Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II) complexes as a biological active agent

Article abstract of DOI:10.1080/15257770.2018.1498510

Square planar mononuclear platinum(II) complexes having general formula [Pt(Lⁿ)Cl₂], (where, Lⁿ = L^1–4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The syn

Full text of DOI:10.1080/15257770.2018.1498510

Nucleosides, Nucleotides and Nucleic Acids
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Evolution of 1, 3, 5-trisubstituted bipyrazole
scaffold based platinum(II) complexes as a
biological active agent
Miral V. Lunagariya, Khyati P. Thakor, Bharat H. Pursuwani & Mohan N. Patel
To cite this article: Miral V. Lunagariya, Khyati P. Thakor, Bharat H. Pursuwani & Mohan
N. Patel (2018): Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II)
complexes as a biological active agent, Nucleosides, Nucleotides and Nucleic Acids, DOI:
10.1080/15257770.2018.1498510
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1498510
Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold
based platinum(II) complexes as a biological
active agent
Miral V. Lunagariya, Khyati P. Thakor, Bharat H. Pursuwani, and
Mohan N. Patel
Department of Chemistry, Sardar Patel University, Gujarat, India
ABSTRACT
ARTICLE HISTORY
Received 22 June 2017
Accepted 22 June 2018
Square planar mononuclear platinum(II) complexes having gen-
eral formula [Pt(Ln)Cl2], (where, Ln ¼ L1–4) were synthesized with
neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole
based ligands. The synthesized compounds were characterized
by physicochemical method such as TGA, molar conductance,
micro-elemental analysis and magnetic moment, and spectro-
scopic method such as, FT-IR, UV–vis, 1H NMR, 13C NMR and
mass spectrometry. Biological applications of the compounds
were carried out using in vitro brine shrimp lethality bioassay,
in vitro antimicrobial study against five different pathogens,
and cellular level cytotoxicity against Schizosaccharomyces
pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA
interaction activities using electronic absorption titration, vis-
cosity measurements study, fluorescence quenching technique
and molecular docking assay. Binding constants (Kb) of ligands
and complexes were observed in the range of
0.23–1.07 ꢀ 105 Mꢁ1 and 0.51–3.13ꢀ 105 Mꢁ1, respectively. Pt(II)
complexes (I–IV) display an excellent binding tendency to bio-
molecule (DNA) and possess comparatively high binding con-
stant (Kb) values than the ligands. The DNA binding study
indicate partial intercalative mode of binding in complex-DNA.
The gel electrophoresis activity was carried out to examine
DNA nuclease property of pUC19 plasmid DNA.
KEYWORDS
Neutral N, S –donor
bidentate 1, 3,
5-trisubstituted bipyrazole
derivatives ligands; Pt(II)
complexes; DNA binding
study; Cellular level
cytotoxicity; DNA
nuclease activity
CONTACT Mohan N. Patel
jeenen@gmail.com
Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar, Gujarat 388 120, India
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn
Supplemental data for this article can be accessed https://doi.org/10.1080/15257770.2018.1498510.
ß 2018 Taylor & Francis Group, LLC

2
M. LUNAGARIYA ET AL.
Introduction
Heterocyclic compounds namely pyrazole, bipyrazole and thiophene are of
great importance because they possess wide range of physical, chemical,
pharmacological and biological properties having great stability and reactiv-
ity.[1] The pyrazole ring is present in many important drugs, such as
Zometapine[2] and Viagra[3] and in agrochemicals namely Tolfenpyrad and
Fenpyroximate.[4] Most of natural products, vitamins, alkaloids and agro-
chemicals constitute heterocycles bearing nitrogen donor atom play an
important role in coordination chemistry.[5] Compounds having pyrazole
moiety behave as potential cytotoxic agents.[6]
Nucleic acids are important cellular targets for many anticancer and
antiviral drugs. Identification of new agents that can interact with DNA
molecules effectively is considered important for development of new
chemotherapeutic agents.[7] Rosenberg showed that cis-plantin can inter-
act non covalently with nucleoside and nucleotide.[8] As one of the
important anticancer drugs, cisplatin has been successfully implemented
in chemotherapy against various solid tumors including genitourinary,
colorectal, testicular, ovarian, head, neck and lung cancers. Cisplatin has
limitation towards drug resistance and on other hand possess severe
side effects that include high toxicity, mainly nephrotoxicity, neurotox-
icity, ototoxicity, and emetogenesis.[9] So, better platinum coordinated
complexes are needed which can overcome this limitations.[10] Platinum
complexes such as oxaliplatin, nedaplatin, and lobaplatin are appropriate
for clinical use and possesses similar anticancer activities like cisplatin
and comparatively low toxicity. These drugs are second generation plat-
inum anticancer agents that interact with DNA in a similar manner as
cisplatin.[11] The development of new methods for the synthesis of
bipyrazole derivatives are of great interest.[12] The synthesis of ligands
(L1–L4) include three step reaction using conventional methods.
Whereas, mononuclear homoleptic platinum (II) complexes (I–IV) are
synthesized by single step one-pot two-component reaction. In addition,
we report the biological activities of the synthesized 1,3,5-tri-substituted
bipyrazole derivatives ligands (L1–L4) and mononuclear platinum(II)
complexes (I–IV). The biological studies include DNA binding, DNA
nuclease, antibacterial, cellular level in vitro cytotoxicity against S.
pombe cells and in vitro cytotoxicity against brine shrimp lethality bio-
assay. For the biological significance, we have developed new approach
for structural diversity of heterocycles incorporating scaffold.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Experimental section
Chemicals and materials
All the chemicals used were of analytical grade. Solvents were dried and
distilled previously before use.[13] Potassium tetrachloro platinate(II) salt, 5-
chloro-2-acetylthiophene, 5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one,
5-bromo-2-acetylthiophene, 3-bromo-2-acetylthiophene, hydrazine hydrate
were purchased from Sigma Chemical Co. (India) and used as received
without further purification. Ethidium bromide (EB), bromophenol blue,
agarose and Luria Broth (LB) were purchased from Hi-media Laboratories
Pvt. Ltd., India. S. pombe Var. Paul Linder 3360 was purchased from
IMTECH, Chandigarh, India. Culture of pUC19 bacteria (MTCC 47) i.e.
Staphylococcus aureus (S. aureus) (MTCC-3160), Bacillus subtilis (B. subti-
lis) (MTCC-7193), 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).
Physical measurements
The micro-elemental analysis (C, H, N and S) of the compounds was car-
ried out with a Euro Vector EA3000 elemental analyzer (240). Thermo-
gravimetric analysis (TGA) was performed with a model SDT Q600 V20.9
Build 20, TA instrument (USA), with condition of recording curves under
a nitrogen (N2) atmosphere with a heating rate of 20 ꢂC minꢁ1 in the tem-
perature range from 25 ꢂC to 800 ꢂC. Infrared spectra were recorded on
Perkin-Elmer FT IR Shimadzu model spectrophotometer using the KBr
disc technique in the range of 4000–400 cmꢁ1. Electronic absorption spec-
tra were recorded using a UV-160A UV–Vis spectrophotometer Shimadzu,
Kyoto (Japan), with a 10 mm path length quartz cell in the range of
200–800 nm wavelength. The magnetic moments were measured by Gouys
method using mercury tetrathiocyanatocobaltate(II) as the calibrate
(vg ¼ 16.44 ꢀ 10ꢁ6 cgs units at 20 ꢂC). Melting points were determined on
Buchi 540 melting point apparatus. Fluorescence spectra were recorded on
1
a FluoroMax-4, spectrofluorometer, HORIBA (Scientific). The H NMR
and 13C NMR spectra were recorded on a Bruker Avance (400 MHz) and
(100 MHz), respectively in presence of DMSO-d6 (35 ꢂC) having inert prop-
erties w.r.t solvent. The LC–MS spectra were recorded by Model Q-ToF
Micro Waters. Minimum inhibitory concentration study was carried out
using laminar air flow cabinet, Toshiba, Delhi, (India). Photo quantization
image of the gel after electrophoresis was prepared by AlphaDigiDocTM

4
M. LUNAGARIYA ET AL.
RT. Version V.4.0.0 PC–Image software, CA (USA). Hydrodynamic chain
length study was carried out by viscometric measurement bath.
General synthesis of the ligands (L1–L4)
General procedure for the synthesis of 1-(5-chloro-3-methyl-1-phenyl-1H-
pyrazol-4-yl)ethan-1-one via vilsmeier–haack reaction (2)
The 1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)ethan-1-one (2) was
prepared
from
5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one
by
Vilsmeier Haack reaction. To a solution of 5-methyl-2-phenyl-2,4-dihydro-
3H-pyrazol-3-one (1) (1 mmol) in dry dimethyl formamide (DMF)
(3 mmol) at 0–5 ꢂC handled with mechanical stirrer, phosphoryl trichloride
(POCl3) (10 mmol) was added dropwise equipped with 100 mL round bot-
tom flask. After addition of these compounds, the reaction mixture was
heated at 80–90 ꢂC in water bath for 2–3 hours. The progress of the reac-
tion was monitored by TLC. The completion of reaction was confirmed by
TLC and mixture was poured into the cold water containing crushed ice
with stirring for 10 minutes and the resulting product was washed thor-
oughly with water, filtered and dried. The 1-(5-chloro-3-methyl-1-phenyl-
1H-pyrazol-4-yl)ethan-1-one (2) product was recrystallized from ethanol to
obtain a pale yellow solid.
Synthesis of the a, b unsaturated carbonyl compounds (4a–4d)
The a; b unsaturated carbonyl compounds analogs have been synthesized
using Claisen-Schmidt condensation. To a methanolic solution of substi-
tuted 2-acetyl thiophene (3a–3d) (1 mmol) in round bottom flask, solution
of potassium hydroxide (KOH) was dropwise added which abstract acidic
hydrogen from the 2-acetyl thiophene, after completion of above step,
methanolic solution of aromatic 1-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)ethan-1-one (2) in the reaction mixture was added. The reaction mix-
ture was stirred at room temperature until formation of precipitate. The
formed chalcone product was isolated by filtration, washed with cold etha-
nol and recrystallized from methanol.
General synthesis of the 1, 3, 5-tri-substituted bipyrazole derivative
ligands (L1–L4)
The 3-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-
2-en-1-one derivatives enone (4a–4e) (1.0 mmol) and hydrazine hydrate
(1.0 mmol) were thoroughly mixed in ethanol (5 mL) with potassium ter-
tiary butoxide (t-Buoꢁ Kþ) (1 mmol) as a basic condition in a 50 mL round
bottom flask. The reaction mixture was refluxed for 5 hours. After the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme 1. Schematic representation of the preparation of a series of the 1, 3, 5-trisubstituted
bipyrazole derivatives ligands (L1–L4) via Vilsmeier–Haack reagent.
completion of the reaction as monitored by TLC (ethyl acetate:hexane: 1:5),
the reaction mixture was cooled to room temperature washed with water
and dried over Na2SO4. Upon purification by column chromatography (sil-
ica gel 60–120 mesh, eluent 20% EtOAc/hexane) the desired compound
was obtained as white solid (Scheme 1).
Analytical data of 50-chloro-5-(5-chlorothiophen-2-yl)-30-methyl-10-phenyl10H,
4H-3,40-bipyrazole (L1, C17H14Cl2N4S). Yield: 90%; Colour: yellowish powder;
mol. wt.: 377.29 g/mol; M.p.: 162 ꢂC; 1H NMR (400 MHz, DMSO-d6):

6
M. LUNAGARIYA ET AL.
d ¼ 9.514 (1H, s, N–H), 8.106–7.011 (7H, m, Ar-H3’,4’,2”’,6”’,3”’,5”’,4”’), 5.229
(1H, dd, 3J1 ¼ 7.2 Hz, 3J2 ¼ 7.2 Hz, H5), 3.705 (1H, dd, 3J1 ¼ 11.2 Hz,
3
3
3J2 ¼ 10.8 Hz, Hb), 2.934 (1H, dd, J1 ¼ 9.6 Hz, J2 ¼ 9.6 Hz, Ha), 1.692 (3H,
s, pyrazole-CH3) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 155.61 (C3,
Cquat.), 147.12 (C3’’, Cquat.), 137.29 (C1”’, Cquat.), 130.70 (C5’, Cquat.),
129.34 (C3”’,5”’,–CH), 129.00 (C5”, Cquat.), 128.00 (C4’, –CH), 126.22
(C4”’,–CH), 125.90 (C3’, –CH), 125.28 (C2’, Cquat.), 124.87 (C2”’,6”’, –CH),
119.00 (C4”, Cquat.), 42.88 (C4, –CH2), 40.90 (C5, –CH), 12.28 (–CH3,
pyrazole) ppm. [Total signal observed: 15 ¼ signal of C and CH2 ¼ 8
(thiophene-C ¼ 2, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole-CH2 ¼ 1),
signal of CH and CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH ¼1, phenyl
ꢀ
ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr):V¼ 2923 m(¼C–H)ar (w), 1542
m(C ¼ N) (s), 1442–1419 m(C ¼ C)conjugated alkenes (m), 1342 m(C–H)banding
(m), 3294 m(N–H), 1319 m(C–N), 1257 m(C–C)alkanes (s), 1080
m(C–S–C)stre.
(w) cmꢁ1; UV–Vis (DMSO, c ¼ 10ꢁ4 mol
(thiophene ring)
dmꢁ3): kmax (e) ¼ 257 (35,700), 334 (10,610) nm (molꢁ1 dm3 cmꢁ1); MS
(70 eV): m/z ¼ 377 (M)þ, 379 (M þ 2), 381 (M þ 4); Anal. Calc. For
C17H14Cl2N4S: C, 54.12; H, 3.74; N, 14.85; S, 8.50%; Found: C, 54.06; H,
3.65; N, 14.70; S, 8.30%.
Analytical data of 5-(5-bromothiophen-2-yl)-50-chloro-30-methyl-10-phenyl-10H,
4H-3,40-bipyrazole (L2, C17H14BrClN4S). Yield: 94%; Colour: yellowish pow-
1
der; mol. wt.: 421.74 g/mol; M.p.: 160 ꢂC; H NMR (400 MHz, DMSO-d6):
d ¼ 9.518 (1H, s, N–H), 8.111–7.012 (7H, m, Ar-H3’,4’,2”’,3”’,4”’,5”’,6”’), 5.230
(1H, dd, 3J1 ¼ 5.6 Hz, 3J2 ¼ 7.2 Hz, H5), 3.707 (1H, dd, 3J1 ¼ 11.2 Hz,
3
3
3J2 ¼ 9.6 Hz, Hb), 2.936 (1H, dd, J1 ¼ 9.6 Hz, J2 ¼ 8.8 Hz, Ha), 1.670 (3H, s,
pyrazole-CH3) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 155.60 (C3,
Cquat.), 147.13 (C3”, Cquat.), 137.20 (C1”’, Cquat.), 130.36 (C4’, –CH), 129.39
(C3”’,5”’,–CH), 129.00 (C5”, Cquat.), 128.92 (C3’, –CH), 127.00 (C2’, Cquat.),
126.25 (C4”’, –CH), 124.86 (C2”’,6”’, –CH), 119.00 (C4”, Cquat.), 114.25 (C5’,
Cquat.), 42.85 (C4, –CH2), 40.90 (C5, –CH), 12.24 (–CH3, pyrazole) ppm
[Total signal observed ¼15: signal of C and CH2 ¼ 8 (thiophene-C ¼ 2, pyr-
azole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole-CH2 ¼ 1), signal of CH and
CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH ¼1, phenyl ring-CH ¼3, pyra-
ꢀ
zole-CH3 ¼ 1]; IR (KBr): V¼ 2923 m(¼C–H)ar (w), 1566 m(C ¼ N) (s),
1450-1504 m(C ¼ C)conjugated
(m), 1373 m(C–H)banding (m), 3271
m(N–H), 1334 m(C-N), 1211 m(C–C)alkanes (s), 1064 m(C-S-C)stre. (thiophene
alkenes
(w) cmꢁ1; UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 264
ring)
(7,040), 340 (3,950) nm (molꢁ1 dm3 cmꢁ1); MS (70 eV): m/z ¼ 421 (M)þ,
423 (M þ 2); Anal. Calc. For C17H14BrClN4S: C, 48.42; H, 3.35; N, 13.28; S,
7.60%; Found: C, 48.16; H, 3.31; N, 13.22; S, 7.23%.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Analytical data of 5-(3-bromothiophen-2-yl)-50-chloro-30-methyl-10-phenyl-10H,
4H-3,40-bipyrazole (L3, C17H14BrClN4S). Yield: 91%; Colour: yellowish pow-
1
der; mol. wt.: 421.74 g/mol; M.p.: 165 ꢂC; H NMR (400 MHz, DMSO-
d6): d ¼ 9.513 (1H, s, N–H), 8.117–7.023 (7H, m, Ar-H4’,5’,2”’,3”’,4”’,5”’,6’’’),
3
3
3
5.231 (1H, dd, J1 ¼ 9.6 Hz, J2 ¼ 5.2 Hz, H5), 3.708 (1H, dd, J1 ¼ 11.6 Hz,
3J2 ¼ 6.4 Hz, Hb), 2.937 (1H, dd, 3J1 ¼ 10.4 Hz, 3J2 ¼ 7.6 Hz, Ha), 1.679
(3H, s, pyrazole-CH3) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 155.66
(C3, Cquat.), 147.13 (C3”, Cquat.), 137.26 (C1”’, Cquat.), 129.37 (C3”’,5”’,
–CH), 129.00 (C5”, Cquat.), 126.27 (C4”’, –CH), 124.84 (C2”’,6’’’, –CH),
123.80 (C5’, –CH), 122.90 (C4’, –CH), 122.26 (C2’, Cquat.), 119.00 (C4”,
Cquat.), 110.46 (C3’, Cquat.), 42.18 (C4, –CH2), 40.97 (C5, –CH), 12.23
(–CH3, pyrazole) ppm [Total signal observed ¼ 15: signal of C and
CH2 ¼ 8 (thiophene-C ¼ 2, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole-
CH2 ¼ 1), signal of CH and CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH
ꢀ
¼1, phenyl ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr): V¼ 3031
m(¼C–H)ar (w), 1581 m(C ¼ N) (s), 1488-1519 m(C ¼ C)conjugated
alkenes
(m), 1396 m(C–H)banding (m), 3294 m(N–H), 1280 m(C-N), 1195
m(C–C)alkanes (s), 1064 m(C-S-C)stre.
(w) cmꢁ1; UV–Vis
(thiophene ring)
(DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 256 (21,940), 335 (13,010) nm
(molꢁ1 dm3 cmꢁ1). MS (70 eV): m/z ¼ 421 (M)þ, 423 (M þ 2); Anal.
Calc. For C17H14BrClN4S: C, 48.42; H, 3.35; N, 13.28; S, 7.60%; Found:
C, 48.36; H, 3.29; N, 13.10; S, 7.39%.
Analytical data of 50-chloro-30-methyl-10-phenyl-5-(thiophen-2-yl)-10H,4H-3,40-
bipyrazole (L4, C17H15ClN4S). Yield: 96%, Colour: yellowish powder; mol.
1
wt.: 342.85 g/mol; M.p.: 160 ꢂC; H NMR (400 MHz, DMSO-d6): d ¼ 9.520
(1H, s, N–H), 8.110–7.020 (8H, m, Ar-H3’,4’,5’,2”’,6”’,3”’,5”’,4”’), 5.231 (1H, dd,
3
3
3
3J1 ¼ 9.2 Hz, J2 ¼ 6.0 Hz, H5), 3.707 (1H, dd, J1 ¼ 11.6 Hz, J2 ¼ 7.6 Hz,
3
3
Hb), 2.938 (1H, dd, J1 ¼ 10.4 Hz, J2 ¼ 4.4 Hz, Ha), 1.688 (3H, s, pyrazole-
CH3) ppm; 13C NMR (100 MHz, DMSO-d6): d ¼ 155.60 (C3, Cquat.), 147.14
(C3”, Cquat.), 137.28 (C1”’, Cquat.), 129.35 (C3”’,5”’, –CH), 129.00 (C5”, Cquat.),
127.40 (C3’, –CH), 127.00 (C4’, –CH), 126.27 (C4”’, –CH), 125.84 (C5’,
–CH), 124.80 (C2”’,6”’, –CH), 124.00 (C2’, Cquat.), 119.00 (C4”, Cquat.), 42.88
(C4, –CH2), 40.93 (C5, –CH), 12.25 (–CH3, pyrazole) ppm [Total signal
observed ¼15: signal of C and CH2 ¼ 7 (thiophene-C ¼ 1, pyrazole-C ¼ 4,
phenyl ring-C ¼ 1, pyrazole-CH2 ¼ 1), signal of CH and CH3 ¼ 8 (thio-
phene-CH ¼3, pyarazole-CH ¼1, phenyl ring-CH ¼3, pyrazole-CH3 ¼ 1];
ꢀ
IR (KBr): V¼ 2923 m(¼C–H)ar (w), 1558 m(C ¼ N) (s), 1481–1518
m(C ¼ C)conjugated alkenes (m), 1380 m(C–H)banding (m), 3263 m(N–H), 1280
m(C-N), 1118 m(C–C)alkanes (s), 1064 cmꢁ1 m(C-S-C)stre. (thiophene ring) (w);
UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 256 (28,120), 334 (15,760)

8
M. LUNAGARIYA ET AL.
Scheme 2. Synthesis of the 1, 3, 5-trisubstituted bipyrazole derivatives ligands coordinated to
platinum metal to formed platinum(II) complexes (I–IV).
nm (molꢁ1 dm3 cmꢁ1); MS (70 eV): m/z ¼ 342 (M)þ, 344 (M þ 2); Anal.
Calc. For C17H15ClN4S: C, 59.56; H, 4.41; N, 16.34; S, 9.35%; Found: C,
59.06; H, 4.21; N, 16.20; S, 9.19%.
General synthesis of platinum(II) complexes (I–IV)
Mononuclear heteroleptic platinum(II) complexes (I–IV) with aromatic 1,
3, 5-trisubstituted bipyrazole derived bidentate ligands having –N, S
-donor atoms were synthesized using method proposed by Hodges and
Rund.[14] The neutral platinum(II) complexes (I–IV) were synthesized by
refluxing 1:1 ratio of ligand (L1–L4) (0.25 mmol) and K2PtCl4 (0.25 mmol)
in water–methanol system (50 mL) at 60 ꢂC with 1–2 drops of hydro-
chloric acid (free acid is to avoid displacement of Cl\bar by –OH) until
the solution became colorless (0.5–6 hours). Reaction mixture was allowed
to cool at room temperature. The obtained product was washed with hot
water and dried under vacuum. This procedure was reported in the litera-
ture.[15] The proposed reaction of platinum(II) complexes is represented
in Scheme 2.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Analytical data of complex [pt(L1)Cl2] (I, C17H14Cl4N4PtS)
Yield: 91%; Colour: brown powder; mol. wt.: 641.26 g/mol; M.p.: >300 ꢂC;
1H NMR (400 MHz, DMSO-d6): d ¼ 10.460 (1H, s, N–H), 7.560–6.816 (7H,
m, Ar-H3’,4’,2”’,6”’,3”’,5”’,4”’), 5.378 (1H, dd, 3J1 ¼ 8.0 Hz, 3J2 ¼ 7.2 Hz, H5),
3
3
3
3.783 (1H, dd, J1 ¼ 12.8 Hz, J2 ¼ 11.2 Hz, Hb), 3.193 (1H, dd, J1 ¼ 7.6 Hz,
3J2 ¼ 7.2 Hz, Ha), 2.191 (3H, s, pyrazole-CH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d ¼ 152.83 (C3, Cquat.), 147.15 (C3”, Cquat.), 139.80 (C3’, –CH),
147.57 (C5’, Cquat.), 137.24 (C1”’, Cquat.), 133.74 (C4’, –CH), 129.37 (C3”’,5”’,
–CH), 129.00 (C5”, Cquat.), 126.25 (C4”’, –CH), 124.85 (C2”’,6”’, –CH), 116.00
(C2’, Cquat.), 54.00 (C5, –CH), 43.02 (C4, –CH2), 119.00 (C4”, Cquat.), 12.29
(–CH3, pyrazole) ppm [Total signal observed ¼15: signal of C and CH2 ¼ 8
(thiophene-C ¼ 2, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole–CH2 ¼ 1),
signal of CH and CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH ¼1, phenyl
ꢀ
ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr): V ¼ 3078 m(¼C–H)ar (w),
1627–1674 m(C ¼ N) (s), 1527–1481 m(C ¼ C)conjugated alkenes (m), 1411–1380
m(C–H)banding (m), 3404 m(N–H), 1296 m(C-N), 1257 m(C–C)alkanes (s), 1018
m(C-S-C)stre.(thiophene
(w), 616 m(N-Pt) (w), 555 m(S-Pt) (s) cmꢁ1;
ring)
UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 266 (37,750), 288
(36,540), 359 (15,940) nm (molꢁ1 dm3 cmꢁ1); MS (70 eV): m/z ¼ 641.38
(M)þ, 643.62 (M þ 2), 645.35 (M þ 4), 647.82 (M þ 6), 649.03 (M þ 8);
Anal. Calc. (%) For C17H14Cl4N4PtS: C, 31.74; H, 2.19; N, 8.71; Pt, 30.33;
S, 4.98. Found (%): C, 31.70; H, 2.30; N, 8.67; Pt, 30.28; S, 4.89; Molar con-
ductance: 20 Xꢁ1 cm2 molꢁ1.
Analytical data of complex [pt(L2)Cl2] (II, C17H14BrCl3N4PtS)
Yield: 80%; Colour: brown powder; mol. wt.: 687.73 g/mol; M.p.: >300 ꢂC;
1H NMR (400 MHz, DMSO-d6): d ¼ 10.650 (1H, s, N–H), 8.050–7.079 (7H,
m, Ar-H3’,4’,2”’,6”’,3”’,5”’,4”’), 5.791 (1H, dd, 3J1 ¼ 5.2 Hz, 3J2 ¼ 4.8 Hz, H5),
3
3
3
3.818 (1H, dd, J1 ¼ 12.0 Hz, J2 ¼ 11.6 Hz, Hb), 3.174 (1H, dd, J1 ¼ 5.2 Hz,
3J2 ¼ 5.2 Hz, Ha), 2.177 (3H, s, pyrazole-CH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d ¼ 152.82 (C3, Cquat.), 147.17 (C3”, Cquat.), 139.80 (C3’, –CH),
138.93 (C4’, –CH), 137.25 (C1”’, Cquat.), 135.53 (C5’, Cquat.), 129.30 (C3”’,5”’,
–CH), 129.00 (C5”, Cquat.), 126.25 (C4”, Cquat.), 124.84 (C2”’,6”’, –CH), 119.00
(C4”, Cquat.), 116.02 (C2’, Cquat.), 54.09 (C5, –CH), 43.04 (C4, –C), 12.23
(–CH3, pyrazole) ppm [Total signal observed ¼15: signal of C and CH2 ¼ 8
(thiophene-C ¼ 2, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole-CH2 ¼ 1),
signal of CH and CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH ¼1, phenyl
ꢀ
ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr): V¼ 3075 m(¼C–H)ar (w),
1620–1670 m(C ¼ N) (s), 1520–1450 m(C ¼ C)conjugated alkenes (m), 1400–1380
m(C–H)banding (m), 3415 m(N–H), 1290 m(C-N), 1250 m(C–C)alkanes (s), 1028
m(C-S-C)stre.(thiophene
(w), 606 m(N-Pt) (w), 556 m(S-Pt) (s) cmꢁ1;
ring)
UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 255 (21,770), 298

10
M. LUNAGARIYA ET AL.
(21,010), 358 (6,350) (molꢁ1 dm3 cmꢁ1); Anal. Calc. For
C17H14BrCl3N4PtS: C, 29.69; H, 2.05; N, 8.15; Pt, 28.37; S, 4.66%; Found:
C, 29.56; H, 2.18; N, 8.37; Pt, 28.18; S, 4.24%; Molar conductance: 21 Xꢁ1
cm2 molꢁ1.
Analytical data of [pt(L3)Cl2] (III, C17H14BrCl3N4PtS)
Yield: 85%; Colour: brown powder; mol. wt.: 687.73 g/mol; M.p.: >300 ꢂC;
1H NMR (400 MHz, DMSO-d6): d ¼ 10.496 (1H, s, N–H), 7.564–6.818
3
3
(7H, m, Ar-H4’,5’,2”’,6”’,5”’,3”’,4”’), 5.378 (1H, dd, J1 ¼ 9.6 Hz, J2 ¼ 7.6 Hz, H5),
3
3
3
3.783 (1H, dd, J1 ¼ 14.8 Hz, J2 ¼ 9.2 Hz, Hb), 3.194 (1H, dd, J1 ¼ 6.8 Hz,
3J2 ¼ 6.8 Hz, Ha), 2.198 (3H, s, pyrazole-CH3) ppm; 13C NMR (100 MHz,
DMSO-d6): d ¼ 152.84 (C3, Cquat.), 147.16 (C3”, Cquat.), 139.87 (C4’, –CH),
137.20 (C1”’, Cquat.), 129.68 (C3’, Cquat.), 129.30 (C3”’,5”’, –CH), 129.00 (C5”,
Cquat.), 126.28 (C4”’, –CH), 124.89 (C2”’,6”’, –CH), 119.00 (C4”, Cquat.), 116.00
(C5’, –CH), 115.00 (C2’, Cquat.), 54.05 (C5, –CH), 42.04 (C4, –CH2), 12.26
(–CH3, pyrazole) ppm [Total signal observed ¼15: signal of C and CH2 ¼ 8
(thiophene-C ¼ 2, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyrazole-CH2 ¼ 1),
signal of CH and CH3 ¼ 7 (thiophene-CH ¼2, pyarazole-CH ¼1, phenyl
ꢀ
ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr): V¼ 3078 m(¼C–H)ar (w),
1627–1670 m(C ¼ N) (s), 1527–1480 m(C ¼ C)conjugated alkenes (m), 1411–1370
m(C–H)banding (m), 3408 m(N–H), 1296 m(C-N), 1255 m(C–C)alkanes (s), 1010
m(C-S-C)stre.(thiophene
(w), 615 m(N-Pt) (w), 555 m(S-Pt) (s) cmꢁ1;
ring)
UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 256 (37,070), 296
(32,940), 366 (7,840) nm (molꢁ1 dm3 cmꢁ1); Anal. Calc. For
C17H14BrCl3N4PtS: C, 29.69; H, 2.05; N, 8.15; Pt, 28.37; S, 4.66(%); Found:
C, 29.60; H, 2.08; N, 8.07; Pt, 28.23; S, 4.20%; Molar conductance: 19 Xꢁ1
cm2 molꢁ1.
Analytical data of [pt(L4)Cl2] (IV, C17H15Cl3N4PtS)
Yield: 82%; Colour: brown powder; mol. wt.: 606.81 g/mol; M.p.: >300 ꢂC;
1H NMR (400 MHz, DMSO-d6): d ¼ 10.858 (1H, s, N–H), 8.054–7.078
3
3
(8H, m, Ar-H3’,4’,5’,2”’,6”’,3”’,5”’,4”’), 5.794 (1H, dd, J1 ¼ 10.0 Hz, J2 ¼ 6.4 Hz,
H5), 3.818 (1H, dd, 3J1 ¼ 10.0 Hz, 3J2 ¼ 13.2 Hz, Hb), 3.171 (1H, dd,
3
3J1 ¼ 7.2 Hz, J2 ¼ 5.2 Hz, Ha), 2.157 (3H, s, pyrazole-CH3) ppm; 13C NMR
(100 MHz, DMSO-d6): d ¼ 152.88 (C3, Cquat.), 147.10 (C3”, Cquat.), 139.85
(C3’, –CH), 139.06 (C4’, –CH), 137.23 (C1”’, Cquat.), 129.32 (C3”’,5”’, –CH),
129.05 (C5”, Cquat.), 126.28 (C4”’, –CH), 124.87 (C2”’,6”’, –CH), 119.03 (C4”,
Cquat.), 118.01 (C2’, Cquat.), 116.14 (C5’, –CH), 54.00 (C5, –CH), 43.05 (C4,
–CH2), 12.22 (–CH3, pyrazole) ppm [Total signal observed ¼15: signal of
C and CH2 ¼ 7 (thiophene-C ¼ 1, pyrazole-C ¼ 4, phenyl ring-C ¼ 1, pyra-
zole-CH2 ¼ 1), signal of CH and CH3 ¼ 8 (thiophene-CH ¼3, pyarazole-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
ꢀ
CH ¼1, phenyl ring-CH ¼3, pyrazole-CH3 ¼ 1]; IR (KBr): V¼ 3070
m(¼C–H)ar (w), 1643–1674 m(C ¼ N) (s), 1527–1465 m(C ¼ C)conjugated alkenes
(m), 1411 m(C–H)banding (m), 3425 m(N–H), 1296 m(C-N), 1218
m(C–C)alkanes (s), 1010 m(C-S-C)stre.(thiophene ring) (w), 612 m(N-Pt) (w), 555
m(S-Pt) (s) cmꢁ1; UV–Vis (DMSO, c ¼ 10ꢁ4 mol dmꢁ3): kmax (e) ¼ 240
(31,850), 296 (13,100), 357 (3,740) nm (molꢁ1 dm3 cmꢁ1). Anal. Calc. For
C17H15Cl3N4PtS: C, 33.65; H, 2.16; N, 9.23; Pt, 32.15; S, 5.28%; Found (%):
C, 33.50; H, 2.03; N, 9.16; Pt, 32.02; S, 5.12; Molar conductance: 24 Xꢁ1
cm2 molꢁ1.
Biological application of the synthesized compounds
In vitro antibacterial study
To study the antibacterial activity of compounds under investigation, we
used five microorganism: two Gram(þve) (Bacillus subtilis (MTCC 7193)
and Staphylococcus aureus (MTCC 3160)) and three Gram(–ve) (Escherichia
coli (MTCC 433), Pseudomonas aeruginosa (MTCC P-09) and Serratia mar-
cescens (MTCC 7103)). DMSO was used as medium to acquire preferred
concentration of compounds to check upon microbial strains. MIC was
performed by broth dilution technique. A prelature of bacteria was used to
prepare bacterial cultures in Luria Broth. The test compounds of specific
concentration were added to the corning tube containing Luria Broth solu-
tion and sterilized before the addition of previously cultured bacterial spe-
cies. After adding different bacterial species to the tubes containing a
specific concentration of test compounds, it was allowed to incubate for
24 hours at optimum temperature. The experiment is repeated till only a
faint turbidity appears in the tube. The lowest concentration, which showed
no visible growth after 24 hours subculture was considered as MIC for
each compound.[15]
In vitro cytotoxicity against brine shrimp lethality bioassay
The treatment of cancer is limited to surgery, radiotherapy and the use of
cytotoxic agents, regardless of their well-known side effects. However, as no
curative therapy is available and the discovery and development of novel,
active chemotherapeutic agents is desperately needed. For the development
of new drugs, primary study involves the brine shrimp lethality bioassay.
Cytotoxicity assays are widely used by the pharmaceutical industry to screen
for cytotoxicity in compound libraries. Researchers can look for cytotoxic
compounds, if they are interested in developing a therapeutic agent that tar-
gets cancer cells. The brine shrimp lethality assay is a method by which one
can check the toxicity of the compounds on brine shrimp. The experiment
was carried out following the protocol of Meyer et al.[16] Brine shrimp

12
M. LUNAGARIYA ET AL.
(Artemia cysts) eggs were hatched in a shallow rectangular plastic dish
(22 ꢀ 32 cm), filled with artificial seawater, which was prepared with com-
mercial salt mixture and double distilled water. An unequal partition was
made in the plastic dish with the help of a perforated device. A set of 2, 4, 8,
16, 20 mg/cm3 in the test tube was prepared from a stock solution of
1000 mg/cm3 of the test compounds. The final volume in each test tube was
adjusted to 2500 mdm3 (1000 mdm3 sea salt solution, 450 mdm3 double dis-
tilled water, 50 mdm3 DMSO þ complex and remaining 1000 mdm3 were
added as 10 nauplii in a sea salt solution). After 24 hours number of dead
nauplii were counted and from the survival % mortality was calculated.[17]
In vitro cellular level cytotoxicity against Schizosaccharomyces pombe cells
Cellular level bioassay was carried out using S. pombe cells, which were
grown in liquid yeast extract media in 150 cm3 Erlenmeyer flask containing
30 cm3 of yeast extract media. The flask was incubated at 30 ꢂC on a shaker
at 160 rpm till the exponential growth of S. pombe obtained (24–30 hours).
Then the cell culture was treated with the differ concentrations (20, 40, 60,
80, 100 mM) of synthesized complexes, free ligands and also with DMSO as
a control and further allowed to grow for 20–24 hours. Next day, by centri-
fugation at 12,000 rpm 10 minutes; treated cells were collected and dissolved
in 500 cm3 of PBS (Phosphate Buffered Saline). The 80 cm3 of yeast culture
dissolved in PBS and 20 cm3 of 0.4% trypan blue prepared in PBS were
mixed and cells were observed in a compound microscope (40ꢀ). The try-
pan 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.[18]
DNA binding studies
Electronic absorption titration spectroscopy. Electronic absorption titration
was employed to study the relative binding mode of HS-DNA with mole-
cules. The stock solutions of HS-DNA were prepared in phosphate buffer
and stored at 4 ꢂC for <4 days. The UV absorbance of HS-DNA at 260 and
280 nm in phosphate buffer (Na2HPO4/NaH2PO4, pH ¼7.2) solution at
room temperature gave a ratio of 1.8–1.9:1, signifying that the DNA was
suitably free from proteins.[19] The molar absorption coefficient of HS-
DNA was taken as 12858 Mꢁ1 cmꢁ1. Absorption titration experiments were
performed by maintaining constant complex concentration and varying the
concentration of HS-DNA. Complex-DNA solutions were allowed to be
incubated for 10 minutes at room temperature before measurements were

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
taken. While measuring the absorption spectra, an equal quantity of HS-
DNA was added to both the complex solution and the reference solution to
eliminate the absorbance of HS-DNA itself. A compound due to have anti-
cancer activity binds the DNA in following distinct binding modes. (i)
Electrostatic binding in which compound attacks the phosphate groups
attached to DNA backbone on its external helical side. (ii) Groove binding
in which compound attacks two grooves of DNA double helix and (iii)
intercalation mode of binding in which compound is inserted between the
stacked base pairs of DNA.[20] Upon interaction of compound with DNA,
alteration arises in the helical structure of DNA. This causes hyperchromic
effect and hypochromic effect which are the spectral features of DNA con-
cerning its double helix structure. The intrinsic binding constant Kb can be
obtained using the following equation in literature.[21]
Viscosity measurements. Viscosity experiment was made using an Ubbelohde
viscometer, engrossed in a water bath at 25.0 0.5 ꢂC. The viscosity of a
200 mM solution of HS-DNA was determined in the presence of the com-
plexes using different [complex]/[DNA] ratios in the range of 0.00–2.00.
The flow time was measured in triplicate with a digital stopwatch and then
averaged. The relative viscosity ratio (/0) was plotted against the r-bound
(where and were the relative viscosity for DNA in the presence or
0
absence of complexes, respectively). The hydrodynamic length of DNA gen-
erally increases upon partial intercalation while it does not lengthen upon
groove binding.[22] 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.[23]
The data are represented as the plot of the relative viscosity, i.e. (/0)1/3 ver-
sus [complex]/[DNA].
Fluorescence quenching analysis. Fluorescence spectra were recorded using a
FluoroMax-4, spectrofluorometer, HORIBA (Scientific) at 25ꢂC in the wave-
length range 500–800 nm. Excitation wavelength of the sample is 610 nm
with scan speed of 300 nm/min and dimensions of excitation and emission
slit was 1.45nm in width. Fluorescence titration studies were performed in
phosphate buffer solution by keeping the concentration of each compound
constant (10ꢁ5 M) while varying the DNA concentration (0–10ꢁ3 M) to
achieve ratios R ¼ [compound]/[DNA] of 0.3, 0.6, 1.0, 1.33, 1.66, 2.0, 2.33,
2.66, 3.0, 3.33 order . This procedure is reported in literature.[15]
Molecular docking 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. Pt(II)

14
M. LUNAGARIYA ET AL.
complexes were taken from their enhanced structure as a molecule and
converted to .pdb (Protein Data Bank) format using CHIMERA 1.5.1 soft-
ware. 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 irre-
placeable 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 compu-
tational 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
[24]
from the Protein Data Bank (http://www.rcsb.org/pdb).
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 conform-
ation was selected with the lowest binding energy (kJ/mol).[25]
Gel electrophoresis bioassay
Gel electrophoresis study was performed using pUC19 DNA with synthe-
sized compounds. The samples were incubated for 0.5 hour at 37 ꢂC. The
samples were analyzed by 1% agarose gel electrophoresis
[Tris–acetate–ethylenediaminetetraacetic acid, (TAE) buffer, pH 8.0] for
3 hours at 100 mV. The gel was stained with (0.5 mg mLꢁ1) 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.[26]
ð
Þ
ð
Þ
% of SC DNA controlꢁ % of SC DNA sample
DNA cleavage activity % ¼
ꢀ 100
ð
Þ
% of SC DNA control
Results and discussion
UV–visible spectroscopy, magnetic moment measurements and molar
conductivity measurements
The UV–vis spectra of the synthesized compounds were recorded in DMSO
solvent. The absorption bands in the range of 289–313 nm which is attrib-
uted to metal to ligand charge transfer (MLCT) and d–d transition.[27] The
d–d transition band of platinum (II) complexes are observed in the range of
356–396 nm. The UV–Visible spectrum of the platinum(II) complex IV is

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Figure 1. UV–Visible spectrum of the platinum(II) complex (IV).
represented in Figure 1. Square planner geometry is confirmed by UV–Vis
spectroscopy. Magnetic susceptibility (meff.) measurements of the
platinum(II) complexes (I–IV) are carried out in solid state and shows dia-
magnetic character. The molar conductivity of platinum(II) complexes were
performed in DMSO solvent at room temperature which indicate that all the
complexes have non-electrolytic nature (Km ¼ 19–24 Xꢁ1 cm2 molꢁ1).
FT-IR spectroscopy
In FT-IR spectral study, the frequency of the free ligands (L1–L4) contain-
ing azomethine m(>C ¼ N) and m(>C ¼ C)conjugated alkene functionality are
assign in between 1616 and 1480 cmꢁ1. Upon the complexes these bands
are shifted in the range of 1542–1419 cmꢁ1. The N–H band of the free
ligands (L1–L4) are obtained in between 3263 and 3294 cmꢁ1. Whereas
platinum complexes shows N–H bands in the range of 3404–3425 cmꢁ1. In
platinum(II) complexes, the m(N-Pt) and m(S-Pt) bands are in the range of
606–616 and 555–556 cmꢁ1, respectively.
LC-MS spectrum
The mass spectrum of the platinum(II) complex (I) shows molecular ion peaks
at m/z equal to 641.38, 643.62, 645.35, 647.82 and 649.03 assigned to Mþ,
(Mþ 2), (M þ 4), (M þ 6) and (M þ 8) values, respectively due to the presence
of four chlorine atoms. It also confirms that two chlorine atoms are attached
to platinum metal atom through covalent bond and other two chlorine atoms
are present in thiophene and pyrazole ring. Mass spectral data of free ligands
(L1–L4) are represented in experimental section and supplementary material 1.

16
M. LUNAGARIYA ET AL.
Figure 2. LC-MS spectrum of platinum(II) complex (I).
Mass spectral graph of the complex (I) is presented in Figure 2. The peak
appearing at m/z value equal to 605.63, 607.26, 609.56, 611.27 are assigned to
(M), (M þ 2), (M þ 4) and (M þ 6) respectively which indicate that the loss of
the one chlorine atom. The peak observed at m/z value equal to 570.80, 572.63
and 574.28 is assigned to (M), (M þ 2) and (M þ 4) respectively that indicate
the loss of two chlorine molecule. The peak of the ligands are obtained at m/z
value equal to 375.20 (M), 377.18 (M þ 2) and 379.78 (M þ 4) due to loss of
the platinum metal ion. Some other fragments of compounds are observed at
258.28, 260.38 and 68.15 m/z values.
1H NMR spectroscopy
1H NMR spectra of the ligands (L1–L4) and its metal complexes (I–IV)
1
were measured in DMSO d6 solvent. The H NMR spectrum of the ligand
shows signal in range of d 1.670–1.692 ppm corresponding to the signals of
pyrazole(–CH3) and these peak is shifted downfield in the range of d
2.157–2.198 ppm in case of the platinum(II) complexes. The sharp singlet
proton peak is observed at about d 9.513–9.520 ppm due to the N–H pro-
ton of the ligands (L1–L4) whereas, N–H proton peaks are shifted in down-
field at d 10.460–10.858 ppm values in case of complexes. The signal of
pyrazole moiety containing Ha, Hb and H5 protons are appearing in the
range of d 3.707–5.231 ppm and in case of complexes these are observed at
d 3.783–3.818 ppm value. The signal for the aromatic proton of
platinum(II) complexes (I–IV) and ligands (L1–L4) appears at d
6.818–8.054 and d 7.011–8.117 ppm, respectively. The aromatic protons of
phenyl and thiophene rings are shifted upfield with respect to free ligands,
1
indicating that the Pt-N and Pt-S bonds are formed in the complexes. H

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
NMR spectra of the ligands and complexes are represented in supplemen-
1
tary material 2 and H NMR spectral data of the compounds (L1–L4 and
I–IV) are shown in experimental section.
13C NMR spectroscopy
In 13C NMR spectrum of ligand, pyrazole containing –CH2 signal is
observed at d 42.18–42.88 ppm due to the ligands and in case of the com-
plexes it is observed at d 42.02–43.04 ppm. In ligands, the imine (>C ¼ N)
carbon atom attached with C3 position of thiophene ring is observed at d
155.60–155.66 ppm and is shifted downfield at d 155.82–155.88 ppm value
in case of complexes. The aromatic carbon atoms of the ligands and com-
plexes are in range of d 147.12–110.46 and 147.15–115.00 ppm, respectively.
13C NMR spectra of the ligands and complexes are represented in supple-
mentary material 3 and spectral data are illustrated in experimental section.
Thermo-gravimetric analysis (TGA)
The thermal behavior of the platinum(II) complex (I) was studied by the
thermo-gravimetric analysis (TGA) under dinitrogen atmosphere at a scan-
ning rate of 10 ꢂC minꢁ1. Furthermore thermal curve is represented in sup-
plementary material 4. There is no weight loss up to temperature 210 ꢂC it
means that there is absence of water molecule in complex. The decompos-
ition of the platinum(II) complex(I) is divided in two steps, first loss occur-
ring at temperature range of 220–260 ꢂC showing 10.28% weight loss
corresponding to chlorine molecule (Cl2) whereas second loss occurring in
between temperature range of 290–500 ꢂC is observed at 61.46% due to the
decomposition of quinoline based 1,3,5-tri-substituted pyrazole ligand and
leaving behind platinum metal (Pt) as a residue.[28] These results suggest
the platinum(II) complexes have high thermostability.
Biological application of synthesized compounds
In vitro antibacterial assay
The in vitro antibacterial activity of all the newly synthesized ligands
(L1–L4) and complexes (I–IV) were evaluated by broth dilution tech-
nique.[29] The minimum inhibitory concentration (MIC, mM) were deter-
mine, and concentration of ligands and complexes were taken in the range
of 215–260 and 25–90 mM, respectively. The in vitro antibacterial activities
of the synthesized ligands and complexes (L1–L4 and I–IV) are carried out
using three Gram(ꢁve) and two Gram(þve) microorganism as listed in
Figure 3 and data of these compounds are given in supplementary material
5. From the Figure 3, it is clear that all the platinum(II) complexes exhibit

18
M. LUNAGARIYA ET AL.
Figure 3. Minimum inhibitory concentration of synthesized ligands and platinum(II) complexes
against Gram(ꢁve) and Gram(þve) five different microorganism. Error bar are a representation of
the experiment repeated in three times and STD 5%.
Figure 4. Plot of LC50 values of different compounds in mg/cm3 using brine shrimp to deter-
mine the cytotoxic effects. Error bars represent the standard deviation of three replicates 5%.
excellent activity compared to synthesized ligands and K2PtCl4 salt. MIC
values of complexes increases due to chelation effect.[30] Furthermore, all
the compounds are more potent against Gram(ꢁve) bacteria than the
Gram(þve) bacteria because most of pathogenic bacteria represent

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
Gram(ꢁve) group. Complexes (I, II, III) containing 5-chloro, 5-bromo and
3-chloro functional group have good potency as compared to other com-
plex (IV).
In vitro cytotoxicity against brine shrimp lethality bioassay
The in vitro cytotoxicity study of the synthesized ligands (L1–L4) and com-
plexes (I–IV) was carried out using the protocol of Meyer et al.[16]
Platinum(II) complexes (I–IV) exhibit excellent activity as compared to
1,3,5-tri-substituted bipyrazole derived ligands (L1–L4). The mortality rate of
nauplii is found to increase with increasing concentration of synthesized
ligands and complexes. The 50% lethal concentration (LC50, mg/cm3) values
of ligands, complexes, cisplatin and transplatin are represented in Figure 4.
The in vitro cytotoxicity order of the synthesized compounds is
cisplatin > I > II > III > IV > transplatin > L1 > L2 > L3 > L4. Cisplatin and
transplatin LC50 values are obtained at 3.133 and 14.45 mg/cm3, respectively.
In vitro cellular level cytotoxicity against S. Pombe cells
The in vitro cellular level cytotoxicity of the synthesized ligands and com-
plexes was carried out using S. Pombe cells. From the results, cell death
due to toxicity of the synthesized compounds could be easily monitored by
trypan blue dye as a staining agent. The toxicity is found to vary with the
different type of functional group (–Cl, –Br, –H) present and differing con-
centrations of the synthesized ligands and platinum(II) complexes.
Complexes I, II and III are found to have excellent toxicity as compared to
Figure 5. The percentage viability of compounds at different concentrations with error uncer-
tainty in the value 5%.

20
M. LUNAGARIYA ET AL.
Figure 6. Electronic absorption spectra of ligand (L1) (400 mM) and platinum(II) complex
(I) (400 mM) phosphate buffer (Na2HPO4/NaH2PO4, pH ¼7.2) in the absence and in the presence
of increasing amounts of HS-DNA. Arrows show the changes in absorbance with increasing
HS-DNA concentration. Inset: plot of [DNA]/(eaꢁef) versus [DNA].
complex IV. Platinum(II) complexes are more toxic in nature as compared
to 1,3,5-tri-substituted bipyrazole derivative ligands (L1–L5). The toxicity of
synthesized platinum(II) complexes are comparable to cisplatin and trans-
platin drug. The cytotoxicity potency order of the synthesized compounds
are found to be cisplatin > transplatin > I > II > III > IV > L1 > L2 > L3 > L4
(Figure 5). After 17–20 hours of the treatment, many of the S. Pombe cells
are dead due to toxic nature of the complexes. Cellular level cytotoxicity
data are represented in supplementary material 6.
DNA binding studies
Electronic absorption titration. The electronic absorption spectra of the syn-
thesized ligands (L1–L4) and platinum(II) complexes (I–IV) with HS-DNA
are shown in Figure 6. Electronic absorption spectra of the synthesized
ligands (L1–L4) and platinum(II) complexes (I–IV) are studied upon add-
ition of increasing amounts of HS-DNA to determine the binding modes
and binding extent of metal complexes with DNA. The absorption intensity
of the compounds are decreases (hypochromism) with a slight increase in
absorption wavelength (bathochromism) upon the addition of HS-DNA.
Overall, it is assumed that hypochromism and bathochromism are related
with the partial intercalative due to stacking interaction between aromatic
chromophore and the base pair of DNA. The intrinsic binding constant
(Kb) for the association of the compound-DNA is determined from the

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
Figure 7. (a) The effect of increasing amounts of ligands (L1–L4) on the relative viscosity of HS-
DNA at 27 ( 0.1)ꢂC in phosphate buffer at pH ¼7.2. (b) The effect of increasing amounts of
complexes (I–IV) on the relative viscosity of HS-DNA at 27 ( 0.1)ꢂC in phosphate buffer at pH
¼7.2. Error bars represent the standard deviation of three replicates.
spectroscopic titration data using the following Eq. (1).[14]
½
ꢃ
½
ꢃ
DNA
DNA
1
¼
þ
(1)
ð
Þ
ð
Þ
ð
Þ
ea ꢁ ef
eb ꢁ ef
Kb eb ꢁ ef
where, eb and ef are the extinction coefficients of fully bound and free
(unbound) compounds, respectively. ea is the apparent extinction coeffi-
cient. A plot of [DNA]/(eaꢁef) versus [DNA] will give a slope of 1/(ebꢁef)
and an intercept of 1/Kb(ebꢁef) (inset of the Figure 4). Binding constant
(Kb) and Gibb’s free energy (DG0) of the synthesized compounds are
observed in the range of 0.231–3.130 ꢀ 105 Mꢁ1 and ꢁ31.351 to ꢁ24.893
kJmolꢁ1, respectively. Percentage hypochromism values of synthesized com-
pounds are found to be 17.56–33.94%. The Kb value suggests modest DNA
partial intercalative binding affinity for the complex.
Viscosity measurement study. To explore further the interaction between the
ligands (L1–L4) and platinum(II) complexes (I–IV) and DNA, viscosity
measurements were carried out with HS-DNA by varying the concentration
of the compounds. The effects of the ligands (L1–L4) and platinum(II)
complexes (I–IV) on the viscosity of HS-DNA solution are given in
Figure 7. The plot ((g/g0)1/3 versus [Complex]/[DNA]) shows that the
relative viscosity of the HS-DNA increases upon the addition of varying
concentration of complexes suggesting partial intercalative mode of bind-
ing. In general, a partial intercalation model lengthens the DNA helix since
the base pairs of DNA are separated on binding. The binding ability of
compounds with increase in the viscosity of DNA follows the order
I > II > III > IV > L1 > L2 > L3 > L4, which is in agreement with electronic
absorption spectra and fluorescence quenching results.

22
M. LUNAGARIYA ET AL.
Figure 8. Fluorescence emission spectra of EB binds to HS-DNA in the presence of complex (I).
[EB] ¼ 33.3 lM, [DNA] ¼ 10 lM; [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 mM; kex¼ 510nm. The arrows show the intensity changes
upon increasing the concentrations of complex.
Fluorescence quenching analysis. To develop additional information concern-
ing the HS-DNA interaction properties of the synthesized platinum(II)
complexes were carried out using fluorescence improvement experiments.
The binding ability of the synthesized platinum(II) complexes (I–IV) with
HS-DNA are confirmed by ethidium bromide (EB) displacement tests
which confirms competitive binding of platinum(II) complexes with HS-
DNA. Ethidium bromide (EB) is non-fluorescent, but EB-DNA system
shows strong emission intensity being a good intercalating agent. DNA
induced EB fluorescence emission could be quenched after addition of the
complexes capable for strong interactions with HS-DNA replacing EB.[31]
The emission spectra of EB bound to DNA in the absence and presence of
platinum(II) complex (I) are shown in Figure 8. A competitive interaction
of the metal complexes to HS DNA results in the displacement of the
bound EB and inner filter effect (IFE) were carried out by equation in lit-
erature.[32] Therefore, it is needed to eliminate IFE interference with the
results. A competitive interaction of the metal complexes to HS DNA
results in the displacement of the bound EB.[33] Fluorescence quenching
data (Stern–Volmer binding constant, Ksv) of the synthesized platinum(II)
Complexes (I–IV) are examined using the Stern-Volmer Eq. (2) whose val-
ues are given in Table 1 and are presented in supplementary material 7.[34]
I0
¼ 1 þ Ksv ½Qꢃ
(2)
I
where, I0 and I is indicate that the absence and presence of the com-
plexes, Ksv is a Stern–Volmer binding constant, [Q] is the total

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
Table 1. The binding constant (Kb,
M
ꢁ1), % hypochromicity and change in Gibb’s free energy
(DG0, Jmolꢁ1) of platinum(II) complexes (I–IV) and 1,3,5-tri-substituted bipyrazole derivatives
ligands (L1–L4) with HS-DNA at different temperatures.
kmax (nm)
Bound Free
Compounds
Dk (nm)
Kb (Mꢁ1) ꢀ 105
H%
DG (Jmolꢁ1
)
L1
L2
L3
L4
I
II
III
IV
304,302
268,264
265,264
259,257
266,265
266,265
265,260
306,302
2
4
1
2
1
1
5
4
0.686
0.674
0.510
0.231
3.134
1.198
1.070
0.826
33.94
20.48
24.57
32.54
18.93
18.44
17.56
18.89
ꢁ27,590.36
ꢁ27,546.63
ꢁ26,855.84
ꢁ24,893.62
ꢁ31,351.09
ꢁ28,971.68
ꢁ28,691.73
ꢁ28,050.48
H% ¼ [(AfreeꢁAbound)/Afree] ꢀ 100%.
Kb ¼ Intrinsic DNA binding constant determined from the UV–vis absorption spectral titration.
Dk 5 Difference between bound wavelength and free wavelength.
Table 2. Linear Stern–Volmer quenching constants (Ksv,
M
ꢁ1), number of binding sites (n),
change in Gibb’s free energy (DG0, Jmolꢁ1) and associative binding constant (Ka,
complexes (I–IV) with HS-DNA.
M
ꢁ1) of PtII
Complexes
Ksv (Mꢁ1
)
Ka (Mꢁ1
)
n
DG(J molꢁ1
)
I
II
III
IV
4.9 ꢀ 103
3.0 ꢀ 103
2.2 ꢀ 103
1.9 ꢀ 103
3.06 ꢀ 105
2.17 ꢀ 105
1.06 ꢀ 104
0.69 ꢀ 103
1.4216
1.4490
1.2120
0.9406
ꢁ31,297.79
ꢁ30,447.20
ꢁ22,976.73
ꢁ16,209.11
Figure 9. (a) Stern–Volmer plots of the fluorescence titration of platinum(II) complexes (I–IV)
with HS-DNA. Inset graph: plots of I0/I versus [Q], with ꢄ for the experimental data points and
the full line for the linear fitting of the data. (b) Scatchard plots of the fluorescence titration of
platinum(II) complexes (I–IV) with HS-DNA. Comparative plot of log[I0 ꢁ I/I] versus log[complex]
for the titration of HS-DNA EB system with complexes (I) in (Na2HPO4/NaH2PO4, pH ¼7.2) phos-
phate buffer medium.
concentration of quencher (complexes), Ksv can be got by a slope from
the plot of I0/I versus [Q] (Table 2). All plots signify a good linear rela-
tionship, indicating a static quenching mechanism, the formation of a
complex between quencher and fluorophore.[35] Binding constant (Ksv)

24
M. LUNAGARIYA ET AL.
of the complexes are obtained in the range of 4.9–1.9 ꢀ 103 Mꢁ1 and also
represented in Table 2.
The associative binding constant (Ka) and number of binding sites (n)
are calculated by the Scatchard equation Eq. (3).[36]
logðI0ꢁIÞ
¼ logKa þ nlog½Qꢃ
(3)
I
The number of binding site (n) and associative binding constant (Ka)
can be calculated by the slope and the intercept of the double logarithm
curve of log[(I0 ꢁ I)/I] versus log[Q] (Figure 9 and Table 2) The associative
binding constant (Ka) values of the synthesized complexes (I–IV) are in the
range of 0.69 ꢀ 103 to 3.06 ꢀ 105 Mꢁ1. The values of the Ksv and Ka indicate
strong interaction between the HS-DNA and synthesized platinum(II) com-
plexes (I–IV). The calculated value of number of binding site (n) is around
1.0 for all of the platinum(II) complexes which indicate single binding site
in DNA with complexes.
The interaction forces between drugs and biomolecules are namely
hydrogen bonding, hydrophobic interaction, electrostatic attraction and
Van der Waals forces, etc. In order to evaluation the interaction between
HS-DNA and platinum(II) complexes, the change in standard Gibb’s
energy (DG0) for the binding method were calculated by the Van’t Hoff
Eq. (4).[37]
DG0 ¼ ꢁ RTlnKa
(4)
where, R is the gas constant 8.314 J molꢁ1K and T is the temperature
(298 K here). The values of the change in Gibb’s free energy of the
Figure 10. Molecular docking of ligand (L1) and complex (I) (ball and stick) with the DNA
duplex (VDW spheres) of sequence d(ACCGACGTCGGT)2. The complex is docked into the DNA,
complexes and ligands are partial intercalative mode of binding with the DNA base pairs (A ¼ T
and GꢅC).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
25
synthesized platinum(II) complexes (I–IV) are calculated in the range of
ꢁ16.209 to ꢁ31.297 ꢀ 103 kJmolꢁ1 and also summarized in Table 2. The
negative sign for DG0 means that the binding process was spontaneous.
Molecular docking study. Docking study is also in good favor with complex
(I) which shows that the complex (I) energetically and geometrically fits for
binding. Docking energies of ligands (L1–L4) are ꢁ268.37, ꢁ266.91,
ꢁ272.89 and ꢁ264.45 kJmolꢁ1 and that of corresponding complexes (I–IV)
are ꢁ299.78, ꢁ303.29, ꢁ309.87 and ꢁ287.78 kJmolꢁ1, respectively.
Docking images of complex (I) and ligand (L1) binding with B-DNA are
Figure 11. Photogenic view of cleavage of pUC19 DNA (300 mg/cm3) with series of ligands
(L1–L4) and Pt(II) complexes (200 mM) using 1% agarose gel containing 0.5 mg/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 ꢂC.
Figure 12. Plot of nuclease cleavage (% of form I, form II and DNA cleavage) assay of the
ligands (L1–L4) and complexes (I–IV). Error bars represent standard deviation of three replicates.

26
M. LUNAGARIYA ET AL.
shown in Figure 10 whereas docking structures of ligands and complexes
are shown in supplementary material 8 which indicate partial intercalative
binding mode of interaction with DNA. Each complex has good ability of
interaction with DNA comparison to each respective ligand which is clearly
indicated by the energy values. The reason of more effective value of energy
could be attributed to presence of platinum in the complex which interact
effectively with the base pairs of DNA.
Gel electrophoresis bioassay
The DNA cleavage activities of the compounds were examined by pUC19
plasmid DNA in phosphate buffer (pH ¼7.2). Figure 11 shows that upon
increasing complex concentrations, the amount of the supercoiled form of
DNA (Form I) is decreased while the nicked form of DNA (Form II) with
single-strand breaks significantly. The quantification of cleavage experiment
is done from percentage of supercoiled form in test compounds with
respect to control Figure 12. The ratio of percentage of supercoiled form in
test compounds to control is a quantitative measure of % cleavage of
pUC19 DNA. The data of electrophoresis are represented in the supple-
mentary material 9. The order of cleavage efficiency of the compounds is
cisplatin > I > II > III > IV > L1 > L2 > L3 > L4 > transplatin > K2PtCl4.
Conclusion
In summary, a series of new compounds containing 4, 5 dihydropyrazole
center with a phenyl pyrazole moiety have been synthesized and evaluated
for their biological activities such as in vitro cytotoxicity against brine
shrimp lethality bioassay, in vitro cellular level cytotoxicity against S.
Pombe cells, in vitro antibacterial activity, these studies indicate that the
platinum(II) complexes have good toxicity values as compared to ligands
and salts. From the results of these studies it is concluded that complexes
(I, II and III) shows excellent potency compare to complex (IV). In anti-
bacterial study, IC50 values of the synthesized ligands and complexes
exhibit good potency against Gram(ꢁve) bacteria as compared to Gram(þve)
bacteria. DNA interaction stud0ies of the synthesized compounds such as
absorption titration spectroscopy, viscosity measurements, fluorescence
quenching analysis and molecular docking study suggest that the com-
pounds bind with HS-DNA via partial intercalation mode. Binding con-
stant (Kb) of the complexes (I-IV) is higher than the ligands (L1–L4). In
addition, complexes are able to displace EB from the DNA–EB complex
which shows partial intercalative mode of binding.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
27
Acknowledgments and Funding
The authors are thankful to the U.G.C., New Delhi for providing financial assistance of a
UGC BSR grant no. C/2013/BSR/Chemistry/7489 and “BSR UGC One Time Grant”, vide
UGC letter no. F.19-119/2014 (BSR). Head, Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar, Gujarat, India, for providing the laboratory facilities, DST-PURSE, Sardar
Patel University, Vallabh Vidyanagar for mass spectra analysis, P.G. Department of Bioscience,
Vallabh Vidyanagar, Gujarat, India, for providing the biochemistry laboratory facilities, to car-
ried out cellular level cytotoxicity and gel electrophoresis techniques.
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