Design, synthesis, MTT assay, DNA interaction studies of platinum(II) complexes

Article abstract of DOI:10.1080/07391102.2016.1268071

The square planar Pt(II) complexes of the type [Pt(Lⁿ)(Cl₂)] (where Lⁿ?=?L^1?3?=?thiophene-2-carboxamide derivatives and L^4?6?=?thiophene-2-carbothioamide derivatives) have been synthesized and characterized by physicochemical and various spectroscopic studies. MIC method was employed to inference the antibacterial potency of complexes in reference to free ligands and metal salt. Characteristic binding constant (K_b) and binding mode of complexes with calf thymus DNA (CT-DNA) were determined using absorption titration (0.76–1.61?×?10⁵?M^?1), hydrodynamic chain length assay and fluorescence quenching analysis, deducing the partial intercalative mode of binding. Molecular docking calculation displayed free energy of binding in the range of –260.06 to –219.63 kJmol^?1. The nuclease profile of complexes towards pUC19 DNA shows that the complexes cleave DNA more efficiently compared to their respective metal salt. Cytotoxicity profile of the complexes on the brine shrimp shows that all the complex exhibit noteworthy cytotoxic activity with LC₅₀ values ranging from 7.87 to 15.94?μg/mL. The complexes have been evaluated for cell proliferation potential in human colon carcinoma cells (HCT 116) and IC₅₀ value of complexes by MTT assay (IC₅₀?=?125–1000?μg/mL).

Full text of DOI:10.1080/07391102.2016.1268071

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Design, synthesis, MTT assay, DNA interaction
studies of platinum(II) complexes
Miral V. Lunagariya, Khyati P. Thakor, Bhargav N. Waghela, Foram U. Vaidya,
Chadramani Pathak & Mohan N. Patel
To cite this article: Miral V. Lunagariya, Khyati P. Thakor, Bhargav N. Waghela, Foram U. Vaidya,
Chadramani Pathak & Mohan N. Patel (2016): Design, synthesis, MTT assay, DNA interaction
studies of platinum(II) complexes , Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2016.1268071
To link to this article: http://dx.doi.org/10.1080/07391102.2016.1268071
Accepted author version posted online: 04
Dec 2016.
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Date: 07 December 2016, At: 17:01

1
Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://dx.doi.org/10.1080/07391102.2016.1268071
Design, synthesis, MTT assay, DNA interaction studies of platinum(II) complexes
a
a
b
Miral V. Lunagariya , Khyati P. Thakor , Bhargav N. Waghela ,
b
b
a
Foram U. Vaidya , Chadramani Pathak and Mohan N. Patel *
aDepartment of Chemistry, Sardar Patel University,
Vallabh Vidyanagar–388 120, Gujarat, India.
Corresponding author. Tel.: +91 2692 226856 E-mail: jeenen@gmail.com
bDepartment of Cell biology,
Indian institute of advanced Research,
Gandhinagar, Gujarat, India.
E-mail: cmpathak@iiar.res.in
Abstract
n
n
1-3
The square planar Pt(II) complexes of the type [Pt(L )(Cl )] (where L = L = thiophene-2-
2
4
-6
carboxamide derivatives and L = thiophene-2-carbothioamide derivatives) have been
synthesized and characterized by physicochemical and various spectroscopic studies. MIC
method was employed to inference the antibacterial potency of complexes in reference to free
ligands and metal salt. Characteristic binding constant (K ) and binding mode of complexes
b

2
with calf thymus DNA (CT-DNA) were determined using absorption titration (0.76 – 1.61 ×
5
-1
1
0 M ), hydrodynamic chain length assay and fluorescence quenching analysis, deducing
the partial intercalative mode of binding. Molecular docking calculation displayed free
-1
energy of binding in the range of -260.06 to -219.63 kJmol . The nuclease profile of
complexes towards pUC19 DNA shows that the complexes cleave DNA more efficiently
compared to their respective metal salt. Cytotoxicity profile of the complexes on the brine
shrimp shows that all the complex exhibit noteworthy cytotoxic activity with LC values
5
0
ranging from 7.87 to 15.94 µg/mL. The complexes have been evaluated for cell proliferation
potential in human colon carcinoma cells (HCT 116) and IC value of complexes by MTT
5
0
assay (IC = 125 - 1000 µg/mL).
5
0
Keywords: Platinum(II) complexes, Cell proliferation assay, Antibacterial study, Brine
shrimp cytotoxicity assay, DNA interaction study.
1
.
Introductions
Medicinal inorganic chemistry has been strengthened by the discovery
of the anticancer properties of the coordination compound cis-platin by Rosenberg et.
al in 1969, owing to its great impact in the treatment of cancer.(N. Farrell (Ed.), 1989;
Rosenberg, Vancamp, Trosko, & Mansour, 1969) Since its approval in 1979, it has
been used for therapeutic agent, diagnostic agent, mineral supplement and chelation
therapy. Platinum metal has also been frequently incorporated into
pharmaceuticals.(Guo & Sadler, 1999; Mjos & Orvig, 2014; Thompson & Orvig,
2
003) For the treatment of ovarian, testicular, lung and bladder cancers, it has been an
important component in chemotherapeutic treatments.(Kelland, 2007; Wheate,
Walker, Craig, & Oun, 2010) Although it is found successful in the treatment of
various cancers, it has several side effects like nausea, vomiting, ototoxicity and
neurotoxicity that limit treatment with cis-platin.(Kelland, 2007; Wheate et al., 2010)

3
As anticancer agent’s carboplatin, oxaliplatin, nedaplatin, lobaplatin and heptaplatin
enclosing platinum metal have been reported.(I.H.Krakoff, 1988,; Loehrer, Williams,
&
Einhorn, 1988) In cancer chemotherapy, cis-platin and other platinum-based drugs
have been greatly inspired by their clinical use for the treatment of hypercalcaemia and
skeletal metastases and in geminal bisphosphonates (BPs) drug, which shows the
affinity for bones and other calcified tissues(Kelland, 2007) and also being active in
the development of biosensors.(Aslanoglu, Isaac, Houlton, & Horrocks, 2000) Initial
investigations of the mechanism of cisplatin, as an anticancer drug, proposed that
metal based drug interact with DNA, due to the covalent binding of platinum
compounds to the N7 of guanine residue. Hence, it affects the replication and
generation of cellular events and finally leads to the death of cancer cell.(Boulikas,
2
003; Wang & Lippard, 2005) However, limitation of cisplatin is that, it has
nephrotoxicity and is only partially soluble in water in its clinical application. It was
observed that reaction between sulphur-containing proteins and platinum metal
induces poisonous effect to carcinogenic cells and inactivate some enzymes.(Florea &
Büsselberg, 2011; Kostova, 2006; N. Farrell, 2009; P. Kalyani, 2012) Hence, the
synthesis of new types of platinum based complexes, whose structure and mode of
action differ from those of cisplatin and bearing oxygen and sulphur donor atoms are
important to prevent adverse reactions. Several platinum complexes with S-
heterocyclic ligands such as thiophene, benzo[b]thiophene, benzothiazole, 2-acetyl
thiophene and thiophene-2-carboxaldehyde were reported.(Ji, Shigeta, Niko,
Watanabe, & Konishi, 2013; S. B. Kumar et al., 2012; M. Zala, 2011; Neto, de Lima,
&
Beraldo, 2006)
A series of Pt(II) complexes have been synthesized using -S, O/ -S, S donor
containing heterocyclic thiophene-2-carboxamide/thiophene-2-carbothioamide

4
derivative ligands and characterized by elemental analysis, electronic spectra,
conductance measurements, TGA, FT-IR and LC-MS spectroscopy and Pt(II)
complexes to explore them as molecules of biological significance. Some of its
derivatives have potent biological activities as in vitro cytotoxicity, antimicrobial and
anticancer agents.
2
.
Experimental
2
.1
Material and Instrumentation
All solvents and reagents used were of analytical grade. Potassium
tetrachloroplatinate(II) chloride salt was purchased from S. D Fine-Chem Ltd.
(
(
SDFCL). Nutrient broth (NB), agarose, ethidium bromide (EtBr), tris-acetyl-EDTA
TAE), bromophenol blue was purchased from Himedia (India). Calf thymus (CT)
DNA, thiophene-2-carbonyl chloride were purchased from Sigma Aldrich (India).
Thin layer chromatography (TLC) was performed using Merck aluminium sheets
coated with silica gel 60 F . Purification by flash chromatography was performed
254
using Merck silica gel 60. The compounds were visualized under UV light. All the
bacterial cultures used were purchased from MTCC, Institute of Microbial Technology
and Chandigarh, India. An Artemia cyst was purchased from local aquarium store.
GenElute mini Pre Kit for pUC19 DNA isolation was purchased from Sigma Aldrich
(
India). MilliQ™ (18.2 mƱ, Millipore) was used for the preparation of all deionized
water. HPLC grade DMSO was used to dissolve the platinum compounds.
Micro elemental analysis (C, H, N and S) of the synthesized compounds
was performed with a EuroEA elemental analyser. Room temperature magnetic
measurement for the complexes was made using Gouy’s magnetic balance. Melting
points were determined on Buchi 540 melting point apparatus.

5
1
13
H NMR and C NMR spectra were obtained on a 400 MHz and 100
MHz Bruker avance nuclear magnetic resonance spectrometer, either in DMSO-d (35
6
13
°
C), referenced internally to the solvent. The attached proton test in a C APT
(
Attached Proton Test) characterisation showed CH, CH and CH signals unattached
2
3
to protons. The APT experiment yields methine (CH) and methyl (CH ) signals
3
negative and quaternary (C) and methylene (CH ) signals positive. The chemical shift
2
of each resonance was quoted as an approximate midpoint of its multiplicity.
The electronic spectra were recorded on a UV-160A UV-Vis
spectrophotometer, Shimadzu, Kyoto (Japan). Infrared spectra were recorded on a FT-
IR ABB Bomen MB-3000 spectrophotometer (Canada) in the range of 4,000 to 400
-1
cm . The magnetic moments were measured by Gouy’s method using mercury
–6
tetrathiocyanatocobaltate(II) as the calibrate (χg=16.44 × 10 cgs units at 20 °C). The
LC-MS spectra were recorded using thermo mass spectrophotometer (USA). MIC
study was carried out using laminar air flow cabinet, Toshiba, Delhi, (India).
Fluorescence spectroscopy was carried out by FluoroMax-4, spectrofluorometer,
HORIBA (Scientific). Photo quantization of the gel after electrophoresis was done
TM
using AlphaDigiDoc RT. Version V.4.0.0 PC–Image software, CA (USA).
Hydrodynamic chain length study was carried out by viscometric bath.
1
-6
2.2
General synthesis of the ligands (L )
Synthesis was achieved by using an adaptation of the published
1-3
method.(Hodges & Rund, 1975) For synthesis of ligands (L ), thiophene-2-carbonyl
chloride (0.015 mol) in chloroform (CHCl ) was reacted with substituted aniline
3
(
0.015 mol) derivatives. The reaction mixture was stirred for 2 h at 0 °C. Pyridine was
used as a catalyst to promote the quick synthesis of ligands. After stirring, aq. HCl was
added (3 - 4 drops) and the mixture was extracted with chloroform using brine solution

6
as demulsifier. Extracted organic layer was dried over MgSO and evaporated to
4
obtain the precipitate. The residue obtained was purified by column chromatography
on silica using (hexane: ethyl acetate = 4:1) system. Solid obtained as a fine crystalline
4-6
product was filtered and dried in vacuum. For the synthesis of ligands (L ), ligands
1-3
(
L ) (0.015 mol) and Lawesson’s reagents (0.015 mol) were refluxed in toluene for 3
h. After refluxing, water was added to the reaction mixture. Obtained precipitate was
extracted with chloroform using brine as demulsifier, dried over MgSO and
4
evaporated to dryness. The residue was then purified by column chromatography on
4-6
silica gel using (hexane: ethyl acetate = 4:1) system, and the finally ligands (L ) were
obtained as yellow crystalline product, which were filtered and dried in vacuum.
2
.2.1 N-Phenylthiophene-2-carboxamide (L1)
1
The ligand (
L
) was synthesized using thiophene-2-carbonyl chloride
and aniline following the general process described in experimental section. Colour:
white crystal, Yield: 66 %, mol. wt. 203.26 g/mol, m.p. 251 °C; Anal. Calc. (%) For
C H NOS: C, 65.00; H, 4.46; N, 6.89; S, 15.77. Found (%): C, 64.98; H, 4.40; N,
11
9
-1
-1
1
6
.86; S, 15.20. UV-Vis: λ (nm) (ε, M cm ): 301 (13,850), 357 (4,275). H NMR
(
400 MHz, DMSO-d ) δ/ppm: 8.036 (d, 1H, J = 3.6 Hz, H ), 7.862 (d, 1H, J = 4.8
6
2’’
Hz, H ), 7.729 (d, 2H, J = 8.4 Hz, H ), 7.361 (t, 2H, J = 7.6 Hz, H ), 7.228 (dd, 1H,
4’’
2,6
3,5
13
J = 4.8 Hz, J = 4.4 Hz, H ), 7.112 (t, 1H, J = 7.2 Hz, H ), 10.220 (s, 1H, H ).
C
3”
4
1’
NMR (100 MHz, DMSO-d ) δ/ppm: 137.0 (C ), 121.6 (C ), 128.9 (C ), 128.0
6
1
2,6
3,5
(
C ), 130.3 (C ), 129.0 (C ), 131.9 (C ), 139.4 (C ), 161.8 (C2”). [Signal observed =
4
2’
3’
4’
5’
9
: Ar-CH = 3, Ar-C = 1 carbonyl = 1, thiophene-C = 1, thiophene-CH = 3]. Mass
+
(
m/z): 202.26 [M] .
2
.2.2 N-(4-Chlorophenyl) thiophene-2-carboxamide (L2
)

7
2
The ligand (L ) was synthesized using thiophene-2-carbonyl chloride
and 4-chloroaniline. Colour: white crystal, Yield: 70 %, mol. wt. 237.71 g/mol, m.p.
1
63 °C; Anal. Calc. (%) For C H ClNOS: C, 55.58; H, 3.39; N, 5.89; S, 13.49.
11
8
-1
-1
Found (%): C, 55.30; H, 3.20; N, 5.69; 13.10. UV-Vis: λ (nm) (ε, M cm ): 300
1
(
13,950), 359 (4,295). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.368 (d, 2H, J = 7.2
6
Hz, H ), 8.274 (dd, 1H, J = 4.0 Hz, J = 4.4 Hz, H ), 7.926 (d, 2H, J = 7.6 Hz, H ),
3,5
3”
2,6
13
7
.361 (d, 1H, J = 3.6 Hz, H ), 7.030 (d, 1H, J = 4.4 Hz, H ), 10.765 (s, 1H, H ). C
2” 4” 1’
NMR (100 MHz, DMSO-d ) δ/ppm: 136.0 (C ), 121.6 (C ), 129.0 (C ), 133.3
6
1
2,6
3,5
(
C ), 130.3 (C ), 129.0 (C ), 131.9 (C ), 139.4 (C ), 161.8 (C2”); [Signal observed =
4 2’ 3’ 4’ 5’
9
: Ar-CH = 2, Ar-C = 2, carbonyl = 1, thiophene-C = 1, thiophene-CH = 3]. Mass
+
m/z(%): 236.70 [M] , 238.70 [M + 2].
2
.2.3 N-(4-Nitrophenyl) thiophene-2-carboxamide (L3)
3
The ligand (L ) was synthesized using thiophene-2-carbonyl chloride
and 4-nitroaniline as described in general process. Colour: light yellow crystal, Yield:
8
5
2 %, mol. wt. 248.26 g/mol, m.p. 253 °C; Anal. Calc. (%) For C H N O S: C,
11
8
2
3
3.22; H, 3.25; N, 11.28; S, 12.91. Found (%): C, 53.01; H, 3.12; N, 11.08; S, 12.36.
-1
-1
1
UV-Vis: λ (nm) (ε, M cm ): 298 (13,600), 360 (4,290). H NMR (400 MHz,
DMSO-d ) δ/ppm: 8.270 (d, 2H, J = 8.0 Hz, H ), 8.171 (d, 2H, J = 8.0 Hz, H ),
6
3,5
2,6
8
.052 (d, 1H, J = 4.0 Hz, H ), 7.939 (d, 1H, J = 4.0 Hz, H ), 7.260 (dd, 1H, J = 4.0
2” 4”
13
Hz, J= 4.0 Hz, H ), 10.442 (s, 1H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm:
3”
1’
6
1
1
44.0 (C ), 119.9 (C ), 124.1 (C ), 143.5 (C ), 130.3 (C ), 129.0 (C ), 131.9 (C ),
1 2,6 3,5 4 2’ 3’ 4’
39.4 (C ), 161.8 (C ); [Signal observed = 9: Ar-CH = 2, Ar-C = 2, carbonyl = 1,
5’
2”
+
thiophene-C = 1, thiophene-CH = 3]. Mass m/z: 247.26 [M] .
2
.2.4 N-Phenylthiophene-2-carbothioamide (L4)

8
4
1
The ligand (L ) was synthesized, using ligand (L ) and Lowsson’s
reagent, according to the general process described in experimental section. Colour:
light yellow crystal, Yield: 87 %, mol. wt. 219.33 g/mol, m.p. 167 °C; Anal. Calc.
(
%) For C H NS : C, 60.24; H, 4.14; N, 6.39; S, 29.24. Found (%): C, 60.01; H,
11
9
2
-1
-1
1
4
.08; N, 6.15; S, 28.94. UV-Vis: λ (nm) (ε, M cm ): 280 (13,700), 388 (4,285).
H
NMR (400 MHz, DMSO-d ) δ/ppm: 8.036 (d, 1H, J = 3.6 Hz, H ), 7.862 (d, 1H, J
6
2”
=
4.8 Hz, H ), 7.719 (d, 2H, J = 8.0 Hz, H ), 7.323 (t, 1H, J = 6.8, H ), 7.248 (dd,
4” 2,6 4
1
H, J = 4.0 Hz, J = 4.8 Hz, H ), 7.112 (t, 2H, J = 7.2 Hz, H ), 10.232 (s, 1H, H ).
3” 3,5 1’
13
C NMR (100 MHz, DMSO-d ) δ/ppm: 139.0 (C ), 123.8 (C ), 129.1 (C ), 128.4
6
1
2,6
3,5
(
C ), 129.1 (C ), 127.2 (C ), 126.3 (C ), 144.0 (C ), 197.0 (C ); [Signal observed =
4 2’ 3’ 4’ 5’ 2”
9
: Ar-CH = 3, Ar-C = 1, thiocarbonyl = 1, thiophene-C = 1, thiophene-CH = 3]. Mass
+
m/z: 218.32 [M] .
2
.2.5 N-(4-Chlorophenyl) thiophene-2-carbothioamide (L5)
5
2
The ligand (L ) was synthesized using ligand (L ) and Lowsson’s
reagent. Colour: dark yellow crystal, Yield: 85 %, mol. wt. 253.35 g/mol, m.p. 240
C; Anal. Calc. (%) For C H ClNS : C, 52.06; H, 3.18; N, 5.52; S, 25.27. Found
°
11
8
2
-1
-1
(
%): C, 51.77; H, 3.12; N, 5.12; S, 25.07. UV-Vis: λ (nm) (ε, M cm ): 278
1
(
13,935), 387 (9,085). H NMR (400 MHz, DMSO-d ) δ/ppm: 7.882 (d, 2H, J = 7.6
6
Hz, H ), 7.733 (d, 2H, J = 7.2 Hz, H ), 7.520 (d, 1H, J = 3.6 Hz, H ), 7.506 (d, 1H,
3,5
2,6
2”
13
J = 4.4 Hz, H ), 7.280 (dd, 1H, J = 4.4 Hz, J = 4.4 Hz, H ), 11.565 (s, 1H, H ).
C
4”
3”
1’
NMR (100 MHz, DMSO-d ) δ/ppm: 137.1 (C ), 125.0 (C ), 125.4 (C ), 133.7
6
1
2,6
3,5
(
C ), 129.1 (C ), 127.2 (C ), 126.3 (C ), 144.0 (C ), 197.0 (C ); [Signal observed =
4 2’ 3’ 4’ 5’ 2”
9
: Ar-CH = 2, Ar-C = 2, thiocarbonyl = 1, thiophene-C = 1, thiophene-CH = 3]. Mass
+
m/z: 252.76 [M] , 254.95 [M + 2].
2
.2.6 N-(4-Nitrophenyl) thiophene-2-carboxamide (L6)

9
6
3
The ligand (L ) was synthesized using ligand (L ) and Lowsson’s
reagent. Colour: brown powder, Yield: 88 %, mol. wt. 264.32 g/mol, m.p. 270 °C;
Anal. Calc. (%) For C H ClNS : C, 49.99; H, 3.05; N, 10.60; S, 24.26. Found
11
8
2
-1
-1
(
%): C, 51.17; H, 3.02; N, 9.13; S, 25.97. UV-Vis: λ (nm) (ε, M cm ): 281
1
(
13,935), 389 (9,000). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.023 (d, 1H, J = 4.0
6
Hz, H ), 7.870 (d, 1H, J = 4.0 Hz, H ), 7.779 (d, 2H, J = 7.6 Hz, H ), 7.405 (d, 2H, J
2”
4”
3,5
13
=
7.2 Hz, H ), 7.237 (dd, 1H, J = 3.6 Hz, J = 3.2 Hz, H ), 10.336 (s, 1H, H ). C
2,6 3” 1’
NMR (100 MHz, DMSO-d ) δ/ppm: 145.1 (C ), 124.8 (C ), 124.2 (C ), 143.9
6
1
2,6
3,5
(
C ), 129.1 (C ), 127.2 (C ), 126.3 (C ), 144.0 (C ), 197.0 (C ); [Signal observed =
4 2’ 3’ 4’ 5’ 2”
9
: Ar-CH = 2, Ar-C = 2, carbonyl = 1, thiophene-C = 1, thiophene-CH = 3]. Mass
+
m/z: 263.32 [M] .
1-6
2
.3
General synthesis of [Pt(L )(Cl )] complexes (1a-1f)
2
Herein, we report six mononuclear Pt(II) complexes (1a-1f) with the
1-3
aromatic bidentate thiophene-2-carboxamide (L ) and thiophene-2-carbothioamide
4-6
(
L ) derivative ligands having -S, O/ -S, S donor atoms. The complexes were
synthesized using method proposed by Hodges and Rund.(Wolfe, Shimer, & Meehan,
1-6
1-6
1
987) Complexes [Pt(II)(L )Cl ] were synthesized by refluxing 1:1 ratio of (L )
2
(
0.2 mmol) and K PtCl (0.2 mmol) in water-methanol system (50 mL) at 60 °C with
2 4
¯
-
1
-2 drops of hydrochloric acid (free acid is to avoid displacement of Cl by OH) until
the solution became colourless (0.5 - 6 h). Reaction mixture was allowed to cool at
room temperature. The obtained product was washed with hot water and dried under
vacuum.
1
2
.3.1 Synthesis of [Pt(L )(Cl )] (1a)
2
1
It was synthesized using ligand (L )
. Colour: brown powder, Yield: 70
%,
mol. wt. 469.25 g/mol, m.p. ≥ 300 °C, Anal. Calc. (%) For C H Cl NOPtS: C,
11
9
2

1
0
2
6
8.16; H, 1.93; N, 2.99; S, 6.83; Pt, 41.57. Found (%): C, 28.01; H, 1.71; N, 2.71; S,
-1
2
-1
-1
-1
.54; Pt, 41.04. Conductance: 24 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ): 292
1
(
13,460), 387 (6,100), 419 (5,505). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.025
6
(
d, 1H, J = 3.6 Hz, H ), 7.860 (d, 1H, J = 4.8 Hz, H ), 7.723 (d, 2H, J = 7.6 Hz, H ),
2” 4” 2,6
7
.361 (t, 2H, J = 7.6 Hz, H ), 7.224 (t, 1H, J = 7.6 Hz, H ), 7.114 (dd, 1H, J = 4.0 Hz,
3,5 4
13
J = 3.6 Hz, H ), 10.204 (s, 1H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 140.0
3”
1’
6
(
(
C ), 122.3 (C ), 129.5 (C ), 128.2 (C ), 135.1 (C ), 129.9 (C ), 132.0 (C ), 145.0
1 2,6 3,5 4 2’ 3’ 4’
C ), 165.5 (C ); [Signal observed = 9: Ar-CH = 3, Ar-C = 1, carbonyl = 1,
5’
2”
thiophene-C = 1, thiophene-CH = 3].
2
2
.3.2 Synthesis of [Pt(L )(Cl )] (1b)
2
2
It was synthesized using ligand (L )
. Colour: brown powder, Yield: 75
%,
mol. wt. 503.70 g/mol, m.p. 300 °C, Anal. Calc. (%) For C H Cl NOPtS: C,
≥
11
8
3
2
6
6.23; H, 1.60; N, 2.78; S, 6.37; Pt, 38.73. Found (%): C, 25.89; H, 1.63; N, 2.67; S,
-1
2
-1
-1
-1
.02; Pt, 38.05. Conductance: 22 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ): 292
1
(
13,500), 376 (6,000), 418 (5,670). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.033
6
(
d, 1H, J = 3.6 Hz, H ), 7.974 (dd, 1H, J = 4.4 Hz, J = 4.4 Hz, H ), 7.842 (d, 1H, J =
2”
3”
4
1
.8 Hz, H ), 7.418 (d, 2H, J = 7.2 Hz, H ), 7.273 (d, 2H, J = 8.0 Hz, H ), 10.348 (s,
4” 3,5 2,6
13
H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 140.1 (C ), 123.6 (C ), 128.0
1’
6
1
2,6
(
C ), 134.0 (C ), 138.0 (C ), 129.3 (C ), 135.9 (C ), 142.0 (C ), 164.0 (C ); [Signal
3,5 4 2’ 3’ 4’ 5’ 2”
observed = 9: Ar-CH = 2, Ar-C = 2, carbonyl = 1, thiophene-C = 1, thiophene-CH =
3
].
.3.3 Synthesis of [Pt(L )(Cl )] (1c)
3
2
2
3
It was synthesized using ligand (L )
. Colour: brown powder, Yield: 82
%,
mol.wt. 514.25 g/mol, m.p. ≥ 300 °C, Anal. Calc. (%) For C H C N O PtS: C,
11
8
l2
2
3
2
7.22; H, 1.87; N, 2.89; S, 6.23; Pt, 40.20. Found (%): C, 25.29; H, 1.40; N, 5.40; S,

1
1
-1
2
-1
-1
-1
6
.08; Pt, 39.82. Conductance: 20 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ): 292
1
(
13,355), 389 (5,950), 417 (5,500). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.282
6
(
d, 2H, J = 7.2 Hz, H ), 8.124 (d, 1H, J = 4.0 Hz, H ), 8.034 (d, 2H, J = 9.2 Hz, H ),
3,5 2” 2,6
7
1
.953 (d, 1H, J = 4.0 Hz, H ), 7.271 (dd, 1H, J = 3.6 Hz, J = 3.6 Hz, H ), 10.791 (s,
4” 3”
13
H. H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 148.0 (C ), 121.5 (C ), 126.3
1’
6
1
2,6
(
C ), 149.8 (C ), 131.0 (C ), 129.2 (C ), 123.0 (C ), 145.0 (C ), 167.0 (C ); [Signal
3,5 4 2’ 3’ 4’ 5’ 2”
observed = 9: Ar-CH = 2, Ar-C = 2, carbonyl = 1, thiophene-C = 1, thiophene-CH =
3
].
.3.4 Synthesis of [Pt(L )(Cl )] (1d
4
2
)
2
4
It was synthesized using ligand (L )
. Colour: brown powder, Yield: 75
%,
mol.wt. 485.32 g/mol, m.p. ≥ 300 °C, Anal. Calc. (%) For C H Cl NPtS : C,
11
9
2
2
2
1
7.22; H, 1.87; N, 2.89; S, 13.21; Pt, 36.89. Found (%): C, 27.01; H, 1.67; N, 2.78; S,
-1
2
-1
-1
-1
3.11; Pt, 38.50. Conductance: 25 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ): 292
1
(
10,555), 405 (4,720), 419 (4,955). H NMR (400 MHz, DMSO-d ) δ/ppm: 8.046
6
(
d, 1H, J = 4.8 Hz, H ), 7.875 (d, 1H, J = 4.0 Hz, H ), 7.739 (d, 2H, J = 7.2 Hz, H ),
2”
4”
2,6
7
.361 (t, 1H, J = 8.0 Hz, H ), 7.166 (dd, 1H, J = 3.6 Hz, J = 3.6 Hz, H ), 7.062 (t, 2H,
4 3”
13
J = 8.0 Hz, H ), 10.969 (s, 1H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 143.0
3,5
1’
6
(
(
C ), 124.0 (C ), 130.0 (C ), 128.5 (C ), 133.5 (C ), 131.4 (C ), 127.8 (C ), 155.2
1 2,6 3,5 4 2’ 3’ 4’
C ), 199.0 (C ); [Signal observed = 9: Ar-CH = 3, Ar-C = 1, thiocarbonyl = 1,
5’
2”
thiophene-C = 1, thiophene-CH = 3].
5
2
.3.5 Synthesis of [Pt(L )(Cl )] (1e)
2
5
It was synthesized using ligand (L ). Colour: light brown powder, Yield:
8
2
1
0 %, mol.wt. 519.76 g/mol, m.p. ≥ 300 °C, Anal. Calc. (%) For C H Cl NPtS : C,
11
8
3
2
5.42; H, 1.55; N, 2.69; S, 12.34, Pt, 37.53. Found (%): C, 25.40; H, 1.51; N, 2.58; S,
-1
2
-1
-1
-1
2.25; Pt, 37.17. Conductance: 19 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ): 294

1
2
1
(
10,570), 405 (4,740), 422 (4,970). H NMR (400 MHz, DMSO-d ) δ/ppm: 7.902
6
(
d, 2H, J = 7.2 Hz, H ), 7.881 (d, 2H, J = 7.6 Hz, H ), 7.739 (d, 1H J = 4.0 Hz, H ),
3,5 2,6 2”
7
1
.598 (d, 1H, J = 4.0 Hz, H ), 7.224 (dd, 1H, J = 4.8 Hz, J = 3.6 Hz, H ), 11.590 (s,
4” 3”
13
H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 140.2 (C ), 128.1 (C ), 130.3
1’
6
1
2,6
(
C ), 136.4 (C ), 133.5 (C ), 128.0 (C ), 127.1 (C ), 148.0 (C ), 198.3 (C ); [Signal
3,5 4 2’ 3’ 4’ 5’ 2”
observed = 9: Ar-CH = 2, Ar-C = 2, thiocarbonyl = 1, thiophene-C = 1, thiophene-CH
3].
.3.6 Synthesis of [Pt(L )(Cl )] (1f)
=
6
2
2
6
It was synthesized using ligand (L ). Colour: brown powder, Yield: 78
%,
mol.wt. 530.31 g/mol, m.p. ≥ 300 °C, Anal. Calc. (%) For C H Cl N O PtS :
11
8
2
2
2
2
C, 24.91; H, 1.52; N, 5.28; S, 12.09; Pt, 36.79. Found (%): C, 24.92; H, 1.50; N, 5.29;
-1
2
-1
-1
-1
S, 12.01; Pt, 35.30. Conductance: 20 Ω cm mol . UV-Vis: λ (nm) (ε, M cm ):
1
2
8
92 (10,550), 404 (4,710), 421 (4,995). H NMR (400 MHz, DMSO-d ) δ/ppm:
6
.030 (d, 2H, J = 7.2 Hz, H ), 7.860 (d, 1H, J = 4.8 Hz, H ), 7.723 (d, 2H, J = 7.6
3,5
2”
Hz, H ), 7.374 (d, 1H, J = 4.4 Hz, H ), 7.203 (dd, 1H, J = 4.8 Hz, J = 4.4 Hz, H ),
2,6
4”
3”
1
3
1
1
0.204 (s, 1H, H ). C NMR (100 MHz, DMSO-d ) δ/ppm: 148.3 (C ), 126.0 (C ),
1’
6
1
2,6
25.2 (C ), 145.2 (C ), 130.2 (C ), 128.2 (C ), 127.9 (C ), 149.0 (C ), 197.8 (C );
3,5
4
2’
3’
4’
5’
2”
[
Signal observed = 9: Ar-CH = 2, Ar-C = 2, thiocarbonyl = 1, thiophene-C = 1,
thiophene-CH = 3,]. LC-MS m/z(%): 530.31 [M], 532.42 [M + 2], 534.16 [M + 4].
2
.4
.4.1 In vitro antibacterial activity
The MIC informs about the degree of resistance of certain bacterial species towards
Biological screening of synthesized compounds
2
the test compounds. MIC value was performed by serially two fold dilution of the test
(-ve)
compound added to three gram
microorganisms namely Escherichia coli (MTCC 433),
Pseudomonas aeruginosa (MTCC P-09), Serratia marcescens (MTCC 7103) and two

1
3
(+ve)
gram
bacteria namely Bacillus subtilis (MTCC 7193), Staphylococcus aureus (MTCC
3160). The lowest compound concentration inhibiting visible bacterial growth is reported as
MIC. (Patra et al., 2015), (Sadana, Mirza, Aneja, & Prakash, 2003)
2.4.2 Nucleotide binding experiments
All the experiments involving the interaction of the Pt(II) complexes (1a-1f) with CT
DNA were used for nucleotide binding study. The stock solution was prepared by dissolving
CT-DNA in a Tris-HCl buffer (pH 7.2) containing 5% DMSO at 4 °C for complete
dissolution and used within 4 days. Ratio of UV absorbance kept at 260 and 280 nm was
about 1.89:1 for CT DNA in the buffer, suggesting the CT DNA sufficiently free from
protein.(Reichmann, Rice, Thomas, & Doty, 1954) The nucleotide binding experiments were
completed at room temperature. All experiments were carried out by keeping the
concentration of platinum complexes constant (400 µM) while Concentrations of CT-DNA
–
1
–1
were determined spectrophotometrically by assuming ꢀ = 6600 M cm .(Marmur, 1961)
260
(H. Paul, 2012; Ramakrishnan, Suresh, Riyasdeen, Akbarsha, & Palaniandavar, 2011; Y. Li,
2010).
2.4.3 Hydrodynamic chain length study
Viscosity experiments were carried out using reported method (Gajera, Mehta, &
Patel, 2015). The flow time of solutions in a Tris-HCl buffer (pH 7.2) was recorded in
triplicate for each sample with a digital stopwatch, and the average flow time was calculated.
⁰
1/3
Data are presented as (ƞ/ƞ ) versus the binding ratio.
2.4.4 Molecular modelling study
The rigid molecular modelling study was performed by using HEX 8.0 software
(Mustard & Ritchie, 2005) and the most stable configuration was chosen as input for
investigation. Pt(II) complexes were .mol format converted to (Protein Data Bank) .pdb
format using CHIMERA 1.5.1 software. CT-DNA used in the experimental work was too

1
4
large for current computational resources to dock, therefore, the structure of the DNA of
sequence (5'-D(CPGPCPGPAPAPTPTPCPGPCPG)-3') (1BNA) for groove binding and an
octamer (5'-D(CPGPCPGPAPAPTPTPCPGPCPG)-3') (1BNA) for intercalation(Ricci &
Netz, 2009) (PDB ID: 1BNA, a familiar sequence used in oligodeoxynucleotide study)
obtained from the Protein Data Bank (http://www.rcsb.org/pdb). All calculations were using
on an Intel CORE i5, 2.5 GHz based machine running MS Windows 8, 64 bit as the operating
system. The by default parameters were used for the docking calculation with correlation type
shape only, FFT mode at 3D level, grid dimension of 6 with receptor range 180 and ligand
range 180 with twist range 360 and distance range 40.
2.4.5 Quenching of DNA–ethidium bromide fluorescence by the Pt(II) complexes.
In the emission quenching experiment, ethidium bromide (EB) was used as a common
fluorescent probe for the DNA in order to examine the mode and process of metal complex
binding to the DNA.(C. V. Kumar, Barton, & Turro, 1985) A 33.3 µM of the EB and 10 µM
of DNA solution added to 1 M Tris-HCl buffer (pH 7.2) solution. Before measurements, the
system was shaken and incubated at ambient temperature for 10 min. The emission was
recorded at 500–800 nm. The fluorescence spectra of EB bound to DNA were recorded (with
excitation wavelength of 510 nm), emission at 610 nm (λmax) and slit wavelength 1.45 nm in
the FluoroMax-4, HORIBA (Scientific) spectrofluorometer. Then the solution of each of the
Pt(II) complexes was titrated into the CT-DNA EB system and diluted in 1 M Tris-HCl
buffer to 3.0 mL to get the solution with the [complex]/[DNA] (r = 0 to 3.33) mole ratio. All
measurements were performed at room temperature.
2
.4.6 DNA mobility shift assay
In gel-electrophoresis method, DNA cleavage assay was carried out which works on
the principle that migration of DNA takes place under the influence of electric potential,
which is negatively charged at neutral pH, and migrates toward the positively charged anode.

1
5
The sample were incubated for 30 min at 37 °C and the samples were analysed by 1 %
agarose gel electrophoresis containing 0.5 mg/mL ethidium bromide, [Tris-acetate-ethylene
diaminetetraacetic acid (EDTA) (TAE) buffer, pH 8.0] for 3 h at 100 mV. The oxidative
cleavage of pUC19 plasmid DNA was monitored using agarose gel electrophoresis. The gel
was visualized by UV light and photographed on UV transilluminator. The extent of cleavage
of the SC DNA was determined by measuring the intensities of the bands using
TM
AlphaDigiDoc RT.
2
.4.7 In vitro cytotoxicity against brine shrimp lethality bioassay (BSLB)
The cytotoxicity assay was performed on brine shrimp nauplii by Meyer method.(Ferrigni,
1
982) The lethal concentrations of compounds resulting in 50% mortality (LC ) of the brine shrimp
50
from the 24 h and the dose–response data were transformed into a straight line by means of a trend
line fit linear regression analysis; the LC was determine from the best-fit line, a graph of mortality
50
vs. concentration of nauplii.(M.R. Islam, 2007) The LC value is obtained the antilogarithm of log
5
0
[
complex] vs. 50% mortality (LC ). All data has been collected from three independent experiments
50
and the LC determined using OriginPro 8 software.
5
0
2.4.9 In vitro cell proliferation assay
Determination of half maximal inhibitory concentration (IC ) value of complexes (1a-
5
0
1f) was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assay. Briefly, HCT 116 cells were treated with a series of concentrations (control, DMSO, 1,
0, 50, 100, 500, 1000 μg/mL) of complexes (1a-1f) for 24 h. The cells were washed twice
1
with DPBS and incubated with 0.5 mg/ml MTT solution for 4 h at 37 °C. Thereafter, 0.1 ml
of SDS-HCl (10 % SDS in 0.01 M HCl) was added to each well, mixed thoroughly and
allowed for incubation in the dark for 20 min at 37 °C. Finally, the absorbance of each well
was recorded at 570 nm with a reference wavelength of 650 nm using a multimode micro
plate reader (Spectra Max M2e, Molecular devices, USA). The results are represented in
terms of percentage inhibition of cell proliferation compared to that of vehicle control. Cell

1
6
viability was evaluated by trypan blue exclusion assay. HCT 116 cells were treated with 1,
1
0, 50, 100, 500, 1000 μg/ml of complexes (1a-1f) conjugate for 24 h. After completion of
incubation, cells were harvested and washed once with DPBS. Equal amount of cell
suspension was mixed with trypan blue. Subsequently, live and dead cells were counted and
percentage of cell death was determined by the following formula (Percentage of cell death =
Number of dead cells/Total number of cells x 100. In addition, cell proliferation of Human
colorectal carcinoma (HCT 116) was examined by MTT assay. The results are represented as
percentage of cell proliferation in different treatment group.
3
.
Results and discussion
1
-6
3
.1
Synthesis of substituted ligands (L ) and complexes (1a-1f)
The synthesized ligands and complexes schemes are represented in
supplymetry material 1 and 2, we focused upon the effect of change in hetero atom of
1-6
1-3
ligands (L ) i.e. oxygen atom in ligands (L ) is replaced by sulphur atom in ligands
4-6
13
1
(
L ). Ligands and complexes have been fully characterized by C and H NMR
spectroscopy as well as by mass spectrometry.
3
.2
1H NMR spectrum
1
1-6
H NMR spectra of free ligands (L ) and Pt(II) complexes (1a-1f) were recorded in
1
DMSO-d solution. H NMR data are shown in the experimental section and spectra are
6
1
represented in supplementary material 3 and 4. In the H NMR spectra of compounds, the
doublet (d), doublet of doublet (dd), triplet (t) are observed in the range of 7.12 - 8.50 ppm,
which are correspond to the aromatic protons of the phenyl and thiophene rings. The signal
appears between 10.00 - 12.00 ppm corresponds to the amide and thioamide hydrogen of
1
-6
ligands (L ) and Pt(II) complexes (1a-1f).
3
.3
13C NMR spectrum

1
7
1
3
1-6
C NMR spectra of ligands (L ) and Pt(II) complexes (1a-1f) are shown in
1
3
supplementary material 5. C NMR data are reported in the experimental section. Peak of
Pt(II) complexes (1a-1c) shifted to downfield from 161.8 ppm to 164.0 - 167.0 ppm than their
1
-3
respective ligands (L ) due to presence of carbonyl group and peaks of Pt(II) complexes
1d-1f) shifted to downfield from 197.0 ppm to 197.8 - 199.0 ppm than their respective
(
4
-6
ligands (L ) due to presence of thiocarbonyl group. Thiocarbonyl group containing
compounds have been shifted to more downfield than the carbonyl group containing
compounds.
3
.4
LC - MS spectrum
The mass fragments and mass spectra of bidentate ligands (L ) are represented in
1
-6
supplementary material 6. The mass spectra of complexes showed a single molecular peak in
the LC-MS spectrum indicating stability of the complexes. The isotopic pattern confirmed the
presence of platinum metal ion. The representative LC-MS spectrum and probable mass
fragmentation pattern of complex (1f) [C H Cl N O PtS ] (mol.wt. = 530.31 g/mol) are
11
8
2
2
2
2
shown in figure 1. The mass spectrum of the complex (1f) shows molecular ion peak at
C H Cl N O PtS ] [M] 530.31, [M+2] 532.42, [M+4] 534.16 m/z. Peak at 459.41 m/z is
[
1
1
8
2
2
2
2
due to bidentate ligand attached with platinum metal. The fragment of bidentate ligand give
peak at 264.32 m/z due to loss of platinum metal ion.

1
8
Fig. 1. LC-MS spectrum of complex (1f)
.5 Electronic spectra and conductance measurements
3
1-6
The electronic spectra of the ligands (L ) and complexes (1a-1f) were
recorded in the UV-visible region using DMSO solution at room temperature. The
1-6
appropriate data of complexes (1a-1f) and ligands (L ) are shown in experimental
section. Absorption spectrum obeys the Lambert-Beer’s law and are taken in the range
of 100-150 µM concentration of Pt(II) complexes. UV –Visible absorption and
reflectance spectra of platinum(II) complexes are taken in solution state and solid
state.( supplymetry material 7) We observed that the wavelength of Pt(II) complexes
in solid state and in solution remain same, which indicate that platinum(II) complexes
are stable in solid state as well as in solution state. This observation ruled out the
possibility of hydrolysis of complexes in plasma. The electronic spectra of the
mononuclear Pt(II) complexes show three different high-energy absorption bands with
*
varied intense peaks at wavelengths 280-300 nm is consigned to the π-π typically
parallels to a intra ligand charge transfer (ILCT) transition.(A. B. P. Lever, 1984.) In

1
9
addition, the peak observed with broad bands at lower frequency (310-395 nm) is
assigned metal to ligand charge transfer (MLCT) transitions.(Shiju, Arish,
Bhuvanesh, & Kumaresan, 2015) Further addition, the peak observed with broad
bands at very lower frequency (397-420 nm) is consigned to d-d transition. Pt(II)
complexes having
Pt(II) complexes having
the stronger electron-pushing ability of the substituent -NO on Pt(II) complexes (1c,
p
-substituted -NO ligands (1c, 1f) show large red-shift than the
2
p
-substituted -Cl ligands (1b, 1e), which can be attributed to
2
1
f) resulting in the energy of the MLCT transition being higher than -Cl on Pt(II)
complexes (1b, 1e)
All complexes are soluble in DMSO and non-electrolytes (Ʌ ≤ 19 – 25
.
-1
2
-1
Ω cm mol ).
3.6
Thermogravimetric analysis (TGA)
The content of particular components in a complex changes in weight
loss and extent of conversion patterns with increasing temperature. In order to
determine such thermal stability and decomposition mechanism of the solid complexes
examined via thermogravimetric analyse technique. The objective of this analysis is to
analyse weather the water molecule is present in the lattice space or coordinated to the
metal in complex. TGA was carried out for Pt(II) complex (1d) at a heating rate of 10
°
C per minute in the range of 20-800 °C under N atmosphere.
2
The TGA graph representing loss in mass of complex (1d) with respect to temperature
is shown in supplementary material 8, which indicates that complex decomposes in
two main well define steps. There is no loss in weight up to 110 °C, suggesting the
absence of water molecule in the lattice. Further increment of the temperature causes
decomposition of complex in two steps. First mass loss occurring between 120 °C to
3
10 °C (14%) corresponds to two chlorine molecules, second mass loss occurring in

2
0
the temperature range of 310-557 °C (45%) is attributed to loss of bidentate ligand and
leaving behind the platinum as residue. A similar type of thermal degradation trend
was observed in the literature for platinum metal complexes.(Abdel Ghani & Mansour,
2
011; Li et al., 2011)
3.7
Infrared spectra
The characterization of FT-IR spectra recorded for the free ligands (L )
1-6
are vary from their respective complexes (1a-1f) and provided significant indications
1-6
of the bonding sites of amide and thioamide ligands (L ) to the platinum metal ion.
The main bands with assignments are listed in supplementary material 9 (Table 1).
1-3
-1
The band of ν(-NHC=O) in ligands (L ) is observed at (1642 - 1649) cm (Abdel Ghani
4-6
&
Mansour, 2011), which is disappear in ligands (L ). A new peak is observed in
4-6
-1
ligands (L ) in the range of (1250-1270) cm , which clearly indicate presence of
4-6
thiocarbonyl group in ligands (L ).(Abdel Ghani & Mansour, 2011) The ν
of
(N-H)
1-3
amide functional group of the ligands (L ) is observed in the range of (3200-3363)
-1
4-6
cm , while ν
band of thioamide functional group of the ligands (L ) is observed
N-H)
(
-1
in the range of (3273-3364) cm . The band ν
is observed in the range of (1100-
(-C-S-C)
-
1
1-6
1
1
300) cm in ligands (L ). The band of ν
in complexes (1a-1c) is observed at
-NHC=O)
(
-1
630 - 1639 cm (Li et al., 2011; Zhang et al., 2009) frequencies indicate that the
oxygen atom of carbonyl group coordinated with platinum metal ion . The band of ν(-
-1
is observed in the range of (1300-1380) cm in complexes (1d-1f), it indicate
NHC=S)
that sulphur atom of thiocarbonyl group are coordinated with platinum metal ion.
These are further confirmed by the presence of ν(-C=O-Pt) band in the range of (460-500)
-1
-1
cm and the band of ν
in the range of (530-550) cm , in the far IR frequency
-C=S-Pt)
(
region.(Zhang et al., 2009) The ν(N-H) of amide functional group of the complexes (1a-
-1
1
c) is observed in the range of (3296 - 3315) cm , while ν
of thioamide functional
(N-H)

2
1
-1
group of the complexes (1d-1f) is observed in the range of (3080-3213) cm . The
coordination of chelating group (>C=O) of ligand in complexes (1a-1c), (>C=S) group
1-6
in complexes (1d-1f) and sulphur atom of thiophene ring of ligand (L ) coordinate to
platinum metal ion and form the five membered chelate ring. The sulphur atom of
thiophene ring coordinated to platinum metal is further confirmed by appearance of
-1
new peak in the range of 415-450 cm .(Veysel T. Yilmaza, 2014)
.8 Biological screening of synthesized compounds
3
3
.8.1 Antimicrobial activity
Antibacterial activity of the Pt(II) complexes against various bacterial strains
estimated by the MIC (μM) are given in supplementary material 10 and shown in figure 2. In
this study five pathogen bacterial species are considered some of the most virulent
microorganisms for the human population. Effect of the solvent used in the antibacterial tests
is tested and found that the solvent has no effect on bacterial growth. The MIC values of the
Pt(II) complexes (1a-1f) are in the range of 35 μM to 125 μM for all bacterial strains tested.
(-ve)
The highest antibacterial activity is observed for complexes (1d-1f) against Gram
and
(+ve)
Gram
bacteria having MIC value of in the range of 30-45 μM concentration. The MIC’s
of complexes (1c-1d) are generally higher than those for complexes (1a-1c). The significant
antibacterial activities of complexes (1d-1f) may be correlated with their DNA binding
affinities. The antimicrobial activity of the platinum complexes are greater than those of the
respective free ligand, this indicates that the complexation to metal enhances the activity of
the ligands (Shiju et al., 2015; Tysoe, Morgan, Baker, & Strekas, 1993).

2
2
1
-6
Fig. 2. Effect of different concentration of ligands (L ) and synthesized Pt(II) complexes
(+ve)
(-ve)
(1a-1f) on gram
and gram
bacteria.
3.8.2 Nucleotide binding experiments
The study of change in electronic state of a molecule by the action of UV-Visible
light is a significant tool to determine the interactions of complexes with CT-DNA. Figure 3
shows the absorption spectra of complex (1f) in the presence and absence of CT-DNA at
different concentrations. The figure shows that complex has a very strong absorption band at
315 nm. As the CT-DNA concentration is increased, the absorption spectrum of complex (1f)
shows clear hypochromism and bathochromic shift at the maximum peak. Hypochromism
with red shift (2-8) nm are observed at 315 nm, indicating that the complexes (1a-1f) bind to
CT-DNA via partial intercalation mode of binding. This could be attributed to the strong
stacking interaction between the planar aromatic chromophore and the base pairs of DNA.
Also, the degree of the hypochromism is in agreement with the strength of partial
intercalative action(Ameta, Singh, & Kale, 2013; Mansouri-Torshizi, Moghaddam, Divsalar,
&
Saboury, 2009; Millar, Ho, & Aroney, 1988; "NCCLS, National Committee for Clinical
Laboratory Standards, Performance Standards for Antimicrobial Disk Susceptibility Test,"
997; Temple, McFadyen, Holmes, Denny, & Murray, 2000). The binding constant value of
the complexes is measured using the following eqn (1):(Pyle et al., 1989)
1

2
3
[
ꢁꢂꢃ]
[ꢁꢂꢃ]
ꢉ
=
+ ꢊ ꢈꢋꢌ − ꢌ ꢏ
(1)
ꢍ
ꢎ
ꢄꢅꢆꢄ
ꢇ
ꢄꢅꢆꢈꢄ
ꢇ
ꢇ
where, [DNA] is the concentration of DNA, ꢌ ꢌ and ꢌ correspond to Aobsd/[complex], the
ꢐ
ꢎ
ꢍ
extinction coefficient for the free complex, and the extinction coefficient for the complex in
the fully bound form, respectively. Complexes (1a-1f), however, show significant
hypochromicity when treated with CT-DNA. The intrinsic nucleotide binding constant (Kb)
5
-1
values of all the complexes are found in the range of 0.768 – 1.611 × 10 M and through an
increasing concentration of CT-DNA, significant hypochromicity (H%) ranging from 16.66%
to 21.13% were observed in the bands of compounds, (Table 1) these indicate the partial
intercalating mode. Complex (1f) has the highest binding constant value, which is attributed
to the different positions of the substituents of -NO functional group on the phenyl
2
ring.(Křikavová, Vančo, Šilha, Marek, & Trávníček, 2016; Özel, Barut, Demirbaş, &
Biyiklioglu, 2016) The interactions of complex (1f) with DNA have shown, that the
complexes are electron-acceptors, while the DNA molecule is an electron donor. Since, -NO2
functional group is a powerful electron-withdrawing group, which can accept electrons from
DNA, complex (1f) shows strong binding affinity towards CT-DNA compared to other
synthesized complexes, binding constant values are found in the order of 1f > 1e > 1c > 1d >
1
b > 1a. The K value of synthesized complexes (1a-1f) are found higher binding affinity than
b
3
-1
3
-1
the oxaliplatin (5.3 × 10 M ) and carboplatin (0.33 × 10 M ) (Soori H1, 2015 Jan 7) and
6
-1
lower than the intercalator ethidium bromide (EB) (5.52 × 10 M ).(Meenongwa et al., 2016)
From the values of the binding constant (K ), ΔGꢈisꢈfree energy changes for the binding
b
process have been calculated using equation (2):
ΔGꢈ= -RT ln Kb
(2)
Binding constants are the measure of the complex stability while the free energy indicates the
spontaneity/non-spontaneity of compound–DNA binding. The free energy of the complexes

2
4
-1
(
1a-1f) were calculated and observed in the range of -27.87 to -29.70 kJmol showing the
spontaneity of compound–DNA interaction. ΔGꢈandꢈK ꢈvaluesꢈareꢈrepresented in Table 1.
b
Fig. 3. Absorption spectral changes on addition of CT-DNA to the solution of complex (1f)
after incubating it for 10 min at room temperature in phosphate buffer (Tris-HCl,
pH=7.2). Inset: plot of [DNA]/(Ɛ -Ɛ ) vs. [DNA]. (Arrow shows the change in
a
b
absorption with increase in concentration of DNA).
Table 1. Binding constant (K ), % hypochromicity and standard free energy changes (ΔG)
b
values of synthesized Pt(II) complexes.
a
b
-1
-1
Complexes
H%
K (M )
ΔG(J mol )
b
1
a
19.23 ± 0.78
0.768 ± 0.05 × 105
-27873.0
5
1
1
1
1
b
c
d
e
f
16.66 ± 0.69
20.70 ± 0.91
16.77 ± 0.73
20.92 ± 0.08
21.13 ± 0.57
0.875 ± 0.10 × 10
1.463 ± 0.08 × 10
0.939 ± 0.12 × 10
1.540 ± 0.05 × 10
-28193.3
-29466.8
-28368.7
5
5
5
-29593.9
-29705.6
5
1
1.611 ± 0.06 × 10
a
H% = [(Afree - Abound)/Afree] × 100%.
b
Kb = Intrinsic DNA binding constant determined from the UV-Visible absorption spectral titration.
3.8.3 Hydrodynamic chain length study

2
5
It is well known that viscosity measurement is an effective tool to study such binding
models in solution, in the absence of crystallographic structure data, visual or photo physical
probes generally provide necessary but not sufficient information to support a partial
intercalative binding model. Ethidium bromide interact to DNA via classical intercalation,
increases the axial chain length of the DNA and it becomes more rigid, resulting in an
increase in the relative hydrodynamic volume. When the DNA helix is partially intercalated
by small planar molecules, base pairs separate to accommodate the binding molecules,
resulting in the increase of viscosity of the DNA sample solution. On the other hand, a
decrease in viscosity will be observed with groove binding of the compound due to the
tightening of the CT-DNA helix, and no significant changes on viscosity via hydrogen
bonding.(Colmenarejo et al., 1995; Rajput, Rutkaite, Swanson, Haq, & Thomas, 2006)
As complex to DNA concentration ratio increase, the viscosity of the solution
increase significantly. Values of (ƞ/ƞ⁰)1/3 for complex (1f) show in figure 4. These results are
agree with those of the spectral analyses, suggesting that complex (1f) has a sensible partial
intercalating ability to DNA. Results suggest that among these complex (1f) is the most
powerful intercalation(Davis et al., 2012) to CT-DNA concluded from the viscosity test,
which is in agreement with the spectral analysis.

2
6
Fig. 4. Viscosity measurements of the complexes (1a-1f)
.8.4 Molecular modelling study
Molecular modelling study has played important roles both in the
3
molecular recognition of nucleic acid as well as in the rational design of new
chemotherapeutic drugs,(Rohs, Bloch, Sklenar, & Shakked, 2005; Zhu et al., 2015)
discovery as well as in the mechanistic study by placing a small molecule into the
binding site of the target specific area of DNA. The molecular docking studies of
complexes with DNA duplex of sequence (5'-D(CPGPCPGPAPAPTPTPCPGPCPG)-
') dodecamer were performed in order to predict the binding site along with preferred
3
orientation of the molecules inside the DNA. Complex (1f) is illustrate in Figure 5 and
other complexes are shown in supplementary material 11. The study shown that the
complexes under investigation interact with DNA via an intercalation mode involving
outside edge stacking interaction with oxygen atom of the phosphate backbone. From
the resultant molecular docked structures, it is clear that the planarity of ligand core is
comfortable for strong π-π stacking interactions and the complexes fit well into the
intercalative mode of the targeted DNA and A−T rich region stabilized by van der
Waal’s interaction and hydrophobic contacts.(Mehta, Gajera, & Patel, 2016; Pettersen
et al., 2004; Tabassum, Zaki, Afzal, & Arjmand, 2013) The resulting binding energies
of docked complexes (1a-1f) are found to be -224.33 (1a), -251.87 (1b), -349.31(1c), -
−
1
2
19.63 (1d), -231.37(1e), -238.47 (1f) kJ mol , respectively. The more negative the
relative binding values, the more potent is the binding between DNA and target
molecules. Thus, we can conclude that there is a mutual complement between
spectroscopic techniques and molecular docked model, which substantiate our
spectroscopic results, and provide further evidence of intercalative binding. Therefore,
molecular docking together with absorption spectral and viscosity studies indicate that

2
7
the complex interacts with the DNA through the partial intercalation mode mainly
inside the DNA helix.
Fig. 5. Molecular docking of the complexes 1f (ball and stick) with the DNA duplex
(
VDW spheres with solid surface) of sequence DNA (5'-
D(CPGPCPGPAPAPTPTPCPGPCPG)-3'). The complex is docked in to the
DNA showing partial intercalation between the DNA base pairs.
3
.8.5 Quenching of DNA–ethidium bromide fluorescence by the Pt(II) complexes.
Fluorescence spectroscopy is an effective method used to explore
the small molecules bind independently to a set of equivalent sites on a
biomacromolecules. The binding tendency of platinum complexes to CT-DNA has
been examined by the steady state quenching probes with the emission intensity of
ethidium bromide (EB).(Lakowicz, 2006) It is well known that ethidium bromide (3,8-
diamino-5-ethyl-6-phenyl-phenanthridinium bromide) is typical indicator of
intercalation, establishing soluble complexes with nucleic acids, causing a emitting
intense fluorescence at about 610 nm presence of DNA, due to its intercalation of the
planar Pt(II) complexes among adjacent DNA base pairs on the double helix.
(
Arjmand & Aziz, 2009) Addition of a second molecule, which may bind to DNA
more strongly than EB, two mechanisms have been suggested to account for this
reduction in the emission intensity: the replacement of EB and/or electron

2
8
transfer.(Pasternack et al., 1991) Compounds competing with EB to intercalation in
DNA will induce displacement of bound EB and in a decrease in the fluorescence
intensity of the CT-DNA EB system. The extent of decrease in the fluorescence
intensity of CT-DNA EB reflects the extent of interaction of the complex with CT-
DNA.(Lepecq & Paoletti, 1967) In the present study, the fluorescence intensity of the
CT-DNA EB system (with excitation wavelength of 510 nm) is partial replaced by the
increasing concentration of the complexes and caused by EB migration from a
hydrophobic to an aqueous environment.(Zeng, Yang, Liu, & Tang, 2003) The
observed changes clearly indicate partial intercalation mode of binding with CT-DNA.
Quenching of an EB-DNA can arise due to inner-filter effect. The mechanism of
quenching is obtained from the emission intensity of EB. To rule out the influence of
an inner filter effect, a correction factor was carried out to all the fluorescence data,
using equation :(1) (J.R. Lakowicz, 2006)
Icoor. = Iobs.
×
ꢑ(ꢃꢒꢓꢔꢈꢕꢃꢒꢖꢔ)/2
(1)
I and I are the observed and corrected emission intensities, and A and Aemi are
obs
corr
exi
the solution absorption at the excitation and emission wavelengths. Although overall
inner-filter effects were negligible, corrected values were used in all the data treatment
and analysis.
The quenching of EB bound to DNA by Pt(II) complexes (1a-1f) is in good agreement
(
[complex]/[DNA] = 3.33) with the linear Stern–Volmer equation (2):(Eftink &
Ghiron, 1981; Mahendiran, Kumar, Viswanathan, Velmurugan, & Rahiman, 2016)
/I = 1 + Kq [Q]
I
₀
(
2)
where, I
quencher (complexes), respectively. Kq is a linear Stern–Volmer quenching constant,
₀
and I represent the fluorescence intensities in the absence and presence of

2
9
Q is the concentration of the quencher (complexes). In the quenching plot for complex
1b) (insets of Figure 6.) of I /I vs. [complex], indicating that complex (1b) reveals the
(
₀
highest binding tendency. Kq is given by the ratio of the slope to the intercept. The
quenching abilities of Pt(II) complexes, evaluated by the linearity Stern–Volmer
3
4
-1
quenching constant (Kq) are found in the range of 2.6 × 10 – 9.0 × 10 M . These
values show that the partial replacements of EB bound to DNA base pairs by the
complexes result in a decrease of the fluorescence intensity, which is corresponding
with the Kb values (Table 1).
The titration data found from the fluorescence quenching experiment is helpful for the
calculation of number of binding sites (n) and the associative binding constant (Ka
)
using the following equation (3):(Kathiravan & Renganathan, 2009)
(
ꢚ₀ꢆꢚ)
ꢚ
ꢗꢘꢙꢈ
= log ꢛꢜꢈ + ꢝꢗꢘꢙ[ꢞ]
(
3)
where, n and Ka are represent the number of binding sites and associative binding
constant of Pt(II) complexes, respectively. The number of binding sites n, per
nucleotide is obtained from the slope of log[(I
₀ - I)/I] vs. log [Q] (Figure 6). The
associative binding constant (Ka) values of Pt(II) complexes are found in the range of
2
4
-1
0
.34 ×10 - 3.1 × 10 M and the results of all complexes (1a -1f) are represented in
supplementary material 12. The value of n is nearly 1, suggesting that the
complex bond to CT-DNA base pair according to the molar ratio of 1:1. Thus, the
fluorescence study lead us to a conclusion that the complexes can bind to DNA via
partial intercalative mode.
In order to evaluation the interaction force of CT DNA with complexes, the standard
free energy changes (ΔG) for the binding process have been calculated using the van’t
Hoff equation (4):(Hong, Chang, Li, & Niu, 2016)