Cytotoxic, antibacterial and nucleic acid interaction studies of square planar palladium(II) complexes

Article abstract of DOI:10.1016/j.ica.2014.04.037

A series of substituted N, O-donor bidentate ligands(Lⁿ) and their square planar mononuclear palladium(II) complexes of the type [PdCl ₂(Lⁿ)] were synthesized and characterized by elemental analysis, conductivity measureme

Full text of DOI:10.1016/j.ica.2014.04.037

Accepted Manuscript
Cytotoxic, antibacterial and nucleic acid interaction studies of square planar
palladium(II) complexes
Mohan N. Patel, Anshul P. Patidar, Parag S. Karia, Pankaj A Vekariya
PII:
S0020-1693(14)00242-4
http://dx.doi.org/10.1016/j.ica.2014.04.037
ICA 15976
DOI:
Reference:
Inorganica Chimica Acta
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Accepted Date:
16 December 2013
28 April 2014
30 April 2014
Please cite this article as: M.N. Patel, A.P. Patidar, P.S. Karia, P.A. Vekariya, Cytotoxic, antibacterial and nucleic
acid interaction studies of square planar palladium(II) complexes, Inorganica Chimica Acta (2014), doi: http://
dx.doi.org/10.1016/j.ica.2014.04.037
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Cytotoxic, antibacterial and nucleic acid interaction studies of square planar
palladium(II) complexes
Mohan N. Patel,* Anshul P. Patidar, Parag S. Karia, Pankaj A Vekariya
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar-388 120, Gujarat, India
Phone number: (+912692) 226856*218
Corresponding address: jeenen@gmail.com
Abstract
A series of substituted N, O –donor bidentate ligands(Ln) and their square planar
mononuclear palladium(II) complexes of the type [PdCl2(Ln)] were synthesized
and characterized by elemental analysis, conductivity measurement, TGA, FTIR,
1H-NMR, C-NMR, LC-MS and electronic spectroscopy. Antibacterial activities
13
of the complexes were evaluated against five pathogenic bacteria. The
antimicrobial test results reveal that all the compounds have good antibacterial
activity against both Gram(+ve) and Gram(–ve) bacterial species compare to ligands
and metal salt. Interaction of a palladium complex with DNA was investigated by
absorption titration and viscosity measurement. Hypochromism and
bathochromism were observed as a result of interaction between palladium(II)
complexes with DNA. It suggests an intercalative binding mode. Furthermore,
viscosity measurements data support the intercalation mode of binding. The
palladium(II) complexes were found to promote cleavage of plasmid pUC19 DNA
from the supercoiled form to the open circular form. Cytotoxic activity of
complexes was carried out to calculate LC50 values.
Key words: Square planar palladium(II) complexes, DNA interaction,
Cytotoxicity
1

1.
Introduction
During the last decades, much attention has been paid to the research in the
area of inorganic antitumor agents. Cisplatin [cis-diamminedichloroplatinum(II)] is
the most effective anticancer drugs [1], in the treatment of a variety of human solid
tumors [2]. Unfortunately, its usefulness is limited due to the development of
resistance to tumor cells and the significant side effects [3]. In order to overcome
the shortcomings, a number of metal complexes have been synthesized, which
show anticancer activity distinctly from cisplatin and bind to DNA in a manner
distinctly different from that of cisplatin [4-8]. Amongst these complexes, the main
interest has been paid to Pd(II), mainly due to the evident structural analogy
between Pt(II) and Pd(II) complexes. Some derivatives of cis-[PdCl2(L)2] have
been described, where L＝pyrazole [9] or inosine derivatives [10], with significant
antitumour activity. The palladium complexes has drawn broad attention, because
of the similar coordination modes and chemical properties of palladium(II) and
platinum(II). Over the past decades, the studies on the interaction of metal
complexes with deoxyribonucleic acid (DNA) attracted much attention due to their
potential applications as anticancer medications and stereo selective probes of
nucleic acid structures [11-13]. Investigation of the interactions of DNA with
transition metal complexes is the base to design new types of the pharmaceutical
molecules to elucidate the mechanism involved in the site specific recognition of
DNA and to determine the principles governing the recognition [14].
In general, most components have three distinct modes of non-covalent interaction
with DNA [15].
1) Intercalative association in which a planar aromatic moiety slides between the
DNA base pairs.
2) DNA groove binding and
3) External binding by electrostatic attraction.
2

Herein, continuation of earlier work [16], we have studied the mode of
coordination and biological properties of Pd(II) complexes with substituted N, O -
donor ligands.
2.
Experimental
2.1. Materials and Instrumental details
All the chemicals and solvents were of reagent grade and used as purchased.
Sodium tetrachloropalladate(II) was purchased from Sd fine chemicals (India). CT-
DNA was purchased from Sigma Chemical Co. (India). 2-Acetylfuran was
purchased from Alfa Aesar (England). p-Chloro acetophenone and p-bromo
acetophenone were purchased from Spectrochem (India). All substituted aldehydes
were purchased from sigma Aldrich (USA). Ethidium bromide and Luria Broth
were purchased from Himedia (India).
C, H, and N elemental analyses were performed with a Perkin–Elmer 240
elemental analyzer. Infrared spectra were recorded on a FT–IR ABB Bomen MB
1
3000 spectrophotometer as KBr pellets in the range 4000–400 cm-1. The H and
13C NMR were recorded on a Bruker Avance (400 MHz). TGA was carried out
using a model mettler toledo TGA/DSC 1 thermogravimetric analyzer. The LC-MS
were recorded using Thermo mass spectrophotometer (USA). Molar conductance
was measured using conductivity meter model no. EQ-660A, Mumbai (India). The
electronic spectra were recorded on a UV–160A UV–Vis spectrophotometer,
Shimadzu (Japan). The minimum inhibitory concentration (MIC) study was carried
out by means of laminar air flow cabinet (Toshiba, Delhi, India). Photo
quantization of the gel after electrophoresis was carried out on AlphaDigiDocTM
RT. Version V.4.0.0 PC–Image software.
2.2. Synthesis of Ligands
Ligands L1 to L7 were synthesized using modified Kröhnke synthesis [17].
Detail mechanism for the synthesis of ligand is shown in Scheme 1.
3

2.2.1. 4-(4-Fluorophenyl)-2-(furan-2-yl)-6-phenylpyridine (L1)
An excess amount of ammonium acetate (approximately 10 equiv) was
added to a mixture of 3-(4-fluorophenyl)-1-phenylprop-2-en-1-one and 1-(2-oxo-2-
(furan-2-yl)ethyl)pyridinium iodide (2.88 mmol) in methanol (20 mL). The
mixture refluxed for 6–7 h on a sand bath. The product obtained by keeping the
solution in an ice bath and purified by crystallization in n-hexane. Ligands were
1 13
characterized by elemental analysis, H and C NMR spectroscopy. Yield: 60 %,
m.p. 112 °C, mol. wt. 315.34. Anal. Calc for C21H14FNO(%): C, 79.98; H, 4.47; N,
1
4.44. Found(%): C, 79.93; H, 4.40; N, 4.39. H NMR (CDCl3, 400 MHz): δ (ppm)
8.169-8.148 (dd, 2H, H2'′,6'′), 7.850-7.847 (d, 1H, H3), 7.780-7.734(complex, 3H,
H2′′′,6′′′,5'), 7.594-7.592 (1H, H4''aromatic), 7.553-7.516 (t, 2H, H3'',5'′), 7.490-7.454 (d,
13
1H, H5), 7.274-7.214 (complex, 3H, H3′′′,5′′′,4'), 6.611-6.598 (dd, 1H, H3′). C NMR
(CDCl3, 100 MHz): δ (ppm) 156.28 (C4′′′), 153.03 (C6), 151.26 (C2′), 149.13 (C2),
145.02 (C4), 137.31 (C5′), 137.09 (C1′′′), 135.44 (C1′′), 129.38 (C4′′), 128.94 (C2′′′,6′′′),
128.40 (C3′′,5′′), 128.29 (C2′′,6′′), 128.06 (C5), 127.98 (C3′′′,5′′′), 124.89 (C3), 116.20
(C4′), 115.29 (C3′).
2.2.2. 2-(4-Chlorophenyl)-4-(4-fluorophenyl)-6-(furan-2-yl)pyridine (L2)
It was synthesized using 1-(4-chlorophenyl)-3-(4-fluorophenyl)prop-2-en-1-
one (2.88 mmol) Yield: 58 %, m.p. 120 °C, mol. wt. 349.79. Anal. Calc for
C21H13ClFNO(%): C, 72.11; H, 3.75; N, 4.00. Found(%): C, 72.21; H, 3.83; N,
1
3.96. H NMR (CDCl3, 400 MHz): δ (ppm) 8.114-8.086 (dd, 2H, H2'′,6'′), 7.853-
7.850 (dm-coupled, 1H, H3), 7.761-7.726 (complex, 3H, H3′′,5′′,5'), 7.596-7.593 (d, 1H,
H5), 7.513-7.485 (dd, 2H, H2''′,6'''), 7.264-7.216 (complex, 3H, H3′′',5'′′,4'), 6.612-6.599
13
(dd, 1H, H3′). C NMR (CDCl3, 100 MHz): δ (ppm) 156.28 (C4′′′), 153.03 (C6),
151.26 (C2′), 149.13 (C2), 145.02 (C4), 137.31 (C5′), 137.09 (C1′′′), 135.44 (C1′′),
129.38 (C4′′), 128.94 (C2′′′,6′′′), 128.40 (C3′′,5′′), 128.29 (C2′′,6′′), 128.06 (C5), 127.98
(C3′′′,5′′′), 124.89 (C3), 116.20 (C4′), 115.29 (C3′).
4

2.2.3. 2,4-Bis(4-chlorophenyl)-6-(furan-2-yl)pyridine (L3)
It was synthesized using 1,3-bis(4-chlorophenyl)prop-2-en-1-one.Yield: 59
%, m.p. 129 °C, mol. wt. 366.24. Anal. Calc for C21H13Cl2NO(%): C, 68.87; H,
3.58; N, 3.82. Found(%): C, 68.93; H, 3.47; N, 3.89. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.112-8.078 (dd, 2H, H2'′,6′'), 7.848-7.844 (d, 1H, H3), 7.734-7.730 (d, 1H,
H5), 7.703-7.676 (dd, 2H, H2''',6'''), 7.596-7.592 (t, 1H, H4′), 7.534-7.476 (complex,
13
4H, H3′′',5′'′,3'',5''), 7.282-7.256 (dd, 1H, H5′), 6.611-6.598 (dd, 1H, H3′). C NMR
(CDCl3, 100 MHz): δ (ppm) 158.12 (C4′′′), 154.53 (C6), 150.30 (C2′), 149.10 (C2),
144.00 (C4), 139.86 (C5′), 137.85 (C1′′), 135.89 (C1′′′), 129.90 (C2′′′,6′′′), 129.82
(C3′′,5′′), 129.16 (C2′′,6′′), 128.86 (C4′′), 127.82 (C5), 117.23 (C3′′′,5′′′), 115.38 (C3),
112.88 (C4′), 109.93 (C3′).
2.2.4. 2-(4-Bromophenyl)-4-(4-chlorophenyl)-6-(furan-2-yl)pyridine (L4)
It was synthesized using 1-(4-bromophenyl)-3-(4-chlorophenyl)prop-2-en-1-
one. Yield: 61 %, m.p. 131 °C, mol. wt. 410.69. Anal. Calc for C21H13BrClNO(%):
1
C, 61.41; H, 3.19; N, 3.41. Found(%): C, 61.45; H, 3.23; N, 3.49. H NMR
(CDCl3, 400 MHz): δ (ppm) 8.034-8.012 (d, 2H, H2'′,6'′), 7.836-7.833 (d, 1H, H3),
7.718-7.698 (d, 2H, H2′′′,6′′′), 7.632-7.596 (complex, 4H, H3'',5'',5',5), 7.473-7.460 (d,
2H, H3''′,5'''), 7.284-7.252 (dd, 1H, H4′), 6.609-6.597 (dd, 1H, H3′). 13C NMR
(CDCl3, 100 MHz): δ (ppm) 156.56 (C6), 153.81 (C2'), 149.94 (C2), 148.87 (C4),
143.44 (C5'), 138.00 (C1′''), 136.92 (C1′′), 135.40 (C4′′′), 131.86 (C4′′), 129.34
(C3′′′,5′′′), 128.64 (C3′′,5′′), 128.36 (C2′′,6′′), 123.71 (C2′′',6′′'), 116.16 (C5), 114.94 (C3),
112.19 (C4′), 109.37 (C3′).
2.2.5. 4-(4-Bromophenyl)-2-(4-chlorophenyl)-6-(furan-2-yl)pyridine (L5)
It was synthesized using 3-(4-bromophenyl)-1-(4-chlorophenyl)prop-2-en-1-
one. Yield: 65 %, m.p. 135 °C, mol. wt. 410.69. Anal. Calc for C21H13BrClNO(%):
1
C, 61.41; H, 3.19; N, 3.41. Found(%): C, 61.47; H, 3.22; N, 3.45. H NMR
(CDCl3, 400 MHz): δ (ppm) 8.104-8.083 (dd, 2H, H2'′,6'′), 7.850-7.846 (d, 1H, H3),
5

7.735-7.731 (d, 1H, H5), 7.689-7.663 (dd, 2H, H3'',5''), 7.638-7.616 (dd, 2H, H3''′,5'''),
7.596-7.593 (d, 1H, H5′), 7.505-7.484 (dd, 2H, H2''′,6'''), 7.266-7.258 (dd, 1H, H4'),
13
6.612-6.599 (dd, 1H, H3'). C NMR (CDCl3, 100 MHz): δ (ppm) 155.15 (C6),
152.53 (C2'), 148.30 (C2), 147.48 (C4), 142.08 (C5'), 136.79 (C1′''), 136.09 (C1′′),
130.79 (C4′′), 130.61 (C2'′′,6'''), 130.44 (C3′′,5′′), 128.70 (C2′′,6′′), 128.53 (C4'′′), 127.31
(C3′′',5′′'), 122.62 (C5), 122.09 (C3), 114.79 (C4′), 113.57 (C3′).
2.2.6. 2-(4-Chlorophenyl)-4-(3-fluorophenyl)-6-(furan-2-yl)pyridine (L6)
It was synthesized using 1-(4-chlorophenyl)-3-(3-fluorophenyl)prop-2-en-1-
one. Yield: 55 %, m.p. 139 °C, mol. wt. 349.79. Anal. Calc for C21H13ClFNO(%):
1
C, 72.11; H, 3.75; N, 4.00. Found(%): C, 72.19; H, 3.70; N, 4.11. H NMR
(CDCl3, 400 MHz): δ (ppm) 8.122-8.088 (dd, 2H, H2'′,6'′), 7.865-7.861 (d, 1H, H3),
7.752-7.748 (d, 1H, H5), 7.601-7.597 (dd, 1H, H5'), 7.557-7.481 (complex, 3H,
H3'′,5'',5'''), 7.476-7.442 (complex, 2H, H2''',6′''), 7.271-7.261 (dd, 1H, H4′), 7.221-7.171
13
(complex, 1H, H4'''), 6.614-6.601 (dd, 1H, H3'). C NMR (CDCl3, 100 MHz): δ
(ppm) 160.17 (C3'''), 156.73 (C6), 151.15 (C2'), 149.91 (C2), 144.78 (C4), 144.60
(C1′''), 141.77 (C5′), 139.38 (C1′′), 133.54 (C4′′), 133.36 (C3′′,5′′), 131.99 (C2′′,6′′),
131.59 (C5'′′), 129.97 (C6′′'), 126.90 (C5), 124.96 (C4'''), 124.78 (C2''′), 117.70 (C3),
116.28 (C4'), 113.10 (C3').
2.2.7. 2-(4-Bromophenyl)-6-(furan-2-yl)-4-p-tolylpyridine (L7)
It was synthesized using 1-(4-bromophenyl)-3-p-tolylprop-2-en-1-one.Yield:
56 %, m.p. 143 °C, mol. wt. 390.27. Anal. Calc for C22H16BrNO(%): C, 67.71; H,
4.13; N, 3.59. Found(%): C, 67.74; H, 4.19; N, 3.51. 1H NMR (CDCl3, 400 MHz):
δ (ppm) 8.049-8.028 (d, 2H, H2'′,6'′), 7.894-7.892 (d, 1H, H3), 7.778-7.776 (d, 1H,
H5), 7.679-7.637 (complex, 4H, H3'',5'',3''',5'''), 7.593 (d, 1H, H5′), 7.362-7.342 (d, 2H,
H2′'′, 6′'′), 7.251-7.243 (ddpoor resolve, 1H, H4′), 6.606-6.594 (dd, 1H, H3′), 2.466 (s, 3H,
13
-CH3). C NMR (CDCl3, 100 MHz): δ (ppm) 156.40 (C6), 154.06 (C2'), 150.05
(C2), 143.30 (C4), 139.32 (C5'), 138.33 (C1''′), 135.56 (C1′′), 131.81 (C4'′′), 130.01
6

(C3′′,5''), 129.84 (C2′′′,6′′′), 129.77 (C2′′,6′′), 128.68 (C3'′′,5'′′), 126.92 (C4''), 123.50 (C5),
116.35 (C3), 115.07 (C4′), 112.11 (C3′), 21.27 (-CH3).
2.3. Synthesis of Complexes
2.3.1. [PdCl2(L1)] [1]
The complexes were synthesized using following literature procedure [18].
Complex
1
was synthesized using 4-(4-fluorophenyl)-2-(furan-2-yl)-6-
phenylpyridine (L1) (0.5 mmol) as a ligand and Na2PdCl4 (0.5 mmol). General
reaction scheme for the synthesis of complexes is given in Scheme 2. Yield:
69.00%, m.p.: 217 ºC, Λm: 3.4 ohm-1 mole-1 cm2. Anal. Calc. for:
C21H14Cl2FPdNO(%) (492.67): C, 51.20; H, 2.86; N, 2.84; Pd, 21.60. Found (%):
C, 51.30; H, 2.78; N, 2.90; Pd, 21.50. FT–IR (KBr, 4000–400 cm–1): 3055 ν(C–
H)ar; 1590, ν(C=C); 1510 ν(C=N); 1384, 1269 (pyridine skeleton band); 1220 ν(C–
F); 920, 820 (p–substituted aromatic ring); 718 δ(C–H); 552 ν(Pd–N); 465 ν(Pd–
O).
2.3.2. [PdCl2(L2)] [2]
It was synthesized using 2-(4-chlorophenyl)-4-(4-fluorophenyl)-6-(furan-2-
yl)pyridine (L2) (0.5 mmol). Yield: 67.3%, m.p.: 215 ºC, Λm: 2.7 ohm-1 mole-1 cm2.
Anal. Calc. for: C21H13Cl3FPdNO (%) (527.11): C, 47.85; H, 2.49; N, 2.66; Pd,
20.19. Found (%): C, 47.79; H, 2.43; N, 2.71; Pd, 20.14. FT–IR (KBr, 4000–400
cm–1): 3063 ν(C–H)ar; 1600, ν(C=C); 1489 ν(C=N); 1381, 1273 (pyridine skeleton
band); 1227 ν(C–F); 1050 ν(C–Cl); 1022, 825 (p–substituted aromatic ring); 752
δ(C–H); 555 ν(Pd–N); 480 ν(Pd–O).
2.3.3. [PdCl2(L3)] [3]
It was synthesized using 2,4-bis(4-chlorophenyl)-6-(furan-2-yl)pyridine (L3)
(0.5 mmol). Yield: 64.52%, m.p.: 209 ºC, Λm: 6.9 ohm-1 mole-1 cm2. Anal. Calc.
for: C21H13Cl4PdNO (%) (543.57): C, 46.40; H, 2.41; N, 2.58; Pd, 19.58. Found
7

(%): C, 46.29; H, 2.49; N, 2.51; Pd, 19.49. FT–IR (KBr, 4000–400 cm–1): 3090
ν(C–H)ar; 1591, ν(C=C); 1522 ν(C=N); 1375, 1268 (pyridine skeleton band); 1044
ν(C–Cl); 910, 821 (p–substituted aromatic ring); 732 δ(C–H); 548 ν(Pd–N); 463
ν(Pd–O).
2.3.4. [PdCl2(L4)] [4]
It was synthesized using 2-(4-bromophenyl)-4-(4-chlorophenyl)-6-(furan-2-
yl) pyridine (L4) (0.5 mmol). Yield: 71.09%, m.p.: 221 ºC, Λm: 13.6 ohm-1 mole-1
cm2. Anal. Calc. for: C21H13BrCl3PdNO (%) (588.02): C, 42.89; H, 2.23; N, 2.38;
Pd, 18.10. Found (%): C, 42.80; H, 2.31; N, 2.47; Pd, 18.20. FT–IR (KBr, 4000–
400 cm–1): 3102 ν(C–H)ar; 1593 ν(C=C); 1496 ν(C=N); 1370, 1270 (pyridine
skeleton band); 1022 ν(C–Br);1044 ν(C–Cl); 905, 815 (p–substituted aromatic
ring); 745 δ(C–H); 550 ν(Pd–N); 472 ν(Pd–O).
2.3.5. [PdCl2(L5)] [5]
It was synthesized using 4-(4-bromophenyl)-2-(4-chlorophenyl)-6-(furan-2-
yl) pyridine (L5) (0.5 mmol). Yield: 69.17%, m.p.: 217 ºC, Λm: 4.9 ohm-1 mole-1
cm2. Anal. Calc. for: C21H13BrCl3PdNO (%) (588.02): C, 42.89; H, 2.23; N, 2.38;
Pd, 18.10. Found (%): C, 42.78; H, 2.30; N, 2.44; Pd, 18.17. FT–IR (KBr, 4000–
400 cm–1): 3095 ν(C–H)ar; 1595 ν(C=C); 1532 ν(C=N); 1380, 1274 (pyridine
skeleton band); 1025 ν(C–Br); 1040 ν(C–Cl); 1010, 820 (p–substituted aromatic
ring); 733 δ(C–H); 547 ν(Pd–N); 482 ν(Pd–O).
2.3.6. [PdCl2(L6)] [6]
It was synthesized using 2-(4-chlorophenyl)-4-(3-fluorophenyl)-6-(furan-2-
yl) pyridine (L6) (0.5 mmol). Yield: 66.88%, m.p.: 207 ºC, Λm: 9.6 ohm-1 mole-1
cm2. Anal. Calc. for: C21H13Cl3FPdNO (%) (527.11): C, 47.85; H, 2.49; N, 2.66;
Pd, 20.19. Found (%): C, 47.77; H, 2.53; N, 2.72; Pd, 20.11. FT–IR (KBr, 4000–
400 cm–1): 3070 ν(C–H)ar; 1590 ν(C=C); 1493 ν(C=N); 1365, 1269 (pyridine
8

skeleton band); 1237 ν(C–F); 1052 ν(C–Cl); 920, 790, 779 (m–substituted aromatic
ring); 778 δ(C–H); 541 ν(Pd–N); 477 ν(Pd–O).
2.3.7. [PdCl2(L7)] [7]
It was synthesized using 2-(4-bromophenyl)-6-(furan-2-yl)-4-p-tolylpyridine
(L7) (0.5 mmol). Yield: 72.00%, m.p.: 225 ºC, Λm: 22.0 ohm-1 mole-1 cm2. Anal.
Calc. for: C22H16BrCl2PdNO (%) (567.60): C, 46.55; H, 2.84; N, 2.47; Pd, 18.75.
Found (%): C, 46.60; H, 2.78; N, 2.55; Pd, 18.64. FT–IR (KBr, 4000–400 cm–1):
3105 ν(C–H)ar; 1597 ν(C=C); 1542 ν(C=N); 1367, 1265 (pyridine skeleton band);
1020 ν(C–Br); 920, 818 (p–substituted aromatic ring); 719 δ(C–H); 546 ν(Pd–N);
465 ν(Pd–O).
2.4 Biological Elucidation
2.4.1. Antibacterial activities
Antibacterial activity was performed by determining the minimum inhibitory
concentration (MIC) in 2% Luria Broth (LB) as a medium. The complexes were
dissolved in DMSO. Cultured for Gram(+ve) and Gram(–ve) were incubated at 37 °C.
Control test was performed with no active ingredient [19]. MIC was determined
using two fold serial dilution in liquid media containing 0.2–2,000 µM variation of
test complex concentration. A pre-culture of bacteria was grown in LB overnight at
the optimal temperature of each species. Bacterial growth was monitored by
measuring turbidity of the culture after 18 h. If a certain concentration of a
complex inhibits the bacterial growth, half the concentration of the complex was
tested. This procedure was carried on to a concentration that bacteria grow
normally. The lowest concentration that inhibits the bacterial growth was
determined as the MIC value. All equipment and culture media were sterile.
2.4.2. DNA Binding Study by Absorption Titration
The UV absorbance at 260 and 280 nm of the CT DNA solution in 5 mM
Tris–HCl buffer (pH 7.2) gave a ratio of 1.9, indicating that the DNA was free of
9

protein. The concentration of CT DNA was measured from the band intensity at
260 nm with a known value (ε = 6600 M−1 cm−1) [20]. Absorption titration
measurements were carried out by varying the concentration of CT DNA, keeping
the metal complex concentration constant in 5 mM Tris–HCl/5 Mm NaCl buffer
(pH 7.2). Samples were kept for equilibrium before recording spectrum. The
intrinsic binding constant (Kb) was determined from a plot of the [DNA]/(εa − εf )
versus [DNA] using equation [21].
ꢀ
ꢁ
ꢀ
ꢁ
DNA
DNA
1
ꢄ=ꢄ
ꢄ+ꢄ
ꢂ
ꢅ
ꢂ
ꢅ
ꢂ
ꢅ
ꢃa– ꢄꢃf
ꢃb– ꢄꢃf
ꢆb ꢃb– ꢄꢃf
where, [DNA] = Concentration of DNA in base pairs,
εa = the apparent extinction coefficient = (Aobs/[Complex])
εf = the extinction coefficient for free metal complex
εb = the extinction coefficient for free metal complex in the fully bound form
2.4.3. DNA Binding Study by Viscosity Measurement
The viscosity measurement was performed with an Ubbelodhe automated
viscometer. The viscometer was thermostated at 27
0.1 ºC in a constant
temperature bath. The flow times were measured with a digital timer. Flow time
for each sample was measured in triplicate and an average time was considered for
further calculations. Changes in relative viscosity of DNA for increasing amounts
of complexes were represented graphically as (η/ηo)1/3 versus concentration ratio of
complex to DNA, i.e. [Complex]/[DNA], where, η is viscosity of DNA in the
presence of complex and ηo is the viscosity of DNA alone [22]. Viscosity values
were calculated using the relation η ∝ t-t0; where t0 is the flow time of buffer alone
and t is the flow time for buffer containing DNA.
2.4.4. DNA Cleavage Study by Gel Electrophoresis
Cleavage of pUC19 DNA (50 µM) by complexes (200 µM) was measured
by the conversion of supercoiled pUC19 DNA to open circular (OC) and linear
10

(L). Gel electrophoresis of pUC19 DNA was carried out in TAE buffer (0.04 M
Tris–Acetate, pH 8.2, 0.001 MEDTA). The reaction mixture (15 mm3) contained
plasmid DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and 200 µM
complex. Reaction mixture was incubated for 3h at 37 ºC. After the incubation, all
reactions were quenched by addition of 2 mm3 loading buffer (0.25% bromophenol
blue, 40% sucrose, 0.25% xylene cyanole, and 200 mM EDTA). The aliquots were
loaded directly onto 1% agarose gel and electrophoresed at 50 V in 1X TAE
buffer. Gel was stained with 0.5 µg/mL of EtBr and was photographed on a UV
illuminator. After electrophoresis, the percentage of each form of DNA was
estimated quantitatively from the intensity of the bands using AlphaDigiDoc™ RT.
The degree of DNA cleavage activity was expressed in terms of the percent of
cleavage of the SC-DNA according to the following equation [23].
ꢐꢑꢒꢓꢔꢑꢕꢌꢂ%ꢄꢈꢉꢄꢊꢋꢌꢍꢎꢏꢅꢖꢗꢘꢙꢕꢚ
% DNA cleavage activity = ꢇꢂ%ꢄꢈꢉꢄꢊꢋꢌꢍꢎꢏꢅ
ꢛ X100
ꢂ%ꢄꢈꢉꢄꢊꢋꢌꢍꢎꢏꢅꢐꢑꢒꢓꢔꢑꢕ
2.4.5. Cytotoxic Activity
Brine shrimp assay was carried out according to the method given by Mayer
et al. [24]. The eggs of the brine shrimp were hatched in a shallow rectangular
plastic dish (22 × 32 cm), filled with 3.33% artificial sea water. Artificial sea water
was prepared using commercial salt mixture and double distilled water. An
unequal partition was made in the plastic dish with the help of a perforated device.
Approximately 50 mg eggs were sprinkled into the large compartment, which was
open to ordinary light. After hatching, active nauplii free from egg shells were
collected from the brighter portion of the hatching chamber and used for the assay.
1mL seawater and 10 shrimps were added to each vial (30 shrimps per dilution)
and the volume was adjusted with sea water to 2.5 mL per vial. A sample of the
test compound was prepared by dissolving 10mg of each compound in 10 mL
DMSO. Solutions were transferred from the stock solutions to 18 vials to make
11

final concentrations of 4, 8, 12, 16 and 20 µgmL–1 (three sets for each dilution
were run for all test samples and a mean of three sets was taken for calculation of
LC50). One vial was kept as a control, having the same amount of DMSO only.
When the shrimp nauplii were ready, 1 mL artificial sea water and 10 shrimps were
added to each vial (30 shrimps per dilution). The volume was adjusted to 2.5 mL
per vial using solution of artificial sea water. The number of survivors was counted
after 24 h. Data were analyzed by a simple method to determine the LC50 values, in
which the log of sample concentration was plotted against percentage mortality of
nauplii [25].
3.
Result and Discussion
3.1. Electronic spectra and magnetic moments
For low-spin square-planar d8-metal complexes, three d−d transitions are
expected corresponding to the transitions from three lower lying d-levels to the
2
2
2 2
empty dx –y orbital. These transitions are designated as dz2 (a1g) → dx –y (b1g), dxy
2
2
(b2g) → dx –y2 (b1g) and dxz,yz (eg) → dx –y2 (b1g) [26-29].
In the synthesized Pd(II) complexes, only one band at ~430 (23,256 cm–1)
2
2
nm is observed [30], due to dz2 → dx –y transition, where as other two bands are
masked by high intensity charge transfer bands. The bands observed at ~362
(~27,400 cm−1) and ~330 nm (30,120 cm–1) correspond to the charge transfer
transitions [31]. All these bands indicate the possibility of low spin complex of d8–
system with square planar geometry.
Room temperature magnetic moment value of the synthesized complexes are
found to be zero, suggesting d8 system with low-spin configuration i.e. dsp2
hybridization.
3.2. IR spectra
Generally the aromatic C-H stretching and out of plane bending vibration
give the band at ~3050 and ~740 cm–1. Major changes occur in the bands of C═C
12

(~1550) and C═N (~1450 cm–1) pyridine-ring stretching of the ligands. These
bands are appreciably shifted by 30-40 cm–1 to higher frequencies after
complexation. These shifting support the binding of metal via nitrogen atoms of
ligand. The coordinate covalent bonding of metal via nitrogen atoms of ligand (Pd–
N), which has been supported by a sharp band at ~550 cm-1 [32,33]. The
characteristic band at ~470 cm-1 suggests Pd–O bond [34].
3.3. Molar Conductance Measurements
The Pd(II) complexes were dissolved in DMSO (10–3 M) and molar
conductivities of the solutions were measured at room temperature. The molar
conductance (Λm) values (3.4-22.0 ohm-1 mole-1 cm2) of Pd(II) complexes support
their non ionic and non electrolytes nature.
3.4. Thermogravimetric analysis
Thermogravimetric analysis was carried out for complex1 at a heating rate
of 10 ºC per minute in the range of 20–800 ºC under N2 atmosphere. TGA data of
the complexes show no weight loss between 80 to 240 °C indicates the absences of
coordinated or lattice water molecules. A weight loss (76.00%) between 250 to
500°C corresponds to decomposition of ligands and leaving behind PdO as a
residue.
3.5. Mass spectrometry
Mass spectrum of the complex 1 (Fig.1) shows molecular ion peak [M+] at
490.95m/z, [M+2] at 492.90m/z and [M+4] at 494.95m/z where, the M+2 and M+4
37
peaks stem from isotopic Cl. Peak at 456.00m/z is due to loss of one chlorine
atom from the complex. Peak at 421.00m/z is due to ligand attached with palladium
metal i.e. due to loss of two chlorine atom from the complex. Peak at 315.10m/z is
due to ligand i.e. loss of palladium metal ion. Other peaks at 249.05, 173.05 and
13

155.04m/z are due to the fragmentation of ligand. The probable mass fragmentation
pattern is shown in scheme 3.
3.6 Biological Elucidation
3.6.1. Antibacterial activity
The free ligands (L1–L7), metal salt and their palladium(II) complexes were
screened against pathogenic bacteria, namely (Escherichia coli (MTCC 433),
Pseudomonas aeruginosa (MTCC 1688), Serratia marcescens (MTCC 7103),
Bacillus subtilis (MTCC 7193) and Staphylococcus aureus (MTCC 3160)) to
assess their antimicrobial activity. The antibacterial activity data (Table 1) reveal
that palladium(II) complexes are more potent than the parent ligands (L1–L7) and
Na2PdCl4. The enhanced activity of the metal complexes may be attributed to the
increased lipophilic nature of the complexes arising due to the chelation [35].
Complex 1-3 are found to be more potent and complex 4-5 are comparable than
those of reported [Pd(CEIMAP)], [Pd(CIMPAP)] [36] against Bacillus subtilis,
Staphylococcus aureus and Escherichia coli. Complex 1-4 are more potent than
those of reported [Pd(Tu)2(PPh3)2]Cl2 [37] against Staphylococcus aureus, Bacillus
subtilis, Escherichia coli and Pseudomonas aeruginosa.
3.6.2. DNA Binding Study by Absorption Titration
The absorption spectra of the complex 1 with CT-DNA are shown in Fig. 2.
All complexes show hypochromism with a red shift of 2 nm. The change in the
absorbance values for all complexes with increasing amounts of CT-DNA were
used to evaluate the intrinsic binding constants (Kb) value. This observation gives
good evidence of an interaction between complexes and DNA [38,39]. The
hypochromism with bathochromic shift spectrum of the complexes indicated a
stacking interaction between the aromatic group and the base pairs of CT-DNA, It
14

means that the complexes can intercalate into the double helix structure of DNA
[40,41].
The intrinsic binding constant Kb of the synthesized complexes with CT-
DNA has been found in range of 9.93-3.24×104 M-1 (Table 2), which is lower than
Kb value of classical intercalator ethidium bromide and higher than that of reported
complexes [Pd(L1)Cl2·2H2O] [42] (L1= 2,2′-bipyridyl-5,5′-dicarboxylic acid) and
free ligands (~103 M–1).
3.6.3. DNA Binding Study by Viscosity Measurement
Further clarification of the interactions between the complexes and DNA
was carried out by viscosity measurements. The viscosity of DNA solution is
sensitive to the addition of metal complexes which can bind to DNA. Intercalation
leads to increase in viscosity because the DNA base pairs are pushed apart to
accommodate the bound ligand, a partial, non-classical mode of binding could
bend or kink the DNA helix, reduce its effective length and thereby its viscosity.
The effect of addition of increasing concentrations of complexes on the specific
relative viscosity of DNA is shown in Fig. 3. The viscosity of DNA bound to
complexes reveals a steady increase with increase in complex concentration. The
increase in DNA viscosity suggests a classical intercalative mode of complexes.
From the figure it is confirm that complex 2 binds to DNA effectively than rest of
them.
3.6.4. DNA Cleavage Study by Gel Electrophoresis
The ability of complexes to perform DNA cleavage is generally monitored
by agarose gel electrophoresis using pUC19 plasmid DNA, pBR322 plasmid DNA
and pUC18 plasmid DNA [43–45]. Herein, the ability of complexes to perform
DNA cleavage is monitored by agarose gel electrophoresis using pUC19 plasmid
DNA in the presence of synthesized complexes and metal salt. DNA cleavage
15

efficiency of complexes can be achieved by formation of the naturally occurring
supercoiled (SC) form to open circular relaxed (OC) form. OC form produced
when one of the strands of the plasmid is nicked, and determined by gel
electrophoresis of the plasmid. If both strands of the plasmid are nicked, linear (L)
form will be generated [46]. SC form migrates faster, OC form migrates slowly
while L form migrates in between the SC form and the OC form [47].
Hence, DNA cleavage ability of complexes was quantified by measuring the
transformation of the SC form into OC and linear forms (Fig.4). It clearly shows
that the relative binding efficacy of complexes to DNA is much higher than the
binding efficacy of metal salt (Table 3). Difference in DNA cleavage efficiency of
complexes was due to the difference in binding affinity of complexes to pUC19
plasmid DNA. The percentage cleavage of synthesized complexes is found in
range of 65-79%. It is indicates the synthesized complexes cleave to DNA
efficiently.
3.6.5. Cytotoxic Activity
Percentage mortality was calculated and plotted against log dose. The LC50
values are found in the range of 6.83–15.93 µg/mL (Table 2). It is found that the
complexes show considerable cytotoxicity in the brine shrimp (Artemia cysts)
lethality bioassay. All complexes were compare for their cytotoxic activity and
from the result it was found that synthesized complexes show higher cytotoxicity
than reported one [48,49]. Among all the complexes, complex 1 and 2 shows
maximum activity due to presence of less crowded structure unit (fluorine).
4.
Conclusion
All the synthesized N, O –donor ligands L1–L7 act as bidentate ligands, and are
coordinated to the metal ion. All the synthesized Pd(II) complexes possesses an
square planar geometry. The findings of biological studies indicate that the Pd(II)
16

complexes exhibit significant activities against different bacterial strains, which
might be due to heteroatoms and presence of more electronegative environment.
The hypochromism and bathochromism (~3-4 nm) observed in absorption titration
is consistent with the intercalation mode of binding. The increase in the relative
viscosity of DNA in the presence of metal complexes suggests a classical
intercalation binding mode. The cleavage of plasmid pUC19 DNA by compounds
observed in gel electrophoresis study suggests sufficient interaction of compounds
with DNA. Cytoxicity assessment shows a potent lethality (LC50) of metal
complexes against brine shrimp but this investigation is a primary one and further
tests are required to investigate its actual mechanism of cytotoxicity and its
probable effects on higher animal models and on cancer cell lines. It is suggested
that the complexes can be used as potent cytotoxic agents with the hope of adding
to the arsenal of weapons used against the fatal disease of cancer.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
Acknowledgments
Authors thank Head, Department of Chemistry Sardar Patel University,
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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21

Figure 1: LC-MS mass spectra of complex [PdCl2(L1)] [1].
Figure 2: Electronic absorption spectra of complex 1 in Tris- HCL buffer (pH 7.2) in the
absence and presence of increasing amount of CT-DNA. Inset: Plot of [DNA]/(εa– εf) vs. [DNA].
Arrow shows the absorbance change upon increasing DNA concentrations.
Figure 3: Effect on relative viscosity of CT- DNA under the influence of increasing amount of
complexes at 27 0.1 oC in Tris- HCL buffer (pH 7.2).
Figure 4: Photogenic view of cleavage of pUC19 DNA (300 µg/mL) with series of palladium(II)
complexes (200 µM) using 1% agarose gel containing 0.5 µg/mL ethidium bromide. All
o
reactions were incubated in TE buffer (pH 8) in a final volume of 15 µL, for 24 h. at 37 C. :
Lane 1, DNA control; Lane 2, Na2PdCl4; Lane 3, [PdCl2(L1)]; Lane 4, [PdCl2(L2)]; Lane 5,
[PdCl2(L3)]; Lane 6, [PdCl2(L4)]; Lane 7, [PdCl2(L5)]; Lane 8, [PdCl2(L6)]; Lane 9, [PdCl2(L7).
Scheme 1: Detail mechanism for the synthesis of ligands.
Scheme 2: Synthesis of substituted N,O -donor ligands and their Pd(II)complexes.
Scheme 3: Fragmentation pattern of complex 1.
22

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24

25

26

27

28

29