Synthesis, characterization and biological application of cyclometalated heteroleptic platinum(II) complexes

Article abstract of DOI:10.1002/aoc.4045

A series of square planar cyclometalated heteroleptic platinum(II) complexes of the type [(C^N)Pt(O^O)] [where, O^O is a β-diketonato ligand of acetylacetone (acac), C^N = cyclometalating 7-(4-fluorophenyl)-5-phenylpyrazolo[1,5-a]pyrimidine (L¹

Full text of DOI:10.1002/aoc.4045

Received: 8 May 2017
DOI: 10.1002/aoc.4045
Revised: 21 July 2017
Accepted: 23 July 2017
F U L L P A P E R
Synthesis, characterization and biological application of
cyclometalated heteroleptic platinum(II) complexes
Miral V. Lunagariya | Khyati P. Thakor | Nikita J. Patel | Mohan N. Patel
Department of Chemistry, Sardar Patel
Abstract
University, Vallabh Vidyanagar 388 120
A series of square planar cyclometalated heteroleptic platinum(II) complexes of
the type [(C^N)Pt(O^O)] [where, O^O is a β‐diketonato ligand of acetylacetone
(acac), C^N = cyclometalating 7‐(4‐fluorophenyl)‐5‐phenylpyrazolo[1,5‐a]
pyrimidine (L1), 7‐(4‐chlorophenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L2), 7‐
Gujarat, India
Correspondence
Mohan N. Patel, Department of
Chemistry, Sardar Patel University,
Vallabh Vidyanagar 388 120, Gujarat,
India.
(4‐bromophenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine
(L3),
7‐(4‐
methoxyphenyl)‐5‐phenylpyrazolo[1,5‐a]pyrimidine (L4), 5‐phenyl‐7‐(p‐tolyl)
pyrazolo[1,5‐a]pyrimidine (L5)] have been design, synthesized and character-
ized. All compounds have been screened for biological studies like in vitro anti-
bacterial, in vitro cytotoxicity, cellular level cytotoxicity, absorption titration,
viscosity measurements, fluorescence quenching analysis, molecular docking
and DNA nuclease. The intrinsic binding constants (Kb) of compounds with
HS‐DNA has been obtained in range of 2.892–0.242 × 105 M−1. All the com-
pounds bound with HS DNA by partial intercalative mode of binding. MIC
study has been carried out against Gram(+ve) and Gram(−ve) bacterial species.
In vitro cytotoxicity against brine shrimp lethality bioassay has been also carried
out. The LC50 values of the ligands and complexes have been found in range of
56.49–120.22 μg/mL and 6.71–11.96 μg/mL, respectively.
Email: jeenen@gmail.com
KEYWORDS
Cyclometalated heteroleptic Pt(II) complexes, DNA interaction studies, In vitro antibacterial assay, In
vitro cytotoxicity assay, N –donor bidentate ligands, Pyrazolo[1,5‐a]pyrimidine based neutral C
1 | INTRODUCTION
biological screening of platinum(II) complexes was
revealed by Rosenberg and co‐workers showing the
Cisplatin is one of the most effective antitumor drugs
widely used in cancer The
treatments.[1]
ability of cisplatin to relate non‐covalently with
nucleoside and nucleotide.
anti‐carcinogenic study of cisplatin has been discovered
in 1960s. Cisplatin has established for clinical use in
the late 1970s and platinum(II) compounds are now
the important anticancer drugs on the market and used
in treatment of solid tumours such as testicular,
ovarian, head, neck, cervical and bladder carcinoma.[2,3]
Although its treatment with cisplatin is restricted due
to unwanted side effects like nephrotoxicity, nausea,
vomiting, myelosuppression and ototoxicity.[1] The
Pyrazolo[1,5‐a]pyrimidines based compounds such as
ocinaplon, zaleplon and indiplon are consider as an
important skeleton in a class of heterocycles and widely
used in material science, natural products and
pharmaceutical chemistry due to their effective
bioactivities.[4] The pyrazolo[1,5‐a]pyrimidine drugs are
also used in the treatment of some neurological disorders
containing schizophrenia, attention deficit disorder, and
Parkinson's disease.[5]
Appl Organometal Chem. 2017;e4045.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
1 of 18
https://doi.org/10.1002/aoc.4045

2 of 18
LUNAGARIYA ET AL.
In present work, the synthesis, characterization and
study was carried out by laminar airflow cabinet Toshiba,
Delhi (India). The electronic spectra were recorded on a
UV‐160A UV–vis spectrophotometer, Shimadzu, Kyoto
(Japan). The magnetic moments were measured by Gouy's
method using mercury tetrathiocyanatocobaltate(II) as the
calibrate (χg = 16.44 × 10−6 cgs units at 20 °C) citizen
balance. The thermogram of complexes was recorded with
a Mettler Toledo TGA Thermogravimetric analyser. Photo
quantization of the gel after electrophoresis was done using
AlphaDigiDocTM Version V.4.0.0 PC‐Image software, Cali-
fornia (USA). Conductance measurement was carried out
using conductivity meter model number E‐660A. Fluores-
cence spectroscopy was carried out by FluoroMax‐4, spec-
trofluorometer, HORIBA (Scientific).
their biological application of C, N –donor new class of
different substituted heterocyclic pyrazolo[1,5‐a]pyrimi-
dine based bidentate ligands (L1‐L5) and O, O –donor of
acetylacetone have been used in cyclometalated
heteroleptic platinum(II) complexes (I‐V).
2 | EXPERIMENTAL SECTION
2.1 | Chemicals and materials
MilliQ™ (18.2 m, Millipore) was used for the preparation
of deionized water. K2PtCl4 salts was purchased from S. D
Fine‐Chem Ltd. (SDFCL). Herring sperm (HS) DNA,
3‐amino pyrazole, 4‐methoxy benzaldehyde, 4‐fluoro
benzaldehyde, 4‐chloro benzaldehyde, 4‐bromo benzalde-
hyde, 4‐methyl benzaldehyde and acetophenone were
purchased from Sigma Aldrich Chemical Co. (India). S.
pombe Var. Paul Linder 3360 was obtained from
IMTECH, Chandigarh, India. An Artemia cyst was
purchased from local aquarium store. Nutrient broth
(NB), agarose, ethidium bromide (EtBr), tris‐acetyl‐EDTA
(TAE), bromophenol blue was purchased from Himedia
(India). The bacterial cultures were purchased from
MTCC, Institute of Microbial Technology and Chandi-
garh, India. Thin layer chromatography (TLC) was
performed using Merck aluminium sheets coated with
silica gel plates (silica gel 60 F254 0.25 mm). Purification
by flash chromatography was performed using Merck
silica gel 60. and components were visualized by observa-
tion under UV light. GenElute mini Pre Kit for pUC19
DNA isolation was purchased from Sigma Aldrich (India).
HPLC grade DMSO was used to dissolve the platinum(II)
complexes.
2.3 | Synthesis of α,β unsaturated
carbonyl compounds (3a – 3e)
The α , β unsaturated carbonyl compounds (3a‐3f) have
been synthesized using literature procedure.[2] The
proposed reaction mechanism for the synthesis of
chalcone is shown in Scheme 1.
2.4 | Synthesis of pyrazolo[1,5‐a]
pyrimidine derivatives ligands (L1‐L5)
Synthesis of the pyrazolo[1,5‐a]pyrimidines based ligands
(L1‐L5) have been carried out using Lipson and
co‐workers reported method[3]. To a solution of the α , β
unsaturated carbonyl compounds (3a‐3f) (2.95 mmol) in
2.2 | Physical measurements
Infrared spectra were recorded on a FT‐IR ABB Bomen
MB‐3000 spectrophotometer (Canada) in the range of
1
4,000 to 400 cm−1. H NMR and 13C NMR spectra were
recorded on Bruker Avance nuclear magnetic resonance
spectrometer, either in D2O (25 °C) or DMSO‐d6 (35 °C),
and TMS as an internal orientation. The APT experiment
yields methine (CH) and methyl (CH3) signals negative
and quaternary (C) and methylene (CH2) signals positive.
The mass spectra were obtained on a Thermo scientific
mass spectrophotometer (USA) using the positive
electrospray ionisation mode. Micro elemental analysis
C, H, N and S of the synthesized compounds was
performed with a model Euro EA elemental analyser.
Melting points (0C, uncorrected) were determined in open
capillaries on thermoCal10 melting point apparatus
(Analab Scientific Pvt. Ltd, Vadodara, India). Antibacterial
SCHEME 1 Reaction scheme of the synthesized different
substituted pyrazolo[1,5‐a] pyrimidine based ligands (L1 – L5)

LUNAGARIYA ET AL.
3 of 18
10 mL of DMF, 1H–pyrazol‐3‐amine (4a) (0.245 g,
2.95 mmol) and KOH (0.25 g, 0.1 mmol) solution were
added. The reaction mixture was refluxed for 30 min.
Upon completion of reaction as indicated by TLC plates,
the excess of solvent was removed under reduced pressure
and the reaction mixture was cooled on an ice bath. The
reaction mixture was extracted with ethylacetate
(50 mL × 3) and washed thoroughly with water
(30 mL × 3). The brine solution of sodium chloride was
added to it and dried over sodium sulphate. The resulting
mixture was concentrated under vacuum. The residue
was purified by column chromatography on silica gel
system (ethylacetate: hexane, 4:1) to obtain the
pyrazolo[1,5‐a]pyrimidine based ligands as a product.
The proposed reaction mechanism for the synthesis of
amine (4a) (245 mg, 2.958 mmol). Colour: lemon yellow
powder, Yield: 90%, mol. wt.: 305.77 g/mol, m.p.: 170 °C,
Anal. Calc. (%) For C18H12ClN3: C, 70.71; H, 3.96; N,
1
13.74 Found (%): C, 70.39; H, 3.83; N, 13.67. H NMR
3
(400 MHz, DMSO‐d6) δ/ppm: 8.307 (3H, t, J1 = 6.8 Hz,
3
J2 = 6.8 Hz, H3”,4″,5″), 8.259 (1H, d, J = 8.0 Hz, H3),
8.157 (2H, d, J = 8.4 Hz, H3’,5′), 7.746 (1H, s, H7), 7.544
4
3
(2H, dd, J1 = 8.0 Hz, J2 = 2.4 Hz, H2”,6″), 7.426 (2H, d,
J = 8.0 Hz, H2’,6′), 6.837 (1H, d, J = 8.0 Hz, H4).
13C NMR (100 MHz, DMSO‐d6) δ/ppm: 155.64 (C6, Cquat.),
149.76 (C5a, Cquat.), 146.48 (C8, Cquat.), 145.44 (C3, −CH),
141.46 (C1”, Cquat.), 137.31 (C4’, Cquat.), 130.86 (C1’,
Cquat.), 130.07 (C5’,3′, −CH), 129.37 (C3”,5″, −CH),
129.30 (C2’,6′, −CH), 128.47 (C4”, −CH), 127.76 (C2”,6″,
−CH), 104.91 (C7, −CH), 97.10 (C4, −CH). [Total signal
observed = 14: signal of C = 6 (p‐Cl‐phenyl ring‐C = 2,
pyrazolo[1,5‐a]pyrimidine‐C = 3, phenyl ring‐C = 1),
signal of CH = 8 (pyrazolo[1,5‐a]pyrimidine‐CH = 3,
p‐F‐phenyl ring‐CH = 2, phenyl ring‐CH = 3)]. FT‐IR:
(KBr) (cm−1): 2931 ν(=C‐H)ar, 1550 ν(C=N), 1504 ν(C‐H)
banding, 1218 ν(C‐N), 1605 ν(C=C)conjugated alkenes, 764 ν(Ar‐H)2
1
ligands (3a‐3f) is shown in Scheme 1. The H NMR and
13C NMR spectra of ligands are shown in supplementary
material 1 and 2, respectively.
2.5 | Characterization of 7‐(4‐
fluorophenyl)‐5‐phenylpyrazolo[1,5‐a]
pyrimidine (L1)
hydrogen. Mass (m/z): 306 [M]+, 307 [M + 2],
adjacent
195, 119.
This ligand (L1) has prepared through the addition of
enone (3a) (816 mg, 2.958 mmol) and 1H–pyrazole‐3‐
amine (4a) (245 mg, 2.958 mmol). Colour: lemon yellow
powder, Yield: 87%, mol. Wt.: 289.31 g/mol, m.p.:
180 °C, Chemical formula C18H12FN3. 1H NMR
2.7 | Characterization of 7‐(4‐
bromophenyl)‐5‐phenylpyrazolo[1,5‐a]
pyrimidine (L3)
3
(400 MHz, DMSO‐d6) δ/ppm: 8.297 (3H, t, J1 = 6.8 Hz,
3
J2 = 6.8 Hz, H3”,4″,5″), 8.257 (1H, d, J1 = 7.6 Hz, H3),
This ligand (L3) has prepared through the addition of
enone (3c) (999 mg, 2.958 mmol) and 1H–pyrazole‐3‐
amine (4a) (245 mg, 2.958 mmol). Colour: pale yellow
powder, Yield: 94%, mol. wt.: 350.22 g/mol, m.p.: 173 °C,
Anal. Calc. (%) For C18H12BrN3: C, 61.73; H, 3.45; N,
8.153 (2H, d, J1 = 8.0 Hz, H3’,5′), 7.742 (1H, s, H7), 7.534
3
4
(2H, dd, J1 = 8.0 Hz, J2 = 2.0 Hz, H2”,6″), 7.410 (2H, d,
J = 8.0 Hz, H2’,6′), 6.830 (1H, d, J = 8.0 Hz, H4). 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 162.72 (C4’, Cquat.),
155.71 (C6, Cquat.), 149.70 (C5a, Cquat.), 145.53 (C8, Cquat.),
1
12.00 Found (%): C, 61.49; H, 3.32; N, 12.07. H NMR
3
145.46 (C3, −CH), 137.22 (C1”, Cquat.), 132.88 (C2’,6′
,
(400 MHz, DMSO‐d6) δ/ppm: 8.299 (3H, t, J1 = 6.8 Hz,
3
−CH), 132.79 (C3”,5″, −CH), 130.96 (C4”, −CH), 129.34
(C2”,6″, −CH), 127.81 (C1’, Cquat.), 116.01 (C5’,3′, −CH),
105.36 (C7, −CH), 97.28 (C4, −CH). [Total signal
observed = 14: signal of C = 6 (p‐F‐phenyl ring‐C = 2,
pyrazolo[1,5‐a]pyrimidine‐C = 3, phenyl ring‐C = 1), sig-
nal of CH = 8 (pyrazolo[1,5‐a]pyrimidine‐CH = 3, p‐F‐
phenyl ring‐CH = 2, phenyl ring‐CH = 3)]. FT‐IR: (KBr)
J2 = 6.8 Hz, H3”,4″,5″), 8.235 (1H, d, J = 8.0 Hz, H3),
8.136 (2H, d, J = 8.0 Hz, H3’,5′), 7.740 (1H, s, H7), 7.539
4
3
(2H, dd, J1 = 8.0 Hz, J2 = 3.6 Hz, H2”,6″), 7.428 (2H, d,
J = 8.0 Hz, H2’,6′), 6.833 (1H, d, J = 8.0 Hz, H4). 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 155.66 (C6, Cquat.),
149.77 (C5a, Cquat.), 146.52 (C8, Cquat.), 145.47 (C3, −CH),
141.49 (C1”, Cquat.), 137.32 (C3’,5′, −CH), 130.89 (C3”,5″,
−CH), 130.10 (C4”, −CH), 129.40 (C2’,6′, −CH), 129.33
(C2”,6″, −CH), 128.49 (C1’, Cquat.), 127.78 (C4’, Cquat.),
104.94 (C7, −CH), 97.12 (C4, −CH). [Total signal
observed = 14: signal of C = 6 (p‐Br‐phenyl ring‐C = 2,
pyrazolo[1,5‐a]pyrimidine‐C = 3, phenyl ring‐C = 1), sig-
nal of CH = 8 (pyrazolo[1,5‐a]pyrimidine‐CH = 3, p‐F‐
phenyl ring‐CH = 2, phenyl ring‐CH = 3)]. FT‐IR: (KBr)
(cm−1): 2923 ν(=C‐H)ar, 1550 ν(C=N), 1504 ν(C‐H)banding
,
1218 ν(C‐N), 1604 ν(C=C)conjugated alkenes, 763 ν(Ar‐H)2 adjacent
hydrogen. Mass (m/z): 290 [M]+, 195, 119.
2.6 | Characterization of 7‐(4‐
chlorophenyl)‐5‐phenylpyrazolo[1,5‐a]
pyrimidine (L2)
This ligand (L2) has prepared through the addition of
enone (3b) (868 mg, 2.958 mmol) and 1H–pyrazole‐3‐
(cm−1): 2931 ν(=C‐H)ar, 1551 ν(C=N), 1505 ν(C‐H)banding
,
1226 ν(C‐N), 1604 ν(C=C)conjugated alkenes, 771 ν(Ar‐H)2 adjacent
hydrogen. Mass (m/z): 351 [M]+, 195, 119.

4 of 18
LUNAGARIYA ET AL.
C = 1), signal of CH and CH3 = 9 (pyrazolo[1,5‐a]
pyrimidine‐CH = 3, p‐F‐phenyl ring‐CH = 2, phenyl
ring‐CH = 3, −CH3 = 1)]. FT‐IR: (KBr) (cm−1): 2923
2.8 | Characterization of 7‐(4‐
methoxyphenyl)‐5‐phenylpyrazolo[1,5‐a]
pyrimidine (L4)
ν
(=C‐H)ar, 1542 ν(C=N), 1488 ν(C‐H)banding, 1234 ν(C‐N)
,
This ligand (L4) has prepared through the addition of
enone (3d) (854 mg, 2.958 mmol) and 1H–pyrazole‐3‐
amine (4a) (245 mg, 2.958 mmol). Colour: pale yellow
powder, Yield: 93%, mol. wt.: 301.35 g/mol, m.p.:
174 °C, Anal. Calc. (%) For C19H15N3O: C, 75.73; H,
5.02; N, 13.94; Found (%): C, 75.49; H, 5.56; N, 13.47.
1H NMR (400 MHz, DMSO‐d6) δ/ppm: 8.310 (3H, t,
3J1 = 6.0 Hz, 3J2 = 6.0 Hz, H3”,4″,5″), 8.266 (1H, d,
J = 6.4 Hz, H3), 8.168 (2H, d, J = 8.0 Hz, H3’,5′), 7.766
1605 ν(C=C)conjugated alkenes, 763 ν(Ar‐H)2
.
adjacent hydrogen
Mass (m/z): 286 [M]+, 195, 119.
2.10 | Preparation of heteroleptic
cycloplatinated complexes (I‐V)
2.10.1 | General procedure for synthesis
of organometallic platinum(II) complexes
(I‐V)
4
3
(1H, s, H7), 7.495 (2H, dd, J1 = 8.0 Hz, J2 = 5.6 Hz,
2”,6″), 7.430 (2H, d, J = 8.0 Hz, H2’,6′), 6.837 (1H, d,
Synthesis of the platinum(II) μ‐dichloro‐bridged dimers
have been derived following a modified method of
Lewis.[6,7] To a solution of 5,7‐diphenylpyrazolo[1,5‐a]
pyrimidine derivatives ligand (L1‐L5) (58.6 mg, 0.4 mmol)
and K2PtCl4 (83 mg, 0.2 mmol) in ethoxyethanol, stirred
vigorously under nitrogen (N2) atm. And refluxed at
100 °C for 24–35 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 °C under vacuum. The crude
product was used for next step without further purifica-
tion and characterization.
H
J = 8.0 Hz, H4), 2.440 (3H, s, −OCH3). 13C NMR
(100 MHz, DMSO‐d6) δ/ppm: 155.72 (C4’, Cquat.),
149.68 (C6, Cquat.), 145.57 (C3, −CH), 145.26 (C8, Cquat.),
137.17 (C5a, Cquat.), 136.20 (C1”, Cquat.), 132.07 (C3’,5′,
−CH), 131.00 (C3”,5″, −CH), 130.12 (C1’, Cquat.), 129.36
(C4”, −CH), 128.93 (C2’,6′, −CH), 127.81 (C2”,6″, −CH),
105.50 (C7, −CH), 97.36 (C4, −CH), 55.50 (−O‐CH3).
[Total signal observed = 15: signal of C = 6 (p‐Br‐phe-
nyl ring‐C = 2, pyrazolo[1,5‐a] pyrimidine‐C = 3, phe-
nyl ring‐C
= 1), signal of CH and CH3 = 9
(pyrazolo[1,5‐a]pyrimidine‐CH = 3, p‐F‐phenyl ring‐
CH = 2, phenyl ring‐CH = 3, −OCH3 = 1)]. FT‐IR:
(KBr) (cm−1): 2931 ν(=C‐H)ar, 1550 ν(C=N), 1488 ν(C‐H)
banding, 1218 ν(C‐N), 1604 ν(C=C)conjugated alkenes, 763 ν(Ar‐
H)2 adjacent hydrogen. Mass (m/z): 302 [M]+, 195, 119.
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 80–90 °C under nitrogen (N2) atmosphere for 24–48 h.
The mixture was poured into water for extraction through
separating funnel using solvent dichloromethane
(CH2Cl2). The organic extracts were washed with water
and dried over anhydrous sodium sulphate. After the
solvent was completely evaporated, than the obtained
residue was purified by column chromatography on silica
gel with dichloromethane/petroleum ether (1:1) system as
the eluent to obtained pure product. The proposed
reaction mechanism for the synthesis of cyclometalated
heteroleptic platinum(II) complexes (I‐V) are represented
in Scheme 2. The 1H NMR and 13C NMR spectra of
platinum(II) complexes are shown in supplementary
material 1 and 2, respectively.
2.9 | Characterization of 5‐phenyl‐7‐(p‐
tolyl)pyrazolo[1,5‐a]pyrimidine (L5)
This ligand (L5)has prepared through the addition of
enone (3e) (807 mg, 2.958 mmol) and 1H–pyrazole‐3‐
amine (4a) (245 mg, 2.958 mmol). Colour: pale yellow
powder, Yield: 90%, mol. wt.: 285.35 g/mol, m.p.:
176 °C, Anal. Calc. (%) For C19H15N3: C, 79.98; H,
5.30; N, 14.73; Found (%): C, 79.79; H, 5.16; N, 14.44.
1H NMR (400 MHz, DMSO‐d6) δ/ppm: 8.306 (3H, t,
3J1 = 6.0 Hz, 3J2 = 6.0 Hz, H3”,4″,5″), 8.263 (1H, d,
J = 6.4 Hz, H3), 8.164 (2H, d, J = 8.0 Hz, H3’,5′), 7.762
4
3
(1H, s, H7), 7.492 (2H, dd, J1 = 8.0 Hz, J2 = 6.4 Hz,
2”,6″), 7.430 (2H, d, J = 8.0 Hz, H2’,6′), 6.839 (1H, d,
H
2.10.2 | Characterization of μ‐dichloro
bridge platinum(II) complex (I) (dimer)
J = 8.0 Hz, H4), 1.640 (3H, s, −CH3). 13C NMR
(100 MHz, DMSO‐d6) δ/ppm: 155.36 (C6, Cquat.), 149.73
(C5a, Cquat.), 146.92 (C8, Cquat.), 145.47 (C3, −CH),
141.49 (C1”, Cquat.), 137.39 (C1’, Cquat.), 130.89 (C3’,5′,
−CH), 130.10 (C3”,5″, −CH), 129.80 (C4”, −CH), 129.33
(C2”,6″, −CH), 128.48 (C4’, Cquat.), 127.78 (C2’,6′, −CH),
104.94 (C7, −CH), 97.12 (C4, −CH), 21.56 (−CH3). [Total
signal observed = 15: signal of C = 6 (p‐Br‐phenyl ring‐
C = 2, pyrazolo[1,5‐a] pyrimidine‐C = 3, phenyl ring‐
Dimer of platinum(II) complex (I) has been prepared
according to the above general procedure. Characteriza-
1
tion of this dimer by spectroscopic method like H NMR
and 13C NMR spectra are represented in supplementary
material 1 and 2, respectively. Yield: 25%, mol. wt.:
1
1051.71 g/mol, chemical formula: C37H22Cl2F2N6Pt2, H
NMR (400 MHz, DMSO‐d6) δ/ppm: 8.713 (2H, d,

LUNAGARIYA ET AL.
5 of 18
procedure. Colour: brown powder, Yield: 40%, mol. wt.:
582.50 g/mol, m.p.: >300 °C, Chemical formula:
C23H18FN3O2Pt. UV–Vis: λ (nm) (ε, M−1 cm−1): 299
1
(12,230), 360 (3,900). Conductance: 22 Ω−1cm2mol−1. H
NMR (400 MHz, DMSO‐d6) δ/ppm: 8.295 (2H, t,
3
3J1 = 6.8 Hz,
J2 = 6.8 Hz, H3”,4″), 8.256 (1H, d,
J = 8.0 Hz, H3), 8.151 (2H, d, J = 8.0 Hz, H3’,5′), 7.752
4
3
(1H, s, H7), 7.533 (2H, dd, J1 = 8.0 Hz, J2 = 2.0 Hz,
2”,5″), 7.420 (2H. d, J = 8.0 Hz, H2’,6′), 6.822 (1H, d,
H
J = 8.0 Hz, H4), 6.020 (1H, s, H3”’), 1.997 (3H, s, H5”’),
0.880 (3H, s, H1”’). 13C NMR (100 MHz, DMSO‐d6) δ/
ppm: 191.00 (C2”’, C = O), 181.89 (C4”’, C‐O‐Pt), 162.89
(C4’, Cquat.), 155.98 (C6, Cquat.), 149.80 (C5a, Cquat.), 145.57
(C3, −CH), 145.46 (C8, Cquat.), 137.72 (C1”, Cquat.), 132.88
(C2’,6′, −CH), 132.79 (C3”, −CH), 130.96 (C4”, −CH),
129.74 (C5”, −CH), 128.80 (C1’, Cquat.), 128.01 (C2”,
−CH), 127.81 (C6”, Cquat.), 121.21 (C7, −CH), 116.23
(C2’,6′, −CH), 97.32 (C4, −CH), 81.33 (C3”’, −CH), 24.43
(C5”’, −CH3), 16.00 (C1”’
,
−CH3). [Total signal
observed = 21: signal of C = 9 (p‐F‐phenyl ring‐C = 2,
pyrazolo[1,5‐a]pyrimidine‐C = 3, phenyl ring‐C = 2,
acetyl acetone (C = O) = 2), signal of CH = 12
(pyrazolo[1,5‐a] pyrimidine‐CH
=
3, p‐F‐phenyl
ring‐CH = 2, phenyl ring‐CH = 4, acetyl acetone‐CH = 1,
acetylacetone‐CH3 = 2)]. FT‐IR: (KBr) (cm−1): 3062 ν(=C‐
H)ar, 1552 ν(C=N), 1496 ν(C‐H)banding, 1789 ν(C=O), 1226 ν(C‐
1606 ν(C=C)conjugated alkenes, 432 ν(O‐Pt), 779 ν(Ar‐H)2 adja-
cent hydrogen, 501 ν(N‐Pt), 416 ν(C‐Pt).
N),
SCHEME
2
Synthesis of the cyclometalated heteroleptic
platinum(II) complexes (I‐V) via μ‐dichloro bridge platinum(II)
complex (dimer) as an intermediate
2.10.4 | Structure characterization of
J = 8.0 Hz, H3,3), 8.536 (2H, d, J = 8.0 Hz, H4,4), 8.407 (4H,
d, J = 7.6 Hz, H3’,5′,3′,5′), 8.178 (2H, s, H7,7), 7.857 (4H, d,
J = 7.6 Hz, H2’,6′,2′,6′), 7.708 (4H, dd, 3J1 = 8.0 Hz,
cycloplatinated complex [(L2)Pt(acac)] (II)
It has prepared according to the synthetic procedure of
complex (II) but ratio among the [Pt(L2)Cl]2 dimer and
acetylacetone, which is 1:3 mole to the described in
procedure. Colour: brown powder, Yield: 35%, mol. wt.:
598.95 g/mol, m.p.: >300 °C, Anal. Calc. (%) For
C23H18ClN3O2Pt: C, 46.12; H, 3.03; N, 7.02; Pt, 32.57;
Found (%): C, 46.79; H, 3.16; N, 7.44; Pt, 32.53.
Conductance: 22 Ω−1cm2mol−1. UV–vis: λ (nm) (ε, M
4
3
J2 = 5.6 Hz, H2”,5″,2″,5″), 7.634 (2H, t, J1 = 8.0 Hz,
3J2 = 8.0 Hz, H3”,3″), 7.448 (2H, t, 3J1 = 8.0 Hz,
3J2 = 8.0 Hz, H4”,4″). 13C NMR (100 MHz, DMSO‐d6) δ/
ppm: 162.79 (C4’, Cquat.), 155.91 (C6, Cquat.), 149.70 (C5a,
Cquat.), 145.53 (C8, Cquat.), 145.46 (C3, −CH), 137.22 (C1”,
Cquat.), 132.88 (C2’,6′, −CH), 132.79 (C5”, −CH), 130.96
(C4”, −CH), 129.94 (C2”, −CH), 128.80 (C1’, Cquat.), 128.01
(C3”, −CH), 127.81 (C6”, Cquat.), 116.21 (C5’,3′, −CH),
105.36 (C7, −CH), 97.22 (C4, −CH). [Total signal
observed = 16: signal of C = 7 (p‐F‐phenyl ring‐C = 2,
pyrazolo[1,5‐a]pyrimidine‐C = 3, phenyl ring‐C = 2),
signal of CH = 9 (pyrazolo[1,5‐a]pyrimidine‐CH = 3,
p‐F‐phenyl ring‐CH = 2, phenyl ring‐CH = 4)].
−1
1
cm−1): 295 (22,770), 357 (6,610). H NMR (400 MHz,
DMSO‐d6) δ/ppm: 8.299 (2H, t, 3J1
=
8.0 Hz,
3J2 = 8.0 Hz, H3”,4″), 8.253 (1H, d, J = 8.0 Hz, H3), 8.150
(2H, d, J = 8.0 Hz, H3’,5′), 7.750 (1H, s, H7), 7.540 (2H,
dd, 4J1 = 8.0 Hz, 3J2 = 6.0 Hz, H2”,5″), 7.424 (2H, d,
J = 8.0 Hz, H2’,6′), 6.826 (1H, d, J = 8.0 Hz, H4), 6.022
(1H, s, H3”’), 1.998 (3H, s, H5”’), 0.890 (3H, s, H2”’). 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 190.00 (C2”’, C = O),
181.89 (C4”’, C‐O‐Pt), 155.94 (C6, Cquat.), 149.76 (C5a,
Cquat.), 146.48 (C8, Cquat.), 145.44 (C3, −CH), 141.46 (C1”,
Cquat.), 137.31 (C4’, Cquat.), 130.86 (C3’,5′, −CH), 130.07
(C5”, −CH), 129.37 (C3”, −CH), 129.30 (C4”, −CH),
128.87 (C1’, Cquat.), 128.67 (C2”, −CH), 128.47 (C2’,6′,
2.10.3 | Structure characterization of
cycloplatinated complex [(L1)Pt(acac)] (I)
It has prepared according to the synthetic procedure of
complex (I) but the ratio among [Pt(L1)Cl]2 dimer and
acetyl acetone, which is 1:3 mole to the described in

6 of 18
LUNAGARIYA ET AL.
−CH), 127.16 (C6”, Cquat.), 121.19 (C7, −CH), 97.90 (C4,
−CH), 81.33 (C3”’, −CH), 24.43 (C5”’, −CH3), 16.00 (C1”’,
−CH3). [Total signal observed = 21: signal of C = 9 (p‐
Cl‐phenyl ring‐C = 2, pyrazolo[1,5‐a]pyrimidine‐C = 3,
phenyl ring‐C = 2, acetyl acetone (C = O) = 2), signal of
CH and CH3 = 12 (pyrazolo[1,5‐a] pyrimidine‐CH = 3,
p‐Cl‐phenyl ring‐CH = 2, phenyl ring‐CH = 4, acetyl
acetone‐CH = 1, acetylacetone‐CH3 = 2)]. FT‐IR: (KBr)
(cm−1): 3060 ν(=C‐H)ar, 1551 ν(C=N), 1488 ν(C‐H)banding,
1728 ν(C=O), 1280 ν(C‐N), 1605 ν(C=C)conjugated alkenes, 433
ν(O‐Pt), 764 ν(Ar‐H)2 adjacent hydrogen, 509 ν(N‐Pt), 417 ν(C‐Pt).
LC–MS (m/z): 599.80 [M]+, 601.85 [M + 2].
acetyl acetone, which is 1:3 mole to the described in
procedure. Colour: brown powder, Yield: 35%, mol. wt.:
594.53 g/mol, m.p.: >300 °C, Anal. Calc. (%) For
C24H21N3O3Pt: C, 48.49; H, 3.56; N, 7.07; Pt, 32.81; Found
(%): C, 48.39; H, 3.76; N, 7.44; Pt, 32.73. Conductance: 22
Ω
−1cm2mol−1. UV–vis: λ (nm) (ε, M−1 cm−1): 292
1
(38,830), 362 (7,390). H NMR (400 MHz, DMSO‐d6) δ/
ppm: 8.302 (2H, t, J1 = 4.8 Hz, J2 = 4.8 Hz, H3”,4″),
3
3
8.251 (1H, d, J = 8.0 Hz, H3), 8.154 (2H, d, J = 8.0 Hz,
4
H
3’5′), 7.745 (1H, s, H7), 7.535 (2H, dd, J1 = 8.0 Hz,
J2 = 2.0 Hz, H2”,5″), 7.427 (2H, d, J = 8.0 Hz, H2’,6′),
4
6.829 (1H, d, J = 8.0 Hz, H4), 6.017 (1H, s, H3”’), 2.890
(3H, s, −OCH3), 1.990 (3H, s, H5”’), 0.891 (3H, s, H1”’).
13C NMR (100 MHz, DMSO‐d6) δ/ppm: 190.00 (C2”’,
C = O), 181.89 (C4”’, C‐O‐Pt), 155.58 (C6, Cquat.), 149.56
(C5a, Cquat.), 146.48 (C8, Cquat.), 145.44 (C3, −CH), 141.48
(C1”, Cquat.), 137.31 (C4’, Cquat.), 130.82 (C3’,5′, −CH),
130.07 (C5”, −CH), 129.57 (C3”, −CH), 129.20 (C4”,
−CH), 128.87 (C1’, Cquat.), 128.62 (C2”, −CH), 128.47
(C2’,6′, −CH), 127.15 (C6”, Cquat.), 121.59 (C7, −CH), 97.52
(C4, −CH), 81.33 (C3”’, −CH), 55.98 (−OCH3), 24.43
(C5”’, −CH3), 16.00 (C1”’, −CH3). [Total signal
observed = 22: signal of C = 9 (p‐OCH3‐phenyl
ring‐C = 2, pyrazolo[1,5‐a] pyrimidine‐C = 3, phenyl
ring‐C = 2, acetyl acetone (C = O) = 2), signal of CH
and CH3 = 13 (pyrazolo[1,5‐a]pyrimidine‐CH = 3, p‐Br‐
phenyl ring‐CH = 2, phenyl ring‐CH = 4, acetyl
acetone‐CH = 1, acetylacetone‐CH3 = 2, −OCH3 = 1)].
FT‐IR (KBr): (cm−1): 3031 ν(=C‐H)ar, 1551 ν(C=N), 1512
ν(C‐H)banding, 1773 ν(C=O), 1266 ν(C‐N), 1604 ν(C=C)conjugated
2.10.5 | Structure characterization of
cycloplatinated complex [(L3)Pt(acac)] (III)
It has prepared according to the synthetic procedure of
complex (III) but the ratio among [Pt(L3)Cl]2 dimer and
acetyl acetone, which is 1:3 mole to the described in
procedure. Colour: brown powder, Yield: 38%, mol. wt.:
643.40 g/mol, m.p.: >300 °C, Anal. Calc. (%) For
C23H18BrN3O2Pt: C, 42.94; H, 2.82; N, 6.53; Pt, 30.32;
Found (%): C, 42.49; H, 2.56; N, 6.34; Pt, 30.13. Conduc-
tance: 22 Ω−1cm2mol−1. UV–Vis: λ (nm) (ε, M−1 cm−1):
1
292 (12,570), 353 (2,660). H NMR (400 MHz, DMSO‐d6)
3
3
δ/ppm: 8.309 (2H, t, J1 = 7.6 Hz, J2 = 7.6 Hz, H3”,4″),
8.258 (1H, d, J = 8.0 Hz, H3), 8.159 (2H, d, J = 8.0 Hz,
4
H
3’,5′), 7.742 (1H, s, H7), 7.533 (2H, dd, J1 = 8.0 Hz,
3J2 = 2.0 Hz, H2”,6″), 7.417 (2H, d, J = 8.0 Hz, H2’,6′), 6.822
(1H, d, J = 8.0 Hz, H4), 6.021 (1H, s, H3”’), 1.990 (3H, s,
H
5”’), 0.891 (3H, s, H2”’). 13C NMR (100 MHz, DMSO‐d6)
439 ν(O‐Pt), 764 ν(Ar‐H)2 adjacent hydrogen, 501 ν(N‐Pt),
alkenes,
δ/ppm: 190.00 (C2”’, C = O), 181.89 (C4”’, C‐O‐Pt), 155.98
(C6, Cquat.), 149.76 (C5a, Cquat.), 146.48 (C8, Cquat.), 145.44
(C3, −CH), 141.48 (C1”, Cquat.), 137.31 (C4’, Cquat.), 130.82
(C3’,5′, −CH), 130.07 (C5”, −CH), 129.37 (C3”, −CH),
129.30 (C4”, −CH), 128.87 (C1’, Cquat.), 128.62 (C2”, −CH),
128.47 (C2’,6′, −CH), 127.12 (C6”, Cquat.), 121.89 (C7,
−CH), 97.92 (C4, −CH), 81.33 (C3”’, −CH), 24.43 (C5”’,
−CH3), 16.00 (C1”’, −CH3). [Total signal observed = 21: sig-
nal of C = 9 (p‐Br‐phenyl ring‐C = 2, pyrazolo[1,5‐a]pyrim-
idine‐C = 3, phenyl ring‐C = 2, acetyl acetone (C = O) = 2),
signal of CH and CH3 = 12 (pyrazolo[1,5‐a]pyrimidine‐
CH = 3, p‐Br‐phenyl ring‐CH = 2, phenyl ring‐CH = 4, ace-
tyl acetone‐CH = 1, acetylacetone‐CH3 = 2)]. FT‐IR: (KBr)
424 ν(C‐Pt).
2.10.7 | Structure characterization of
cycloplatinated complex [(L5)Pt(acac)] (V)
It has prepared according to the synthetic procedure of
complex (IV) but the ratio among [Pt(L5)Cl]2 dimer and
acetyl acetone, which is 1:3 mole to the described in
procedure. Colour: brown powder, Yield: 35%, mol. wt.:
578.54 g/mol, m.p.: >300 °C, Anal. Calc. (%) For
C24H21N3O2Pt: C, 49.83; H, 3.66; N, 7.26; Pt, 33.72; Found
(%): C, 49.67, H, 3.56 N, 7.44; Pt, 32.73. Conductance: 22
Ω
−1cm2mol−1. UV–Vis: λ (nm) (ε, M−1 cm−1): 296
1
(cm−1): 3039 ν(=C‐H)ar, 1550 ν(C=N), 1496 ν(C‐H)banding
1774 ν(C=O), 1265 ν(C‐N), 1605 ν(C=C)conjugated
,
(19,920), 351 (5,550). H NMR (400 MHz, DMSO‐d6) δ/
ppm: 8.309 (2H, t, J1 = 8.0 Hz, J2 = 8.0 Hz, H3”,4″),
3
3
alkenes,
493 ν(O‐Pt), 763 ν(Ar‐H)2 adjacent hydrogen, 563 ν(N‐Pt), 430 ν(C‐Pt).
8.252 (1H, d, J = 8.0 Hz, H3), 8.158 (2H, d, J = 8.0 Hz,
4
H
3’,5′), 7.755 (1H, s, H7), 7.534 (2H, dd, J1 = 8.0 Hz,
3J2 = 2.4 Hz, H2”,5″), 7.428 (2H, d, J = 8.0 Hz, H2’,6′),
6.824 (1H, d, J = 8.0 Hz, H4), 6.017 (1H, s, H3”’), 2.291
(3H, s, −CH3), 1.912 (3H, s, H5”’), 0.892 (3H, s, H1”’). 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 190.00 (C2”’, C = O),
181.89 (C4”’, C‐O‐Pt), 155.73 (C6, Cquat.), 149.66 (C5a,
2.10.6 | Structure characterization of
cycloplatinated complex [(L4)Pt(acac)] (IV)
It has prepared according to the synthetic procedure of
complex (IV) but the ratio among [Pt(L4)Cl]2 dimer and

LUNAGARIYA ET AL.
7 of 18
Cquat.), 146.38 (C8, Cquat.), 145.64 (C3, −CH), 141.48 (C1”,
Cquat.), 137.36 (C4’, Cquat.), 130.82 (C3’,5′, −CH), 130.37
(C5”, −CH), 129.67 (C3”, −CH), 129.20 (C4”, −CH),
128.87 (C1’, Cquat.), 128.62 (C2”, −CH), 128.47 (C2’,6′,
−CH), 127.12 (C6”, Cquat.), 121.87 (C7, −CH), 97.92 (C4,
−CH), 81.33 (C3”’, −CH), 24.66 (−CH3), 24.43 (C5”’,
−CH3), 16.00 (C1”’, −CH3). [Total signal observed = 22:
signal of C = 9 (p‐CH3‐phenyl ring‐C = 2, pyrazolo[1,5‐
a] pyrimidine‐C = 3, phenyl ring‐C = 2, acetyl acetone
(C = O) = 2), signal of CH and CH3 = 13 (pyrazolo[1,5‐
a] pyrimidine‐CH = 3, p‐CH3‐phenyl ring‐CH = 2, phenyl
ring‐CH = 4, acetyl acetone‐CH = 1, acetylacetone‐
CH3 = 2, −CH3 = 1)]. FT‐IR (KBr): (cm−1): 3062 ν(=C‐H)
extract media. The flask was incubated at 30 °C on a
shaker at 160 rpm till the exponential growth of S. pombe
obtained (24 to 30 h). Then the cell culture was treated
with the differ concentrations (20, 40, 60, 80, 100 μM) of
synthesized complexes, free ligands and also with DMSO
as a control and further allowed to grow for 20–24 h. Next
day, by centrifugation at 12,000 rpm 10 min; treated cells
were collected and dissolved in 500 mL of PBS (Phosphate
Buffered Saline). The 80 mL of yeast culture dissolved in
PBS and 20 mL of 0.4% trypan blue prepared in PBS were
mixed and cells were observed in a compound microscope
(40 X). The trypan blue dye could enter the dead cell only
so they appeared blue, whereas live cells resisted the entry
of dye. The number of dead cells and number of live cells
was counted in one field. Cell counting was repeated in
two more of the microscopic fields and average
percentage of cells died due to synthesized compounds
were calculated.[12]
1550 ν(C=N), 1488 ν(C‐H)banding, 1789 ν(C=O), 1272 ν(C‐N),
ar,
1604 ν(C=C)conjugated alkenes, 455 ν(O‐Pt), 763 ν(Ar‐H)2 adjacent
563 ν(N‐Pt), 430 ν(C‐Pt).
hydrogen,
2.11 | Biological screening of compounds
2.11.1 | In vitro antibacterial activity
3 | DNA INTERACTION STUDIES
The MIC informs about the degree of resistance of certain
bacterial species towards the test compounds. MIC value
was performed by serially two fold dilution of the test
compound added to three Gram(−ve) microorganisms
namely Escherichia coli (MTCC 433), Pseudomonas
aeruginosa (MTCC P‐09), Serratia marcescens (MTCC
7103) and two Gram(+ve) bacteria namely Bacillus subtilis
(MTCC 7193), Staphylococcus aureus (MTCC 3160). The
lowest compound concentration inhibiting visible
3.1 | Electronic absorption titration
spectroscopy
All the experiments involving the interaction of the Pt(II)
complexes (1a‐1f) with HS DNA were used for nucleotide
binding study. The stock solution was prepared by
dissolving HS‐DNA in a phosphate 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 HS DNA in the
buffer, suggesting the HS DNA sufficiently free from
protein.[13] 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
HS DNA were determined spectrophotometrically by
[8,9]
bacterial growth is reported as MIC.
2.11.2 | In vitro brine shrimp lethality
bioassay
The cytotoxicity assay was performed on brine shrimp
nauplii by Meyer method.[10] The lethal concentrations
of compounds resulting in 50% mortality (LC50) of the
brine shrimp 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 LC50 was determine
from the best‐fit line, a graph of mortality vs. concentra-
tion of nauplii.[11] The LC50 value is obtained the antilog-
arithm of log [complex] vs. 50% mortality (LC50). All data
has been collected from three independent experiments
and the LC50 determined using OriginPro 8 software.
assuming ε260 = 12558 M−1 cm−1 [14–17]
.
3.2 | Viscosity measurements
Viscosity experiment was made using an Ubbelohde
viscometer, engrossed in a water bath at 25.0
0.5 °C.
The viscosity of a 200 μM solution of HS‐DNA was
determined in the presence of the complexes using
different [complex]/[DNA] ratios in the range of 0.00
to 2.00. The flow time was measured in triplicate with
a digital stopwatch and then averaged. The relative
viscosity ratio (/) was plotted against the r‐bound
(where and were the relative viscosity for DNA in
the presence or absence of complexes, respectively).
The hydrodynamic length of DNA generally increases
upon partial intercalation while it does not lengthen
2.11.3 | Cellular level cytotoxicity against
S. Pombe cells
Cellular level bioassay was carried out using S. pombe
cells, which were grown in liquid yeast extract media in
150 mL Erlenmeyer flask containing 30 mL of yeast

8 of 18
LUNAGARIYA ET AL.
upon groove binding.[18] Viscosity measurements can
sensitively detect the lengthening of a DNA helix
induced by the binding of intercalators and thus provide
evidence of intercalation for small DNA‐binding
molecules.[19] The data are represented as the plot of
the relative viscosity, i.e.(/)1/3 vs. [complex]/[DNA].
binding. The acting forces between drugs and
biomacromolecules include hydrogen bonds, van der
Waals forces, electrostatic attraction and hydrophobic
interaction, etc. In order to estimate the interaction force
of all compounds, the standard free energy changes (ΔG)
for the binding process have been calculated using the
Van't Hoff equation 3:[26]
ΔG0 ¼ −RTlnKa
(3)
3.3 | Fluorescence quenching analysis
Emission intensity measurements of ethidium bromide
where, T is the temperature (25 °C, 298 K here), Ka is
(EB
= 3,8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium
associative binding constant and R is gas constant 8.314
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 = 3.33
([DNA]/[Complex]) of pre‐treated EB–DNA mixture
([EB] = 33.3 μM, [DNA] = 10 μM) at ambient tempera-
ture and incubate for 10 min before measurement. The
emission intensity was recorded in the range of
Jmol−1
K
−1. The negative sign for ΔG0 means that the
binding process is spontaneous.
3.4 | Molecular modeling study
Docking study was made for Pt(II) complexes with
biomolecule (DNA), to identify the binding mode of
action and the vital functional groups interacting with
the DNA, using Hex 8.0 software. PtII complexes were
taken from their enhanced structure as a molecule and
converted to .pdb (Protein Data Bank) format using
CHIMERA 1.5.1 software. The change of transcription
or replication of DNA may leading to gene mutation, thus
causing a series of diseases, in this way it plays an
irreplaceable role in life. DNA is also the target of many
antibacterial drugs, antiviral, cancer and playing an
important role in the treatment of diseases. HS‐DNA used
in the experimental work was too large for current
computational resources to dock, therefore, the structure
of the DNA of sequence d(ACCGACGTCGGT)2 (1BNA)
is used for interaction study (PDB id: 1BNA, a familiar
sequence used in oligodeoxynucleotide study) obtained
from the Protein Data Bank (http://www.rcsb.org/
pdb).[27] All calculations were done 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. The best
conformation was selected with the lowest binding energy
(kJ/mol).[28]
500–800 nm. The emission intensities at 610 nm (λmax
)
were obtained through excitation at 510 nm and slit
wavelength 1.45 nm in the FluoroMax‐4, HORIBA
(Scientific) spectrofluorometer. The changes in fluores-
cence 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 florescence 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.[20,21]
To examine the fluorescence quenching mechanism,
the Stern–Volmer quenching constant (Ksv) was
determined by equation 1:[22,23]
I0=I ¼ Ksv½Qꢀ 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 lin-
ear 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:[24,25]
3.5 | Photochemical analysis of DNA
nuclease activity
Gel electrophoresis study was performed using pUC19
DNA with synthesized compounds. The samples were
incubated for 0.5 h at 37 ̊C. The samples were analysed
log I0−I=I ¼ logKa þ nlog½Qꢀ
(2)
where, I0 and I are the fluorescence intensities of the EB‐
DNA in the absence and presence of different concentra-
tions of complexes, respectively and n is the number of
by 1% agarose gel electrophoresis [Tris–acetate–ethylene-
diaminetetraacetic acid, (TAE) buffer, pH 8.0] for 3 h at
100 mV. The gel was stained with (0.5 mg mL−1) ethidium

LUNAGARIYA ET AL.
9 of 18
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.[29]
are appeared at 55.50 ppm and 21.56 ppm, respectively.
These peaks are shifted to downfield in complex (IV)
and complex (V) are observed at 55.98 ppm and
24.66 ppm, respectively. The cyclometalated heteroleptic
platinum(II) complexes (I‐V) containing acetylacetone
bidentate ligands having >C = O peak observed in range
of 181.89–190.00 ppm. The peak of ligand (Ln, C6”, −CH)
are observed in the range of 127.76–129.33 ppm. It
indicate that the peak observed in down part of base line
in attached proton test (APT) spectra. In complexes
(Pt‐C6”, Cquat.), the C6” quaternary carbon atom peak is
shifted to upfield in the range of 127.16–127.81 ppm,
and this peak is obtained in the upper part of the base line
in APT. These observation suggests that the complexes
exhibit upfield shift as compared to free ligands.
4 | RESULTS AND DISCUSSION
4.1 | 1H NMR spectra
The 1H NMR spectra of the pyrazolo[1,5‐a]pyrimidine
based ligands (L1‐L5) and cyclometalated platinum(II)
complexes (I‐V) have been obtained in solvent DMSO d6
as illustration in supplementary material 1 and the
resultant physicochemical data of synthesized compounds
are summarized in experimental section. In 1H NMR
spectra of synthesized ligands (L1‐F, L2‐Cl, L3‐Br,
L4‐OCH3, L5‐CH3) the number of aromatic hydrogen
atoms are 12‐H (pyrazolo[1,5‐a]pyrimidine derivative).
While in complexes (I‐V) number of aromatic and
aliphatic hydrogen atoms are 11‐H (pyrazolo[1,5‐a]pyrim-
idine derivative ligand) and 7‐H (acetyl acetone), respec-
tively it indicates that carbon C6” attached to central
4.3 | FT‐IR spectroscopy
FT‐IR spectral data of synthesized pyrazolo[1,5‐a]pyrimi-
dine based ligands (L1‐L5) and cyclometalated
heteroleptic platinum(II) complexes (I‐V) are presented
in experimental section. The peaks of ligands containing
azomethine ν(C = N) group is observed in the range of
1542–1550 cm−1 and it is shifted to higher frequencies
(1550–1522 cm−1) in the complexation, showing the
metal ion. The peak of synthesized ligands (L1‐L5, H2”,6″
)
are found in the range of 7.492 to 7.534 δ ppm. These
hydrogen (H2”,6″) exhibit doublet of doublet (dd). It is
not observed in the complexes, it indicates that platinum
ion coordinated to C6” carbon atom of ligands via covalent
bond. Hence, one aromatic hydrogen atom less in
complexes than ligands. In pyrazolo[1,5‐a]pyrimidine
derivatives ligands (L1‐L5), all the aromatic proton are
observed in the range of ~δ 8.29 to 6.50 ppm and all
aromatic proton of the cyclometalated platinum(II)
complexes are appeared in the range of ~δ 8.309 to
6.8 ppm. Methoxy group of ligand (L4) and complex (IV)
are appeared at about ~δ 2.44 and ~δ 2.89 ppm,
respectively. Methyl (−CH3) group of ligand (L5) and
complex (V) are observed at about ~δ 1.640 and ~δ
2.291 ppm, respectively. All the cyclometalated
platinum(II) complexes contain acetylacetone bidentate
ligands. Therefore methyl group protons are obtained in
range of ~δ 0.892–1.912 ppm.
coordination of the heterocyclic nitrogen atoms
[30,31]
(azomethine group) to platinum metal ion
The
bands νC = Car of the compounds are observed in the of
range 1604–1606 cm−1.The νC–Har. stretching band of
the free ligands are observed in the range of 2926–
2930 cm−1 and it is shifted to higher frequency in the
range of 3050–3060 cm−1 of complexes. The spectra of
all the platinum(II) complexes show bands in the range
of 501–563 cm−1, 432–493 cm−1 and 416–430 cm−1, due
to (Pt‐N), (Pt‐O) and (Pt‐C), respectively. In platinum(II)
complexes
groupcontaining (>C = O) is observed in the range of
1728–1774 cm−1
(I‐V),
the
peak
of
acetylacetone
.
4.4 | Mass spectroscopy
The mass spectra and fragmentation pattern of
synthesized pyrazolo[1,5‐a]pyrimidine based ligands
(L1‐L5) are shown in supplementary material 3 and data
of all the ligands are illustrate in experimental section.
The LC–MS spectrum and possible mass fragmentation
pattern of complex (II) are shown in Figure 1. The mass
spectrum of a synthesized platinum(II) complex (II)
shows a molecular ion peak at 599.80 m/z and
601.85 m/z due to [M]+ and [M + 2], respectively, it
indicate that the one chlorine atom present in the
complexes. The peaks at 499.84 m/z and 501 m/z is due
to pyrazolo[1,5‐a]pyrimidine ligands attached to platinum
4.2 | 13C NMR spectra
The 13C NMR spectra of the synthesized pyrazolo[1,5‐a]
pyrimidine based aromatic ligands, cyclometalated
heteroleptic platinum(II) complexes and μ‐dichloro
bridge complex (I) are shown in supplementary material
2 and data of these compounds are represented in
experimental section. The peak of ligand (L4) containing
methoxy group and ligand (L5) containing methyl group

10 of 18
LUNAGARIYA ET AL.
FIGURE 1 LC–MS spectrum and mass fragmentation pattern of the synthesized cyclometalated heteroleptic platinum(II) complex (II)
metal ion with loss of acetylacetone moiety. The peaks at
305.77 m/z and 307.68 m/z corresponds to pyrazolo[1,5‐a]
pyrimidine based ligands. The peaks at 229.67 m/z and
231.50 m/z are obtained with loss of phenyl ring in this
ligand. The peak at 119.13 m/z is observed of
pyrazolo[1,5‐a]pyrimidine.
4.5 | Electronic spectra, magnetic
behaviour and conductance measurements
The geometry of the cyclometalated heteroleptic
platinum(II) complexes has been confirmed using
electronic spectral analysis. The cyclometalated
platinum(II) complexes (I‐V) exhibit two intense bands,
in the range of 292–299 nm and 307–360 nm due to
charge transfer (CT) and d‐d transitions, respectively.[1,32]
Electronic spectra of all the complexes (I‐V) are
represented in Figure 2. 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 complexes are diamagnetic in
nature.[33] The molar conductivity (Λm) of platinum(II)
complexes are observed in the range of 18–25
FIGURE 2 Electronic spectra of the synthesized cyclometalated
heteroleptic platinum(II) complexes (I‐V).
absence of co‐ordinated and lattice water molecule or
any volatile component. The platinum(II) complexes
decomposed in two steps. First mass loss (17.01%)
occurring in the temperature range of 205–270 °C
corresponds to loss of acetylacetone ligand. Second mass
loss (52.49%) in the range of 280–410 °C corresponds to
the loss of pyrazolo[1,5‐a]pyrimidine based ligand and
leaving behind metallic platinum as residue.[33]
Ω
−1cm2mol−1, which suggests the non‐electrolytic nature
of the complexes.
4.6 | Thermogravimetric analysis (TGA)
4.7 | Biological screening of synthesized
Thermogravimetric curve for cyclometalated heteroleptic
platinum(II) complex (II) is obtained at a heating rate of
10 °C per minute in the range of 0–800 °C under
dinitrogen atmosphere (See Supplementary material 4).
No mass loss is observed up to 200 °C indicating the
compounds
4.7.1 | In vitro antibacterial activity
The study of antimicrobial potency is carried out in terms
of minimum inhibitory concentration (MIC) defined as

LUNAGARIYA ET AL.
11 of 18
the lowest concentration which inhibits the growth of
microorganism, referred by lack of turbidity in the tube.
If specific concentration for a test compounds no turbidity
is observed then a whole experimental procedure is
repeated with the next dilution i.e. half the inhibitory
concentration (IC50) of test compound that has been
previously added. This procedure is repeated till the faint
turbidity by the inoculums itself is observed and this
concentration is termed as MIC (in μM). The result of this
study are represented in the Figure 3 and data of the
compounds are represented in supplementary material
5. The IC50 values of the complexes and ligands are
observed in range of 25–95 μM and 190–270 μM,
respectively. The platinum(II) complexes (I‐V) exhibit
value is calculated from the plot of log of concentration
of samples against percentage of mortality of nauplii and
represented in Figure 4. It is concluded that the
platinum(II) complexes show excellent toxicity as
compared to corresponding ligands. The mortality rate
of nauplii is found to increase with increasing concentra-
tion of compounds. The LC50 values of synthesized
ligands and complexes are observed in the range of
56.49–120.22 μgmL−1 and 6.714–11.96 μg mL−1
,
respectively. The LC50 values of cisplatin and transplatin
are 3.133 and 14.45 μg mL−1. The potency of the
synthesized compounds are represented in order of
L5
< < < < < transplatin <
L4 L3 L2 L1
V < IV < III < II < I < cisplatin. In vitro cytotoxicity study
is a basic one and additional studies are necessary to
investigate its actual mechanism of cytotoxicity and its
probable effects on cancer cell line.
higher antimicrobial activity than metal salt and free
[34]
pyrazolo[1,5‐a]pyrimidine based ligands (L1‐L5).
The
in vitro antimicrobial bioassay of the metal complexes
can be described on the basis of Tweedy's chelation
theory.[35] Chelation decreases the polarity of metal ion
due to partial sharing of its positive charge with donor
groups and possible π‐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 microorgan-
ism which inhibit the further growth of the organism.[36]
4.7.3 | Cellular level in vitro cytotoxicity
against S. Pombe cells
Cellular level in vitro cytotoxicity of the synthesized
ligands and complexes have been tested by S. pombe cells.
From the conclusion, cell death caused by toxicity of the
synthesized 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 concentrations of the synthesized ligands and
platinum(II) complexes. Complexes I, II and III (−F,
−Cl and ‐Br) are found to be excellent toxicity as
compared to other complexes (IV and V), All the
platinum(II) complexes (I‐V) are more toxic in nature as
compared to pyrazolo[1,5‐a]pyrimidine based ligands
4.7.2 | In vitro cytotoxicity against
Artemia cyst lethality bioassay
All the synthesized compounds pyrazolo[1,5‐a]pyrimidine
based ligands(L1‐L5) and cyclometalated platinum(II)
complexes (I‐V) have been tested for in vitro brine shrimp
lethality bioassay using Meyer et al. process.[37] The
percentage mortality of brine shrimp nauplii has been
determined from the number of dead nauplii. The LC50
FIGURE 4 Effect of the substituted pyrazolo[1,5‐a]pyrimidine
based ligands (L1‐L5), cisplatin, transplatin and cyclometalated
heteroleptic platinum(II) complexes (I‐V) on the brine shrimp
lethality bioassay shown with standard deviation for three times
repeat experiments
FIGURE 3 Effect of different concentrations (μM) of free ligands
and platinum(II) complexes on two gram(+ve) and three gram(−ve)
microorganisms. Error bars represent the standard deviation of
three independent 5%

12 of 18
LUNAGARIYA ET AL.
(L1‐L5). The in vitro cytotoxicity potency of the
platinum(II) complexes is comparable to standard drug
such as cisplatin and transplatin. After 17–20 h of the
treatment, many of the S. pombe cells are destroyed due
to toxic nature of the complexes. The order of the synthe-
sized compounds are found to be cisplatin > transplatin >
I > II > III > IV > V > L1 > L2 > L3 > L4 > L5 (Figure 5)
and data are represented in supplementary material 6.
4.8 | DNA interaction studies
4.8.1 | Electronic absorption titration
spectroscopy
Electronic absorption titration spectroscopy is most useful
spectroscopic method for examine the interaction of
substituted pyrazolo[1,5‐a]pyrimidine based ligands
(L1‐L5) and cyclometalated heteroleptic platinum(II)
FIGURE 5 Percentage viability of the synthesized ligands (L1‐L5), cisplatin, transplatin and cyclometalated platinum(II) complexes (I‐V) on
S. pombe cells existing with standard deviation for three independent experiments
FIGURE 6 Electronic absorption spectra of ligand (L1) and complex (I) in phosphate buffer pH =7.2 at 25 °C in presence of increasing
amount of HS‐DNA

LUNAGARIYA ET AL.
13 of 18
[39]
complexes with HS‐DNA. A compounds interaction with
HS‐DNA through intercalative mode of binding is a result
of hypochromism and bathochromism.[38] The electronic
absorption titration spectra of the ligand (L1) and
cyclometalated platinum(II) complex (I) in the absence
and presence of HS‐DNA are represented in Figure 6.
The strong absorption bands of the synthesized ligands
(L1‐L5) and complexes (I‐V) are found in between
wavelength at about 234 to 308 nm.
Therefore, the
experimental obtained hypochromic effect in the
intraligand transition band proposes that the compounds
bind to HS‐DNA via partial intercalative mode. The magni-
tude 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 λmax with increasing con-
centrations of HS‐DNA by the Wolfe‐Shimer equation.[40]
The intrinsic binding constant Kb is determined the ratio
of slope and intercept in plots of [DNA]/(εa ‐ εf) versus
[DNA] (Figure 6). The binding constant (Kb) values of syn-
thesized pyrazolo[1,5‐a]pyrimidine based ligands (L1‐L5)
and cyclometalated platinum(II) complexes (I‐V) are
obtained in range of 0.242–1.459 × 105 M−1 and 2.243–
2.890 × 105 M−1, respectively. It's concluded that the com-
plexes have higher binding affinity as compared to ligands.
Binding constant (Kb) of the ethidium bromide is
7.1 × 105 M−1, it is higher than synthesized compounds.
The Kb values of cisplatin, oxaliplatin and carboplatin are
TABLE 1 The binding constant (Kb,
M
−1), % hypochromicity and
change in Gibb's free energy (ΔG0, Jmol−1) of organometallic
platinum(II) complexes (I‐V) and pyrazolo[1,5‐a]pyrimidine deriv-
atives ligands (L1‐L5) with DNA.
λ
max (nm)
aΔλ bKb (M
dΔG0
Compounds Bound free (nm) −1) × 105 cH% (Jmol−1
)
L1
L2
L3
L4
L5
I
267 265
269 267
275 272
308 302
268 267
243 238
269 267
272 267
273 271
247 244
2
2
3
5
1
1
1
2
2
3
1.459
0.858
0.532
0.286
0.242
2.890
2.820
2.776
2.325
2.243
20.01 −29,460.00
34.50 −28,144.65
27.98 −26,960.47
29.29 −25,422.76
33.86 −25,008.87
37.14 −31,153.44
17.67 −31,092.69
18.55 −31,053.73
30.32 −30,614.47
19.59 −30,525.51
5.73 × 104 M‐1[41], 5.3 × 103 M‐1[42] and 0.33 × 103 M‐1[43]
,
respectively and these Kb values are comparable to
synthesized cyclometalated platinum(II) complexes and
ligands. Moreover, the percentage hypochromism (H %)
of the synthesized ligands (L1‐L5) and complexes (I‐V) are
observed in between 20.01–34.50%. The Gibb's free energy
of the synthesized compounds are found negative values in
the range of −25.00 to −31.15 kJmol−1 representing the
spontaneity of compound‐DNA binding. The results of
the compound bind to DNA more spontaneously in order
of I > II > III > IV > V > L1 > L2 > L3 > L4 > L5. The
DNA binding data of the synthesized compounds are
represented in Table 1.
II
III
IV
V
aΔλ = Difference between bound wavelength and free wavelength.
bKb = Intrinsic DNA binding constant determined from the UV–visible
absorption spectral titration.
cH% = [(Afree ‐ Abound)/Afree] × 100%.
dΔG0 = Change in Gibb's free energy.
FIGURE 7 Effect of increasing amounts of ligands (L1‐L5) and complexes (I‐V) on specific relative viscosity of HS‐DNA. 1/R = [complex]/
[DNA] ratio of 0.0, 0.04, 0.08, 0.12, 0.16, 0.20

14 of 18
LUNAGARIYA ET AL.
via intercalation. Upon EB‐DNA interaction, characteristic
of EtBr shows the changes in absorbance, reflected by an
enhancement in fluorescence intensity by about one order
of magnitude, as compared to free dye in solution. Hence,
when a platinum(II) complexes are added to the EB‐DNA
system, any quenching of the fluorescence will indicate
the replacement of EB by the coordination molecule
(DNA), via intercalative mode, it is expected of its strong
stacking interaction between the adjacent DNA base pairs.
Fluorescence quenching analysis has been carried out
for platinum(II) complexes and the corresponding
emission spectra of the EB‐DNA solutions in the presence
of the increasing amounts of complex concentrations
(r = 0.33 to 3.33), florescence quenching data of all
complexes and graph are represented in supplementary
material 7. Which clearly indicate a dramatic increase in
the fluorescence intensity of the EB‐DNA by adding the
Pt(II) complex. In DNA‐EB system, the increase of the
fluorescence intensity is due to releasing free EB mole-
cules. Therefore, the formation of complex–DNA stops
the binding of EB and the complete metal complex–
DNA formation occurs when the study fluorescence
intensity is sufficient.[25]
4.8.2 | Viscosity measurements
The special effects of the synthesized ligands and
complexes on the relative viscosity of HS‐DNA has been
represented in Figure 7. The viscosity of all the com-
pounds 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 titra-
tion spectroscopic data. The observed relative viscosity of
the synthesized ligands and complexes are in order of
EB > I > II > III > IV > V > L1 > L2 > L3 > L4 > L5.
4.8.3 | Interaction of the complexes with
DNA by fluorescence spectroscopic method
In fluorescence quenching study, ethidium bromide (EtBr)
as a cationic dye that is normally used for DNA interaction
TABLE 2 Linear stern‐Volmer quenching constant (Ksv), binding
sites (n) and association binding constant (Ka) calculated from
double logarithmic plots for the interaction of complex with HS‐
DNA.
Complexes Ksv (M−1
)
Ka (M−1
)
n
ΔG(J mol‐1)
I
1.40 × 102
5.01 × 105 1.4212 −32,517.48
1.76 × 105 1.3505 −29,937.18
1.73 × 104 1.1148 −24,187.30
5.05 × 103 1.1162 −21,127.23
0.93 × 103 0.8124 −16,961.00
Moreover, the Stern‐Volmer quenching constants
(Ksq), change in standard Gibb's free energy (ΔG0) and
associative binding constant (Ka) of platinum(II) com-
II
III
IV
V
7.1 × 103
5.9 × 103
2.6 × 103
6.8 × 103
plexes are observed in range of 1.40 × 102−7.1 × 103 M−1
,
,
−12.37 to −32.51 kJmol−1 and 0.15 × 103–5.05 × 103 M−1
respectively (Table 2). The corresponding plots obtained
FIGURE 8 Fluorescence emission spectra of EB bound to HS‐DNA in the presence of complexes (I). [EB] = 33.3 μM, [DNA] = 10 μM;
[complex] = (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 μM; λex = 510 nm. The arrows
show the intensity changes upon increasing the concentrations of complex. Inset graph: Plots of I/I vs. [Q], with • for the experimental data
points and the full line for the linear fitting of the data. Comparative plot of log[I‐I/I] versus log[complex] for the titration of HS‐DNA EB
system with platinum(II) complexes (I) in phosphate buffer medium

LUNAGARIYA ET AL.
15 of 18
FIGURE 9 Molecular docking model of ligand (L1) and complex (I) (ball and stick) with DNA helix (PDB ID: 1BNA) (VDW spheres) of
sequence d(ACCGACGTCGGT)2. The compounds is docked with DNA base pairs via partial intercalation mode
from the experimental quenching data for the complexes
are shown in Figure 8.
hydrogen bonding with the DNA functional groups that
define the partial intercalation.[46,47] The binding energies
of the docked cyclometalated platinum(II) complexes
(I‐V) with DNA are −278.81 (I), −284.92 (II), −280.91
(III), −286.02 (IV) and −286.08 (V), respectively. The
binding energies of the pyrazolo[1,5‐a]pyrimidine based
ligands (L1‐L5) with DNA are −225.93 (L1), −227.33 (L2),
−228.00 (L3), −233.25 (L4) and −228.12 (L5), respectively.
The platinum(II) complexes exhibit higher binding affin-
ity with DNA as compared to pyrazolo[1,5‐a]pyrimidine
4.8.4 | Molecular modeling study
The synthesized compound can play an important role in
the improvement of new chemotherapeutic drugs that
lead to the recognition of specific sequences and
structures of nucleoside and nucleotide.[44] Molecular
docking study displays
a very significant role to
understanding the mechanistic pathway of complex‐
DNA interactions, by placing the complex penetrate
binding site of the DNA helix. The substituted
pyrazolo[1,5‐a]pyrimidine based ligands (L1‐L5) and
cyclometalated heteroleptic platinum(II) complexes (I‐V)
are docked with in a DNA double helix structure by
molecular docking analysis. Which is represented in
Figure 9 and supplementary material 8. Thus, in order
to check and illuminate the obtained spectroscopic results
and to get a further insight into the intercalation ability,
platinum(II) complexes (I‐V) and ligands (L1‐L5) docked
with DNA double helix structure. The complexes under
investigation bind with B‐DNA (PDB ID: 1BNA) at the
A‐T rich via partial intercalation mode.[45] Furthermore,
the DNA in such a way that the part of the planar hetero-
cyclic ring containing ligands and complexes including
outside edge stacking interaction with oxygen atoms of
the phosphate backbone and further stabilized by Van
der Waals interactions, hydrophobic contacts and
FIGURE 10 Photogenic view of cleavage of pUC19 DNA (300 μg/
cm3) with series of ligands (L1‐L5) and Pt(II) complexes (200 μM)
using 1% agarose gel containing 0.5 μg/cm3 EtBr. Reactions were
incubated in TE buffer solution (pH 8) at a final volume of 15 mm3
for time period of 3 h at 37 °C

16 of 18
LUNAGARIYA ET AL.
FIGURE 11 Plot of nuclease cleavage (% of form I, form II and DNA cleavage) assay of the ligands (L1‐L5) and complexes (I‐V). Error bars
represent standard deviation of three replicates ( 5%)
based ligands. The compound interact with DNA, to
provide greater binding affinity obtained by molecular
docking study is in agreement with the experimental
results developed from electronic absorption titration, vis-
cosity measurements and fluorescence studies.
concentration. The Figure 11 represent percentage
cleavage of compounds.
5 | CONCLUSION
A series of cyclometalated platinum(II) complexes and
pyrazolo[1,5‐a]pyrimidine based ligands have been
synthesized. The synthesized compounds have been
characterized by the spectroscopic and physicochemical
4.8.5 | Photochemical analysis of DNA
nuclease activity
1
techniques such as H NMR, 13C NMR, FT‐IR, electronic
The effect of the compounds on DNA is estimated by
their DNA‐cleavage ability.[48,49] Which can be deter-
mined by the cleavage mechanism. The cleavage effi-
ciency of these molecules has usually examined by
agarose gel electrophoresis.[50,51] The cyclometalated
platinum(II) complexes, cisplatin, transplatin and
pyrazolo[1,5‐a]pyrimidine based ligands are promote the
percentage cleavage of pUC19 DNA from supercoiled
Form I to the open circular Form II (Figure 10). The con-
trol experiment using DNA alone does not show any sig-
nificant cleavage of DNA (Lane 1). A slight cleavage is
observed when K2PtCl4 salt has been added to 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 3–9). The % cleavage graph for all the com-
pounds can be calculated using AlphaDigiDocTM RT
Version V.4.1.0 PC–Image software. And % cleavage data
of the synthesized compounds are represented in supple-
mentary material 9. The % cleavage ability of the all
spectral study, mass spectroscopy, magnetic moment,
molar conductivity, TGA and elemental analysis. The
platinum(II) complexes have square planar geometry
and diamagnetic in nature. DNA binding activities of
the synthesized compounds have been carried out using
electronic absorption titration spectroscopy, fluorescence
quenching analysis, viscosity measurements and
molecular docking study. These studies suggest partial
intercalation mode of binding. The complexes exhibit
highest binding ability and binding strength as compared
to ligands. The binding constant (Kb) of complex (I) is
higher than other compound because of the complex (I)
containing highly electron withdrawing functional group
(−F atom). The complexes and cisplatin exhibit effective
DNA cleavage as compared to ligands, transplatin and
K2PtCl4 salt. The platinum(II) complexes and cisplatin
exhibit excellent in vitro brine shrimp cytotoxicity activity
as compared to ligands and transplatin. In vitro cellular
level cytotoxicity bioassay of the synthesized platinum(II)
complexes, cisplatin, transplatin and ligands have been
carried out using S. Pombe cells. The % viability of the
platinum(II) complexes is comparable to the cisplatin
and higher as compared to transplatin and ligands. The
minimum inhibitory concentration of the synthesized
compounds is arrange in order of cisplatin
>
I > II > III > IV > V > L1 > L2 > L3 > L4 > L5 > transplatin
> K2PtCl4. The complexes (I‐V) and cisplatin exhibits
greater DNA cleavage affinity as compared to ligands
(L1‐L5), transplatin and K2PtCl4 salt at the same

LUNAGARIYA ET AL.
17 of 18
[12] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am.
Chem. Soc. 1954, 76, 3047.
compounds have been carried out using five different
bacterial species. The platinum(II) complexes (I‐V) show
higher potency against two Gram(+ve) and three Gram(−ve)
microorganism. Further studies are necessary to evaluate
precise molecular mechanism of cytotoxicity and the
pharmacological properties to reveal the actual
mechanism of the biological activity.
[13] J. Marmur, J. Mol. Biol. 1961, 3, 208.
[14] T. M. H. Paul, M. G. B. Drew, P. Chattopadhya, J. Coord. Chem.
2012, 65, 1289.
[15] Z.‐Y. Y. Yong Li, M.‐F. Wang, J. Fluoresc. 2010, 891.
[16] S. Ramakrishnan, E. Suresh, A. Riyasdeen, M. A. Akbarsha, M.
Palaniandavar, Dalton Transactions 2011, 40, 3524.
[17] J. B. Chaires, N. Dattagupta, D. M. Crothers, Biochemistry 1982,
21, 3933.
ACKNOWLEDGEMENTS
[18] F. Leng, W. Priebe, J. B. Chaires, Biochemistry 1998, 37, 1743.
[19] J. R. Lakowicz, 2006.
The authors are thankful to the Head, Department of
Chemistry, Sardar Patel University, Vallabh Vidyanagar,
Gujarat, India, for providing necessary research facilities,
DST‐PURSE, Sardar Patel University, Vallabh Vidyanagar
for mass spectral analysis. The authors gratefully
acknowledge the University Grants Commission, New
Delhi, India for meritorious fellowships awarded to them
during year 2014‐2018 and “BSR UGC One Time Grant”,
vide UGC letter no. F.19‐119/2014 (BSR).
[20] L. Zhu, K. Zheng, Y.‐T. Li, Z.‐Y. Wu, C.‐W. Yan, J. Photochem.
Photobiol., B 2016, 155, 86.
[21] O. Stern, M. Volmer, Z. Phys. 1919, 20, 183.
[22] R. Frank, H. Rau, The Journal of Physical Chemistry 1983, 87,
5181.
[23] A. Kathiravan, R. Renganathan, Polyhedron 2009, 28, 1374.
[24] K. P. Thakor, M. V. Lunagariya, M. N. Patel, J. Biomol. Struct.
Dyn. 2016, 1.
[25] M. Hong, G. Chang, R. Li, M. Niu, New J. Chem. 2016, 40, 7889.
[26] C. G. Ricci, P. A. Netz, J. Chem. Inf. Model. 2009, 49, 1925.
[27] J. V. Mehta, S. B. Gajera, M. N. Patel, J. Biomol. Struct. Dyn.
2016, 1.
DECLARATION OF INTEREST
[28] D. Sinha, A. K. Tiwari, S. Singh, G. Shukla, P. Mishra, H.
Chandra, A. K. Mishra, Eur. J. Med. Chem. 2008, 43, 160.
[29] P. R. Reddy, A. Shilpa, Polyhedron 2011, 30, 565.
[30] C. Shiju, D. Arish, N. Bhuvanesh, S. Kumaresan, Spectrochim.
Acta, Part A 2015, 145, 213.
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
[31] V. X. Jin, J. D. Ranford, Inorg. Chim. Acta 2000, 304, 38.
[32] A. A. Soliman, O. I. Alajrawy, F. A. Attaby, W. Linert, J. Mol.
Struct. 2016, 1115, 17.
ORCID
Mohan N. Patel http://orcid.org/0000-0001-9016-9245
[33] R. Prabhakaran, A. Geetha, M. Thilagavathi, R. Karvembu, V.
Krishnan, H. Bertagnolli, K. Natarajan, J. Inorg. Biochem.
2004, 98, 2131.
REFERENCES
[34] B. G. Tweedy, Phytopathology 1964, 55, 910.
[35] P. Kalaivani, R. Prabhakaran, F. Dallemer, P. Poornima, E.
Vaishnavi, E. Ramachandran, V. V. Padma, R. Renganathan,
K. Natarajan, Metallomics 2012, 4, 101.
[1] H. Li, J. Ding, Z. Xie, Y. Cheng, L. Wang, J. Organomet. Chem.
2009, 694, 2777.
[2] A. Guida, M. H. Lhouty, D. Tichit, F. Figueras, P. Geneste,
[36] N. R. F. B. N. Meyer, J. E. Putnam, L. B. Jacobsen, D. E. Nichols,
J. L. McLaughlin, Planta Med. 1982, 45, 31.
Appl. Catal., A 1997, 164, 251.
[3] V. V. Lipson, S. M. Desenko, V. V. Borodina, M. G.
Shirobokova, Chemistry of Heterocyclic Compounds 2007, 43,
1544.
[37] E.‐J. Gao, F. Hong, M.‐C. Zhu, C. Ma, S.‐K. Liang, J. Zhang, L.‐
F. Li, L. Wang, Y.‐Y. Li, J. Wei, Eur. J. Med. Chem. 2014, 82, 172.
[38] C. Janiak, J. Chem. Soc., Dalton Trans. 2000, 3885.
[39] C. Protogeraki, E. G. Andreadou, F. Perdih, I. Turel, A. A.
Pantazaki, G. Psomas, Eur. J. Med. Chem. 2014, 86, 189.
[40] J. Liu, W. Zheng, S. Shi, C. Tan, J. Chen, K. Zheng, L. Ji,
J. Inorg. Biochem. 2008, 102, 193.
[4] J. Sun, J.‐K. Qiu, B. Jiang, W.‐J. Hao, C. Guo, S.‐J. Tu, The Jour-
nal of Organic Chemistry 2016, 81, 3321.
[5] J. J. Mousseau, A. Fortier, A. B. Charette, Org. Lett. 2010, 12, 516.
[6] A. Liang, Y. Li, W. Zhu, Y. Wang, F. Huang, H. Wu, Y. Cao,
Dyes Pigm. 2013, 96, 732.
[41] C. N. N'Soukpoé‐Kossi, C. Descôteaux, É. Asselin, H.‐A. Tajmir‐
Riahi, G. Bérubé, DNA Cell Biol. 2007, 27, 101.
[42] H. Soori, A. Rabbani‐Chadegani, J. Davoodi, Eur. J. Med. Chem.
2015, 89, 844.
[7] C.‐L. Ho, W.‐Y. Wong, B. Yao, Z. Xie, L. Wang, Z. Lin,
J. Organomet. Chem. 2009, 694, 2735.
[8] M. Patra, M. Wenzel, P. Prochnow, V. Pierroz, G. Gasser, J. E.
Bandow, N. Metzler‐Nolte, Chem. Sci. 2015, 6, 214.
[43] A. Paul, S. Anbu, G. Sharma, M. L. Kuznetsov, B. Koch, M. F. C.
Guedes da Silva, A. J. L. Pombeiro, Dalton Transactions 2015,
44 19983.
[9] B. N. M. N. R. Ferrigni, Journal of Medicinal Plant Research
1982, 45, 31.
[10] S. M. R. I. M. R. Islam, A. S. M. Noman, J. A. Khanam, S. M. M.
[44] D. R. Boer, A. Canals, M. Coll, Dalton Transactions 2009, 399.
[45] V. Thamilarasan, P. Karunakaran, N. Kavitha, C. Selvaraju, N.
Sengottuvelan, Polyhedron 2016, 118, 12.
Ali, M. W. Lee, Mycobiology 2007, 35, 25.
[11] S. C. Karad, V. B. Purohit, D. K. Raval, Eur. J. Med. Chem. 2014,
84, 51.
[46] M. Baginski, F. Fogolari, J. M. Briggs, J. Mol. Biol. 1997, 274, 253.

18 of 18
LUNAGARIYA ET AL.
[47] J.‐G. Liu, Q.‐L. Zhang, X.‐F. Shi, L.‐N. Ji, Inorg. Chem. 2001, 40,
SUPPORTING INFORMATION
5045.
Additional Supporting Information may be found online
in the supporting information tab for this article.
[48] A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J.
Turro, J. K. Barton, J. Am. Chem. Soc. 1989, 111, 3051.
[49] J. Qian, W. Gu, H. Liu, F. Gao, L. Feng, S. Yan, D. Liao, P.
Cheng, Dalton Transactions 2007, 1060.
How to cite this article: Lunagariya MV, Thakor
KP, Patel NJ, Patel MN. Synthesis, characterization
and biological application of cyclometalated
heteroleptic platinum(II) complexes. Appl
Organometal Chem. 2017;e4045. https://doi.org/
10.1002/aoc.4045
[50] B. Macías, M. V. Villa, B. Gómez, J. Borrás, G. Alzuet, M.
González‐Álvarez, A. Castiñeiras, J. Inorg. Biochem. 2007, 101,
444.
[51] A. K. Sadana, Y. Mirza, K. R. Aneja, O. Prakash, Eur. J. Med.
Chem. 2003, 38, 533.