02, found: C, 44.98; H, 2.29; N, 2.55; Pd, 19.08%,
FT-IR (KBr, cm−1): ꢀ(C–H), 3069; ꢀ(C C), 1605; ꢀ(C N), 1551;
ꢀ(C–Cl), 1047; ı(C–H), 735; ꢀ(C–S), 691; ꢀ(M–N), 545; ꢀ(M–S), 521.
[DNA]
(εa − εf )
[DNA]
(εb − εf )
=
+ [Kb(εb − εf )]−1
(1)
where [DNA] is the concentration of DNA in base pairs. The apparent
absorption coefficients εa, εf and εb correspond to Aobsd./[Pd], the
extinction coefficient for the free palladium(II) complex and extinc-
tion coefficient for the palladium(II) complex in the fully bound
form, respectively [24]. The Kb is given by the ratio of slope to the
intercept.
2.4.4. [Pd(L4)Cl2] [4]
Complex 4 was prepared using 2,2ꢀ-bisbenzimidazole by fol-
lowing above method. Yield: 62.5%, m.p.: 249 ◦C, anal. calc. for:
C14H9Cl2N4Pd (410.57): C, 40.95; H, 2.21; N, 13.65; Pd, 25.92, found:
C, 40.88; H, 2.16; N, 13.61; Pd, 25.86%, FT-IR (KBr, cm−1): ꢀ(NH),
3371; ꢀ(C–H), 741; ꢀ(C C), 1582; ꢀ(C N), 1551; ı(C–H), 741; ꢀ
(M–N), 540.
2.6.2. Hydrodynamic measurement
Viscosity experiments were carried on an Ubbelohde viscome-
ter, immersed in a thermostated water-bath maintained at a
constant temperature at 27 ◦C ( 0.1). DNA samples approximately
200 base pairs in average length were prepared by sonication in
order to minimize complexities arising from DNA flexibility [25].
Data were presented as (Á/Á0)1/3 versus binding ratio [26], where
Á is the viscosity of DNA in the presence of complex and Á0 is the
viscosity of DNA alone.
2.4.5. [Pd(L5)Cl2] [5]
Complex 5 was prepared using 4,5-diazafluorene-9-one by
following above method. Yield: 53.7%, m.p.: 233 ◦C, anal. calc.
for: C11H6Cl2N2OPd (359.50): C, 36.75; H, 1.68; N, 7.79; Pd,
29.60, found: C, 36.71; H, 1.77; N, 7.72; Pd, 29.63%, FT-IR (KBr,
4000–626 cm−1): ꢀ(C–H), 3063; ꢀ(C O), 1720, ꢀ(C C), 1589;
ꢀ(C N), 1491; ı(C–H), 694 (s); ꢀ(M–N), 532.

M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
511
Scheme 1. Reaction scheme of complex 1.
2.6.3. Thermal denaturation studies
shown in Fig. 1. The peaks at the 136, 137, 154, 289 and 307 m/z
values were due to the usage of matrix. Peaks at 372 and 374 m/z in
spectra were assigned to (M) and (M+2) of complex molecule. There
also existed peaks at 337, 300 m/z values which also showed a pat-
tern as if the compound had Cl atom, hence it could be concluded
that these fragments consisted of two Cl atom. The bands at m/z
195, 119 and 108 are assigned to the peak of 2-(pyridin-2-yl)-1H-
benzo[d]imidazole, benzo[d]imidazole and o-phenylenediamine,
respectively. The isotopic pattern due to presence of chlorine and
palladium is appeared in the spectrum.
DNA melting experiments were carried out by monitoring
the absorption intensity of CT DNA at 260 nm at various tem-
peratures, both in the absence and presence of the ternary
palladium(II) complexes. Measurements were carried out using
a Perkin-Elmer Lambda 35 spectrophotometer equipped with a
Peltier temperature-controlling programmer (PTP 6) ( 0.1 ◦C). The
absorbance at 260 nm was continuously monitored for solutions of
CT DNA (100 ␮M) in the absence and presence of the palladium(II)
complexes (15 ␮M) on increasing the temperature of the solution
by 0.5 ◦C per min.
3.4. Antibacterial activity
2.7. Gel electrophoresis technique
Results from in-vitro antibacterial study (minimum inhibitory
concentration: MIC) of palladium salt, ligands (L1–L6) and the syn-
thesized complexes against S. aureus, B. subtilis, S. marcescens, P.
aeruginosa and E. coli are shown in Table 1. Data of antibacterial
activity showed that the complexes of palladium are more potent
than ligands and metal salt against all the microorganisms. The
complexes show better antibacterial activity than Pd(II) complexes
reported by Radic´ et al. [31] and Juribasˇic´ et al. [32], while have simi-
lar antibacterial activity with Pd(II) complexes reported by Nadeem
et al. [33]. It is well reported that the ligands with nitrogen, oxy-
gen and sulphur donor inhibit enzyme production, the enzymes
which require these groups for their activity appear to be especially
more susceptible to deactivation by the metal ions upon chelation
[34]. Chelation reduces the polarity of the metal ion considerably,
mainly because of the partial sharing of its positive charge with
donor groups and possible ꢁ-electron delocalization on the whole
chelate ring, which in turn increases the lipophilic character of the
chelate. Thus, the interaction between metal ion and the lipid is
favoured. This may lead to breakdown of the permeability barrier
of the cell, resulting in interference with normal cell processes [35].
Other factors contributing to activity are nature of the metal ion,
nature of the ligand, coordinating sites and geometry of the com-
plex, concentration, hydrophilicity, lipophilicity and the presence
of co-ligands.
For the gel electrophoresis experiments, pUC 19 plasmid DNA
(300 ␮M) was treated with the complexes (150 ␮M) in Tris buffer
(50 mM Tris–acetate, 18 mM NaCl buffer, pH 7.2), and the contents
were incubated for 1 h at room temperature. The samples were
electrophoresed for 3 h at 90 V on 0.8% agarose gel in Tris–acetate
buffer. After electrophoresis, the gel was stained with 1 ␮g/mL
ethidium bromide and photographed under UV light.
3. Result and discussion
3.1. Electronic spectra and magnetic moments
In the synthesized Pd(II) complexes, only one d–d band at
∼400 nm is observed, which is assigned to dz → dx − dy tran-
2
2
2
sition, where as other two bands are masked by charge transfer
bands. The bands observed at ∼350 and ∼270 nm correspond to
the charge transfer transitions. From the transitions all these bands
points towards the low spin complex of d8-system with square
planar geometry.
3.2. IR spectra
The infrared spectra provide some important information
regarding the skeleton of the complexes. The IR spectrum of the lig-
and 1 shows characteristic bands for ꢀ(N–H) at 3399 cm−1, ꢀ(C C)
at 1535 and ꢀ(C N) at 1532 cm−1. In the complexation, the ꢀ(C C)
and ꢀ(C N) bands were shifted by 40–30 cm−1 to higher frequen-
cies, due to participation of the azomethine groups in coordination
[27,28]. On the other hand, the stretching vibration of ꢀ(NH) was
not affected in complexes 1, which indicates that the secondary
amine groups are not involved in coordination to the metal ion.
The coordination of nitrogen to the metal atom is supported by
the appearance of a new band in the region 532 cm−1 assignable to
ꢀ(M–N) vibration [29,30].
3.5. DNA-binding studies
3.5.1. Absorption titration
The absorption spectra are one of the most important technique
for complexes binding with DNA [36,37]. The absorption spectra of
the complexes with and without CT DNA are illustrated in Fig. 2.
In the UV region, the intense absorption bands around 225 and
320 nm exist in complex 1, due to intraligand ꢁ–ꢁ* transition of
the coordinated groups. Addition of increasing amounts of CT DNA
resulted in a reduction in absorbance and red shift in the UV spectra
of the complex. This phenomenon is attributed to the strong stack-
ing interaction between aromatic group and the base pairs of CT
DNA, when the complexes intercalate to the CT DNA. The intrinsic
binding constant Kb (Fig. 3) of complex 1 with CT DNA was deter-
mined according to Eq. (1) [24], through a plot of [DNA]/(εa–εf)
3.3. FAB mass spectra
The FAB-mass spectrum of the representative complex 1, that
is [Pd(L1)Cl2], obtained using m-nitro benzyl alcohol as matrix is

512
M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
Fig. 1. FAB MS of [Pd(L1)Cl2] [1] using Argon/Xenon (6 kV, 10 mA) as the FAB gas.
Table 1
Minimum inhibitory concentration of metal salt, ligands and complexes (␮M).
Compounds
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
Na2PdCl4
Ligand 1
Ligand 2
Ligand 3
Ligand 4
Ligand 5
Ligand 6
2238.00
390
560
535
680
120
350
71
47
57
82
15
2490.00
430
650
490
710
135
395
72
42
4
81
22
2145.00
490
610
455
675
105
310
81
49
66
93
31
1940.00
510
565
515
735
110
370
75
43
58
88
18
2820.00
450
590
525
710
120
370
79
46
59
89
22
[Pd(L1)Cl2] (1)
[Pd(L2)Cl2] (2)
[Pd(L3)Cl2] (3)
[Pd(L4)Cl2] (4)
[Pd(L5)Cl2] (5)
[Pd(L6)Cl2] (6)
30
45
50
65
70
is raised, and offer information about the interaction extent of com-
pounds with DNA. When the temperature in the solution increases,
the double-stranded DNA will gradually dissociate to single strands,
and generate a hyperchromic effect on the absorption spectra of
DNA bases (ꢃmax = 260 nm) (Fig. 4). The melting temperature Tm
,
which is defined as the temperature where half of the total base
pairs are unbounded, is determined from the thermal denatura-
tion curves of DNA, and considerably increase when intercalative
binding mode occurs [40]. The present thermal denaturation stud-
ies show an increase in the melting temperature (ꢂTm) of ∼5 ◦C
suggesting primarily intercalative binding mode of the complexes
(Table 2).
Fig. 2. Electronic absorption spectra of [Pd(L1)Cl2] [1] in absence and in presence of
increasing amount of DNA 50–150 ␮M in Tris–HCl buffer (50 mM Tris–HCl, pH 7.2),
[complex] = 20 ␮M, with incubation period of 30 min. at 37 ◦C.
3.5.3. Viscosity measurements
Further clarification of the interactions between the complexes
and DNA was carried out by viscosity measurements. Hydro-
dynamic measurements that are sensitive to length change (i.e.
versus [DNA]. Intrinsic binding constant (Kb) of complexes 1–6 were
calculated and found in the range of 0.998 × 104–4.281 × 104 M−1
(Table 2). The complexes show higher and similar binding constant
value than Pd(II) complexes reported by H. Mansouri-Torshizi et al.
[38,39]. This result shows that interaction is strong between the
complexes and CT DNA.
3.5.2. Thermal denaturation studies
Thermal behaviours of DNA in the presence of compounds can
give insight into their conformational changes when temperature
Table 2
Binding constants and melting temperature of palladium complexes.
Compounds
Binding constant (Kb) (M−1
)
Tm (◦C)
ꢂTm (◦C)
CT-DNA
–
74.2
78.1
77.3
79.6
78.5
78.9
77.8
-
[Pd(L1)Cl2] (1)
[Pd(L2)Cl2] (2)
[Pd(L3)Cl2] (3)
[Pd(L4)Cl2] (4)
[Pd(L5)Cl2] (5)
[Pd(L6)Cl2] (6)
1.139 × 104
0.998 × 104
4.281 × 104
1.149 × 104
2.077 × 104
1.025 × 104
3.9
3.1
5.4
4.3
4.7
3.6
Fig. 3. Plot of [DNA]/(εa–εf) versus [DNA].

M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
513
Fig. 6. Photogenic view of interaction of pUC19 DNA (300 ␮g/mL) with series of pal-
ladium(II) complexes (200 ␮M) using 1% agarose gel containing 0.5 ␮g/mL ethidium
bromide. All reactions were incubated in TE buffer (pH 8) in a final volume of 15 ␮L,
for 3 h. at 37 ◦C.: Lane 1, DNA control; Lane 2, Na2PdCl4; Lane 3, [Pd(L1)Cl2] [1]; Lane
4, [Pd(L2)Cl2] [2]; Lane 5, [Pd(L3)Cl2] [3]; Lane 6, [Pd(L4)Cl2] [4]; Lane 7, [Pd(L5)Cl2]
[5]; Lane 8, [Pd(L6)Cl2] [6].
Table 3
DNA cleavage data.
Compounds
% SC
% OC
% Cleavage
DNA control
DNA + metal salt
DNA + 1
DNA + 2
DNA + 3
DNA + 4
DNA + 5
DNA + 6
74
26
–
63.1
27.4
29.2
17.8
23.5
19.5
28.9
36.9
72.6
70.8
82.2
76.5
80.5
71.1
14.72
62.97
60.54
75.94
68.24
73.64
60.94
Fig. 4. DNA melting temperature curves of CT DNA and palladium complexes in
Tris–HCl buffer (50 mM Tris–HCl, pH 7.2).
complexes can induce the obvious cleavage of the plasmid DNA
at the concentration of 300 ␮M and it shows that the cleavage
efficiency is higher than metal salt (Table 3). The complexes show
quite similar cleavage nature with the complexes reported by G.
Enjun et al. [45] and W. Chuan-sheng et al. [46].
3.7. Effect of ligands on the biological activities of palladium(II)
complexes
Complex 3 is highly potent in all activities due to presence of
sulphur containing bipyridine ring and having two pyridine ring
in the ligand 3. So, the resonance stabilization and inductive effect
might be the reasons for better activity compare to other ligands.
Other complexes show rather poor biological activity compare to
complexes 3 due to absence of above active motifs. Presence of
phenanthroline ring in the ligand 5 makes complex 5 better biolog-
ical active. The poor activity of other complexes can be attributed to
the presence of less active benzimidazole and phenyl ring in their
backbone.
Fig. 5. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 27 0.1 ◦C in Tris–HCl buffer (50 mM Tris–HCl, pH 7.2), as a medium.
viscosity and sedimentation) are regarded as the least ambigu-
ous and the most critical tests of binding model in solution in the
absence of crystallographic structural data [41,42]. A classical inter-
calation model results in lengthening the DNA helix, as base pairs
are separated to accommodate the binding ligand, leading to the
increase of DNA viscosity. However, a partial and/or non-classical
intercalation of ligand may bend (or kink) DNA helix, resulting in
the decrease of its effective length and, concomitantly, its viscos-
ity. The effects of complexes 1–6 on the viscosities of rod-like CT
DNA are shown in Fig. 5. From Fig. 5, it could be seen that all these
complexes show increase the relative viscosity of CT DNA but less
compare to ethidium bromide. The observed increase in the rela-
tive viscosity of DNA could be as a result of a length increase of the
duplex on intercalation of the complex. The viscosity results may
reflect the tendency of each ligand to intercalate into DNA base
pairs, and also parallel to the above spectral results.
Acknowledgements
We wish to express our gratitude to Head, Department of Chem-
istry and Head, B & R Doshi School of Biosciences, Sardar Patel
University, Vallabh Vidyanagar, Gujarat, India, for providing the
necessary laboratory facilities. U.G.C. for providing financial sup-
port under “UGC Research Fellowship for Meritorious Students”
scheme.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.10.077.
3.6. DNA cleavage activity
References
In DNA cleavage study, the six complexes which could function
as DNA cleavage agents, were examined using supercoiled pUC19
DNA as the target. The efficiency of cleavage of these complexes
was probed using agarose gel electrophoresis [43,44]. All the
complexes are found to promote the cleavage of pUC19 plasmid
DNA from supercoiled Form (I) to the nicked Form (II) (Fig. 6). The
[1] P.J. O’Dwyer, P. Stevenson, S.W. Johnson, Clinical status of cisplatin, carboplatin,
and other platinum-based antitumor drugs, in: B. Lippert (Ed.), Chemistry and
Biochemistry of a Leading Anticancer Drug, Verlag Helvetica Chimica Acta,
Zurich, 1999, pp. 31–72.
[2] M. Groessl, E. Reisner, C.G. Hartinger, R. Eichinger, O. Semenova, A.R. Timerbaev,
M.A. Jakupec, V.B. Arion, B.K. Keppler, J. Med. Chem. 50 (2007) 2185–2193.

514
M.N. Patel et al. / Spectrochimica Acta Part A 86 (2012) 508–514
[3] D.S. Gill, in: M.P. Hacker, E.B. Douplr, I.H. Krakoff (Eds.), Platinum Coordination
[28] I.C. Mendes, J.P. Moreira, N.L. Speziali, A.S. Mangrich, J.A. Takahashi, H. Beraldo,
J. Braz. Chem. Soc. 17 (2006) 1571–1577.
[29] A.P. Rebolledo, G.M. de Lima, L.N. Gambi, N.L. Speziali, D.F. Maia, C.B. Pin-
heiro, J.D. Ardisson, M.E. Cortes, H. Beraldo, Appl. Organomet. Chem. 17 (2003)
945–951.
Complexes in Cancer Chemotherapy, Nijhoff, Boston, 1984, pp. 267–278.
[4] L.S. Hollis, in: S.B. Howell (Ed.), Platinum and Other Metal Coordination Com-
pounds in Cancer Chemotherapy, Plenum Press, New York, 1991, pp. 115–125.
[5] J. Kuduk-Jaworska, A. Puszko, M. Kibiak, M. Pelczynska, J. Inorg. Biochem. 98
(2004) 1447–1456.
[6] G. Zhao, H. Lin, Curr Med. Chem.-Anticancer Agents 5 (2005) 137–147.
[7] P. Kafarski, B. Lejczak, in: V.P. Kukhar, H.R. Hudson (Eds.), Aminophosphonic
and Aminophosphinic Acids. Chemistry and Biological Activity, John Wiley &
Sons, Chichester, 2000, pp. 407–442.
[30] A.P. Rebolledo, M. Vieites, D. Gambino, O.E. Piro, E.E. Castellano, C.L. Zani, E.M.
Souza-Fagundes, L.R. Teixeira, A.A. Batista, H. Beraldo, J. Inorg. Biochem. 99
(2005) 698–706.
ˇ
[31] G.P. Radic´, V.V. Glodovic´, I.D. Radojevic´, O.D. Stefanovic´, L.R. Comic´,
Z.R. Ratkovic´, A. Valkonen, K. Rissanen, S.R. Trifunovic´, Polyhedron
[8] X.J. Yang, F. Drepper, B. Wu, W.H. Sun, W. Haehnel, C. Janiak, J. Chem. Soc.,
Dalton Trans. (2005) 256.
[9] E.K. Efthimiadou, N. Katsaros, A. Karaliota, G. Psomas, Inorg. Chim. Acta 360
(2007) 4093–4102.
[10] D. Lawrence, V.G. Vaidyanathan, B.U. Nair, J. Inorg. Biochem. 100 (2006)
1244–1251.
doi:10.1016/j.poly.2011.08.042.
[32] M. Juribasˇic´, K. Molcˇanov, B. Kojic´-Prodic´, L. Bellotto, M. Kralj, F. Zani, L. Tusˇek-
Bozˇic´m, J. Inorg. Biochem. 105 (2011) 867–879.
[33] S. Nadeem, M. Bolte, S. Ahmad, T. Fazeelat, S.A. Tirmizi, M.K. Rauf, S.A.
Sattar, S. Siddiq, A. Hameed, S.Z. Haider, Inorg. Chim. Acta 363 (2010)
3261–3269.
[11] Y.J. Liu, H. Chao, Y.X. Yuan, H.J. Yu, L.N. Ji, Inorg. Chim. Acta 359 (2006)
3807–3814.
[34] Z.H. Chohan, M. Arif, M.A. Akhtar, C.T. Supuran, Bioinorg. Chem. Appl. 131
(2006) 1–13.
[12] M. Kalek, A.S. Madsen, J. Wengel, J. Am. Chem. Soc. 129 (2007) 9392–9400.
[13] A. Bagno, G. Saielli, J. Am. Chem. Soc. 129 (2007) 11360–11361.
[14] D. Bohme, N. Dupre, D.A. Megger, J. Muller, Inorg. Chem. 46 (2007)
10114–10119.
[15] E. Almaraz, Q.A. Paula, Q. Liu, J.H. Reibenspies, M.Y. Darensbourg, N.P. Farrell, J.
Am. Chem. Soc. 130 (2008) 6272–6280.
[35] P.K. Panchal, P.B. Pansuriya, M.N. Patel, J. Enz. Inhib. Med. Chem. 21 (2006)
203–209.
[36] R.K. Rao, A.K. Patra, P.R. Chetana, Polyhedron 26 (2007) 5331–5338.
[37] Y. Li, Y. Wu, J. Zhao, P. Yang, J. Inorg. Biochem. 101 (2007) 283–290.
[38] H. Mansouri-Torshizi, M. Saeidifar, F. Khosravi, A. Divsalar, A.A. Saboury, Z.Y.
Ghasemi, Bull. Korean Chem. Soc. 32 (2011) 947–955.
[39] H. Mansouri-Torshizi, M. Saeidifar, A. Divsalar, A.A. Saboury Nucleosides,
Nucleotides Nucleic Acids 30 (2011) 405–422.
[16] G.A. Cisneros, L. Perera, R.M. Schaaper, L.C. Pedersen, R.E. London, L.G. Pedersen,
T.A. Darden, J. Am. Chem. Soc. 131 (2009) 1550–1556.
[17] L.N. Ji, X.H. Zou, J.G. Liu, Coord. Chem. Rev. 216–217 (2001) 513–536.
[18] F.R. Keene, J.A. Smith, J.G. Collins, Coord. Chem. Rev. 253 (2009) 2021–2035.
[19] A.M. Pizarro, P.J. Sadler, Biochimie 91 (2009) 1198–1211.
[20] M.N. Patel, P.A. Parmar, D.S. Gandhi, Bioorg. Med. Chem. 18 (2010) 1227–1235.
[21] K.D. Hodges, J.V. Rund, Inorg. Chem. 14 (1975) 525.
[22] J. Marmur, J. Mol. Biol. 3 (1961) 208–218.
[23] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047–3053.
[24] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392–6396.
[25] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
[26] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45.
[40] T. Uno, K. Hamasaki, M. Tannigawa, S. Shimabayashi, Inorg. Chem. 36 (1997)
1676–1683.
[41] P.K. Sasmal, A.K. Patra, A.R. Chakravarty, J. Inorg. Biochem. 102 (2008)
1463–1472.
[42] C.-W. Jiang, J. Inorg. Biochem. 98 (2004) 497–501.
[43] E.J. Gao, K.H. Wang, X.F. Gu, Y. Ying, Y.G. Sun, W.Z. Zhang, H.X. Yin, Q. Wu, M.C.
Zhu, X.M. Yan, J. Inorg. Biochem. 101 (2007) 1404–1409.
[44] Q.L. Zhang, J.G. Liu, J.Z. Liu, H. Li, Y. Yang, H. Xu, H. Chao, L.N. Ji, Inorg. Chim.
Acta 339 (2002) 34–40.
[45] G. Enjun, L. Lei, S. Chunyue, Y. Hongxia, Z. Mingchang, W. Qiong, L. Qitao, Chinese
J. Chem. 27 (2009) 1061–1066.
[27] I.C. Mendes, J.P. Moreira, A.S. Mangrich, S.P. Balena, B.L. Rodrigues, H. Beraldo,
Polyhedron 26 (2007) 3263–3270.
[46] W. Chuan-sheng, W. Ni, L. Lie, Z. Ming-Chang, G. Enjun, J. Chin. Clin. Med. 3
(2008) 554–556.