Synthesis, characterization and biological applications of substituted pyrazolone core based platinum(II) organometallic compounds

Article abstract of DOI:10.1016/j.jorganchem.2017.11.012

New series of organometallic platinum(II) compounds (I-VI) containing the 2-phenyl-5-methyl pyrazole-3-one derivatives ligands (L¹-L⁶) were synthesized and characterized by spectroscopic and physicochemical techniques. The synthesize

Full text of DOI:10.1016/j.jorganchem.2017.11.012

Journal of Organometallic Chemistry 854 (2018) 49e63
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and biological applications of substituted
pyrazolone core based platinum(II) organometallic compounds
Miral V. Lunagariya, Khyati P. Thakor, Darshana N. Kanthecha, Mohan N. Patel*
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of organometallic platinum(II) compounds (I-VI) containing the 2-phenyl-5-methyl pyrazole-
3-one derivatives ligands (L1-L6) were synthesized and characterized by spectroscopic and physico-
chemical techniques. The synthesized compounds were screen for their in vitro antibacterial activity
against two Gram(þve) and three Gram(ꢀve) bacterial species. In vitro cytotoxicity against brine shrimp
lethality bioassay and in vitro cellular level cytotoxicity against S. pombe cells were carried out for the
synthesized compounds. Complexes (I-VI) exhibit excellent cytotoxicity as compared to ligands (L1-L6).
DNA interaction and DNA cleavage study of the synthesized ligands and complexes were carried out
using absorption titration spectroscopy, fluorescence quenching analysis, viscosity measurements, mo-
lecular docking study and agarose gel electrophoresis. The interactions of compounds towards herring
Received 22 June 2017
Received in revised form
8 November 2017
Accepted 11 November 2017
Available online 15 November 2017
Keywords:
Organometallic platinum(II) compounds
2-Phenyl-5-methyl pyrazolone based
ligands
sperm DNA (HS-DNA) was examined. The binding constant (Kb), Stern-Volmer quenching constant (Ksv
)
and associative binding constant (Ka) of the synthesized ligands and complexes were observed in the
In vitro cytotoxicity
DNA interaction study
DNA cleavage assay
range of 0.138e5.580 ꢁ 105
M
ꢀ1, 1.1 ꢁ 102 to 7.0 ꢁ 103 Mꢀ1 and 1.871 ꢁ 103 to 1.711 ꢁ 105 Mꢀ1
,
respectively. In molecular docking study, binding energy of compounds were observed in the range
of ꢀ240.74 to ꢀ325.79 kJmol-1. The results propose that all the compounds could interact with HS-DNA
via the partial intercalative mode of binding.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
change in biological properties of ligands as well as metal moiety
and in many cases it causes synergistic effect of metal ion and
Since the discovery by Rosenberg, cisplatin (cis-diamminedi-
chloroplatinum(II)) is one of the most general anticancer drugs
encouraged the search for novel metal-based therapeutic agents in
cancer [1,2]. There is still scope for the elimination of side effects
(e.g. nausea, hearing loss, vomiting, etc.), higher solubility and the
ability to use it in combination with other drugs [3]. Coordination
compounds of transition metals describe a great history of their
usefulness in the cure of various diseases [4e6]. The major appli-
cation of these compounds finds use in the field of discovery of new
anticancer agent. Various oxidation states exhibited by transition
metals, find use as compounds interacting with a number of
negatively charged molecules and thus leads to the development of
metal-based drugs with surprising pharmacological purpose and
may present exceptional curative opportunities. Metals can play an
important role in modifying the pharmacological properties of li-
gands after its coordination to a ligand [7]. Chelation causes drastic
ligand both. Organometallic platinum(II) complexes have been re-
ported as anti-diabetic, anti-inflammatory, antimanic, antimicro-
bial, antiparasitic, antiulcer, antihypertensive agents [8].
Many pyrazole derivatives are well approved to possess a wide
range of medicinally and biologically activities [9]. The heterocyclic
molecules, which are present in many important drugs and agro-
chemical such as Celecoxib (antiarthritic), Mavacoxib (antiar-
thritic), Razaxaban (anticoagulant), Fluazolate (herbicide) and
Penthiopyrad (fungicide) [10,11]. Despite of an extensive amount of
literature on metal complexeDNA interaction, the information
regarding the nature of binding of these complexes to DNA has
remained a subject of extreme debate. The structure, size and
relative nature of the ligands in the coordination complex has a
direct influence on DNA binding [12].
Considering these aspects in mind, in present work, the syn-
thesis of pyrazole derivatives ligands were carried out by conven-
tional methods. And also synthesized organometallic platinum(II)
complexes (I-VI) via dichloro bridge dimer as an intermediate.
Biological activity of the synthesized ligands and complexes were
* Corresponding author.
E-mail address: jeenen@gmail.com (M.N. Patel).
https://doi.org/10.1016/j.jorganchem.2017.11.012
0022-328X/© 2017 Elsevier B.V. All rights reserved.

50
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
carried out by performing in vitro cytotoxicity against brine shrimp
lethality bioassay, in vitro cellular cytotoxicity against S. pombe
cells, in vitro antibacterial activity against five different pathogen,
electronic absorption titration spectroscopy, viscosity measure-
ments, fluorescence quenching analysis, molecular docking and
DNA nuclease activity.
with catalytic amount acetic acid (5 mL) was added in round bot-
tom flask using ethanol solvent. The mixture was refluxed under
stirring for 3 h. After completion of the reaction, the reaction mass
was cooled, and then evaporated to dryness in vacuum. The reac-
tion mixture extracted with ethyl acetate (3 ꢁ 5 mL). The combined
ethyl acetate layers were back-extracted with saturated sodium
bicarbonate (3 ꢁ 5 mL) and brine (3 ꢁ 5 mL), dried over MgSO4,
filtered, and evaporated in vacuum. The residue was crystallized
from ethanol to obtain the target compounds. The proposed reac-
tion scheme for the synthesized uninegative bidentate ligands (L1-
L6) are represented in Scheme 1.
2. Experimental section
2.1. Chemicals and reagents
Solvents were dried and distilled previous to their use following
standard process [13]. All the chemicals were using of analytical
grade. Ethidium bromide (EB), bromophenol blue, agarose and
Luria Broth (LB) were purchased from Hi-media Laboratories Pvt.
Ltd., India. Potassium tetrachloro platinate(II) salt, 4-tolualdehyde,
4-methoxy benzaldehyde, 4-hydroxy benzaldehyde, 4-chloro
benzaldehyde, 4-bromo benzaldehyde, 4-nitro benzaldehyde and
2-phenyl-5-methyl-pyrazole-3-one were purchased from Sigma
Chemical Co. (India) and were used as received without further
2.3.1. Structural characterization of (E)-5-methyl-4-(4-
methylbenzylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L1)
This ligand (L1) was prepared through the addition of 2-phenyl-
5-methyl pyrazole-3-one (1a) (522 mg, 3 mmol) and p-methyl
benzaldehyde (2a) (360 mg, 3 mmol) and reflux for 5e6 h in
presences of catalytic amount AcOH and EtOH solution as described
in general process. Colour: yellowish powder, Yield: 85%, mol. wt.:
276.34 g/mol, m.p.: 125 ꢂC; Anal. Calc. (%) For C18H16N2O: C, 78.24;
H, 5.84; N, 10.14. Found (%): C, 78.06; H, 5.65; N, 10.10. UV-vis:
purification. Culture of pUC19 bacteria (MTCC 47), two Gram(þve)
,
i.e. Staphylococcus aureus (S. aureus) (MTCC-3160) and Bacillus
subtilis (B. subtilis) (MTCC-7193), and three Gram(ꢀve), i.e. Serratia
marcescens (S. marcescens) (MTCC-7103), Pseudomonas aeruginosa
(P. aeruginosa) (MTCC-1688) and Escherichia coli (E. coli) (MTCC-
433), were purchased from Institute of Microbial Technology
(Chandigarh, India). S. pombe Var. Paul Linder 3360 was obtained
from IMTECH, Chandigarh. Ethidium bromide (EB) and pUC19
bacteria (MTCC 47) were used for all DNA binding and cleavage
studies.
l
(nm) (ε, M¡1 cm¡1): 256 (34,070), 288 (7160). MS m/z (%): 276.70
[M]. 1H NMR (400 MHz, DMSO-d6)
d
0
/ppm: 8.71 (2H, d, J ¼ 8.8 Hz,
00 00
0
H2 ,6 ), 7.92 (2H, d, J ¼ 8.0 Hz, H2 ,6 ), 7.76 (1H, s, H6), 7.43 (3H, t,
3J1 ¼7.6 Hz, 3J2 ¼ 7.6 Hz, H3 ,4 ,5 ), 7.12e7.10 (2H, m, H3 ,5 ), 3.89 (3H,
00 00 00
0
0
s, pyrazole-CH3), 2.33 (3H, s, toluene ring-CH3). 13C NMR (100 MHz,
DMSO-d6)
d
/ppm: 165.52 (C¼O, Cquat.), 147.58 (C3, Cquat.), 143.45
00
0
0
0
(C6,-CH), 140.34 (C1 , Cquat.), 137.64 (C4 , Cquat.), 134.43 (C2 ,6 , -CH),
0
0
0
00 00
00
129.93 (C1 ,Cquat.),128.99 (C3 ,5 , -CH),127.69 (C3 ,5 , -CH),127.00 (C4 ,
00
-CH), 126.27 (C4, -Cquat.), 118.50 (C2 ,6” -CH), 21.34 (-CH3, phenyl
2.2. Physical measurements
ring), 15.05 (-CH3, pyrazole). [Total signal observed ¼ 14: signal of
C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-C ¼ 3), signal of CH and CH3 ¼ 8
(substituted pyarazole-CH ¼ 1, phenyl ring-CH ¼ 5, pyrazole-
CH3 ¼ 1, phenyl ring-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3078 n(¼C-H)ar
(w), 1689 n(-N-C¼O), 1514e1604 n(C¼N) (s), 1426 n(C¼C)conjugated alkenes
(m), 1373 n(C-H)banding (m), 1319 n(C-N), 1195 n(C-C)alkanes (s), 763 n(Ar-H)
Thermogravimetric analysis (TGA) was performed with a model
SDT Q600 V20.9 Build 20, TA instrument (USA), to record curves
under a nitrogen (N2) atmosphere with a heating rate of 20 ꢂC
minꢀ1 in the temperature range from 25 ꢂC to 800 ꢂC. The micro-
elemental analysis (C, H, N and S) content of the compounds was
carried out with a Euro Vector EA3000 elemental analyser (240).
The magnetic moments were measured by Gouys method using
(s).
2 adjacent hydrogen
mercury
(
tetrathiocyanatocobaltate(II)
as
the
calibrate
2.3.2. Structural characterization of (E)-4-(4-methoxybenzylidene)-
5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L2)
cg ¼ 16.44 ꢁ 10ꢀ6 cgs units at 20 ꢂC). Melting points were deter-
mined on Buchi 540 melting point apparatus. Infrared spectra were
recorded on a Perkin-Elmer FT IR Shimadzu model spectropho-
tometer using the KBr disc technique in the range of
4000e400 cmꢀ1. In the range of 200e800 nm, electronic absorp-
tion spectra were recorded using a UV-160A UVeVis spectropho-
tometer Shimadzu, Kyoto (Japan), with a 10 mm path length quartz
cell. Fluorescence spectra were recorded on a FluoroMax-4, spec-
trofluorometer, HORIBA (Scientific). The LCeMS spectra were
recorded by thermo mass spectrophotometer (USA). Minimum
concentration inhibitory study was carried out using laminar air
flow cabinet, Toshiba, Delhi, (India). The 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance (400 MHz) and
(100 MHz), respectively either in DMSO-d6 (35 ꢂC), referenced
internally to the solvent. Photo quantization of the gel after elec-
trophoresis was prepared by AlphaDigiDocTM RT. Version
V.4.0.0 PCeImage software, CA (USA). Hydrodynamic chain length
study was carried out by viscometric measurement bath.
This ligand (L2) was prepared through the addition of 2-phenyl-
5-methyl pyrazole-3-one (1a) (522 mg, 3 mmol) and p-methoxy
benzaldehyde (2b) (408 mg, 3 mmol) and reflux for 5e6 h in
presences of catalytic amount AcOH and EtOH solution as described
in general process. Colour: pale yellowish powder, Yield: 92%, mol.
wt.: 292.34 g/mol, m.p.: 130 ꢂC; Anal. Calc. (%) For C18H16N2O2: C,
73.95; H, 5.52; N, 9.58. Found (%): C, 73.10; H, 5.45; N, 9.30. UV-vis:
l
(nm) (ε, M¡1 cm¡1): 257 (37,260), 330 (17,690). MS m/z (%):
292.37 [M]. 1H NMR (400 MHz, DMSO-d6)
d
/ppm: 8.73 (2H, d,
00 00
0
0
J ¼ 8.0 Hz, H2 ,6 ), 7.93 (2H, d, J ¼ 7.6 Hz, H2 ,6 ), 7.92 (1H, s, H6), 7.43
(3H, t, 3J1 ¼ 7.2 Hz, 3J2 ¼ 5.6 Hz, H3 ,4 ,5 ), 7.21e7.11 (2H, m, H3 ,5 ),
00 00 00
0
0
4.41 (3H, phenyl ring-CH3), 2.34 (3H, pyrazole-CH3). 13C NMR
(100 MHz, DMSO-d6)
d
/ppm: 165.52 (C¼O, Cquat.), 147.58 (C3, Cquat.),
0
00
0
0
159.84 (C4 ,Cquat.), 143.45 (C6,-CH.), 140.34 (C1 , Cquat.), 130.23 (C2 ,6
,
0
00 00
-CH), 126.20 (C4,Cquat.), 125.24 (C1 , -CH), 128.99 (C3 ,5 , -CH), 128.00
00
00 00
0
(C4 , -CH), 118.50 (C2 ,6 , -CH), 114.22 (C3 ,5’ -CH), 55.81 (-OCH3,
phenyl ring), 15.05 (-CH3, pyrazole). [Total signal observed ¼ 14:
signal of C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-C ¼ 3), signal of CH and
CH3 ¼ 8 (substituted pyarazole-CH ¼ 1, phenyl ring-CH ¼ 5, pyr-
azole-CH3 ¼ 1, phenyl ring-OCH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3078
n(¼C-H)ar (w), 1674 n(-N-C¼O), 1550e1589 n(C¼N) (s), 1488 n(C¼C)conju-
gated alkenes (m), 1365 n(C-H)banding (m), 1311 n(C-N), 1265 n(C-C)alkanes (s),
756 n(Ar-H)2 adjacent hydrogen (s).
2.3. General synthesis of the substituted 2-phenyl-5-methyl
pyrazole-3-one based ligands (L1eL6)
To the solution of 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-
3-one (1 mmol) and different substituted benzaldehyde (1 mmol)

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
51
Scheme 1. Synthesis of the different substituted 2-phenyl-5-methyl-pyrazol-3-one derivatives ligands (L1 e L6).
2.3.3. Structural characterization of (E)-4-(4-hydroxybenzylidene)-
278.57 [M]. 1H NMR (400 MHz, DMSO-d6)
d
/ppm: 8.74 (2H, d,
5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L3)
J ¼ 8.0 Hz, H2 ,6 ), 7.93 (2H, d, J ¼ 7.6 Hz, H3 5 ), 7.77 (1H, s, H6), 7.42
00 00
0 0
This ligand (L3) was prepared through the addition of 2-phenyl-
5-methyl pyrazole-3-one (1a) (522 mg, 3 mmol) and p-hydroxy
benzaldehyde (2c) (366 mg, 3 mmol) and reflux for 5e6 h in
presences of catalytic amount AcOH and EtOH solution as described
in general process. Colour: orange powder, Yield: 88%, mol. wt.:
278.31 g/mol, m.p.: 135 ꢂC; Anal. Calc. (%) For C17H14N2O2: C,
73.37; H, 5.07; N, 10.07. Found (%): C, 73.31; H, 5.05; N, 10.30. UV-
(3H, t, 3J1 ¼ 8.0 Hz, 3J2 ¼ 8.0 Hz, H3 ,4 ,5 ), 7.21e7.10 (2H, m, H2 ,6 ),
00 00 00
0
0
2.37 (3H, s, pyrazole-CH3). 13C NMR (100 MHz, DMSO-d6)
d/ppm:
165.56 (C¼O, Cquat.), 147.83 (C3, Cquat.), 143.40 (C6,-CH), 140.32
00
0
0
0
0
(C1 ,Cquat.), 134.98 (C2 ,6 , -CH), 133.56 (C4 , Cquat.), 131.08 (C1 , Cquat.),
00 00
0
0
00
128.90 (C3 ,5 , -CH), 128.50 (C3 ,5 , -CH), 128.00 (C4 , -CH), 126.23 (C4,
00 00
Cquat.), 118.57 (C2 ,6 , -CH), 15.26 (-CH3, pyrazole). [Total signal
observed ¼ 13: signal of C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-C ¼ 3),
signal of CH and CH3 ¼ 7 (substituted pyarazole-CH ¼ 1, phenyl
ring-CH ¼ 5, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3078 n(¼C-H)ar
(w), 1674 n(-N-C¼O), 1589 n(C¼N) (s), 1488 n(C¼C)conjugated alkenes (m),
1365e1411 n(C-H)banding (m), 1311 n(C-N), 1149 n(C-C)alkanes (s), 756 n(Ar-
H)2 adjacent hydrogen (s).
vis: l
(nm) (ε, M¡1 cm¡1): 254 (36,470), 288 (6390). MS m/z (%):
278.57 [M]. 1H NMR (400 MHz, DMSO-d6)
d/ppm: 9.95 (1H, s, -OH),
00 00
0
0
8.74 (2H, d, J ¼ 8.0 Hz, H2 ,6 ), 7.93 (2H, d, J ¼ 8.0 Hz, H3 ,5 ), 7.77 (1H,
s, H6), 7.43 (3H, t, 3J1 ¼ 8.4 Hz, 3J2 ¼ 7.6 Hz, H3 ,4 ,5 ), 7.21e7.11 (2H,
00 00 00
m, H2 ,6 ), 2.34 (3H, pyrazole-CH3). 13C NMR (100 MHz, DMSO-d6)
d
/
0
0
0
ppm: 165.54 (C¼O, Cquat.), 157.71 (C4 , Cquat.), 147.53 (C3,Cquat.),
00
0
0
143.40 (C6,-CH.), 140.32 (C1 , Cquat.), 130.68 (C2 ,6 , -CH), 128.96
2.3.5. Structural characterization of (E)-4-(4-bromobenzylidene)-5-
methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L5)
00 00
00
0
(C3 ,5 ,-CH), 128.00 (C4 , -CH), 126.25 (C4, -CH), 125.55 (C1 , -CH),
00 00
0
118.51 (C2 ,6 , -CH), 115.86 (C3 ,5’ -CH), 15.22 (-CH3, pyrazole). [Total
signal observed ¼ 13: signal of C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-
C ¼ 3), signal of CH and CH3 ¼ 7 (substituted pyarazole-CH ¼ 1,
phenyl ring-CH ¼ 5, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3209
n(-O-H), 3055 n(¼C-H)ar (w), 1658 n(-N-C¼O), 1550e1596 n(C¼N) (s), 1504
n(C¼C)conjugated alkenes (m), 1380 n(C-H)banding (m), 1334 n(C-N), 1296 n(C-
This ligand (L5) was prepared through the addition of 2-phenyl-
5-methyl pyrazole-3-one (1a) (522 mg, 3 mmol) and p-bromo
benzaldehyde (2e) (555 mg, 3 mmol) and reflux for 5e6 h in
presences of catalytic amount AcOH and EtOH solution as described
in general process. Colour: orange powder, Yield: 81%, mol. wt.:
341.21 g/mol, m.p.: 130 ꢂC; Anal. Calc. (%) For C17H13BrN2O: C,
59.84; H, 3.84; N, 8.21. Found (%): C, 59.39; H, 3.75; N, 8.34. UV-vis:
(s), 748 n(Ar-H)2 adjacent hydrogen (s).
C)alkanes
l
(nm) (ε, M¡1 cm¡1): 256 (34,400), 364 (15,340). MS m/z (%):
2.3.4. Structural characterization of (E)-4-(4-chlorobenzylidene)-5-
methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L4)
341.17 [M]. 1H NMR (400 MHz, DMSO-d6)
d
/ppm: 8.73 (2H, d,
00 00
0
0
J ¼ 8.0 Hz, H2 ,6 ), 7.93 (2H, d, J ¼ 7.6 Hz, H3 ,5 ), 7.76 (1H, s, H6), 7.42
This ligand (L4) was prepared through the addition of 2-phenyl-
5-methyl pyrazole-3-one (1a) (522 mg, 3 mmol) and p-chloro
benzaldehyde (2d) (421 mg, 3 mmol) and reflux for 5e6 h in
presences of catalytic amount AcOH and EtOH solution as described
in general process. Colour: orange powder, Yield: 85%, mol. wt.:
296.75 g/mol, m.p.: 132 ꢂC; Anal. Calc. (%) For C17H13ClN2O: C,
68.81; H, 4.42; N, 9.44. Found (%): C, 68.31; H, 4.05; N, 9.30. UV-vis:
(3H, t, 3J1 ¼ 8.4 Hz, 3J2 ¼ 8.4 Hz, H3 ,4 ,5 ), 7.21e7.12 (2H, m, H2 ,6 ),
00 00 00
0
0
2.35 (3H, s, pyrazole-CH3). 13C NMR (100 MHz, DMSO-d6)
d/ppm:
165.56 (C¼O, Cquat.), 147.84 (C3, Cquat.), 143.43 (C6,-CH), 140.39
00
0
0
0
0
0
(C1 ,Cquat.), 134.78 (C2 ,6 , -CH), 131.96 (C1 , Cquat.), 131.48 (C3 ,5 , -CH),
00 00
00
0
128.90 (C3 ,5 , -CH), 128.00 (C4 , -CH), 126.25 (C4, Cquat.), 122.32 (C4 ,
00 00
C
quat.), 118.56 (C2 ,6 , -CH), 15.00 (-CH3, pyrazole). [Total signal
observed ¼ 13: signal of C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-C ¼ 3),
l
(nm) (ε, M¡1 cm¡1): 259 (34,740), 306 (25,330). MS m/z (%):
signal of CH and CH3 ¼ 7 (substituted pyarazole-CH ¼ 1, phenyl

52
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
ring-CH ¼ 5, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3070
n
(¼C-H)
completely evaporated, than the obtained residue was purified by
column chromatography on silica gel with dichloromethane/pe-
troleum ether (1:1) system as the eluent to obtained pure product.
The proposed reaction mechanism for the synthesis of cyclo-
metalated heteroleptic platinum(II) complexes (I-VI) are repre-
sented in Scheme 2. The 1H NMR and 13C NMR spectra of
platinum(II) complexes are shown in supplementary material 1 and
2, respectively.
ar (w), 1674
n
(-N-C¼O), 1581
n
(C¼N) (s), 1488
n
(C¼C)conjugated alkenes
(m), 1365e1411
756 (Ar-H)2 adjacent hydrogen (s).
n(C-H)banding (m), 1311 n(C-N), 1141 n(C-C)alkanes (s),
n
2.3.6. Structural characterization of (E)-5-methyl-4-(4-
nitrobenzylidene)-2-phenyl-2,4-dihydro-3H-pyrazol-3-one (L6)
This ligand (L6) was prepared through the addition of 2-phenyl-
pyrazole-3-one (1a) (522 mg, 3 mmol) and p-nitro benzaldehyde
(2f) (453 mg, 3 mmol) and reflux for 5e6 h in presences of catalytic
amount AcOH and EtOH solution as described in general process.
Colour: orange powder, Yield: 82%, mol. wt.: 307.31 g/mol, m.p.:
138 ꢂC; Anal. Calc. (%) For C17H13N3O3: C, 66.44; H, 4.26; N, 13.67.
2.5.1. Synthesis of [(L1)Pt(acac)]-I
It was prepared using [(L1)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
67%, mol. wt.: 569.52 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
Found (%): C, 66.39; H, 4.15; N,13.64. UV-vis:
l
(nm) (ε, M¡1 cm¡1):
C
23H22N2O3Pt: C, 48.51; H, 3.89; N, 4.92; Pt, 34.25. Found (%): C,
48.30; H, 3.60; N, 4.67; Pt, 34.20. Molar conductivity (Lm):
21
ꢀ1cm2molꢀ1. UV-vis: (nm) (ε, M¡1 cm¡1): 308 (35,330), 360
(17,040). 1H NMR (400 MHz, DMSO-d6)
256 (34,400), 364 (15,340). MS m/z (%): 307.88 [M]. 1H NMR
00 00
(400 MHz, DMSO-d6)
d
0
/ppm: 8.74 (2H, d, J ¼ 11.6 Hz, H2 ,6 ), 7.93
U
l
(2H, d, J ¼ 6.0 Hz, H3 ,5 ), 7.76 (1H, s, H6), 7.42 (3H, t, 3J1 ¼ 7.2 Hz,
d/ppm: 7.58e7.50 (9H, m,
0
3J2 ¼ 9.6 Hz, H3 ,4 ,5 ), 7.21e7.11 (2H, m, H2 ,6 ), 2.36 (3H, pyrazole-
Ar-H2 ,3 ,5 ,6 ,6,2 ,3 ,4 ,5 ), 6.57 (1H, s, H3 ), 2.57 (3H, s, acac-CH3 1 ),
00 00 00
0
0
0
0
0
0
00 00 00 00
000
000
CH3). 13C NMR (100 MHz, DMSO-d6)
d
/ppm: 165.47 (C¼O, Cquat.),
1.66 (3H, s, acac-CH3 ), 1.27 (3H, s, pyrazole-CH3), 0.90 (3H, s,
5000
147.80 (C3, Cquat.), 147.13 (C4 ,-Cquat.), 143.49 (C6,-CH), 140.38 (C1 ,
phenylring-CH3). 13C NMR (100 MHz, DMSO-d6)
d/ppm: 191.53
0
00
0
0
0
00 00
000
-CH), 139.00 (C1 , Cquat.), 132.28 (C2 ,6 , -CH), 128.92 (C3 ,5 , -CH),
(C¼O, Cquat.), 181.96 (C-O, C4 ), 169.51 (C¼O, C5), 152.83 (C3, Cquat.
)
00
0
0
00 0 0 0
142.60 (C6, -CH), 141.85 (C1 , Cquat.), 137.63 (C4 , Cquat.), 134.42 (C2 ,6 ,
128.00 (C4 , -CH), 126.27 (C4, Cquat.), 123.83 (C3 ,5 , Cquat.), 118.50
00 00
0 0 0 00
-CH), 129.93 (C1 , Cquat.), 128.90 (C3 ,5 , CH), 128.18 (C3 , -CH), 128.00
00 00 00 00
(C5 , -CH), 124.34 (C4 , -CH), 121.60 (C2 , -CH), 120.00 (C6 ,Cquat.),
(C2 ,6 , -CH), 15.08 (-CH3, pyrazole). [Total signal observed ¼ 13:
signal of C ¼ 6 (pyrazole-C ¼ 3, phenyl ring-C ¼ 3), signal of CH and
CH3 ¼ 7 (substituted pyarazole-CH ¼ 1, phenyl ring-CH ¼ 5, pyr-
azole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3050 n(¼C-H)ar (w), 1681 n(-N-
000
000
,
116.72 (C4, Cquat.), 81.36 (C3 -CH), 24.41 (C5 -CH3), 21.39 (-CH3
,
000
aromatic), 16.09 (CH3, C1 ), 13.90 (CH3, pyrazole). [Total signal
observed ¼ 21: signal of C ¼ 9 (acetyl acetone-C ¼ 2, pyrazole-
C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 12 (acetyl acetone-
CH ¼ 1, pyrazole-CH ¼ 1, phenyl ring-CH ¼ 6, acetyl acetone-
CH3 ¼ 2, pyrazole-CH3 ¼ 1, phenyl ring-CH3 ¼ 1]. FT-IR (KBr):
,
1589 n(C¼N) (s), 1488e1512 n(C¼C)conjugated
(m),
C¼O)
alkenes
1342e1421 n(C-H)banding (m), 1303 n(C-N), 1110e1141 n(C-C)alkanes (s),
748 n(Ar-H)2 adjacent hydrogen (s).
2.4. General synthesis of cycloplatinated(II) chloro bridge dimer
[Pt(L6)Cl]2
(cm¡1): 3105 n(¼C-H)ar (w), 1558 n(C¼N)
, n(C¼O), 1419 n(C¼C)conjugated
alkenes (m),1365 n(C-H)banding (m),1018 n(C-N), 1265 n(C-C)alkanes (s), 609
n(Pt-N), 532 n(Pt-O)
.
Synthesis of the platinum(II) dichloro-bridged dimers was car-
ried out using a modified method of Lewis [14,15]. To the solution of
2-phenyl-5-methyl pyrazole-3-one derivative ligands (L1eL6)
(58.6 mg, 0.4 mmol) and K2PtCl4 (83 mg, 0.2 mmol) in ethox-
yethanol, stirred vigorously under nitrogen (N2) atm. and refluxed
at 100 ꢂC for 24e48 h. The reaction mixture was allowed to cool at
room temperature. The obtained precipitate (chloro-bridged Pt(II)
dimer) was washed with water (20 mL) and dried at 50 ꢂC under
vacuum. The crude product was used for next step without puri-
fication. The characterization data of dimer are as follows.
2.5.2. Synthesis of [(L2)Pt(acac)]-II
It was prepared using [(L2)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
62%, mol. wt.: 585.52 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
C
23H22N2O4Pt: C, 47.18; H, 3.79; N, 4.78; Pt, 33.32. Found (%): C,
47.70; H, 3.62; N, 4.60; Pt, 33.37. Molar conductivity (Lm):
20
ꢀ1cm2molꢀ1. UV-vis: (nm) (ε, M¡1 cm¡1): 294.50 (25,210),
357 (6920). 1H NMR (400 MHz, DMSO-d6)
/ppm: 7.83e7.24 (9H,
m, Ar-H3 ,5 ,2 ,6 ,6,2 ,3 ,4 ,5 ), 6.04 (1H, s, H3 ), 2.33 (3H, s, phenylring-
U
l
d
0
0
0
0
00 00 00 00
000
0
0
0
0
0
0
Colour: brown powder, Yield: 37%, mol.wt.: 1073.67 g/mol,
OCH3), 1.79 (3H, s, acac-CH3 1 ), 0.99 (3H, s, acac-CH3 5 ), 0.59 (3H,
m.p.: >300 ꢂC; Anal. Calc. (%) For C34H24Cl2N6O6Pt2. Conduc-
s, pyrazole-CH3). 13C NMR (100 MHz, DMSO-d6)
d
/ppm: 191.52
ꢀ1cm2molꢀ1
.
1H NMR (400 MHz, DMSO-d6)
d/ppm:
(C¼O, C2 ), 181.90 (-C-O, C4 ), 169.53 (C¼O, C5), 159.80 (C4 , Cquat.),
000
000
0
tance: 22
U
00 0 0
152.86 (C3, Cquat.), 142.65 (C6, -CH), 141.83 (C1 , Cquat.), 130.22 (C2 ,6 ,
7.78e7.20 (16H, m, Ar-H), 6.10 (2H, s, H6,6), 1.28 (6H, s, pyrazole-
CH3). 13C NMR (100 MHz, DMSO-d6)
d
/ppm: 169.50 (C¼O, C5),
00 00 0
-CH), 128.90 (C3 , -CH.), 128.50 (C5 , -CH), 125.28 (C1 , Cquat.), 124.30
00 00 00
(C4 , -CH), 121.64 (C2 , -CH), 120.06 (C6 , Cquat.), 116.70 (C4, Cquat.),
0
00
152.81 (C3, Cquat.), 147.14 (C4 , Cquat), 142.68 (C6, -CH), 141.82 (C1 ,
0
0
0
00
0 0 000
114.20 (C3 ,5 , -CH), 81.32 (C3 , -CH), 55.81 (-OCH3, aromatic), 24.40
Cquat.), 139.00 (C1 , Cquat.), 132.23 (C2 ,6 , -CH), 128.96 (C3 , -CH.),
00
00
00
0
0
000
000
128.00 (C5 , -CH), 128.00 (C5 , -CH), 124.37 (C4 , -CH), 123.82 (C3 ,5 ,
(C5 , -CH3), 16.09 (CH3, C1 ), 13.90 (CH3, pyrazole). [Total signal
observed ¼ 21: signal of C ¼ 9 (acetyl acetone-C ¼ 2, pyrazole-
C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 12 (acetyl acetone-
CH ¼ 1, pyrazole-CH ¼ 1, phenyl ring-CH ¼ 6, acetyl acetone-
CH3 ¼ 2, pyrazole-CH3 ¼ 1, phenyl ring-OCH3 ¼ 1]. FT-IR (KBr):
00
00
-CH), 121.65 (C2 ,-CH.), 120.00 (C6 , Cquat), 116.53 (C4, Cquat.), 13.95
(CH3, pyrazole). [Total signal observed ¼ 15: signal of C ¼ 7 (pyr-
azole-C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 8 (pyrazole-
CH ¼ 1, phenyl ring-CH ¼ 6, pyrazole-CH3 ¼ 1].
(cm¡1): 3103 n(¼C-H)ar (w), 1566 n(C¼N)
, n(C¼O), 1404 n(C¼C)conjugated
alkenes (m),1360 n(C-H)banding (m),1010 n(C-N), 1260 n(C-C)alkanes (s), 555
n(Pt-N), 509 n(Pt-O)
2.5. Preparation of cycloplatinum(II) complexes (I-VI)
.
The crude product (chloro-bridged Pt(II) dimer) was treated
with 3 molar equivalent acetyl acetone in the presence of 10 equiv.
of K2CO3 in 2-ethoxyethanol solvent at 80e90 ꢂC under nitrogen
(N2) atmosphere for 24e48 h. The mixture was poured into water
for extraction through separating funnel using solvent dichloro-
methane (CH2Cl2). The organic extracts were washed with water
and dried over anhydrous sodium sulphate. After the solvent was
2.5.3. Synthesis of [(L3)Pt(acac)]-III
It was prepared using [(L3)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
65%, mol. wt.: 571.50 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
C22H20N2O4Pt: C, 46.24; H, 3.53; N, 4.90; Pt, 34.14. Found (%): C,
46.27; H, 3.52; N, 4.70; Pt, 34.37. Molar conductivity (Lm):

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
53
Scheme 2. Synthesis of the organometallic platinum(II) complexes (I-VI).
0
0
0
0
00
22
U
ꢀ1cm2molꢀ1. UV-vis:
l
(nm) (ε, M¡1 cm¡1): 294.50 (25,210),
/ppm: 10.12 (1H, s,
134.92 (C2 ,6 , -CH), 133.52 (C4 , -CH.), 131.00 (C1 , Cquat.), 128.98 (C3 ,
357 (6920). 1H NMR (400 MHz, DMSO-d6)
d
-CH), 128.70 (C3 ,5 , -CH), 128.05 (C5 , -CH), 124.30 (C4 , -CH), 121.60
0
0
0
0
0
0
0
0
0
0
00 00 00 00
0
0
0
0
000
,
phenyl ring-OH), 7.78e7.20 (9H, m, Ar-H2 ,3 ,5 ,6 ,2 ,3 ,4 ,5 ), 6.19 (1H,
(C2 , -CH), 120.00 (C6 , Cquat.), 116.70 (C4, Cquat.), 81.34 (C3 -CH),
000
000
000
000
000
s, H3 ),1.97 (3H, s, acac-CH3 1 ),1.26 (3H, s, acac-CH3 5 ), 0.89 (3H, s,
24.49 (CH3, C5 ), 16.06 (CH3, C1 ), 13.91 (-CH3, pyrazole). [Total
signal observed ¼ 20: signal of C ¼ 9 (acetyl acetone-C ¼ 2, pyr-
azole-C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 11 (acetyl
acetone-CH ¼ 1, pyrazole-CH ¼ 1, phenyl ring-CH ¼ 6, acetyl
acetone-CH3 ¼ 2, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3001
pyrazole-CH3). 13C NMR (100 MHz, DMSO-d6)
d
/ppm: 191.50 (C¼O,
000
000
0
C2 ), 181.92 (-C-O, C4 ), 169.52 (C¼O, C5), 157.73 (C4 , Cquat.), 152.81
00
0
0
(C3, Cquat.), 142.64 (C6, -CH), 141.80 (C1 , Cquat.), 130.62 (C2 ,6 , -CH),
00
00
0
00
129.90 (C3 , -CH.), 128.90 (C5 , -CH), 125.58 (C1 , Cquat.), 124.30 (C4 ,
-CH), 121.62 (C2 , -CH), 120.06 (C6 , Cquat.), 116.74 (C4, Cquat.), 115.80
00
00
n(¼C-H)ar (w), 1558 n(C¼N)
n(C-H)banding (m), 1010 n(C-N), 1265 n(C-C)alkanes (s), 563
n(Pt-O).
,
n(C¼O), 1404 n(C¼C)conjugated alkenes (m), 1360
0
0
000
000
000
(C3 ,5 , -CH), 81.36 (C3 -CH), 24.42 (C5 -CH3), 16.08 (CH3, C1 ),
n
(Pt-N), 509
,
,
13.96 (CH3, pyrazole). [Total signal observed ¼ 20: signal of C ¼ 9
(acetyl acetone-C ¼ 2, pyrazole-C ¼ 3, phenyl ring-C ¼ 4), signal of
CH and CH3 ¼ 11 (acetyl acetone-CH ¼ 1, pyrazole-CH ¼ 1, phenyl
ring-CH ¼ 6, acetyl acetone-CH3 ¼ 2, pyrazole-CH3 ¼ 1]. FT-IR
2.5.5. Synthesis of [(L5)Pt(acac)]-V
It was prepared using [(L5)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
67%, mol. wt.: 634.08 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
(KBr): (cm¡1): 3340 n(O-H), 3105 n(¼C-H)ar (w), 1558 n(C¼N)
,
n(C¼O)
,
,
1404 n(C¼C)conjugated
(m), 1365 n(C-H)banding (m), 1018 n(C-N)
alkenes
1265 n(C-C)alkanes (s), 532 n(Pt-N), 509 n(Pt-O)
.
C
22H19BrN2O3Pt: C, 41.65; H, 3.02; N, 4.42; Pt, 30.75. Found (%): C,
41.77; H, 3.01; N, 4.40; Pt, 30.17. Molar conductivity (Lm):
19
ꢀ1cm2molꢀ1. UV-vis: (nm) (ε, M¡1 cm¡1): 298 (29,910), 370
(10,050). 1H NMR (400 MHz, DMSO-d6)
2.5.4. Synthesis of [(L4)Pt(acac)]-IV
U
l
It was prepared using [(L4)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
60%, mol. wt.: 589.94 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
d
/ppm: 7.78e7.19 (9H, m,
0
0
0
0
00 00 00 00
000
000
Ar-H2 ,3 ,5 ,6 ,6,2 ,3 ,4 ,5 ), 6.21 (1H, s, H3 ), 2.31 (3H, s, acac-CH3 1 ),
1.66 (3H, s, acac-CH3 ), 0.97 (3H, s, pyrazole-CH3). 13C NMR
5000
000
000
C
22H19ClN2O3Pt: C, 44.79; H, 3.25; N, 4.75; Pt, 33.07. Found (%): C,
44.77; H, 3.42; N, 4.70; Pt, 33.17. Molar conductivity (Lm):
24
ꢀ1cm2molꢀ1. UV-vis: (nm) (ε, M¡1 cm¡1): 293 (27,030), 354
(8000). 1H NMR (400 MHz, DMSO-d6)
(100 MHz, DMSO-d6)
d
/ppm: 191.52 (C¼O, C2 ), 181.94 (-C-O, C4 ),
00
169.54 (C¼O, C5), 152.80 (C3, Cquat.), 142.62 (C6, -CH), 141.82 (C1 ,
0 0 0 0 0
Cquat.), 134.72 (C2 ,6 , -CH), 131.92 (C1 , Cquat.), 131.50 (C3 ,5 , -CH),
U
l
00 00 0 00
128.95 (C3 , -CH), 128.00 (C5 , -CH), 122.30 (C4 , Cquat.), 124.30 (C4 ,
d
/ppm: 8.86e7.56 (9H, m, Ar-
0
0
0
0
00 00 00 00
000
000
00 00
-CH), 121.65 (C2 , -CH), 120.00 (C6 , Cquat.), 116.75 (C4, Cquat.), 81.33
H2 ,3 ,5 ,6 ,6,2 ,3 ,4 ,5 ), 6.18 (1H, s, H3 ), 2.27 (3H, s, acac-CH3 1 ), 1.68
(3H, s, acac-CH3 5 ), 1.06 (3H, s, pyrazole-CH3). 13C NMR (100 MHz,
DMSO-d6)
(C¼O, C5), 152.80 (C3, Cquat.), 142.62 (C6, -CH), 141.82 (C1 , Cquat.),
(C3 , -CH), 24.44 (CH3, C5 ), 16.04 (CH3, C1 ), 13.95 (-CH3, pyrazole).
[Total signal observed ¼ 20: signal of C ¼ 9 (acetyl acetone-C ¼ 2,
pyrazole-C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 11
000
000
000
000
000
000
d
/ppm: 191.52 (C¼O, C2 ), 181.94 (-C-O, C4 ), 169.54
00

54
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
(acetyl acetone-CH ¼ 1, pyrazole-CH ¼ 1, phenyl ring-CH ¼ 6, acetyl
acetone-CH3 ¼ 2, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 3001
number of viability of nauplii (observing several seconds if nauplii
did not exhibit any internal or external movement considered
nauplii dead). Data were analysed by the log concentration of
sample vs percentage mortality of nauplii that gives LC50 values. All
data has been collected from three independent experiments and
the LC50 determined using OriginPro 8 software.
n(¼C-H)ar (w), 1558e1650 n(C¼N), n(C¼O), 1404 n(C¼C)conjugated
alkenes
(m), 1350 n(C-H)banding (m), 1018 n(C-N), 1260 n(C-C)alkanes (s), 640 n(Pt-
N), 478 n(Pt-O)
.
2.5.6. Synthesis of [(L6)Pt(acac)]-VI
It was prepared using [(L6)PtCl]2 dimer and acetyl acetone, ac-
cording to the general procedure. Colour: brown powder, Yield:
69%, mol. wt.: 600.49 g/mol, m.p. >300 ꢂC; Anal. Calc. (%) For
2.6.3. In vitro cellular level cytotoxicity against S. Pombe cells
Eukaryotic Schizosaccharomyces pombe was an important or-
ganism for the study of effects of the metal complexes at cellular
level (cytotoxicity) to the DNA damage (genotoxicity). S. pombe
were grown in liquid yeast extract media in 150 mL Erlenmeyer
flask containing 50 mL of yeast extract media. To acquire the
enough growth of S. pombe, flask was incubated at 30 ꢂC on shaker
at 150 rpm (24e30 h). Then the cell culture was treated with
synthesized ligands and complexes at different concentrations (20,
C
22H19N3O5Pt: C, 44.00; H, 3.19; N, 7.00; Pt, 32.49. Found (%): C,
44.07; H, 3.12; N, 7.01; Pt, 32.47. Molar conductivity (Lm):
18
ꢀ1cm2molꢀ1. UV-vis: (nm) (ε, M¡1 cm¡1): 298 (29,910), 370
(10,050). 1H NMR (400 MHz, DMSO-d6)
U
l
d
/ppm: 7.79e7.22 (9H, m,
0
0
0
0
00 00 00 00
000
000
Ar-H2 ,3 ,5 ,6 ,6,2 ,3 ,4 ,5 ), 6.20 (1H, s, H3 ), 2.26 (3H, s, acac-CH3 1 ),
1.67 (3H, s, acac-CH3 ), 0.97 (3H, s, pyrazole-CH3). 13C NMR
5000
000
000
(100 MHz, DMSO-d6)
d
/ppm: 191.59 (C¼O, C2 ), 181.96 (-C-O, C4 ),
40, 60, 80, 100 mM), which were dissolve in DMSO took as a control
0
169.51 (C¼O, C5), 152.87 (C3, Cquat.), 147.12 (C4 , Cquat.), 142.61 (C6,
and further allowed to growth for 16e18 h. Next day, centrifuge the
treated and control cells at 12,000 rpm for 10 min to remove the
media and wash the cells with phosphate buffer saline (PBS) thrice.
00
0
0
0
-CH), 141.84 (C1 , Cquat.), 139.00 (C1 , Cquat.), 132.22 (C2 ,6 , -CH),
00
0
0
00
00
128.92 (C3 , -CH), 123.80 (C3 ,5 , -CH), 128.00 (C5 , -CH), 124.31 (C4 ,
00
00
-CH), 121.68 (C2 , -CH), 120.00 (C6 , Cquat.), 116.73 (C4, Cquat.), 81.30
Resuspend the cells in 500 mL of PBS. Take the equal volume of cells
000
000
000
(C3 , -CH), 24.40 (CH3, C5 ), 16.03 (CH3, C1 ), 13.93 (-CH3, pyrazole).
[Total signal observed ¼ 20: signal of C ¼ 9 (acetyl acetone-C ¼ 2,
pyrazole-C ¼ 3, phenyl ring-C ¼ 4), signal of CH and CH3 ¼ 11
(acetyl acetone-CH ¼ 1, pyrazole-CH ¼ 1, phenyl ring-CH ¼ 6, acetyl
acetone-CH3 ¼ 2, pyrazole-CH3 ¼ 1]. FT-IR (KBr): (cm¡1): 2993
suspension and 0.4% trypan blue dye in PBS to incubate for 5 min at
room temperature. Cells-dye mixture were put on glass slide and
cells were observed in a compound microscope (40X). Dead cells
were permitting trypan blue dye to appear the blue whereas live
cells resisted the entry of dye seen in colourless. Percentage
viability were count in triplicate where number of dead cells and
number of live cells were counted in three microscopic fields and
calculated average percentage of live cells [17].
n(¼C-H)ar (w), 1558e1650
(m), 1350 n(C-H)banding (m), 1018 n(C-N), 1260 n(C-C)alkanes (s), 555 n(Pt-
N), 501 n(Pt-O)
n
(
, n(C¼O), 1411 n(C¼C)conjugated
C¼N) alkenes
.
2.6. Biological application of the synthesized compounds
2.6.4. DNA interaction studies
2.6.4.1. (a)
Electronic
absorption
titration
spectroscopy.
2.6.1. In vitro antibacterial activity
Binding mode and interaction strength of DNA with metal com-
plexes have been examined effectively by electronic absorption
spectra (UVeVis absorbance titration) using Herring Sperm DNA
(HS-DNA) with ε ¼ 12,858 dm3 molꢀ1 cmꢀ1 in phosphate buffer
solution (pH 7.2). The stock solutions of the complexes were pre-
pared in DMSO. The absorption titration has been performed by the
All of the newly synthesized Pt(II) complexes (I-VI) were
screened for their antibacterial activity using Staphylococcus aureus,
Bacillus subtilis, Serratia marcescens, Pseudomonas aeruginosa and
Escherichia coli micro-organisms. The broth dilution technique has
been used to determine the bactericidal effect by minimum
inhibitory concentration (MIC) in terms of
mM. MIC, lowest con-
concentration of complex keeping constant (20
mM) and contin-
centration that prevents the microbial growth incubated at
uous adding the volume of DNA (100 L), and incubated for
m
37 1 ꢂC for 24 h. MIC was determined in liquid media containing
10 min at room temperature. By using absorption spectral titration
data Kb value have been determined from the ratio of the slope to
intercept from plot of [DNA]/(εaeεf) versus [DNA] [18e21].
0.2e3500
grown in Luria broth overnight at 37 ꢂC. First culture was used as a
control to examine normal growth and second culture 20 L of the
mM of the test compound. A preculture of bacteria was
m
bacteria and compound at the desired concentration were added to
monitor bacterial growth by measuring turbidity of the culture
after 18 h. If a certain concentration of a compound inhibit bacterial
growth, half of the concentration of the compound was tested. This
procedure was carried out up to the concentration that inhibited
the growth of bacteria. All equipment and culture media were
sterilized.
2.6.4.2. (b) Viscosity measurements. An Ubbelohde viscometer
maintained at a constant temperature of 27 0.1 ꢂC in a thermo-
static jacket. It was used to measure the flow time of HS-DNA in
phosphate buffer (pH 7.2) with a digital stopwatch. Flow time
measurements of each compound were carried out three times to
calculate average flow time. Data were presented as relative specific
viscosity ((h/h0) h
1/3) vs binding ratio ([Drug]/[DNA] [22], where
and h0 is the viscosity of DNA in the presence of complex and
viscosity of DNA alone, respectively. Viscosity values have been
calculated from the observed flow time of DNA containing solutions
2.6.2. In vitro cytotoxicity against brine shrimp lethality bioassay
For the determination of toxicity of bioactive compounds in vitro
cytotoxicity technique was used. Brine shrimp (Artemia cysts)
Lethality bioassay method given by Meyer et al. [16] accomplished
with brine shrimp nauplii in artificial seawater (prepared with a
commercial salt mixture in double distilled water) and using
approximately 50 mg eggs in ordinary light. After 24 h, keeping
constant volume 2.5 mL per vial accumulate 1 mL of seawater and
10 shrimps (collected nauplii using a pipette) in each vial, then add
a sample solution (from 10 mg in 10 mL) in it to make final con-
centrations of solution 2, 4, 8, 12, 16 and 20 mg mLꢀ1 (triplicate sets
18 vials) along with control (3 vials) having only DMSO with same
amount finally volume adjust by seawater. After 24 h, counted the
(t > 100 s), corrected for the flow time of buffer alone (t0),
h f t e t0
[23].
2.6.4.3. (c) Fluorescence quenching analysis. Emission intensity
measurements of ethidium bromide (EB ¼ 3,8-diamino-5-ethyl-6-
phenylphenanthridinium bromide) with free HS-DNA in the
absence and presence of Pt(II) complexes were performed in
phosphate buffer. The HS-DNA solution was up to the value of
r ¼ 3.33 ([DNA]/[Complex]) of pre-treated EBeDNA mixture
([EB] ¼ 33.3
m
M, [DNA] ¼ 10
mM) at ambient temperature and
incubate for 10 min before measurement. The emission intensity

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
55
was recorded in the range of 500e800 nm. The emission intensities
2.6.5. DNA nuclease study
at 610 nm (lmax) were obtained through excitation at 510 nm and
slit wavelength 1.45 nm in the FluoroMax-4, HORIBA (Scientific)
spectrofluorometer. The changes in fluorescence intensities of
ethidium bromide and ethidium bromide bound to DNA were
measured with respect to different concentration of the complex.
The ethidium bromide has less-emission intensity in phosphate
buffer solution 7.2 pH due to fluorescence quenching of free
ethidium bromide by the solvent molecules. In the presence of
DNA, EB exhibits higher intensity due to its partial intercalative
binding mode to DNA. Fluorescence quenching of an EB-DNA can
arise owing to inner-filter effect. The mechanism of quenching is
found from the emission intensity of EB. In our study, inner filter
effect was corrected with the following equation in this literature
[24,25].
Gel electrophoresis study was performed using pUC19 DNA with
synthesized compounds. The samples were incubated for 0.5 h at
37ꢂC. The samples were analysed by 1% agarose gel electrophoresis
[Triseacetateeethylenediaminetetraacetic acid, (TAE) buffer, pH
8.0] for 3 h at 100 mV. The gel was stained with (0.5 mg mLꢀ1
)
ethidium bromide. The gels were viewed in an Alpha Innotech
Corporation Gel doc system and photographed using a CCD camera.
The cleavage efficiency of the compounds, the degree of DNA
cleavage activity was measured by determining the ability of the
complex to SC-DNA to OC-DNA equation describe in literature [32].
3. Results and discussion
3.1. UV-vis spectroscopy, molar conductivity measurements and
magnetic moment
To examine the fluorescence quenching mechanism, the
SterneVolmer quenching constant (Ksv) was determined by equa-
tion (1): [26,27]
The geometry of the cyclometalated heteroleptic platinum(II)
complexes has been confirmed using electronic spectral analysis.
The cyclometalated platinum(II) complexes (I-VI) exhibit two
intense bands, in the range of 294e308 nm and 354e370 nm due to
charge transfer (CT) and DMSO-d6 transitions, respectively [33,34].
Electronic spectral data of synthesized ligands (L1-L6) and com-
plexes (I-VI) are represented in experimental section. The magnetic
moments of Pt(II) complexes are zero B.M. and are consistent with
low-spin t2g6 eg2 (d8) configuration square planer geometry having
dsp2 hybridisation. All the cyclometalated Pt(II) complexes are
diamagnetic in nature [35]. The molar conductivity (Lm) of plati-
ꢀ
Iꢂ I ¼ Ksv½Qꢃ þ 1
(1)
where, I0 and I are the emission intensity of EB-DNA in the absence
and presence of quencher (complex), Ksv is the linear Stern-Volmer
quenching constant obtained from the plot of I0/I vs. [Q] and [Q] is
concentration of quencher. To determine the strength of the
interaction of complexes with DNA, the value of the associative
binding constant (Ka) was calculated using the Scatchard equation
(2): [28,29]
num(II)
18e25
the complexes.
complexes
are
observed
in
the
range
of
ꢂ
ꢀ1cm2molꢀ1, which suggests the non-electrolytic nature of
log I ꢀ I=I ¼ log Ka þ nlog½Qꢃ
(2)
U
where, I0 and I are the fluorescence intensities of the EB-DNA in the
absence and presence of different concentrations of complexes,
respectively and n is the number of binding. The acting forces be-
tween drugs and biomacromolecules include hydrogen bonds, van
der Waals forces, electrostatic attraction and hydrophobic inter-
action, etc. In order to estimate the interaction force of all com-
3.2. Fourier transform infrared spectroscopy (FT-IR)
FT-IR spectral data of synthesized ligands (L1-L6) and cyclo-
metalated heteroleptic platinum(II) complexes (I-VI) are presented
in experimental section. The peaks of ligands containing azome-
pounds, the standard free energy changes (
process have been calculated using the Van't Hoff equation (3): [30]
DG) for the binding
thine
n
(C¼N) group is observed in the range of 1514e1604 cmꢀ1
and it is shifted to higher frequencies (1558e1650 cmꢀ1) in the
complexation, showing the coordination of the heterocyclic nitro-
gen atoms (azomethine group) to platinum metal ion [36,37]. The
D
Gꢂ ¼ ꢀRTlnKa
(3)
bands
n
C ¼ Car of the compounds are observed in the of range
where, T is the temperature (25 ꢂC, 298 K here), Ka is associative
binding constant and R is gas constant 8.314 Jmol-1 ꢀ1. The
G⁰ means that the binding process is
1488e1504 cmꢀ1. The
n
CeHar. banding band of the free ligands are
K
observed in the range of 1365e1411 cmꢀ1 and it is shifted to lower
frequency in the range of 1366-1350 cmꢀ1 on complexation. The
spectra of all the platinum(II) complexes show bands in the range of
532e640 cmꢀ1, 478-530 cmꢀ1 and 416e430 cmꢀ1, due to (Pt-N),
(Pt-O) and (Pt-C), respectively. In platinum(II) complexes (I-VI), the
peak of acetylacetone group containing (>C¼O) is observed in the
negative sign for
spontaneous.
D
2.6.4.4. (d) Molecular docking study. Interaction between DNA and
complexes at the molecular level were studied by advanced
computational typical technique like molecular docking. The rigid
molecular docking study has been executed using HEX 8.0 software
to conclude the orientation of the Pt(II) complexes binding to DNA.
The most stable configuration was selected as the input for inves-
tigation. Mole file of coordinates of metal complexes was prepared
for optimized structure and were rehabilitated to.pdb format using
CHIMERA 1.5.1 software. HS-DNA used in the experimental, the
structure of the DNA of sequence (50-d(CGCGAATTCGCG)-30)2 (PDB
id: 1BNA, a familiar sequence used in oligodeoxynucleotide study)
obtained from the Protein Data Bank (http://www.rcsb.org/pdb).
All calculations were carried out on an Intel CORE i5, 2.20 GHz
based machine running MS Windows 8.1 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 [31].
range of 1650 cmꢀ1
.
3.3. 1H NMR spectroscopy
The 1H NMR spectra of the 2-phenyl-5-methyl pyrazolone based
ligands (L1-L6) and cyclometalated platinum(II) complexes (I-VI)
have been obtained in solvent DMSO-d6 and represented in
supplementary material 1. The resultant physicochemical data of
synthesized compounds are summarized in experimental section.
In 2-phenyl-5-methyl pyrazolone based ligands (L1-L6), all the ar-
omatic proton are observed in the range of ~
all aromatic proton of the cyclometalated platinum(II) complexes
are appeared in the range of ~ 7.585 to 7.501 ppm. Methoxy group
of ligand (L2) and complex (II) are appeared at about ~
2.344 and ~
2.338 ppm, respectively. Methyl (-CH3) group of ligand (L1) and
complex (I) are observed at about ~ 2.33 and ~ 0.902 ppm,
d 7.104e8.716 ppm and
d
d
d
d
d

56
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
respectively. All the cyclometalated platinum(II) complexes contain
acetylacetone bidentate ligands. Therefore methyl group protons
pyrazole-3-one derivative ligands attached to platinum metal ion
with loss of acetylacetone moiety. The peak at 307.24 m/z corre-
sponds to 2-phenyl pyeazole-3-one based ligands. The peaks at
174.37 m/z is obtained with loss of nitrobenzene ring in the pres-
ence of 2-phenyl-5-methyl pyrazole-3-one derivative ligand. The
peak at 97.28 m/z is observed phenyl ring in the presence of 2-
phenyl-5-methyl pyrazole-3-one derivative ligand.
are obtained in range of ~
ligand (L3) and complex (III) are observed proton peak at about ~
9.958 ppm and ~ 10.120 ppm, respectively.
d 0.998e2.574 ppm. Hydroxy group of
d
d
3.4. 13C NMR spectroscopy
The 13C NMR spectra of the synthesized 2-phenyl-5-methyl
pyrazolone based aromatic ligands, cyclometalated heteroleptic
3.6. Thermogravimetric analysis (TGA)
platinum(II) complexes and
m
-dichloro bridge complex (I) are
TGA shows the two steps of decomposition (supplementary
material 4). The complex (VI) shows a no weight loss up to tem-
perature 30e200 ꢂC, indicating the absence of lattice and coordi-
nate water molecule. In first step weight loss (16.48%) during
220e250 ꢂC correspond to coordinated acetyl acetone bidentate
ligand. In the second step, weight loss (51.17%) in between 260 and
430 ꢂC correspond to 2-phenyl-5-methyl pyrazole-3-one derivative
ligand. Platinum metallic (Pt) as a residue left behind at the end of
TG curve (>800 ꢂC) was used to calculate the metal content and was
found in close proximity to the expected one.
shown in supplementary material 2 and data of these compounds
are represented in experimental section. The peak of ligand (L2)
containing methoxy group and ligand (L1) containing methyl group
are appeared at 55.81 ppm and 21.34 ppm, respectively. These
peaks are shifted to downfield in complex (II) and complex (I). The
cyclometalated heteroleptic platinum(II) complexes (I-VI) con-
taining acetylacetone bidentate ligands having
observed in range of 181.90e191.52 ppm. The peak of phenyl ring
>
C¼O peak
1
6
00
carbon (-CH, C6 ) of free ligands (L -L ) and phenyl ring carbon
00
(-Cquat, C6 ) of cyclometalated platinum(II) complexes are observed
in the range of 118.50e118.57 ppm and 12.00e120.06 ppm,
respectively. These observation suggest that the complexes exhibit
downfield shift as compared to free ligands.
3.7. Biological applications of synthesized compounds
3.7.1. In vitro antibacterial activity
The antimicrobial activity is carried out in terms of minimum
inhibitory concentration (MIC) defined as the lowest concentration,
which inhibits the growth of microorganism, referred by lack of
turbidity in the tube. The result of this study are represented in
Table 1. The IC50 values of the complexes and ligands are observed
3.5. Mass spectrometry
The LC-MS spectrum and possible mass fragmentation pattern
of complex (VI) are shown in Fig. 1. The mass spectra and mass
fragmentation pattern of the synthesized ligands (L1-L6) are rep-
resented in supplementary material 3. Mass spectral data of the
ligands are shown in experimental section. The mass spectrum of a
synthesized platinum(II) complex (VI) shows a molecular ion peak
at 600.62 m/z indicates that the absence of the chlorine atom in the
complex. The peak at 501.27 m/z is due to 2-phenyl-5-methyl
in range of 22e95 mM and 225e265 mM, respectively. The plati-
num(II) complexes (I-VI) exhibit higher antimicrobial activity than
metal salt and 2-phenyl-5-methyl pyrazole-3-one based ligands
(L1-L6) [38]. The in vitro antimicrobial bioassay of the metal com-
plexes can be described on the basis of Tweedy's chelation theory
[39]. Chelation decreases the polarity of metal ion due to partial
Fig. 1. LC-MS spectrum and fragmentation pattern of the synthesized organometallic platinum(II) complex (VI).

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
57
Table 1
investigate its actual mechanism of cytotoxicity and its probable
effects on cancer cell line.
The effects of inhibitory concentration (MIC, m
M) values of the free ligands (L1-L6)
and synthesized platinum(II) complexes (I-VI) against two Gram(þve) and three
Gram(ꢀve) microorganisms. Error bars represent the standard deviation of three
independent 5%.
3.7.3. In vitro cellular level cytotoxicity against S. Pombe cells
Cellular level in vitro cytotoxicity of the ligands and complexes
have been tested by S. pombe cells. From the conclusion, cell death
caused by toxicity of the compounds could be easily monitored by
trypan blue dye as a staining. The potency has found to vary with
the different type of functional group present and differ concen-
trations of the ligands and platinum(II) complexes. Complexes III,
IV, V and VI (-OH, -Cl, -Br and eNO2) are found to be excellent toxic
as compared to other complexes (I and II), All the platinum(II)
complexes (I-VI) are more toxic in nature as compared to 2-phenyl-
5-methyl pyrazole-3-one based ligands (L1-L6). The in vitro cyto-
toxicity potency of the platinum(II) complexes is comparable to
standard drug cisplatin and transplatin. After 17e20 h of the
treatment, many of the S. pombe cells are destroyed due to toxic
nature of the complexes. The cytotoxicity order of the synthesized
compounds are Cisplatin > VI > IV > V > III > II > I >
Transplatin > L6 > L4 > L5 > L3 > L2 > L1. The % viability data of the
compounds are represented in Table 3.
Compounds Gram(þve) bacteria
Gram(ꢀve) bacteria
S. Aureus B. subtilis S. marcescens P. aeruginosa E. coli
K2PtCl4
2792
248
245
228
230
232
225
89
60
35
50
55
1
2688
245
240
225
227
245
245
85
70
40
45
39
2
2756
240
260
230
230
250
235
90
1
2956
260
236
245
240
245
240
95
1
3289
265
250
255
245
250
236
80
80
40
60
55
1
L1
L2
L3
L4
L5
L6
I
1
2
2
2
2
2
1
1
2
1
1
5
2
1
2
2
2
1
2
2
2
1
1
1
2
2
1
1
1
5
5
2
3
1
1
2
1
2
1
2
1
1
1
1
5
1
3
2
3
2
5
2
1
1
1
2
1
1
2
1
II
75
75
III
IV
V
45
42
42
40
40
60
VI
25
30
22
35
32
sharing of its positive charge with donor groups and possible
p
-
electron delocalization over the whole chelate ring. As a result
lipophilic character of the central metal atom increases, approving
its permeation through the lipid layer of the cell membrane and
quickly attack the metal binding sites on enzymes of microor-
ganism which inhibit the further growth of the organism [40].
3.7.4. Structure-activity relationship (SAR)
The results of the biological screening shown that the activity is
widely affected by introducing different substituted benzyl ring on
2-phenyl-5-methyl pyrazole-3-one nucleus (Fig. 2). The methoxy
group existing at 4th position on benzyl moiety (R1 ¼ -OCH3)
(complex-II and III) exhibits excellent antibacterial activity against
two Gram positive bacteria i.e. B. substiles and S. aureus. The com-
plexes V and VI containing R1 ¼ -NO2 and eBr groups at 4th po-
sition on benzyl moiety, increase antibacterial activity against
B. substiles and S. marcescens. The complex I exhibits the increasing
antibacterial values (MIC) against E. coli bacteria due to presence of
-CH3 group. The complex IV having chloro (R1 ¼ -Cl) group at 4th
position on benzyl moiety shows increasing anti-bacterial activity
against Gram negative bacteria i.e. S. marcescens and P. aeruginosa.
The presence of electron withdrawing functional groups in com-
plexes such as complexes III, IV, V and VI exhibit superior anti-
bacterial activity against Gram(þve) and Gram(ꢀve) micro-organism
as compared to other electron donating group in complexes I and
II. The LC50 value and percentage viability values of the electron
withdrawing functional group containing complexes III, IV, V and
VI exhibit higher potency as compared to other electron releasing
functional groups containing complexes I and II.
3.7.2. In vitro cytotoxicity against brine shrimp lethality bioassay
All the synthesized 2-phenyl-5-methyl pyrazole-3-one based
ligands (L1-L6) and cyclometalated platinum(II) complexes (I-VI)
have been tested for in vitro brine shrimp lethality bioassay using
Meyer et al. process [41]. The percentage mortality of brine shrimp
nauplii has been determined from the number of dead nauplii. The
LC50 value is calculated from the plot of log of concentration of
samples against percentage of mortality of nauplii and data are
represented in Table 2. It is concluded that the platinum(II) com-
plexes show excellent toxicity as compared to corresponding li-
gands. The mortality rate of nauplii is found to increase with
increasing concentration of compounds. The LC50 values of ligands
and complexes are observed in the range of 52.44e99.97
6.373e13.89 g/mL, respectively. The LC50 values of cisplatin and
transplatin are 3.133 and 14.45 g/mL. The potency of the synthe-
mg/mL and
m
m
sized compounds are observed in order of cisplatin > VI > III > IV >
V > II > I > transplatin > L6 > L3 > L4 > L5 > L2 > L1. In vitro cyto-
toxicity study is a basic one and additional studies are necessary to
Table 3
The effect of compounds on % viability of S. Pombe cells at different concentrations
(mM) with error uncertainty in the value 5%.
Table 2
The Lethal Concentration (LC50) values of cisplatin, transplatin and synthesized
ligands (L1-L6) and Pt(II) complexes (I-VI) in
mg/mL using in vitro cytotoxicity
Concentration(
Compounds
mM)
20
40
60
80
100
against brine shrimp lethality bioassay. Error bars represent the standard devia-
tion of three replicates 5%.
% Viability
L1
93
88
80
78
81
72
59
67
65
63
61
58
60
54
96
98
1
1
1
2
2
1
1
1
3
5
1
1
2
2
2
3
90
85
77
75
80
69
56
57
63
60
58
56
57
52
2
2
2
2
2
1
1
4
1
5
2
1
1
2
88
84
74
73
77
67
49
52
60
57
51
49
50
40
2
1
1
1
3
2
4
5
1
2
2
5
1
1
85
81
71
71
76
64
44
48
58
53
48
45
47
39
1
1
2
1
2
2
2
5
3
1
2
1
1
3
82
78
70
70
73
63
37
43
55
49
42
40
41
37
1
2
2
2
2
1
5
1
1
3
2
2
1
1
Compounds
Lethal Concentration (LC50) in mg/mL
L2
Ligand (L1)
Ligand (L2)
Ligand (L3)
Ligand (L4)
Ligand (L5)
Ligand (L6)
Cisplatin
Transplatin
Complex-I
Complex-II
Complex-III
Complex-IV
Complex-V
Complex-VI
99.97
90.26
60.21
68.91
79.43
52.44
3.133
14.45
13.89
11.99
6.45
1
2
5
1
1
1
2
2
2
5
L3
L4
L5
L6
Cisplatin
Transplatin
I
II
III
IV
V
VI
1
9.168
9.212
6.367
2
1
2
DMSO
Untreated cell

58
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
Fig. 2. Structure-activity relationship of complexes against antibacterial activity (MIC), in vitro cytotoxicity against brine shrimp lethality bioassay (LC50) and cellular level cyto-
toxicity against S. Pombe cells (% viability).
3.7.5. DNA interaction studies
3.7.5.1. (a) Electronic absorption
Electronic absorption titration spectroscopy is most useful spec-
troscopic method for examine the interaction of substituted 2-
phenyl-5-methyl pyrazole-3-one based ligands (L1-L6) and cyclo-
metalated heteroleptic platinum(II) complexes with HS-DNA. A
compounds interact with HS-DNA through intercalative mode of
binding is a result of hypochromism and bathochromism [42]. The
electronic absorption titration spectra of the ligand (L6) and
cyclometalated platinum(II) complex (VI) in the absence and
presence of HS-DNA are represented in Fig. 3 and Fig. 4, respec-
tively. The strong absorption bands of the ligands (L1-L6) and
complexes (I-VI) are observed at about 254e380 nm [43]. The
experimental obtained hypochromic effect in the intraligand
transition band proposes that the compounds bind to HS-DNA via
titration
spectroscopy.
Fig. 3. Absorption spectra of ligand (L6) (400
mM) in the absence and presence of
Fig. 4. Absorption spectra of Pt(II) complex (VI) (400 mM) in the absence and presence
increasing amounts of HS-DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH ¼ 7.2)
solution, after incubation at room temperature for 10 min. The arrows show the
absorbance changes upon the addition of the DNA concentrations. Insets: Linear plot of
[DNA]/(εa-εf) vs. [DNA] for the titration of the ligand (L6) with HS-DNA.
of increasing amounts of HS-DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH ¼ 7.2)
solution, after incubation at room temperature for 10 min. The arrows show the
absorbance changes upon the addition of the DNA concentrations. Insets: Linear plot of
[DNA]/(εa-εf) vs. [DNA] for the titration of the Pt(II) complex (VI) with HS-DNA.

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
59
Table 4
Table 5
The binding constant (Kb, Mꢀ1), % hypochromicity and change in Gibb's free energy
Linear Stern-Volmer quenching constant (Ksv, Mꢀ1), no. of binding sites (n) and as-
sociation binding constant (Ka, Mꢀ1) from calculated Stern-Volmer and Scatchard
equation in fluorescence quenching analysis.
(D
G⁰, Jmolꢀ1) of organometallic platinum(II) complexes (I-VI) and 2-phenyl-5-
methyl pyrazole-3-one derivatives ligands (L1-L6) with HS-DNA at different
temperatures.
Complexes
Ksv (Mꢀ1
)
Ka (Mꢀ1
)
n
D )
G(J molꢀ1
Compounds lmax (nm)
Bound Free
Dl
(nm)
Kb (Mꢀ1) ꢁ 105 H%
D )
G⁰ (Jmolꢀ1
I
II
III
IV
V
7.0 ꢁ 103
1.55 ꢁ 102
2.35 ꢁ 102
4.44 ꢁ 102
1.1 ꢁ 102
3.85 ꢁ 102
1.871 ꢁ 103
2.285 ꢁ 103
5.80 ꢁ 104
5.18 ꢁ 104
2.08 ꢁ 104
1.71 ꢁ 105
0.873
ꢀ18,667.88
ꢀ19,162.49
ꢀ27,174.91
ꢀ26,895.95
ꢀ24,642.46
ꢀ29,855.61
0.8061
1.0879
1.0144
1.0837
1.1489
L1
L2
L3
L4
L5
L6
I
304
385
255
261
254
257
297
266
306
266
263
266
302
384
254
259
253
256
293
261
305
265
262
264
2
1
1
2
1
1
4
5
1
1
1
2
0.138
0.172
1.310
0.915
0.327
1.410
1.420
1.482
2.320
1.632
1.500
5.580
27.52 ꢀ23,617.26
50.55 ꢀ24,162.92
18.07 ꢀ29,193.11
17.67 ꢀ28,304.01
27.23 ꢀ25,754.68
18.58 ꢀ29,375.37
17.73 ꢀ29,392.87
22.13 ꢀ29,498.76
23.59 ꢀ30,609.14
21.70 ꢀ29,737.63
18.65 ꢀ29,528.67
21.78 ꢀ32,783.51
VI
II
III
IV
V
VI
H% ¼ [(Afree - Abound)/Afree] ꢁ 100%
Kb ¼ Intrinsic DNA binding constant determined from the UV-vis absorption spectral
titration.
Dl ¼ Difference between bound wavelength and free wavelength.
partial intercalative mode. The magnitude of binding strength of all
the compounds with HS-DNA are considered through the value of
binding constant (Kb), which is calculated by monitoring changes in
the absorbance at the resultant lmax with increasing concentrations
of HS-DNA by the Wolfe-Shimer equation [44]. The intrinsic bind-
ing constant (Kb) values of the compounds is determined by the
ratio of slope and intercept in plots of [DNA]/(εa - εf) versus [DNA]
(Table 4). The binding constant (Kb) values of ligands (L1-L6) and
cyclometalated platinum(II) complexes (I-VI) are obtained in range
of 0.138e1.41 ꢁ 105 Mꢀ1 and 1.42e5.58 ꢁ 105 Mꢀ1, respectively. It's
concluded that the complexes have higher binding affinity as
compared to ligands. Binding constant (Kb) of the ethidium bro-
mide is 7.1 ꢁ 105 Mꢀ1, it is higher than synthesized compounds. The
Kb values of cisplatin, oxaliplatin and carboplatin are
5.73 ꢁ 104 Mꢀ1 [45], 5.3 ꢁ 103 Mꢀ1 [46] and 0.33 ꢁ 103 Mꢀ1 [47],
respectively. These Kb values are comparable to synthesized
cyclometalated platinum(II) complexes and ligands. Moreover, the
percentage hypochromism (H %) of the ligands (L1-L6) and com-
plexes (I-VI) are observed in between 17.67 and 50.55% and
Fig. 6. Fluorescence emission spectra of ethidium bromide (EB) binds to HS-DNA in
the presence of complex (IV). [EB] ¼ 33.3 M, [DNA] ¼ 10 M; [complex] ¼ (i) 3.33, (ii)
6.66, (iii) 10, (iv) 13.33, (v) 16.66, (vi) 20, (vii) 23.33, (viii) 26.66, (ix) 30, (x) 33.3 M;
m
m
m
lex ¼ 510 nm. The arrows show the intensity changes upon increasing the concen-
trations of quencher (complex).
Fig. 5. Effect of increasing amounts of ligands (L1-L6) and platinum(II) complexes (I-VI) on the relative viscosity of HS-DNA at 27 ( 0.1)ꢂC in phosphate buffer at pH ¼ 7.2. Error bars
represent the standard deviation of three replicates.

60
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
Fig. 7. The Stern-Volmer quenching graph of the EB replacement titration. (a) The Stern-Volmer quenching constant (Ksv) ¼ 1.1 ꢁ 102 to 7.0 ꢁ 103 Mꢀ1. Inset graph: plots of I₀/I
versus [Q]. (b) Associative binding constant (Ka) ¼ 1.871 ꢁ 103 to 1.711 ꢁ 105 Mꢀ1. Inset graph: plot of log (I₀-I/I) versus log [Q].
17.73e23.59, respectively. The Gibb's free energy of the synthesized
compounds observed values in the range of ꢀ23.62 to ꢀ32.78
kJmol-1 representing the spontaneity of compound-DNA binding.
The binding constant values of compounds are found in the order of
VI > III > IV > V > II > I > L6 > L3 > L4 > L5 > L2 > L1. Since, -NO2
functional group (complex VI and ligand L6) is a powerful electron-
withdrawing group, which can accept electrons from DNA. There-
fore, complex (VI) and ligand L6 show strong binding affinity to-
wards HS-DNA as compared to other synthesized complexes and
viscosity of all the compounds is slightly increase with HS-DNA as
comparable to EB, suggesting the partial intercalative mode of
binding and this result is similar to the electronic absorption
titration spectroscopic data. The observed relative viscosity of the
synthesized ligands and complexes are in order of
EB > VI > III > IV > V > II > I > L6 > L3 > L4 > L5 > L2 > L1.
3.7.5.3. (c) Fluorescence quenching analysis. Fluorescence quench-
ing analysis has been carried out for cyclometalated platinum(II)
complexes and the corresponding emission spectra of the EB-DNA
solutions in the presence of the increasing amounts of complex
concentrations (r ¼ 0.33 to 3.33), fluorescence quenching data of all
complexes and graph are represented in supplementary material 5
and Table 5. Which clearly indicate a dramatic increase in the
fluorescence intensity of the EB-DNA by adding the Pt(II) complex.
ligands, respectively. The DNA binding data (Kb,
M DG⁰,
ꢀ1, %H and
Jmolꢀ1) of the synthesized compounds are represented in Table 4.
3.7.5.2. (b) Viscosity measurements. The special effects of the syn-
thesized ligands and cyclometalated Pt(II) complexes on the rela-
tive viscosity of HS-DNA has been represented in Fig. 5. The
Fig. 8. Molecular docking of ligand (L6) (ball and stick) with the DNA duplex (VDW spheres) of sequence d(ACCGACGTCGGT)2.

M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
61
Fig. 9. Molecular docking of complex (VI) (ball and stick) with the DNA duplex (VDW spheres) of sequence d(ACCGACGTCGGT)2.
In DNA-EB system, the increase of the fluorescence intensity is due
to releasing free EB molecules (Fig. 6) [29].
study is in agreement with the experimental results developed
from electronic absorption titration, viscosity measurements and
fluorescence studies.
Moreover, the Stern-Volmer quenching constants (Ksv), change
in standard Gibb's free energy (DG⁰) and associative binding con-
stant (Ka) of platinum(II) complexes are observed in range of
3.7.6. DNA nuclease study
1.1 ꢁ 102 to 7.0 ꢁ 103
M
ꢀ1, -18.67 to ꢀ29.85 kJmol-1 and
The effect of the compounds on DNA is estimated by their DNA-
cleavage ability [50,51]. Which can be determined by the cleavage
mechanism. The cleavage efficiency of molecules is usually exam-
ined by agarose gel electrophoresis [52,53]. The cyclometalated
platinum(II) complexes, cisplatin, transplatin and ligands are pro-
mote the percentage cleavage of pUC19 DNA from supercoiled Form
I to the open circular Form II (Fig. 10). The control experiment using
DNA alone does not show any significant cleavage of DNA (Lane 1).
A slight cleavage is observed when K2PtCl4 salt has been added to
1.87 ꢁ 103e1.7 ꢁ 105
M
ꢀ1, respectively (Table 2). Fluorescence
emission spectra and the Stern-Volmer quenching graph are rep-
resented in Figs. 6 and 7, respectively.
3.7.5.4. Molecular docking study. The synthesized compound can
play an important role in the improvement of new chemothera-
peutic drugs that lead to the recognition of specific sequences and
structures of nucleoside and nucleotide [48]. Molecular docking
study displays a very significant role to understanding the mech-
anistic pathway of complex-DNA interactions, by placing the
complex penetrate binding site of the DNA helix. The ligands (L1-L6)
and cyclometalated heteroleptic platinum(II) complexes (I-VI) are
docked with in a DNA double helix structure by molecular docking
study (supplementary material 6). Docked structures of ligand (L6)
and complex (VI) are represented in Fig. 8 and Fig. 9, respectively.
The complexes under investigation bind with B-DNA (PDB ID:
1BNA) at the A-T rich via partial intercalation mode [49]. The
binding energies of the docked ligands and cyclometalated plati-
num(II) complexes (I-VI) with B-DNA are represented in Table 6.
The platinum(II) complexes exhibit higher binding affinity with B-
DNA as compared to ligands. The compound interact with DNA, to
provide greater binding affinity obtained by molecular docking
Table 6
The binding energies of the docked ligands and cyclometalated platinum(II) com-
plexes (I-VI) with B-DNA in kJ/mol.
Ligands
Docking energy (kJ/mol)
Complexes
Docking energy (kJ/mol)
L1
L2
L3
L4
L5
L6
ꢀ240.74
ꢀ253.48
ꢀ242.21
ꢀ243.39
ꢀ247.50
ꢀ256.89
I
II
III
IV
V
ꢀ310.03
ꢀ318.11
ꢀ311.87
ꢀ308.29
ꢀ296.78
ꢀ325.79
Fig. 10. Photogenic view of cleavage of pUC19 DNA (300
m
g/cm3) with ligands (L1-L6)
g/cm3 EtBr. Reactions
and Pt(II) complexes (I-VI) using 1% agarose gel containing 0.5
m
were incubated in TE buffer solution (pH ¼ 8) at a final volume of 15 mm3 for time
VI
period of 3 h at 37 ꢂC.

62
M.V. Lunagariya et al. / Journal of Organometallic Chemistry 854 (2018) 49e63
Fig. 11. Plot of nuclease cleavage activity of the ligands (L1-L6) and Pt(II) complexes (I-VI). Error bars represent standard deviation of three replicates ( 5%).
the DNA (Lane 2). A significant cleavage of the supercoiled form to
open circular form is observed, when cisplatin, transplatin and
platinum(II) complexes is added to the DNA (Lane 3e9). The %
cleavage for all the compounds are calculated using AlphaDigi-
DocTM RT Version V.4.1.0 PCeImage software. And % cleavage data
of the synthesized compounds are represented in supplementary
material 9. The % cleavage ability of the compounds is in order of
Sardar Patel University, Vallabh Vidyanagar, Gujarat, India, for
providing necessary research facilities, DST-PURSE, Sardar Patel
University, Vallabh Vidyanagar for mass spectral analysis. The au-
thors gratefully acknowledge the University Grants Commission,
New Delhi, India for meritorious fellowships awarded to them
during year 2014e2018 and “BSR UGC One Time Grant” (C/2013/
BSR/Chemistry/7489), vide UGC letter no. F.19-119/2014 (BSR).
cisplatin > VI > III > IV > V > II > I > L6 > L3 > L4 > L5 > L2 > L1
>
transplatin > K2PtCl4. The complexes (I-VI) and cisplatin exhibit
greater DNA cleavage affinity as compared to ligands (L1-L6),
transplatin and K2PtCl4 salt at the same concentration. Fig. 11
(supplementary material 7) represent percentage cleavage of
compounds.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.jorganchem.2017.11.012.
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