DNA interaction and cytotoxic activities of square planar platinum(II) complexes with N, S-donor ligands

Article abstract of DOI:10.1016/j.saa.2014.02.053

The platinum(II) complexes with N, S-donor ligands have been synthesized and characterized by physicochemical methods viz. elemental, electronic, FT-IR, ¹H NMR and LC-MS spectra. The binding mode and potency of the complexes with HS DNA (Herrin

Full text of DOI:10.1016/j.saa.2014.02.053

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA interaction and cytotoxic activities of square planar platinum(II)
complexes with N, S-donor ligands
⇑
Mohan N. Patel , Chintan R. Patel, Hardik N. Joshi, Khyati P. Thakor
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
Synthesis of N, S-donor ligands and
their Pt(II) complexes.
Characterization by elemental
analysis, IR, UV–visible, 1H NMR and
mass spectrometry.
ꢀ
ꢀ
All Pt(II) complexes have been tested
for their cytotoxic activity.
The interaction of DNA with
complexes has been performed.
a r t i c l e i n f o
a b s t r a c t
Article history:
The platinum(II) complexes with N, S-donor ligands have been synthesized and characterized by
physicochemical methods viz. elemental, electronic, FT-IR, 1H NMR and LC–MS spectra. The binding mode
and potency of the complexes with HS DNA (Herring Sperm) have been examined by absorption titration
and viscosity measurement studies. The results revealed that complexes bind to HS DNA via covalent
mode with the intrinsic binding constant (Kb) in the range 1.37–7.76 ꢁ 105 Mꢂ1. Decrease in the relative
viscosity of HS DNA also supports the covalent mode of binding. The DNA cleavage activity of synthesized
complexes has been carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA;
showing the unwinding of the negatively charged supercoiled DNA. Brine shrimp (Artemia Cysts) lethality
bioassay technique has been applied for the determination of toxic property of synthesized complexes in
Received 17 October 2013
Received in revised form 30 January 2014
Accepted 11 February 2014
Available online 27 February 2014
Keywords:
Pt(II) complexes
N, S-donor ligand
DNA binding
DNA cleavage
Unwinding
terms of lM.
Ó 2014 Elsevier B.V. All rights reserved.
Cytotoxicity
Introduction
design of new platinum compounds is the synthesis of compounds
that remain active against resistant cell lines, with a wider spec-
trum of antitumor activity and with a lower toxicity than cisplatin.
It is generally accepted that the efficiency of cisplatin is based on its
strong binding to DNA nucleobases resulting in a locally unwound
and kinked helix [2,3].
In order to reduce the toxicity and to modulate activity of the
known anticancer drug, cisplatin, a new strategy is the design of
novel metal complexes containing N, S-donor ligands [4,5]. The
interactions of heavy metals such as platinum, gold with N, S-do-
nor atoms have been recognised for their anti-carcinogenic
In the view of excellent anticancer efficiency of cisplatin, but at
the same toxicity associated with its therapeutic applications,
thousands of new platinum compounds have been synthesized
and investigated. Several of them have been tested in clinical trials,
and up to date only few have been approved for clinical use, e.g.
carboplatin, tetraplatin, or oxaliplatin [1]. The goal behind the
⇑
Corresponding author. Tel.: +91 2692 226856x218.
E-mail address: jeenen@gmail.com (M.N. Patel).
http://dx.doi.org/10.1016/j.saa.2014.02.053
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

262
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
properties with the potential to develop metal-based drug [6,7].
This interest has probably initiated from detoxicant properties of
sulfur-containing ligands against heavy metal intoxication [8].
The chemistry of N, S containing heterocyclic ligands with plati-
num group metals has been of considerable interest both from
structural point of view [9,10] and also for their antitumor activity
[11,12]. High tendency of platinum to bind sulphur and isolation of
thiolate complexes (e.g., [Pt(methionine)2] [13]) from the urine of
cisplatin treated patients have been a driving force to explore plat-
inum complexes with sulphur ligands so as to overcome the side
effects of cisplatin and have improved toxicity and/or their applica-
bility to a wide range of tumours [14].
In continuation of our previous work [15], we describe the syn-
thesis and characterization of some N, S-donor ligand and their
platinum(II) complexes. The mode and extent of interaction of
complexes have been determined by viscosity measurements and
absorption titration using HS DNA. 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 of
the complexes has been carried out by Brine shrimp (Artemia Cysts)
2-(4-Chlorophenyl)-4-(4-fluorophenyl)-6-(thiophen-2-yl)pyridine
(L1)
Anal. Calc. for C21H13ClFNS: Calc. (Found): C, 68.94 (68.80); H,
3.58 (3.50); N, 3.83 (3.70); S, 8.76 (8.65). Yield: 65%, mp: 138–
140 °C, mol. wt. 365.85;1H NMR (CDCl3, 400 MHz) d/ppm:8.14–
0
000,3000,5000,6000
8.11 (complex, 2H, H3,5), 7.74–7.69 (complex, 5H, H5 ,2
),
0
00 00
7.51–7.46 (complex, 3H, H3 ,3 ,5 ), 7.26–7.22 (complex, 2H,
H2 ,6 ), 7.19–7.16 (complex, 1H, H4 ). 13C NMR (CDCl3, 100 MHz)
00 00
0
000
0
d/ppm: 164.75 (C4 ), 156.16 (C6), 152.93(C2), 149.32 (C2 ),
000
00
000,5000
000,6000
145.09 (C1 ), 137.34 (C1 ), 134.76 (C3
), 134.73 (C2
),
0
0
0
00
00
128.94 (C5 ), 128.91 (C3 ), 128.86 (C4 ), 128.30 (C5 ), 128.03 (C3 ),
00
00
127.93 (C6 ), 124.85 (C2 ), 116.07 (C3), 115.32 (C5). IR (KBr,
4000–400 cmꢂ1): 3066, 3056
(CAH)ar; 1545, 1491 (C@C);
1397 (C@N); 1179 (C@S); 1229 (CAF); 1092 (CACl); 1381,
m
m
m
m
m
m
1362 (pyridine skeleton band); 1112, 828 (p-substituted aromatic
ring); 800 d(CAH).
2-(4-Chlorophenyl)-4-phenyl-6-(thiophen-2-yl)pyridine(L2)
Anal. Calc. for C21H14ClNS: Calc. (Found): C, 72.51 (71.95); H,
4.06 (4.02); N, 4.03 (4.01); S, 9.22 (9.14). Yield: 55%, mp: 118–
120 °C, mol. wt. 347.86;1H NMR (CDCl3, 400 MHz) d/ppm:8.16–
lethality bioassay technique to measure the LC50
compounds.
(lM) value of the
000,3000,4000,5000,6000
8.14 (complex, 2H, H3,5), 7.81–7.74 (complex, 5H, H2
),
0
0
00 00 00 00
0
7.58–7.46 (complex, 6H, H3 ,5 ,2 ,3 ,5 ,6 ), 7.18 (t, 1H, H4 , J = 5.2 Hz).
13C NMR (CDCl3, 100 MHz) d/ppm: 156.08 (C2), 152.87 (C6),
0
000
00
0
150.40(C4), 145.24 (C2 ), 138.67 (C1 ), 137.48 (C1 ), 135.29 (C5 ),
Experimental
00
00 00
00 00
000,4000,5000
129.16 (C4 ), 128.89 (C3
), 128.32 (C3 ,5 ), 128.01 (C2 ,6 ),
0
0
000,6000
127.83 (C4 ), 127.13 (C2
), 124.78 (C3 ), 116.55 (C5), 115.56
Materials and instrumental details
(C3). IR (KBr, 4000–400 cmꢂ1): 3066
m
(CAH)ar; 1544, 1490
m
(C@C); 1397
m
(C@N); 1162
m
(C@S); 1099 (CACl); 1381, 1362
m
All solvents, chemicals and reagents used were of analytical re-
agent grade and used as such; double distilled water was used
throughout. Potassium tetrachloroplatinate was purchased from
Chemport India Pvt Ltd Mumbai. 4-Fluorobenzaldehyde, benzalde-
hyde, 4-chlorobenzaldehyde, 4-bromobenzaldehyde, 4-methyl
benzaldehyde and 4-methoxybenzaldehyde were purchased from
SpectrochemPvt. Ltd., Mumbai (India). Agarose, ethidium bromide,
TAE (Tris-Acetyl-EDTA), bromophenol blue and xylene cyanol FF
were purchased from Himedia, India. Herring Sperm DNA was pur-
chased from Sigma Chemical Co. (India). Culture of pUC19 bacteria
(MTCC 47) was purchased from Institute of Microbial Technology
(Chandigarh, India).
(pyridine skeleton band); 1162, 827 (p-substituted aromatic ring);
740 d(CAH).
2,4-Bis(4-chlorophenyl)-6-(thiophen-2-yl)pyridine (L3)
Anal. Calc. for C21H13Cl2NS: Calc. (Found): C, 65.97 (65.88); H,
3.43 (3.35); N, 3.66 (3.50); S, 8.39 (8.30). Yield: 60%, mp: 139–
141 °C, mol. wt. 382.31;1H NMR (CDCl3, 400 MHz) d/ppm:8.13 (d,
0
000,5000
2H, H3,5, J = 8.4 Hz), 7.75 (d, 3H, H5 ,3
, J = 7.2 Hz), 7.68 (d, 2H,
000,6000
0
00 00 00 00
H2
, J = 8.4 Hz), 7.54–7.46 (complex, 5H, H3 ,2 ,3 ,5 ,6 ), 7.18 (dd,
1H, H4 , J = 8.8 and 1.2 Hz). 13C NMR (CDCl3, 100 MHz) d/ppm:
0
0
000
156.28 (C2), 153.03 (C6), 151.26(C4), 149.13 (C2 ), 145.02 (C1 ),
137.54 (C1 ), 137.09 (C4 ), 135.44 (C4 ), 129.38 (C3 ,5 ), 128.94
Elemental analyses (C, H, N and S) of the synthesized complexes
were performed with a model 240 Perkin Elmer elemental ana-
lyzer, Massachusetts (USA). The electronic spectra were recorded
00
000
00
00 00
000,5000
000,6000
00 00
0
0
(C3
), 128.40 (C2 ,6 ), 128.29 (C2
), 128.06 (C5 ), 127.98 (C4 ),
124.89 (C3 ), 116.21 (C5), 115.26 (C3). IR (KBr, 4000–400 cmꢂ1):
0
on
a UV-160A UV–Vis. spectrophotometer, Shimadzu, Kyoto
3061
1202
m
m
(CAH)ar; 1546, 1492
(CACl); 1383, 1304 (pyridine skeleton band); 1123, 830
m(C@C); 1397 m(C@N); 1176 m(C@S);
(Japan). Infrared spectra were recorded on a FT-IR ABB Bomen
MB-3000 (Canada) spectrophotometer as KBr pellets in the range
4000–400 cmꢂ1. The thermogram of complexes was recorded on
a Mettler Toledo TGA/DSC 1 Thermogravimetric analyzer. The
LC–MS spectra were recorded using Thermo mass spectrophotom-
eter (USA). 1H and 13C NMR were recorded on a BrukerAvance
(400 MHz). Photo quantization of the gel after electrophoresis
was done using AlphaDigiDoc™ RT. Version V.4.0.0 PC-Image soft-
ware, California (USA).
(p-substituted aromatic ring); 778 d(CAH).
4-(4-Bromophenyl)-2-(4-chlorophenyl)-6-(thiophen-2-yl)pyridine
(L4)
Anal. Calc. for C21H13ClBrNS: Calc. (Found): C, 59.10 (59.90); H,
3.07 (3.08); N, 3.28 (3.30); S, 7.51 (7.45). Yield: 62%, mp: 122–
124 °C, mol. wt. 426.76;1H NMR (CDCl3, 400 MHz) d/ppm:8.12 (d,
0
000,5000
2H, H3,5, J = 8.0 Hz), 7.73 (t, 3H, H5 ,3
, J = 4.8 Hz), 7.68 (d, 2H,
00 00
000,6000
Synthesis of ligands
H2
0
, J = 8.4 Hz), 7.60 (d, 2H, H2 ,6 , J = 8.4 Hz), 7.48 (dd, 3H,
H3 ,3 ,5 , J = 18.4 and 4.8 Hz), 7.18 (t, 1H, H4 , J = 4.0 Hz). 13C NMR
(CDCl3, 100 MHz) d/ppm: 156.29 (C2), 153.92 (C6), 153.07 (C4),
00 00
0
All ligands were prepared by modified kronke pyridine
synthesis [16]. A halo pyridinium salt of 2 acetyl thiophene and
ammonium acetate was mixed with different chalcones in metha-
nol. The mixture was refluxed for 6–7 h on sand bath. The product
was obtained by keeping solution in ice bath and purified by crys-
tallization in n-hexane Ligands were characterized by elemental
analysis, 1H and 13C NMR spectra. (General synthesis of ligands is
kept in Supplementary material 1).
0
000
00
000
00
149.19 (C2 ), 145.00 (C1 ), 137.54 (C1 ), 137.26 (C4 ), 135.46 (C4 ),
00 00
00 00
000,5000
000,6000
132.34 (C3 ,5 ), 128.93 (C3
), 128.68 (C2 ,6 ), 128.29 (C2
),
0
0
0
128.04 (C5 ), 128.00 (C4 ), 124.92 (C3 ), 116.12 (C5), 115.17 (C3). IR
(KBr, 4000–400 cmꢂ1): 3052
(C@N); 1170 (C@S); 1095
m
(CAH)ar; 1545, 1485
(CACl); 1012 (CABr); 1380, 1360
(pyridine skeleton band); 1150, 815 (p-substituted aromatic ring);
m(C@C); 1390
m
m
m
m
750 d(CAH).

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
263
2-(4-Chlorophenyl)-6-(thiophen-2-yl)-4-p-tolylpyridine (L5)
Anal. Calc. for C22H16ClNS: Calc. (Found): C, 73.02 (73.10); H,
4.46 (4.40); N, 3.87 (3.80); S, 8.86 (8.75). Yield: 64%, mp: 120–
122 °C, mol. wt. 361.89;1H NMR (CDCl3, 400 MHz) d/ppm:8.12 (d,
0
H 2.30 (2.22), N 2.28 (2.39), S 5.22 (5.15). UV–Vis: kmax(nm)
(e dm3 molꢂ1 cmꢂ1): 334 (41,253), 271 (15,260), LC MS: m/
z = 611.95. IR (KBr, 4000–400 cmꢂ1): 3063
m
(CAH)ar; 1643
(C@N); 1381, 1281 (pyridine skeleton band),
(C@S); 1088 (CACl);; 1018, 825 (p-substituted aromatic
ring); 764 d(CAH), 540 (PtAN)sym, 470 (PtAN)asym, 445 (PtAS).
1H NMR (DMSO-d6, 400 MHz) d/ppm:8.32 (d, 2H, H3,5, J = 8.8 Hz),
m(C@C); 1489
m
000,5000
2H, H3,5, J = 8.4 Hz), 7.79 (t, 3H, H5 ,3
, J = 5.2 Hz), 7.64 (d, 2H,
1142
m
m
0
00 00
000,6000
H2
, J = 7.6 Hz), 7.48 (dd, 3H, H3 ,2 ,6 , J = 19.2 Hzand 6 Hz), 7.36
m
m
m
00 00
0
(d, 2H, H3 ,5 , J = 8 Hz), 7.18 (dd, 1H, H4 , J = 8.4 Hz and 0.8 Hz),
2.47 (s, 3H, ACH3). 13C NMR (CDCl3, 100 MHz) d/ppm: 155.90
8.18 (d, 1H, H3 , J = 21.6 Hz), 8.07 (d, 1H, H5 , J = 3.2 Hz), 8.04 (d,
0
0
0
000
00 00 00
00
(C2), 152.59 (C6), 150.53 (C4), 144.70 (C2 ), 139.51 (C1 ), 135.47
3H, H2 ,3 ,5 , J = 7.2 Hz), 7.70 (d, 1H, H6 , J = 4.8 Hz), 7.62–7.53 (com-
00
000
00
00 00
0
000,5000
000,3000,4000,50000,6000
(C1 ), 135.39 (C4 ), 129.91 (C4 ), 128.88 (C3 ,5 ), 128.67 (C3
),
plex, 5H, H2
), 7.23 (t, 1H, H4 , J = 4.8 Hz).
00 00
0
0
000,6000
128.46 (C2 ,6 ), 128.10 (C2
), 127.95 (C5 ), 126.97 (C4 ), 125.25
0
(C3 ), 116.52 (C5), 115.52 (C3), 21.29 (ACH3). IR (KBr, 4000–
Synthesis of [PtCl2(L3)] (3)
It was prepared using 2,4-bis(4-chlorophenyl)-6-(thiophen-2-
yl)pyridine (L3) (0.450 mmol, 0.172 g) and K2PtCl4 (0.450 mmol,
0.186 g). Yield: 80%, mp: 172–174 °C, mol. wt. 648.28, Anal. for
400 cmꢂ1): 3050
1397 (C@N); 1231
skeleton band); 1145, 826 (p-substituted aromatic ring); 800
m
(CAH)ar; 2918 (CAH)al; 1543, 1492
m
m(C@C);
m
m
(C@S); 1091 m(CACl); 1381, 1360 (pyridine
d(CAH).
C
21H13Cl4NPtS, Calc. (found) (%), C 38.91 (38.45), H 2.02 (2.12), N
2.16 (2.10), S 4.94 (4.85). UV–Vis: kmax (nm) (e dm3 molꢂ1 cmꢂ1):
319 (20,689), 271 (17,032), LC MS: m/z = 647.19. IR (KBr, 4000–
2-(4-Chlorophenyl)-4-(4-methoxyphenyl)-6-(thiophen-2-yl)pyridine
(L6)
400 cmꢂ1): 3047
m(CAH)ar; 1643
m
(C@C); 1481
m(C@N); 1373,
Anal. Calc. for C21H16ClNOS: Calc. (Found): C, 69.92 (69.60); H,
4.27 (4.15); N, 3.71 (3.65); S, 8.49 (8.35). Yield: 53%, mp: 114–
116 °C, mol. wt. 377.89;1H NMR (CDCl3, 400 MHz) d/ppm:8.13 (d,
0
1273 (pyridine skeleton band); 1134
m
(C@S); 1080 m
(CACl);
1031, 825 (p-substituted aromatic ring); 750 d(CAH), 548
m
(PtAN)sym, 480
m
(PtAN)asym, 455
m
(PtAS). 1H NMR (DMSO-d6,
0
0
000,3000,5000,6000
2H, H3,5, J = 8.8 Hz), 7.77–7.69 (complex, 5H, H3 ,2
), 7.47
400 MHz) d/ppm:8.19 (d, 3H, H3,5,3 , J = 8.4 Hz), 7.99 (d, 1H, H4 ,
0
00 00
0
0
00 00
(dd, 3H, H5 ,3 ,5 , J = 23.6 Hz and 10.0 Hz), 7.17 (dd, 1H, H4 ,
J = 15.6 Hz), 7.88 (d, 3H, H5 ,2 ,6 , J = 8.4 Hz), 7.75–7.64 (complex,
00 00
00 00
000,3000,5000,6000
J = 8.8 Hz and 0.8 Hz), 7.07 (d, 2H, H2 ,6 , J = 8.8 Hz), 3.91 (s, 3H,
6H, H3 ,5 ,2
).
OCH3). 13C NMR (CDCl3, 100 MHz) d/ppm: 160.64 (C4 ), 156.00
(C2), 152.78 (C6), 149.81 (C4), 145.37 (C2 ), 137.60 (C1 ), 135.19
000
Synthesis of [PtCl2(L4)] (4)
0
00
000
00 00
00 00
00
000,5000
(C1 ), 130.85 (C3 ,5 ), 128.86 (C3
), 128.30 (C2 ,6 ), 128.29 (C4 ),
It was prepared using 4-(4-bromophenyl)-2-(4-chlorophenyl)-
6-(thiophen-2-yl)pyridine (L4) (0.450 mmol, 0.192 g) and K2PtCl4
(0.450 mmol, 0.186 g). Yield: 75%, mp: 173–175 °C, mol. wt.
692.74, Anal. for C21H13 BrCl3NPtS, Calc. (found) (%), C 36.41
(35.95), H 1.89 (1.78), N 2.02 (2.08), S 4.63 (4.65). UV–Vis: kmax
(nm) (e dm3 molꢂ1 cmꢂ1): 319 (19,691), 270 (17,032), LC MS: m/
0
0
0
000,6000
127.97 (C2
), 127.71 (C5 ), 124.67 (C4 ), 116.03 (C3 ), 115.01
(C5), 114.58 (C3), 55.45 (AOCH3). IR (KBr, 4000–400 cmꢂ1): 3040
m
(CAH)ar; 2836
1235 (C@S); 1086
(CAOAC)asy; 1382, 1362 (pyridine skeleton band); 1181, 825
(p-substituted aromatic ring); 796 d(CAH).
m(CAH)al; 1543, 1492
m
(C@C); 1401
m
(C@N);
1030
m
m(CACl); 1261
m
(CAOAC)sy
;
m
z = 689.87. IR (KBr, 4000–400 cmꢂ1): 3155
(C@C); 1481 (C@N); 1389, 1272 (pyridine skeleton band);
1120 (C@S); 1072 (CACl); 1003 (CABr); 910, 810 (p-substi-
tuted aromatic ring); 663 d(CAH), 540 (PtAN)sym 485
(PtAN)asym 465
(PtAS). 1H NMR (DMSO-d6, 400 MHz)
d/ppm:8.19 (d, 3H, H3,5,3 , J = 8.4 Hz), 7.99 (d, 1H, H4 , J = 15.6 Hz),
m(CAH)ar; 1643
m
m
Synthesis of complexes
m
m
m
m
,
Synthesis of [PtCl2(L1)] (1)
m
,
m
Complex 1was synthesized by heating 1:1 ratio of L1(2-(4-chloro-
phenyl)-4-(4-fluorophenyl)-6-(thiophen-2-yl)pyridine) (0.450 mmol,
0.164 g) and K2PtCl4 (0.450 mmol, 0.186 g) in water–methanol sys-
tem (50 mL) at 80 °C. A drop of hydrochloric acid (free acid is to
avoid displacement of Clꢂ by OHꢂ) added until the solution became
colorless (0.5–5 h) Reaction mixture was allowed to cool at room
temperature. The obtained product was washed with hot water
and dried under vacuum. Similarly rest of all the complexes were
prepared. (General synthesis of complexes is kept in Supplemen-
tary material 2).
0
0
0
00 00
7.88 (d, 3H, H5 ,2 ,6
,
J = 8.4 Hz), 7.76–7.64 (complex, 6H,
00 00
H3 ,5 ,2
000,3000,5000,6000
).
Synthesis of [PtCl2(L5)] (5)
It was prepared using 2-(4-chlorophenyl)-6-(thiophen-2-yl)-4-
p-tolylpyridine (L5) (0.450 mmol, 0.162 g) and K2PtCl4
(0.450 mmol, 0.186 g). Yield: 65%, mp: 121–123 °C, mol. wt.
625.87, Anal. for
C22H16 Cl3NPtS, Calc. (found) (%), C 42.08
(41.45), H 2.57 (2.65), N 2.23 (2.15), S 5.11 (5.05). UV–Vis: kmax
Yield: 75%, mp: 148–150 °C, mol. wt. 631.84, Anal. for C21H13-
Cl3FNPtS, Calc. (found) (%), C 39.92 (39.23), H 2.07 (2.12), N 2.22
(2.09), S 5.07 (5.16). UV–Vis: kmax (nm) (e dm3 molꢂ1 cmꢂ1): 333
(10,287), 269 (25,841), LC MS: m/z = 629.59. FT-IR (KBr, 4000–
(nm) (e dm3 mol–1 cmꢂ1): 331 (12,644), 275 (31,350), LC MS: m/
z = 625.97. IR (KBr, 4000–400 cmꢂ1): 3055
m
(CAH)ar; 2916
(C@N); 1381, 1273 (pyridine skele-
(CACl); 1011, 810 (p-substituted
aromatic ring); 756 d(CAH), 540 (PtAN)sym, 485 (PtAN)asym
465
(PtAS). 1H NMR (DMSO-d6, 400 MHz) d/ppm:8.32 (d, 2H,
m(CAH)al; 1643
m(C@C); 1481
m
ton band); 1142
m
(C@S); 1088
m
400 cmꢂ1): 3063
1273 (pyridine skeleton band); 1227
m
(CAH)ar; 1643,
m
(C@C); 1489
m
(C@N); 1381,
(C@S); 1095
m
m
,
m
(CAF); 1157
m
m
0
0
0
m
(CACl); 1101, 825 (p-substituted aromatic ring); 764 d(CAH),
555
d6, 400 MHz) d/ppm:8.37 (d, 2H, H3,5, J = 8.4 Hz), 8.31 (d, 1H, H5 ,
H
3,5, J = 8.0 Hz), 8.17 (t, 2H, H4 ,5 , J = 11.6 Hz), 8.07 (d, 1H, H3 ,
(PtAS). 1H NMR (DMSO-
J = 2.4 Hz), 7.96 (d, 2H, H2 ,6 , J = 7.6 Hz), 7.69–7.60 (complex, 3H,
00 00
m
(PtAN)sym, 475
m(PtAN)asym, 450
m
0
00 00 000
000
000,5000
H3 ,5 ,6 ), 7.39 (d, 2H, H3
, J = 7.6 Hz), 7.23 (d, 1H, H2 ,
00 00
0
000,6000
J = 8.4 Hz), 8.24–8.06 (complex, 4H, H3 ,5 ,2
), 7.69 (d, 1H, H3 ,
J = 4.0 Hz), 2.41 (s, 3H,ACH3).
00 00
000,5000
,
J = 5.2 Hz), 7.61 (d, 1H, H2 ,6 , J = 8.8 Hz), 7.41 (t, 2H, H3
Synthesis of [PtCl2(L6)] (6)
0
J = 8.8 Hz), 7.23 (t, 1H, H4 , J = 4.8 Hz).
It was prepared using 2-(4-chlorophenyl)-4-(4-methoxy-
phenyl)-6-(thiophen-2-yl)pyridine (L6) (0.450 mmol, 0.170 g) and
K2PtCl4 (0.450 mmol, 0.186 g). Yield: 68%, mp: 103–105 °C, mol.
wt. 643.87, Anal. for C22H16 Cl3NOPtS, Calc. (found) (%), C 41.04
(41.15), H 2.50 (2.59), N 2.18 (2.05), S 4.98 (4.90). UV–Vis: kmax
(nm) (e dm3 molꢂ1 cmꢂ1): 293 (50,550), LC MS: m/z = 641.96. IR
Synthesis of [PtCl2(L2)] (2)
It was prepared using 2-(4-chlorophenyl)-4-phenyl-6-(thio-
phen-2-yl)pyridine (L2) (0.450 mmol, 0.156 g) and K2PtCl4
(0.450 mmol, 0.186 g). Yield: 70%, mp: 128–130 °C, mol. wt.
613.84, Anal. for C21H14Cl3NPtS, Calc. (found) (%), C 41.09 (40.95),

264
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
(KBr, 4000–400 cmꢂ1): 3001
(C@C); 1435 (C@N); 1250
skeleton band); 1142 (C@S); 1088
1180, 879 (p-substituted aromatic ring); 818 d(CAH), 540
(PtAN)sym, 480 (PtAN)asym, 460
(PtAS). 1H NMR (DMSO-d6,
400 MHz) d/ppm:8.31 (d, 2H, H3,5, J = 8.8 Hz), 8.17–8.12 (complex,
m
(CAH)ar; 2908
(CAOAC)sy; 1373, 1160 (pyridine
(CACl); 1011 (CAOAC)asy
m
(CAH)al; 1643
tic dish with the help of a perforated device. Approximately 50 mg
of eggs were sprinkled into the large compartment, which was
darkened while the minor compartment was opened to ordinary
light. After two days nauplii were collected by a pipette from the
lighter side. A stock solution of the test complex was prepared in
DMSO. From this stock solution, solutions were transferred to the
m
m
m
m
m
m
;
m
m
m
00 00 00 00
0
0
0
000,6000
6H, H2 ,3 ,5 ,6 ,2
), 8.02 (d, 1H, H4 ,5 , J = 8.8 Hz), 7.61 (d, 1H, H3 ,
vials to make final concentration 2, 4, 6, 8, 10, 12 lM, etc. (three
000,5000
J = 8.8 Hz), 7.12 (d, 2H, H3
, J = 8.8 Hz), 3.86 (s, 3H, AOCH3).
for each dilutions were used for each test sample and LC50 is the
mean of three values) and three vial was kept as control having
of DMSO only. After two days, when the of nauplii were ready,
1 mL of seawater and 10 of nauplii were added to each vial and
the volume was adjusted with seawater to 2.5 mL per vial [22].
After 24 h each vial was observed using a magnifying glass and
the number of survivors in each vial was counted and noted. Data
were analyzed by simple logit method to determine the LC50 val-
ues, in which log of concentration of samples were plotted against
percent of mortality of nauplii [23].
Evaluation of binding constant by absorption titration using HS DNA
The experiment was performed using HS DNA (e = 12858
dm3 molꢂ1 cmꢂ1) in phosphate buffer solution (pH 7.2). The stock
solutions of the complexes were prepared by dissolving the com-
plexes in DMSO. The absorption titration was carried out by keep-
ing the concentration of complex constant (20 lM) and varying the
concentration of DNA and incubated for 10 min at room tempera-
ture. The change in absorbance was recorded after each addition of
DNA aliquot. The Kb value was determined from the ratio of the
slope to intercept [17].
Results and discussion
DNA binding study by viscosity measurement
Thermogravimetric analysis and magnetic moments
Cannon–Ubbelohde viscometer maintained at a constant tem-
perature of (37.0 0.1 °C) in a thermostatic jacket was used to
measure the relative viscosity of DNA solutions in the presence
of platinum(II) complexes. Digital stopwatch with least count of
0.01 s. was used for flow time measurement with accuracy
of 0.1 s. The [Complex]/[DNA] ratio was maintained in the range
of 0–0.2.The flow time of each sample was measured three times
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. The weight loss occurs from 320 to 550 °C
corresponds to loss of ligands and leaving behind the metal
residue.
and an average flow time was calculated. Data are presented as
The magnetic moment value of the synthesized complexes was
found to be zero at room temperature, suggesting d8-system with
low-spin configuration i.e. dsp2 hybridization, pointing toward
square planar Pt(II) complexes.
1/3
(g/g0)
versus [complex]/[DNA], where g is the viscosity of DNA
in the presence of complex and g0 is the viscosity of DNA alone
[18]. Viscosity values were calculated from the observed flow time
of DNA-containing solutions (t) corrected for that of the buffer
alone (t0),
g
/ t–t0 [19].
Electronic absorption analyses
Unwinding of supercoiled pUC19 DNA
For low-spin square-planar d8-metal complexes, three dꢂd spin
Unwinding of supercoiled pUC19 DNA was measured by aga-
rose gel electrophoresis mobility shift assay [20]. The unwinding
angle /, induced per Pt-DNA adduct was calculated by following
allowed transitions are expected corresponding to the transitions
from three lower lying d-levels to the empty dx2
orbital. These
ꢂy2
transitions
are
designated
as
dz2 ða1gÞ ! dx2ꢂy2 ðb1gÞ,
equation,/ = ꢂ18r/rb(c)where,
r
is the superhelical density and
dxz;yzðegÞ ! dx2ꢂy2 ðb1gÞ, dxyðb2gÞ ! dx2ꢂy2 ðb1gÞ and observed at
rb is the bound drug-to-nucleotide ratio, rb(c) is the ratio 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. The superhelical density (r) of
the plasmid was determined from rb(c) values obtained for cisplatin
and its known unwinding angle (/ = 13° for the site-specific 1,2-
intrastrand cross-link) [21].
ꢃ30,000 cmꢂ1
,
ꢃ26,000 cmꢂ1 and ꢃ23,000 cmꢂ1
, respectively
[24,25]. In the synthesized Pt(II) complexes, only one d–d band
ꢃ330 nm (ꢃ30,303 cmꢂ1
) is observed, which is assigned to
dz2 ða1gÞ ! dx2ꢂy2 ðb1gÞ transition, where as one charge transfer tran-
sition band is observed ꢃ270 nm (ꢃ37,037 cmꢂ1) [26], The reason
for only one charge transfer transition band is the masking of high
intensity charge transfer bands. All these bands points toward the
low spin complex of d8-system with square planar geometry.
The IR spectroscopic measurements of the ligands show bands
at ca. 1550 cmꢂ1 and ca. 1400 cmꢂ1 corresponding to C@C and
C@N ring stretching, whereas the bands at 750 cmꢂ1 and
3060 cmꢂ1 are due to aromatic CAH out-of-plane bending and
stretching respectively. The band for CAH out-of-plane ring defor-
mation appears at ca. 763 cmꢂ1 and CACl stretching appears at
1080 cmꢂ1. Major changes occur in the bands of C@C and C@N ring
stretching as they are shifted from 1550, 1400 to 1643 and
1490 cmꢂ1 respectively. This is further supported by the binding
of metal atom to nitrogen and sulphur atom of ligand skeleton,
by a sharp band at ca. 550 and ca. 470 cmꢂ1, characteristic bands
of symmetric and asymmetric stretching of the PtAN bond [27]
and stretching frequency of PtAS bond at ca. 450. The stretching
mode of PtACl is expected in the region below 400 cmꢂ1 i.e. ca.
320 cmꢂ1 [28].
Cytotoxic activity
Brine shrimp (Artemia Cysts) lethality bioassay technique was
applied for the determination of general toxic property of com-
plexes. The in vitro lethality test has been carried out using brine
shrimp eggs i.e. Artemia cysts. Brine shrimp 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 distilled water. An unequal partition was made in the plas-

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
265
Fig. 1. LC–MS spectrum of complex-1.
Fig. 1 represents mass spectrum of complex [PtCl2(L1)] (1). Mass
spectrum of complex 1 shows molecular ion peak [M+] at 629.59
m/z, [M + 2] at 631.69 m/z and [M + 4] at 633.69 m/z. Peak at
560.60 m/z is due to ligand attached with platinum metal ion i.e.
due to loss of two chlorine atoms from the complex. The peak at
365.21 m/z is due to ligand i.e. loss of platinum metal, which is at-
tached to ligand. The other peaks at 283.13, 203.09, 173.97, 98.92,
84.82 and 79.92 m/z are due to the fragmentation of ligand.
The 1H NMR spectra of all the complexes show downfield for
protons. Protons H3,5 in ligands are in the most downfield region,
while for the complexes these protons show more downfield shift
in the spectra. The coordination of ligand results in shifting of all
1H NMR peaks in the downfield region. These suggest that the
coordination occur through N and S atom, which is also supported
by the IR data.
also reveals the nature of binding affinity [29]. The absorption spec-
tra of Pt(II) complex with increasing concentration of HS DNA is
shown in Fig. 2. As the concentration of DNA increases, hyperchro-
mism 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. The Kb values were obtained for Pt(II)
complexes are in the range of 1.37 ꢁ 105–7.76 ꢁ 105 (Table 1).
These values are much lower than that of the classical intercalator
Table 1
Binding constant (Kb) values of synthesized complexes.
Complex
Kb (Mꢂ1
)
[PtCl2(L1)]
[PtCl2(L2)]
[PtCl2(L3)]
[PtCl2(L4)]
[PtCl2(L5)]
[PtCl2(L6)]
7.76 ꢁ 105
2.99 ꢁ 105
6.94 ꢁ 105
5.96 ꢁ 105
2.38 ꢁ 105
1.37 ꢁ 105
Absorption spectral study
Absorption titration is employed universally to determine the
binding mode and binding strength of the complexes with DNA
[17]. Presence of red shift indicates coordination of complex with
DNA through N7 position of guanine. The extent of hyperchromism
Fig. 2. Absorption spectral traces of complex 1 with increasing amount of Herring
Sperm DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2). [complex] = 50
lM,
[DNA] = 0–60 M with incubation period of 15 min at room temperature. Arrow
l
shows the absorbance change upon increasing DNA concentrations. Inset: Plots of
[DNA]/(ea–ef) versus [DNA] for the titration of DNA with platinum(II) complexes.
Fig. 3. Effect on relative viscosity of DNA under the influence of increasing amount
of complexes at 37 0.1 °C in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2).

266
M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
Table 2
cosity of a DNA solution was measured in the presence of com-
pounds is regarded as the least ambiguous and most critical test
of a DNA binding model [33], and affords a stronger argument
for DNA binding mode [34]. 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 viscosity [35]. During the relative specific viscosity
measurement with increase in ratio of [complex]/[DNA] from
0.02 to 0.2, decrease the relative viscosity of DNA solution
(Fig. 3) resulting from bending or kinking of the DNA helix, which
attributes to covalent binding of complexes with DNA bases [36].
Data for determining the unwinding angle of pUC19 DNA under influence of
Complexes.
Complex
Complex-to-nucleotide ratioa rb (C)
Unwinding angle /
1
2
3
4
5
6
0.071
0.092
0.075
0.081
0.098
0.108
14.02°
10.76°
13.20°
12.38°
10.10°
9.17°
a
Complex-to-nucleotide ratio at which supercoiled and nicked forms comigrates.
(ethidium bromide) but these values are higher than intercalative
[PtCl2(4,7-Me2phen)] complex (6.35 0.2 ꢁ 104 Mꢂ1) [30]. While
the Kb values of the synthesized Pt(II) complexes are similar to
the value observed for [Pt(dmphen)(CO3)]ꢄH2O (dmphen = 2,9-di-
methyl-1,10-phenanthroline) (1.8 ꢁ 105 Mꢂ1) [31], [Pt(bpy)(pip)]
(NO3)2 and [Pt(bpy)(hpip)](NO3)2ꢄ 2H2O [bpy = 2,20-bypyridine;
Unwinding of pUC19 DNA
Unwinding of supercoiled pUC19 DNA was measured by aga-
rose gel electrophoresis mobility shift assay [20]. The unwinding
induced in negatively supercoiled pUC19 plasmid determined by
monitoring the degree of supercoiling (Supplementary material
3). The degree of supercoiling decrease upon binding of unwinding
agents, which causes a decrease in the rate of migration through
agarose gel, which makes it possible to observe and to quantify
the mean value of unwinding. Due to binding of unwinding agents
linear form of DNA is not observed and supercoiled form of DNA is
completely converted to open circular form. Under the present
experimental conditions, r was calculated to be ꢂ0.055 on the
basis of the data of cisplatin for which the rb(c) was determined
pip = 2-phenylimidazo[4,5-f]1,10-phenanthroline;
hpip = 2-(2-
hydroxyphenyl) imidazo [4,5-f]1,10-phenanthroline)] [32].
Viscosity measurement
Viscosity measurement study was carried out to confirm the
binding mode of the complexes. Viscosity values were calculated
from the observed flow time of DNA-containing solutions (t)
corrected for that of the buffer alone (t0),
g
/ t–t0 [19,18]. The vis-
Table 3
Data for determining the LC50 values of synthesized complexes.
Compound
1
Conc. (
lM)
Log conc.
No. of nauplii taken
No. of nauplii dead
% of mortality
LC50 (lM)
2
3
4
5
6
8
0.301
0.477
0.602
0.699
0.778
0.903
10
10
10
10
10
10
6
5
4
3
2
1
40
50
60
70
80
90
3.02
2
3
4
5
2
5
6
8
10
12
0.301
0.699
0.778
0.903
1.000
1.079
10
10
10
10
10
10
9
8
7
6
5
4
10
20
30
40
50
60
9.77
2
4
5
8
10
12
0.301
0.602
0.699
0.903
1.000
1.079
10
10
10
10
10
10
7
6
5
4
3
2
30
40
50
60
70
80
5.37
2
4
6
7
10
12
0.301
0.602
0.778
0.845
1.000
1.079
10
10
10
10
10
10
8
7
6
5
4
3
20
30
40
50
60
70
7.08
2
6
0.301
0.778
1.079
1.255
1.301
1.477
1.580
10
10
10
10
10
10
10
9
8
7
6
5
4
3
10
20
30
40
50
60
70
12
18
20
30
38
21.38
6
2
4
6
10
20
30
40
50
0.301
0.602
0.778
1.000
1.301
1.477
1.602
1.699
10
10
10
10
10
10
10
10
10
9
8
7
6
5
4
3
00
10
20
30
40
50
60
70
28.18

M.N. Patel et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 261–267
267
in this study and / = 13° was assumed. Using this approach, the
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.053.