Synthesis, characterization and biological applications of some substituted pyrazoline based palladium (II) compounds

Article abstract of DOI:10.1002/aoc.4523

Palladium (II) complexes of the type [PdLCl₂] (where L?=?substituted pyrazoline ligands) have been synthesized. The metal complexes (5a-5f) have been characterized by various spectroscopic and analytical techniques like ¹H-NMR,

Full text of DOI:10.1002/aoc.4523

Received: 16 April 2018
DOI: 10.1002/aoc.4523
Revised: 26 June 2018
Accepted: 27 June 2018
F U L L P A P E R
Synthesis, characterization and biological applications of
some substituted pyrazoline based palladium (II)
compounds
Khyati P. Thakor | Miral V. Lunagariya | Bhupesh S. Bhatt
| Mohan N. Patel
Department of Chemistry, Sardar Patel
Palladium (II) complexes of the type [PdLCl2] (where L = substituted
pyrazoline ligands) have been synthesized. The metal complexes (5a‐5f) have
been characterized by various spectroscopic and analytical techniques like
1H‐NMR, 13C‐NMR, LC–MS, IR, Energy‐dispersive X‐ray (EDX), electronic
spectroscopy, thermogravimetric analysis (TGA), elemental analysis and
conductance measurement. Complexes to herring sperm DNA (HS‐DNA) bind-
ing has been explored by absorption titration and binding constant (Kb) as well
as Gibb's free energy have been evaluated. Complex 5b shows the highest
binding constant value. So, thermodynamic parameters of 5b‐HS DNA
complex at different temperature have been evaluated. Also, Viscosity
measurement and molecular modeling studies have been performed to know
the binding mode of complexes. Based on the observations, an intercalative
binding mode of DNA has been proposed. Further confirmation of intercalative
mode of DNA binding has been taken by fluorescence spectroscopy and results
are in a good agreement with absorption titration, viscosity measurement and
molecular modeling data. Antibacterial activity of the complexes has been
screened against pathogenic bacteria such as S. aureus, E. coli, B. subtilis, S.
marcescens and P. aeruginosa. Cytotoxicity is performed on brine shrimp and
S. pombe. Gel electrophoresis assay demonstrates that all the complexes can
cleave the pUC19 plasmid DNA. Anti‐tuberculosis activity has been carried
out using mycobacterium tuberculosis H37Rv bacteria by L.J. Medium
conventional method.
University, Vallabh Vidyanagar 388120,
Gujarat, India
Correspondence
Mohan N. Patel and Bhupesh S. Bhatt,
Department of Chemistry, Sardar Patel
University, Vallabh Vidyanagar 388120,
Gujarat, India.
Email: jeenen@gmail.com;
bhupeshbhatt31@gmail.com
KEYWORDS
cytotoxicity, DNA intercalation, ethidium bromide displacement, free energy, mycobacterium
tuberculosis
1 | INTRODUCTION
functional group is one of the approaches to design mul-
tifunctional metal complexes.[3,4] The site specific DNA
The d‐block metal complexes that cleave DNA under
physiological condition are of current interest in the
development of artificial nucleases.[1,2] Phosphodiester
hydrolysis with the cooperation of metal ions and the
cleavage by newly designed metal complexes allow the
development of new antimicrobial agents as well as
chemotherapeutic agents.[5–7] The metal complexes are
well suited as artificial metallonuclease due to the
Appl Organometal Chem. 2018;e4523.
wileyonlinelibrary.com/journal/aoc
© 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4523

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THAKOR ET AL.
applications associated with different geometries and the
possibility to tune their redox potential by choosing
proper ligands.[8] Many organic molecules have been
designed to consolidate biological functional group with
a suitable metal ion to mimic active sites of metallo-
proteins.[9] Thus, the efficiency of DNA cleavage can be
enhanced through promising coordination compound
typically contain a DNA binding group that fit to either
major groove or minor groove, and/or may act as
intercalator, thereby increasing DNA target ability of the
coordination compounds.[10–13] 4, 5‐Dihydropyrazoles, a
small bioactive molecule, are pre‐eminent structural
design found in numerous pharmaceutically active com-
pounds (antifungal, antibacterial, anti‐tumour, anti‐
inflammatory and antiviral) and agrochemical active
agents.[14–17] The combination of transition metals with
biologically active molecules has also been exploited
showing promising activity due to their unique ability to
bind different biological targets.[18]
Platinum based anticancer drug has several side
effects like nephrotoxicity and drug resistance of tumour
cells, which have generated real challenges to
researchers.[19,20] Several side effect along with limited
applicability to certain cell lines associated with cisplatin
administration have triggered the research in the devel-
opment of other transition metal based drugs.[21–24] Mor-
phology, geometry, electronic state of palladium metal
ion is very similar to platinum metal ion. Also, palladium
based metal complexes have been extensively researched
for diverse biological and pharmacological applica-
tions.[25–34] Thus, platinum analogous palladium com-
pounds have been designed and synthesized, which
show promising activity against tumour cell line.[35]
So to explore biochemistry of palladium metal, we
have synthesized 1,3,5‐trisubstituted pyrazoline ligands
and their chelation with palladium (II) has been carried
out. All synthesized compounds have been screened for
the various biological activity like antibacterial activity,
cytotoxicity, DNA binding and DNA cleavage.
bromophenol blue and xylene cyanol FF were purchased
from Himedia (India). S. pombe Var. Paul Linder 3360
was obtained from IMTECH, Chandigarh.
2.2 | Physical measurement
C, H, and N elemental analyses were performed with a
1
model EURO EA3000 Elemental Analyser. The H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were
recorded with a Bruker Avance. IR spectra were recorded
on a FT‐IR Shimadzu spectrophotometer with sample
prepared as KBr pellets in the range 4000–400 cm−1. Pre-
coated silica gel plates (silica gel 0.25 mm, 60 G F 254;
Merck, Germany) were used for thin layer chromatogra-
phy. The LC–MS spectra were recorded using Thermo sci-
entific mass spectrophotometer (USA). The electronic
spectra were recorded on a UV‐160A, UV–vis spectropho-
tometer, Shimadzu, Kyoto (Japan). The magnetic
moments were measured by Gouy's method using mer-
cury tetrathiocyanatocobaltate (II) as the calibrant
(χg = 16.44 × 10−6 cgs units at 20 °C), citizen balance.
Antibacterial study was carried out using laminar air
flow cabinet Toshiba, Delhi (India). The thermogram
of complexes was recorded with a SDT Q600 V20.9
Build 20 thermogravimetric analyser. Conductance
measurement was carried out using conductivity meter
model number E‐660A. Ethidium bromide displace-
ment experiments were performed using Fluoromax‐4
spectrofluorometer, Horiba. Photo quantization of the
gel after electrophoresis was done using Alpha-
DigiDoc™ RT version V.4.0.0 PC‐Image software,
California (USA).
2.3 | Synthesis of the compounds
2.3.1 | General method for synthesis of
α,β‐unsaturated ketones (chalcones)(3a‐3f)
To a solution of 2‐acetyl thiophene (1) (10 mmol,
1.08 mL) in 20 mL of methanol, freshly prepared metha-
nolic KOH solution (10 mmol, 0.56 g) was added, stirred
for 15 min and appropriate aldehyde (2a–2f) (10 mmol)
was added. The reaction mixture was stirred at room tem-
perature for 2 h. The reaction mixture was poured to ice
cooled water and neutralized with dilute hydrochloric
acid. The white precipitate was separated by filtration
and washed with distilled water to give the crude product.
The obtained product was recrystallized from methanol.
The purity of the products was checked on TLC by using
a mixture of ethyl acetate and hexane (20:80) as the
mobile phase.
2 | EXPERIMENTAL
2.1 | Materials and reagents
All the chemicals and solvents were of analytical grade
(Purity >99%) and used as purchased. Na2PdCl4 (98%),
2‐acetyl thiophene (≥98%), p‐fluorobenzaldehyde (98%),
p‐chlorobenzaldehyde (97%), p‐bromobenzaldehyde
(99%), m‐chlorobenzaldehyde (97%), m‐bromobenzaldeh-
yde (97%), m‐fluorobenzaldehyde (97%), potassium tert‐
butoxide (≥98%), HS DNA and EDTA (≥99%) were
purchased from Sigma Aldrich Chemical Co. (India).
Agarose, Luria Broth (LB), ethidium bromide (EB),

THAKOR ET AL.
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1589, υ (C=N); 1311, υ (C=C); 1103, υ (C–N); 1056, υ
(C–Br); 1002, 964, (p‐substituted aromatic ring); 748, υ
(C–H)ar bending; 871, υ (C‐S‐C)tiophene; Mass (m/z%): 383
(100) [M+]; UV–vis: λ (nm) (ε, M−1 cm−1): 263
(155200), 378 (174100).
2.3.2 | General method for synthesis of
1,3,5‐trisubstituted pyrazolines (4a‐4f)
To a solution of the appropriate enones (3a–3f) (5 mmol)
in 10 mL of methanol, phenyl hydrazine (5 mmol,
0.49 mL) and freshly prepared methanolic potassium
tert‐butoxide (5 mmol, 0.56 g) solution were added. The
reaction mixture was refluxed for 5–6 h. The reaction
was monitored in every 60 min interval on precoated sil-
ica TLC plates by using a mixture of ethyl acetate and
hexane (20:80) as the mobile phase. The reaction mixture
was poured into ice cooled water. The products precipi-
tated out at low temperature were washed with an excess
of distilled water, and recrystallized in a minimum
amount of methanol and dried under reduced pressure.
The proposed reaction for the synthesis of ligands 4a–4f
is shown in Scheme 1.
5‐(3‐Bromophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4b)
Prepared by above method using (E)‐3‐(3‐bromophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3b) (5 mmol, 1.47 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 69%;
yellow crystalline solid. mp: 154–156 °C; mol. wt.
383.31 g mol−1; anal. Calc. for: C19H15BrN2S, calc.
(found) (%): C, 59.54 (59.05); H, 3.94 (3.42); N, 7.31
1
(7.25); S, 8.36 (7.28); H NMR (400 MHz, DMSO‐d6) δ/
ppm: 3.17 (1H, dd, J = 6.8 Hz, 17.2 Hz, 4‐Ha), 3.93 (1H,
dd, J = 12.0 Hz, 17.6 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.0 Hz,
11.6 Hz, 5‐H), 6.73–7.51 (11H, m, 3′,4′,2″,3″,4″,6″,2″’,
3″’,4″’,5″’,6″’‐H), 7.62 (1H, d, J = 5.6 Hz, 2’‐H); 13C NMR
(100 MHz, DMSO‐d6) δ/ppm: 44.09 (‐CH2 of pyrazoline),
62.98 (‐CH of pyrazoline), 113.40, 114.44, 119.37, 123.02,,
128.12, 128.20, 128.32, 128.44, 129.13, 129.49, 130.90,
131.80 (‐CH of aromatic region); 122.62, 125.38,
135.89, 144.37, 145.46 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3080, υ (C–H)ar stretching; 1589, υ
(C=N); 1315, υ (C=C); 1109, υ (C–N); 1058, υ (C–Br);
5‐(4‐Bromophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4a)
Prepared by above method using (E)‐3‐(4‐bromophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3a) (5 mmol, 1.47 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 70%;
yellow crystalline solid. mp: 153–155 °C; mol. wt.
383.31 g mol−1; anal. Calc. for: C19H15BrN2S, calc. (found)
(%): C, 59.54 (59.12); H, 3.94 (3.51); N, 7.31 (7.01); S, 8.36
1
(7.56); H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.12 (1H,
890, (m‐substituted aromatic ring); 750, υ (C–H)ar bending
;
867, υ (C‐S‐C)tiophene, Mass (m/z%): 383 (100) [M+];
UV–vis: λ (nm) (ε, M−1 cm−1): 266 (199400), 375 (239900).
dd, J = 7.2 Hz, 17.2 Hz, 4‐Ha), 3.87 (1H, dd, J = 12.4 Hz,
16.8 Hz, 4‐Hb), 5.25 (1H, dd, J = 7.2 Hz, 12.4 Hz, 5‐H),
6.80–7.25,7.47–7.50 (11H, m, 3′,4′, 2″,3″,5″,6″,2″’,3″’,4″’,
5″’,6″’‐H), 7.34 (1H, q, J = 1.2 Hz, 3.6 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 43.99 (‐CH2 of
pyrazoline), 62.95 (‐CH of pyrazoline), 113.09, 114.30,
116.45, 116.98, 119.28, 128.30, 128.34, 128.43, 128.48,
129.26, 129.33, 141.13 (‐CH of aromatic region); 122.34,
132.45, 135.42, 144.03, 144.09 (‐C of aromatic region);
5‐(4‐Fluorophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4c)
Prepared by above method using (E)‐3‐(4‐fluorophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3c) (5 mmol, 1.16 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 80%;
yellow crystalline solid. mp: 158–160 °C; mol. wt.
IR (KBr, 4000–400 cm−1); 3078, υ (C–H)ar
;
1
322.40 g mol−1; H NMR (400 MHz, DMSO‐d6) δ/ppm:
stretching
SCHEME 1 Outline of general
synthesis of compounds (5a‐5f)

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THAKOR ET AL.
3.13 (1H, dd, J = 6.0 Hz, 17.2 Hz, 4‐Ha), 3.92 (1H, dd,
J = 12.4 Hz, 17.6 Hz, 4‐Hb), 5.52 (1H, dd, J = 6.0 Hz,
12.0 Hz, 5‐H), 6.71–7.06 (11H, m, 3′,4′, 2″,3″,5″,6″,2″’,
3″’,4″’,5″’,6″’‐H), 7.62 (1H, d, J = 4.8 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 44.13 (‐CH2 of
pyrazoline), 62.95 (‐CH of pyrazoline), 113.43, 114.40,
116.17, 116.38, 119.23, 128.04, 128.30, 128.37, 128.45,
129.40, 129.58, 131.38 (‐CH of aromatic region);
122.13, 138.88, 144.21, 144.39, 163.05 (‐C of aro-
matic region); IR (KBr, 4000–400 cm−1); 3070, υ
116.17, 116.38, 119.28, 128.04, 128.09, 128.30, 128.34,
129.43, 129.48, 141.67 (‐CH of aromatic region); 122.13,
132.45, 135.98, 144.26, 144.33 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3078, υ (C–H)ar stretching; 1589, υ
(C=N); 1311, υ (C=C); 1149, υ (C–N); 1089, υ (C–Cl); 1003,
964, (p‐substituted aromatic ring); 748, υ (C–H)ar
;
bending
872, υ (C‐S‐C)thiophene; Mass (m/z%): 338 (100) [M+];
UV–vis: λ (nm) (ε, M−1 cm−1): 269 (214800), 364 (262500).
5‐(3‐Chlorophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4f)
(C–H)ar
; 1589, υ (C=N); 1319, υ (C=C);
stretching
1149, υ (C–N); 1060, υ (C–Cl); 1003, 964, (p‐substituted
Prepared by above method using (E)‐3‐(3‐chlorophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3f) (5 mmol, 1.24 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 83%;
yellow crystalline solid. mp: 144–146 °C; mol. wt.
338.85 g mol−1; anal. Calc. for: C19H15ClN2S, calc. (found)
(%): C, 67.35 (67.12); H, 4.46 (4.18); N, 8.27 (8.12); S, 9.46
aromatic ring); 748, υ (C–H)ar bending; 872, υ (C‐S‐C)thiophene
;
Mass (m/z%): 322 (100) [M+]; UV–vis: λ (nm) (ε, M−1 cm−1):
260 (75200), 376 (78800).
5‐(3‐Fluorophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4d)
1
(8.85); H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.17 (1H,
Prepared by above method using (E)‐3‐(3‐fluorophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3d) (5 mmol, 1.16 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 82%;
yellow crystalline solid. mp: 155–157 °C; mol. wt.
dd, J = 5.6 Hz, 17.2 Hz, 4‐Ha), 3.93 (1H, dd,
J = 12.8 Hz, 18.4 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.4 Hz,
12.8 Hz, 5‐H), 6.73–7.41 (11H, m, 3′,4′,2″,3″,4″,6″,
2″’,3″’,4″’,5″’,6″’‐H), 7.62 (1H, d, J = 5.6 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 44.00 (‐CH2 of
pyrazoline), 63.03 (‐CH of pyrazoline), 113.40, 116.31,
116.48, 119.37, 125.01, 126.26, 128.00, 128.11, 128.18,
128.31, 129.48, 131.52 (‐CH of aromatic region); 133.98,
135.90, 136.98, 144.34, 144.36 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3078, υ (C–H)ar stretching; 1589, υ
(C=N); 1311, υ (C=C); 1149, υ (C–N); 1089, υ (C–Cl);
1
383.31 g mol−1; H NMR (400 MHz, DMSO‐d6) δ/ppm:
3.165 (1H, dd, J = 6.0 Hz, 17.2 Hz, 4‐Ha), 3.94 (1H, dd,
J = 12.0 Hz, 17.2 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.4 Hz,
12.4 Hz, 5‐H), 6.72–7.43 (11H,m, 3′,4′,2″,3″,4″,6″,
2″’,3″’,4″’,5″’,6″’‐H), 7.62 (1H, d, J = 5.2 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 42.04 (‐CH2 of
pyrazoline), 63.16 (‐CH of pyrazoline), 113.12, 113.33,
113.42, 114.72, 114.93, 119.34, 128.07, 128.13, 128.30,
128.45, 131.62, 131.70 (‐CH of aromatic region); 122.37,
135.94, 145.62, 145.69, 161.68 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3072, υ (C–H)ar stretching; 1587, υ
(C=N); 1320, υ (C=C); 1150, υ (C–N); 1059, υ (C–Cl);
941, (m‐substituted aromatic ring); 748, υ (C–H)ar bending
;
872, υ (C‐S‐C)thiophene; Mass (m/z%): 338 (100) [M+]; UV–
vis: λ (nm) (ε, M−1 cm−1): 260 (67600), 374 (65800).
2.3.3 | General method of synthesis of
complexes (5a‐5f)
940, (m‐substituted aromatic ring); 747, υ (C–H)ar bending
;
871, υ (C‐S‐C)thiophene; Mass (m/z%): 322 (100) [M+];
UV–vis: λ (nm) (ε, M−1 cm−1): 261 (82100), 373 (65500).
The square planar metal complexes (5a–5f) of type
[Pd(4n)Cl2] were synthesized by the reactions of
Na2PdCl4 with the respective pyrazoline ligands (4a–4f)
in a 1: 1 molar ratio in 1: 1 methanol chloroform system.
5‐(4‐Chlorophenyl)‐1‐phenyl‐3‐(thiophen‐2‐yl)‐4, 5‐dihydro‐
1H‐pyrazole (4e)
Prepared by above method using (E)‐3‐(4‐chlorophenyl)‐
1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one (3e) (5 mmol, 1.24 g)
and phenyl hydrazine (5 mmol, 0.49 mL). Yield: 86%; yel-
low crystalline solid. mp: 149–151 °C; mol. wt.
338.85 g mol−1; anal. Calc. for: C19H15ClN2S, calc. (found)
(%): C, 67.35 (67.05); H, 4.46 (4.11); N, 8.27 (8.01); S, 9.46
[Pd(4a)Cl2] (5a)
A solution of ligand (4a) (0.191 g, 0.5 mmol), in chloro-
form, was added to methanolic solution of Na2PdCl4
(0.147 g, 0.5 mmol). The reaction mixture was refluxed
for half an hour and then stirred 48 h at room tempera-
ture. Greenish brown product was obtained, which is fil-
tered through whatman filter paper and washed with
diethyl ether for several time then dried under reduced
pressure. Proposed reaction is shown in Scheme 1. Yield:
69%; greenish brown solid. Mp: ≥ 300 °C; mol. Wt.
560.63 g mol−1; anal. Calc. for: C19H15BrCl2N2PdS, calc.
(found) (%): C, 40.71 (40.38); H, 2.70 (2.15); N, 5.00
1
(8.95); H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.13 (1H,
dd, J = 6.4 Hz, 18.0 Hz, 4‐Ha), 3.93 (1H, dd,
J = 12.8 Hz, 17.6 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.0 Hz,
12.0 Hz, 5‐H), 6.71–7.43 (11H, m, 3′,4′,2″,3″,5″,6″,
2″’,3″’,4″’,5″’,6″’‐H), 7.62 (1H, d, J = 4.8 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 43.99 (‐CH2 of
pyrazoline), 62.95 (‐CH of pyrazoline), 113.42, 114.40,

THAKOR ET AL.
5 of 15
(4.21); S, 5.72 (5.42); Pd, 18.98 (18.65); 1H NMR
(400 MHz, DMSO‐d6) δ/ppm: 3.12 (1H, dd, J = 7.2 Hz,
17.2 Hz, 4‐Ha), 3.87 (1H, dd, J = 12.4 Hz, 16.8 Hz, 4‐
Hb), 5.25 (1H, dd, J = 7.2 Hz, 12.4 Hz, 5‐H), 6.81–7.35
(11H, m, 3′,4′,2″,3″,5″,6″,2″’,3″’,4″’,5″’,6″’‐H), 7.57 (1H,
d, J = 4.8 Hz, 2’‐H); 13C NMR (100 MHz, DMSO‐d6) δ/
ppm: 43.58 (‐CH2 of pyrazoline), 63.35 (‐CH of pyrazoline),
113.18, 114.31, 116.58, 116.99, 119.29, 128.32, 128.38,
128.53, 128.58, 129.25, 129.36, 143.53 (‐CH of aromatic
region); 122.44, 132.48, 135.49, 144.15, 144.19 (‐C of
aromatic region); IR (KBr, 4000–400 cm−1); 3070, υ
(C–H)ar stretching; 1612, υ (C=N); 1396, υ (C=C); 1172, υ
(C–N); 1072, υ (C–Br); 1010, 980, (p‐substituted aromatic
ring); 779, υ (C–H)ar bending; 902, υ (C‐S‐C)thiophene; con-
ductance: 09 Ω−1 cm2 mol−1; Mass (m/z): 560.6 [M+];
UV–vis: λ (nm) (ε, M−1 cm−1): 269 (91125), 390 (12563).
3070, υ (C–H)ar stretching; 1605, υ (C=N); 1319, υ (C=C);
1157, υ (C–N); 1095, υ (C–Cl); 1003, 964, (p‐substituted
aromatic ring); 771, υ (C–H)ar bending; 895, υ (C‐S‐C)thiophene
;
conductance: 07 Ω−1 cm2 mol−1; UV–vis: λ (nm) (ε,
M−1 cm−1): 272 (71875), 394 (16563).
[Pd(4d)Cl2] (5d)
It was synthesized using solution of ligand (4d) (0.161 g,
0.5 mmol). Yield: 71%; greenish brown solid. Mp: ≥
1
300 °C; mol. Wt. 499.72 g mol−1; H NMR (400 MHz,
DMSO‐d6) δ/ppm: 3.16 (1H, dd, J = 6.0 Hz, 17.2 Hz, 4‐Ha),
3.94 (1H, dd, J = 12.0 Hz, 17.2 Hz, 4‐Hb), 5.53 (1H, dd,
J = 6.4 Hz, 12.0 Hz, 5‐H), 6.72–7.43 (11H, m, 3′,4′,2″,3″,4″,
6″,2″’,3″’,4″’,5″’,6″’‐H), 7.72 (1H, d, J = 5.2 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 44.05 (‐CH2 of
pyrazoline), 63.19 (‐CH of pyrazoline), 113.39, 113.49,
114.76, 114.98, 119.35, 128.09, 128.15, 128.28, 128.43,
131.69, 131.79, 134.74 (‐CH of aromatic region); 122.37,
135.91, 145.68, 145.77, 161.34 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3070, υ (C–H)ar stretching; 1566, υ
(C=N); 1366, υ (C=C); 1157, υ (C–N); 1049, υ (C–Cl); 941,
(m‐substituted aromatic ring); 779, υ (C–H)ar bending; 856, υ
(C‐S‐C)thiophene; conductance: 11 Ω−1 cm2 mol−1; UV–vis: λ
(nm) (ε, M−1 cm−1): 272 (78000), 378 (13365).
[Pd(4b)Cl2] (5b)
It was synthesized using solution of ligand (4b) (0.191 g,
0.5 mmol). Yield: 61%; greenish brown solid. Mp: ≥
300 °C; mol. Wt. 560.63 g mol−1; anal. Calc. for:
C19H15BrCl2N2PdS, calc. (found) (%): C, 40.71 (40.25);
H, 2.70 (2.18); N, 5.00 (4.27); S, 5.72 (5.53); Pd, 18.98
1
(18.44); H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.17 (1H,
dd, J = 6.8 Hz, 17.2 Hz, 4‐Ha), 3.93 (1H, dd,
J = 12.0 Hz, 17.6 Hz, 4‐Hb), 5.52 (1H, dd, J = 6.0 Hz,
11.6 Hz, 5‐H), 6.73–7.51 (11H, m, 3′,4′,2″,3″,4″,6″,
2″’,3″’,4″’,5″’,6″’‐H), 7.72 (1H, d, J = 5.6 Hz,2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 44.25 (‐CH2 of
pyrazoline), 62.66 (‐CH of pyrazoline), 113.49, 114.47,
119.35, 123.32, 128.14, 128.25, 128.32, 128.48, 129.43,
129.62, 130.95, 133.93 (‐CH of aromatic region); 122.66,
125.47, 135.91, 144.47, 145.62 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3070, υ (C–H)ar stretching; 1558, υ
(C=N); 1365, υ (C=C); 1172, υ (C–N); 1095, υ (C–Br);
[Pd(4e)Cl2] (5e)
It was synthesized using solution of ligand (4e) (0.169 g,
0.5 mmol). Yield: 65%; greenish brown solid. Mp: ≥
300 °C; mol. Wt. 516.17 g mol−1; anal. Calc. for:
C19H15Cl3N2PdS, calc. (found) (%): C, 44.21 (43.92); H,
2.93 (2.41); N, 5.43 (5.02); S, 6.21 (5.95); Pd, 20.62 (20.14);
1H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.13 (1H, dd,
J = 6.4 Hz, 18.0 Hz, 4‐Ha), 3.93 (1H, dd, J = 12.8 Hz,
17.6 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.0 Hz, 12.0 Hz, 5‐H),
6.71–7.43 (11H, m, 3′,4′,2″,3″,5″,6″,2″’,3″’,4″’,5″’,6″’‐H),
7.69 (1H, d, J = 4.8 Hz, 2’‐H); 13C NMR (100 MHz,
DMSO‐d6) δ/ppm: 43.94 (‐CH2 of pyrazoline), 62.65 (‐CH
of pyrazoline), 113.41, 114.45, 116.18, 116.34, 119.26,
128.05, 128.08, 128.33, 128.38, 129.47, 129.49, 144.14 (‐CH
of aromatic region); 122.15, 132.47, 135.90, 144.25, 144.36
(‐C of aromatic region); IR (KBr, 4000–400 cm−1); 3070, υ
(C–H)ar stretching; 1596, υ (C=N); 1365, υ (C=C); 1165, υ
(C–N); 1095, υ (C–Cl); 1011, 972, (p‐substituted aromatic
910, (m‐substituted aromatic ring); 779, υ (C–H)ar bending
840, υ (C‐S‐C)thiophene; conductance: 10 Ω−1 cm2 mol−1
UV–vis: λ (nm) (ε, M−1 cm−1): 264 (73563), 389 (16875).
;
;
[Pd(4c)Cl2] (5c)
It was synthesized using solution of ligand (4c) (0.161 g,
0.5 mmol). Yield: 60%; greenish brown solid. Mp: ≥
1
300 °C; mol. Wt. 499.72 g mol−1; H NMR (400 MHz,
DMSO‐d6) δ/ppm: 3.13 (1H, dd, J = 6.0 Hz, 17.2 Hz, 4‐
Ha), 3.92 (1H, dd, J = 12.4 Hz, 17.6 Hz, 4‐Hb), 5.52 (1H,
dd, J = 6.0 Hz, 12.0 Hz, 5‐H), 6.70–7.35 (11H, m,
ring); 741, υ (C–H)ar bending; 933, υ (C‐S‐C)thiophene;
conductance: 07 Ω−1 cm2 mol−1; UV–vis: λ (nm)
(ε, M−1 cm−1): 271 (86938), 387 (12875).
3′,4′,2″,3″,4″,6″,2″’,3″’,4″’,5″’,6″’‐H),
7.72
(1H,
d,
J = 4.8 Hz, 2’‐H); 13C NMR (100 MHz, DMSO‐d6) δ/ppm:
44.26 (‐CH2 of pyrazoline), 62.90 (‐CH of pyrazoline),
113.58, 114.48, 116.23, 116.40, 119.32, 128.11, 128.34,
128.41, 128.50, 129.45, 129.60, 134.33 (‐CH of
aromatic region); 122.19, 138.94, 144.32, 144.47, 163.13
(‐C of aromatic region); IR (KBr, 4000–400 cm−1);
[Pd(4f)Cl2] (5f)
It was synthesized using solution of ligand (4f) (0.169 g,
0.5 mmol). Yield: 63%; greenish brown solid. Mp: ≥
300 °C; mol. Wt. 516.17 g mol−1; anal. Calc. for:
C19H15Cl3N2PdS, calc. (found) (%): C, 44.21 (43.69); H,
2.93 (2.45); N, 5.43 (5.14); S, 6.21 (5.89); Pd, 20.62

6 of 15
THAKOR ET AL.
1
(20.09); H NMR (400 MHz, DMSO‐d6) δ/ppm: 3.17 (1H,
dd, J = 5.6 Hz, 17.2 Hz, 4‐Ha), 3.93 (1H, dd,
J = 12.8 Hz, 17.6 Hz, 4‐Hb), 5.53 (1H, dd, J = 6.4 Hz,
12.8 Hz, 5‐H), 6.73–7.41 (11H, m, 3′,4′,2″,3″,4″,6″,2″’,
3″’,4″’,5″’,6″’‐H), 7.72 (1H, d, J = 5.6 Hz, 2’‐H); 13C
NMR (100 MHz, DMSO‐d6) δ/ppm: 44.10 (‐CH2 of
pyrazoline), 63.43 (‐CH of pyrazoline), 113.36, 116.28,
116.46, 119.52, 125.21, 126.35, 128.05, 128.12, 128.22,
128.30, 129.45, 135.38 (‐CH of aromatic region); 131.62,
135.99, 136.53, 144.44, 144.48 (‐C of aromatic region); IR
(KBr, 4000–400 cm−1); 3070, υ (C–H)ar stretching; 1558, υ
(C=N); 1373, υ (C=C); 1165, υ (C–N); 1095, υ (C–Cl);
2.4.5 | Complex‐DNA binding studies
Different techniques are used to find out complex‐DNA
interaction like spectroscopy, viscosity, circular dichro-
ism, molecular modeling etc. Here, we have used absorp-
tion spectroscopy, viscosity and molecular modeling
techniques for DNA binding studies.
Absorption titration technique
UV visible absorption titration is carried out using known
concentration of Herring sperm DNA (HS DNA) in phos-
phate buffer and synthesized complexes (soluble in
DMSO). HS DNA was dissolved in phosphate buffer and
concentration of DNA is measured at 260 nm wavelength
(ε = 12858 cm−1). The experiment was carried out accord-
ing to our previously published literature.[41]
887, (m‐substituted aromatic ring); 732, υ (C–H)ar bending
925, υ (C‐S‐C)thiophene; conductance: 09 Ω−1 cm2 mol−1
UV–vis: λ (nm) (ε, M−1 cm−1): 271 (110100), 390 (7750).
;
;
Viscosity measurement
2.4 | Biological application of synthesized
compounds
The hydrodynamic volume measurement study was carried
out using an Ubbelohed viscometer kept under the thermo-
static bath at constant temperature 27
0.1 °C. The
2.4.1 | In vitro antibacterial activity
experiment was carried out according to literature.[42–44]
The in vitro antibacterial activity of free ligands and
synthesized palladium (II) complexes was performed
against two Gram positive(+ve): Staphylococcus aureus,
Bacillus subtilis and three Gram negative(−ve): Serratia
marcescens, Escherichia coli, Pseudomonas aeruginosa
microorganisms. The experiment was performed accord-
ing to literature procedure.[36,37]
Ethidium bromide (EB) displacement method
Complexes exhibit no fluorescence at room temperature
in solution or in the presence of HS DNA, and their bind-
ing to DNA could not be directly predicted through the
emission spectra. Intense fluorescent light is emitted from
EB in the presence of HS DNA due to the strong interca-
lation between adjacent DNA base pairs in the double
helix; therefore, EB is considered to be a typical indicator
of intercalation.[45] Hence, a competitive binding study of
each complex with EB was done to understand whether
the complex can displace EB from its HS DNA–EB com-
plex and the mode of DNA interaction with the com-
plexes. The HS DNA–EB complex was prepared by
adding 33.3 μM EB and 10 μM HS DNA in buffer (phos-
phate buffer, pH 7.2). The possible intercalating effect of
the complexes was studied by adding a certain amount
of a solution of the complex step by step into a solution
of the DNA–EB complex. The influence of the addition
of each complex to the DNA–EB complex solution was
obtained by recording the variation of fluorescence emis-
sion spectra with emission wavelength at 610 nm (excita-
tion wavelength at 540 nm). The reaction time has been
studied and the results showed that 4 min was enough
for stabilization. So the change in fluorescence emission
intensity was measured within 4 min after each addition.
2.4.2 | In vitro antimycobacterial activity
Determination of MIC of the test complexes against M.
tuberculosis H37Rv was performed by Lowenstein‐Jensen
agar (MIC) method.[38] The standard strain M. tuberculo-
sis H37Rv was tested with well‐known drug rifampicin
and isoniazide.[38,39]
2.4.3 | In vitro cytotoxicity using brine
shrimp
In vitro cytotoxicity was performed according to our pre-
viously published literature.[40] Data were analyzed by
simple logit method to determine the LC50 values, in
which the log of concentration of samples were plotted
against percentage of mortality of nauplii.
The values of the Stern–Volmer constant (KSV, in M−1
)
were calculated according to the linear Stern–Volmer
equation (equation (2)) and the plots I0/I vs. [Q].
2.4.4 | In vivo cytotoxicity using S. pombe
The in vivo cytotoxicity was performed according to liter-
ature using S. Pombe cells.[41]
I0=I ¼ Ksv ½Qꢀ þ 1
(2)

THAKOR ET AL.
7 of 15
where I0 is the emission intensity of EB‐DNA in the
absence of quencher (complex), I is the emission intensity
of EB‐DNA in the presence of quencher and [Q] is the
quencher concentration.
To determine the strength of the interaction of com-
plexes with DNA, the value of the binding constant (K f )
was calculated using the Scatchard equation (equation (3)):
respectively in metal complex 5a. Similar trends is
observed for IR spectra of all ligands (4a‐4f) and metal
complexes (5a‐5f). The shift in above bands clearly indi-
cates N and S atoms as the coordinating atoms. The IR
spectral data are detailed in the experimental section.
Mass spectra of all ligands show corresponding molec-
ular ion peak and data are embedded in the experimental
section. Supplementary material 3 and 4 represents mass
spectrum and probable fragments of complex 5a [Pd(4a)
Cl2]. Mass spectrum of complex 5a exhibits molecular
ion peak [M+] at 560.60 m/z, [M + 2] at 562.61 m/z,
[M + 4] at 564.61 m/z and [M + 6] at 566.59, suggests
the presence of two chlorine atoms and one bromine
atom in the complex. The peak at 489.89 m/z is due to
loss of two chlorine atoms from the complex 5a and peak
at 383.25 m/z is due to the ligand attached with palla-
dium (II). The peak at 306.18 m/z is due to the removal
of phenyl ring from the fragmented ligand. The com-
plexes have been characterized by EDX spectroscopy also,
which is advantageous over C,H,N‐elemental analysis, as
we can determine the percentage of Cl, Br and metal ion
along with C, H and N atoms. The EDX spectrum for
complex 5a shows the existence of carbon, nitrogen,
sulphur, chlorine, bromine and palladium elements
(Figure 1) and confirmed the weight % of C, N, S, Cl, Br
and Pd as 41.75, 5.47, 5.95, 12.89, 14.49 and 19.45%, as
expected based on the calculated values of 40.71, 5.00,
5.72, 12.65, 14.25 and 18.98%, respectively.
ðI0−IÞ
log
¼ log Kf þ nlog½Qꢀ
(3)
I
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
binding sites.
Molecular docking
To determine the theoretical binding energy of synthe-
sized compounds to DNA, docking study was performed
using HEX 6.0 software. The.pdb files of complex coordi-
nates were obtained by converting their.mol file using
CHIMERA 1.5.1 software. The structure of B‐DNA (1
BNA: 5′‐D(*CP*GP*CP*GP*AP*TP*TP*CP*GP*CP*G)‐3′)
obtained from the Protein Data Bank (http://www.rcsb.
org/pdb). All solvents were removed before docking. Grid
dimension 0.6 with FFT mode 3D and correlation type
shape only were used. The other parameters kept at their
default values.
Gel electrophoresis study
Gel electrophoresis have been performed to monitor
cleavage of pUC19 DNA by synthesized complexes. The
experiment was performed according to previously pub-
lished literature procedure.[46]
3.2 | Thermogravimetric analysis
Thermogravimetric analysis is concerned with the change
in weight of a material as its temperature changes. It
indicates the temperature at which the material loses
weight and weight loss indicates sample decomposition.
The temperature, at which no weight loss occurs, reveals
the stability of the material. TGA was carried out at a
10 °C per minute heating rate in the range of 20–800 °C
3 | RESULTS AND DISCUSSION
3.1 | 1H‐NMR spectra, FT‐IR, LC–MS
spectra and EDX analysis
under
a
nitrogen atmosphere. The characteristic
1H‐NMR of ligands (4a‐4f) and complexes (5a‐5f) are
embedded in supplementary material 1a and 1b respec-
tively. In 1H‐NMR spectra of ligands (4a‐4f), three doublet
of doublet between 3.00 to 5.50 δ ppm suggests the
formation of 1,3,5‐trisubstituted‐4,5‐dihydro‐1H‐pyrazole
moiety. Peak around 7.60 ppm for ligands shifts to down-
field in all complexes (5a‐5f) indicates coordination of
ligands to palladium (II). 13C‐NMR of ligands (4a‐4f)
and complexes (5a‐5f) are embedded in supplementary
material 2a and 2b respectively. In IR spectra of ligand
4a, bands observed at 1589, 1103 and 871 cm−1 are
assigned to υ (C=N), υ (C–N) and υ (C‐S‐C), respectively.
thermogram of complex 5a shows two distinct mass losses
These bands are shifted to 1612, 1172 and 902 cm−1
,
FIGURE 1 EDX spectrum of complex 5a

8 of 15
THAKOR ET AL.
(supplementary material 5). The thermogram of complex
5a shows no weight loss up to ∼200 °C, which infers the
absence of lattice and coordinated water molecules. The
mass loss (about 12%) during the first step between 200
and 350 °C corresponds to loss of two chlorine atoms
from complex 5a. The second step (about 67%) corre-
sponds to the decomposition of ligand and leaving behind
the metal oxide as residue.[37]
Some Bacillus species can cause food poisoning. S.
marcescens causes central nervous system diseases such
as meningitis, urinary tract infection. Toxic shock syn-
drome (TSS) and staphylococcal scalded skin syndrome
(SSSS) are associated with S. aureus.
The MIC (minimum inhibitory concentration) values
are presented in supplementary material 6. The MIC
values of all the ligands are in the range of 200 μM to
575 μM and complexes are in the range of 30 μM to
125 μM (Figure 2). The results reveal that most of the
compounds exhibit significant antibacterial activity. Out
of the twelve compounds, 5c, having electronegative F
as a substituent atom at para position, displayed broad‐
spectrum antimicrobial activity against all tested bacterial
strains with MIC values 30–75 μM. Moreover, compound
5a showed the most potent activity with MIC values of
35 μM for B. subtilis and 70 μM for E. coli. From the
result, we can also conclude that chelation of heterocycle
to metal also reinforce the antimicrobial activity of
heterocyclic molecules.[48,49]
3.3 | Magnetic moments, electronic
spectra and conductance measurement
Magnetic moments measurement has been carried out at
room temperature for all the Pd (II) complexes.
Diamagnetic behaviour with zero B.M. μeff value of Pd
(II) complexes suggests square planar geometry for all
complexes. Electronic spectra of Pd (II) complexes have
been recorded in DMSO. The absorption peaks are
observed at around 300–390 nm and 200–280 nm, which
can be assigned to d‐d transition and metal to ligand
charge‐transfer transition (MLCT), respectively for square
planar complexes.[47] To study the electrolytic nature of
the palladium (II) complexes (5a‐5f), their molar conduc-
tivities have been measured in DMSO. The molar conduc-
tance (ɅM) values for the palladium (II) complexes are found
in the range of 7–11 cm2 Ω−1 mol−1, indicating non electro-
lytic nature and absence of any counter ion outside the coordi-
nation sphere of the complexes. So, we conclude that all Pd
(II) complexes are neutral in nature.
3.4.2 | In vitro antimycobacterial activity
The activity of the complexes against M. tuberculosis viru-
lent strain H37Rv has been determined and results are
presented in supplementary material 7. The fluorine
substituted ligands containing complexes are more active
against H37Rv strain than the other complexes. The
highest MIC is found for the complexes 5c and 5d, having
more electronegative F as a substituent atom. On the
other hand, the least active complexes are the complex
5b (having bulkier and least electronegative Br as a sub-
3.4 | Biological application of compounds
3.4.1 | In vitro antibacterial activity
stituent atom) and 5f, which exhibit MIC >250 μgmL−1
.
But, all the compounds are less active than the standard
drugs rifampicin and isonizaide.
The treatment of infectious diseases has been becoming
an important and challenging problem because of emerg-
ing infectious diseases and emergence of inevitable anti-
biotic‐resistant mutants among bacteria, which results
in decreased efficacy and withdrawal of some antibiotic
from widespread usage. Resistance to available antibiotics
in pathogenic bacteria is currently a global challenge and
hence, the substantial medical need for new classes of
antimicrobial agents has been arising nowadays. The effi-
cacy of the various organic therapeutic agents can often
be enhanced upon coordination with a suitable metal
ion. Hence, we have also evaluated the antibacterial
activity of metal complexes against two Gram‐negative
bacterial strains: E. coli and P. aeruginosa; and against
three Gram positive bacterial strains: B. subtilis, S. aureus
and S. marcescens using the broth dilution method. E. coli
cause gastrointestinal symptoms, ranging from mild to
severe and bloody diarrhea, mostly without fever. P.
aeruginosa can cause infection in lung, blood stream.
3.4.3 | In vitro cytotoxicity using brine
shrimp
Brine shrimp lethality bioassay is a useful tool to carry
out in vitro cytotoxicity of compounds. The advantages
FIGURE
2
Minimal inhibitory concentration values of
synthesized compounds

THAKOR ET AL.
9 of 15
of this method include economical, less time consuming
than other cytotoxicity test and more reliability. The assay
has been carried out according to the protocol of Meyer
et al.[50] A plot of log of sample concentration versus per-
centage of mortality has shown the linear relationship
and the LC50 value of compounds have been calculated
from the plot. The LC50 values of the compounds are in
the range of 5.93 to 17.81 (supplementary material 7),
which is comparable to cytotoxicity of reported Pd (II)
complexes (LC50 = 6.83–15.93μgmL−1).[51] The degree of
mortality is directly proportional to the concentration of
synthesized compounds. From the data recorded, com-
plex 5c is the most potent amongst all the compounds.
From Figure 3, it is concluded that the complexes are
good cytotoxic agent than the ligands. The order of
potency of compounds is 5c > 5d > 5e > 5f > 5a > 5b.
has been carried out, in which trans platin and anticancer
drug cisplatin have been used as the standard drugs.
Result reflect that cytotoxicity is found proportional to
the concentration of the compounds. The complexes
show better cytotoxicity than reported pyrazoline based
Ru (III) complexes.[53] Compounds 5c, 5d and 5e are
found to have maximum cytotoxicity, while compounds
5a, 5b and 5f are found less cytotoxic (Figure 4). After
17 h of the treatment, many of the S. pombe cells died
due to the toxic nature of the compounds (Figure 4).
From experiment, it is observed that cytotoxicity of
ligands are increased upon chelation with palladium (II).
3.4.5 | Complex‐DNA binding
Absorption titration study
Among all the spectroscopic techniques, absorption titra-
tion study is very useful, prominent and reliable tech-
nique to study binding mode of small molecule to
biopolymer like DNA. There are different kind of binding
modes for interaction of small molecule with DNA. In
this study, the binding mode of compounds to HS DNA
has been determined. Absorption spectra are very
sensitive to change in structure of compounds, and the
structural change reflects in the absorption maxima. To
determine the binding constant of synthesized com-
pounds to HS DNA, we have monitoring the absorption
maxima with gradual addition of DNA. For noncovalent
interactioni.e., intercalation, hypochromism with or with-
out red shift takes place.[54,55] While hyperchromism
takes place in electrsostatic binding.[56] The observation
of this study reflects the intercalative binding mode of
compounds to HS DNA. The strong stacking interaction
takes place between the DNA base pair and chromo-
phoric group of the compound. The interaction slightly
break the secondary structure of DNA, which causes the
hypochromism.[57,58] Binding constant values for ligand‐
DNA interaction are in the range of 1.0 × 104 to
3.8 × 105 M−1, while Kb values of complex‐DNA interac-
tion are in the range of 0.94 × 105 to 7.9 × 105 M−1, which
are comparable to antitumor drug cisplatin i.e.
3.4.4 | In vivo cytotoxicity using S. pombe
Schizosaccharomyces pombe, the sixth model eukaryotic
organism, has been used as an important organism in
studying the cellular response to DNA damage and pro-
cess of replication because many genes of this organism
are homologous to human diseases genes.[52] Therefore,
we have used this organism to carry out primary in vivo
cytotoxicity test. The staining trypan blue dye could not
penetrate through the live cell wall, but it could penetrate
through the dead cell wall. Because of this, the dead S.
pombe cells found to be blue in colour while live cells
remain transparent under the microscope. Cellular level
cytotoxicity of synthesized compounds on S. pombe cells
5.51 × 104
M
−1, while higher than trans‐platin i.e.
FIGURE 3 LC50 values in μgmL−1 of all compounds
1.75 × 104 M−1 and lower than classical Intercalator EB
FIGURE 4 Cytotoxic effect of
compounds on S. Pombe cells at five
different concentration (Left) and S.
Pombe cells after treatment with
compound (Right)

10 of 15
THAKOR ET AL.
i.e. 7 × 107 M
.
−1 [59,60] The binding strength of metal com-
TABLE 1 Binding constant (Kb), percentage hypochromicity
(%H), bathochromicity (Δλ), Gibb's free energy of all compounds
plexes with DNA are comparable to reported Pd com-
plexes such as [Pd (H‐Msal‐mtsc) Cl (PPh3)] (Kb = 1.44
x 105 M−1), [Pd (H‐Msal‐metsc) Cl (PPh3)] (Kb = 1.69 x
Compounds
Kb (M−1) (× 105)
% H
ΔG°(kJmol−1
)
4a
4b
4c
4d
4e
4f
0.10
0.10
0.14
0.20
0.72
0.36
1.32
7.49
1.58
1.53
0.94
2.45
11.67
7.71
−22.77
−22.88
−23.66
−24.50
−27.71
−26.00
−29.23
−33.53
−29.67
−29.59
−28.39
−30.76
105 M−1 [61] and [(Pd (H‐Msal‐mtsc))2(μ‐ppm)] (Kb = 1.89
)
x 105 M−1);[62] while Kb values of complexes are higher
than reported complexes such as [PdCl (dapdoH)]
1.07
(Kb = 4.6 x 104 M−1 [28] and [Pd (Msal‐tsc)(PPh3)] (Kb = 5.0
)
9.77
x 104 M−1).[61] The intrinsic binding constant Kb can be
obtained from the ratio of slope to the intercept.
(Figure 5).[63]
5.77
8.31
5a
5b
5c
5d
5e
5f
11.78
22.84
9.73
From the values of the binding constant (Kb), free
energy (ΔG) of the compound–DNA complex has been
calculated using equation (1):
8.82
ΔG ¼ –RT ln Kb
(1)
17.76
15.40
Binding constants are the measure of the compound–
DNA complex stability, while the free energy indicates
the spontaneity or non‐spontaneity of compound–DNA
binding. The free energy value (ΔG = −26.00 to −33.66
kJmol−1) of the Pd (II) complexes are negative. It indi-
cates the spontaneity of compound–DNA interaction.
Binding constant values and Gibb's free energy of com-
plexes are higher than ligands. It indicates that upon
complexation, binding ability of ligands enhances to a
greater extent. Binding constants (Kb), free energy value
(ΔG) and percentage hypochromism (%H = 1.07–20.10)
of all compounds are shown in Table 1.
To calculate the interaction forces between the com-
pound – HS DNA complex, the temperature dependant
thermodynamic parametersi.e., change in enthalpy
(ΔH°), change in entropy (ΔS°) and Gibb's free energy
(ΔG°) have been calculated using van't Hoff equation
(equation (2)),[64] for complex 5b, which exhibit the
highest binding constant.
ΔG° ¼ ΔH°−TΔS°
(3)
where, Kb is the intrinsic binding constant measured at
different absolute temperature (301.5, 303, 308, 313 K)
and R is the gas constant in Jmol−1 K−1. Enthalpy change
and entropy change have been evaluated from the linear
van't Hoff plot (lnKb versus 1/T), where, slope gives
ΔH° and intercept gives ΔS°. The Gibb's free energy has
been calculated from the equation (1), which is also veri-
fied by equation (3). All thermodynamic parameters for
5b‐HS DNA complex are shown in Table 2, where nega-
tive values of ΔH° and ΔG° indicate that the interaction
is thermodynamically favourable. The result shows that
the binding may be driven by van der Waals forces and
hydrogen binding, which is the main evidence for
intercalative binding.[65]
ΔH° ΔS°
lnKb ¼
þ
(2)
Viscosity measurement
RT
R
To support absorption titration, we have carried out rela-
tive viscosity measurement study. The viscosity of macro-
molecules are sensitive to their chain length and change
in chain length. Intercalation and groove binding affect
the length of DNA and hence the viscosity of DNA
increases, while electrostatic binding does not affect the
length of DNA and hence the viscosity of DNA remain
constant.[66–68] Therefore, to clarify the mode of interac-
tion of compounds to DNA, the change in relative viscos-
ity of DNA with successive addition of has been
measured. The relative viscosity of DNA has increased
upon addition of free ligands as well as their respective
complexes (Figure 6). Similar trend is observed for
reported Pd complexes such as Pd (L)(PPh3)], [Pd (L)
(AsPh3)][69] and [PdCl (dapdoH)].[28] The insertion of
FIGURE 5 Absorption spectra upon addition of HS DNA to the
solution of complex 5a after incubating for 10 minutes at room
temperature in phosphate buffer (Na2HPO4/NaH2PO4, pH = 7.2).
Inset: plot of [DNA]/ (εa − εf) vs. [DNA]

THAKOR ET AL.
11 of 15
TABLE 2 Intrinsic binding constant (Kb), relative thermodynamic parameters of 5b – HS DNA complex
T (K)
Kb (×105 M−1
)
ΔG° (kJmol−1
)
ΔH° (kJmol−1
−47.98
)
ΔS° (JK−1 mol−1
−46.64
)
301.5
303
7.49
6.68
5.39
3.60
−33.91
−33.79
−33.79
−33.29
308
313
FIGURE
6
Effect on relative viscosity upon addition of
complexes at 27 °C
FIGURE 7 Emission spectra of EB‐bound DNA solutions in the
absence and presence of increasing concentrations of complex 5b
(3.33–33.3 μM) in Tris–HCl. [EB] = 33.3 μM, [DNA] = 10.0 μM. The
arrow shows the change in intensity upon increasing amounts of
the complex
compound to the base pair of DNA results in lengthening
of DNA chain due to the separation of base pair. This sug-
gests intercalation mode of compounds to DNA bind-
ing.[70–73] Order of increase in viscosity for ligands is
4a < 4b < 4c < 4d < 4f < 4e and for complexes is
5e < 5a < 5d < 5c < 5f < 5b. This order is similar to
the binding constant order of compounds.
TABLE 3 Stern‐Volmer quenching constant (Ksv), binding sites
(n) and binding constant (K f ) from competitive
Complexes
Ksv (M−1
)
Binding sites (n)
K f (M−1
)
Ethidium bromide (EB) displacement method
5a
5b
5c
5d
5e
5f
1.4 × 104
7.0 × 103
1.1 × 103
6.4 × 103
9.4 × 103
9.0 × 103
0.985
1.255
1.021
1.052
0.937
1.144
1.23 × 104
1.22 × 105
1.31 × 104
1.24 × 104
5.10 × 103
4.18 × 104
The successive addition of complexes 5a‐5f to EB‐DNA
system at diverse “r” value [Complex]/ [DNA]) resulted
in a remarkable decrease in fluorescence maxima of
EB‐DNA system (Figure 7). The displacement of EB
by complexes, at 610 nm suggests the intercalative
mode of complex to DNA binding. The extent of fluo-
rescence intensity quenching is the measure of strength
of binding between DNA and complexes.[74,75] In our
study inner filter effect is corrected using Lakowics
equation.[76] The observed fluorescence quenching data
give a linear plot analogous to Stern‐Volmer equation.
The calculated quenching constant (KSV) shows the
ability of complexes to displace the EB.[77] The binding
constant (K f ), quenching constants (KSV), number of
binding sites (n) are listed in Table 3. The K f values
calculated from Scatchard plots indicates that complex
5b exhibits the highest binding propensity and the
order of binding strength of the complexes to HS‐
DNA is 5e < 5a < 5d < 5c < 5f < 5b, is same as
observed in the UV titration study and viscosity mea-
surement study. The K f values of complexes are
comparable to reported Pd complexes such as [Pd
(DMEAImiPr)Cl2] (1.0 x 104 −1) and [Pd (DACH
(ImiPr)2)Cl2] (2.0 x 104 −1);[78] while K f values are
higher than reported Pd complexes such as [Pd (L)
M
M
(PPh3)] (3.92 x 103
M
−1) and [Pd (L)(AsPh3)] (7.04 x
103 M−1).[69] The values of “n” is observed around 1.0
indicates the 1: 1 molar ratio between the complexes
and HS‐DNA.
Molecular docking
Molecular docking can provide some insight of the inter-
actions of macromolecules with ligands and preferred

12 of 15
THAKOR ET AL.
binding mode with the help of a variety of docking
programs. To determine the relative binding energy of
complex to DNA interaction, complexes have been
docked to the B‐DNA (PDB ID: 1BNA). In the docking
method, out of 2000 docking solutions, only the lowest
relative binding energy of docked structure has been
obtained. From the ensuing docked structures it is clear
that all compounds fit well into the rich G‐C minor groove
region of the targeted DNA, which is stabilized by van der
Waals interaction and hydrophobic contacts. The repre-
sentative docked structure is shown in Figure 8 and other
docked structures are embedded in supplementary mate-
rial 8. Relative binding energy of the docked structures of
ligands are −251.16(4a); −255.92(4b); −257.95(4c);
−261.89(4d); −269.11(4e) and − 267.73(4f) eV and of
complexes are −252.61(5a); −260.86(5b); −254.38(5c);
−253.61(5d); −251.60(5e) and − 258.24(5f) eV.
FIGURE 9 Photogenic view of cleavage of pUC19 DNA (300 μM)
with a series of compounds using 1% agarose gel containing
0.5 μM EB, TE buffer (pH 8) at a final volume of 15 μL at 37 °C
Gel electrophoresis
This experiment is carried out to measure the influence of
complexes on the electrophoretic mobility and cleavage of
the supercoiled form of pUC19 DNA in the absence of
reducing agents.[79,80] The working principle of the gel
electrophoresis is when circular plasmid DNA is sub-
jected to electrophoresis, relatively fast migration will be
observed for the intact supercoil form (Form I). If one
strand of DNA is cleaved, the supercoiled form is relaxed
FIGURE 10 Percentage comparison of DNA cleavage from SC to
OC form due to the influence of compounds
to slower moving open circular or nicked form (Form II)
and if both strands are cleaved linear form is generated
which migrates in between the supercoiled (Form‐I) and
open circular form (Form‐II).[81] Complexes have been
found to promote the cleavage of pUC19 DNA from
supercoiled Form I to the nicked Form II. As shown in
Figure 9, the intensity of the circular supercoiled DNA
(Form I) band is found to decrease, while that of the
nicked (Form II) band apparently increase in lane 3 to
10. The gel electrophoretic separations shows the cleav-
age of pUC19 DNA induced by the complexes. The com-
plexes 5c and 5e can induce the cleavage of the pUC19
DNA similar to cisplatin, and each lane has cleavage
effect, comparing lanes 4–10 with lane 1, we can see the
two complexes 5c and 5e have the highest cleavage per-
centage. Percentage cleavage of all compounds are
embedded in supplementary material 9. Percentage com-
parison of DNA cleavage from SC form to OC form is
shown in Figure 10.
4 | CONCLUSION
Series of pyrazoline based palladium (II) complexes have
been synthesized and characterized by various tech-
niques. Chelating effect minimize high labiality and
FIGURE 8 Molecular modeling of the complex 5a (ball and
stick) with the DNA duplex (VDW spheres) of sequence (5′‐
D(*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)‐3′)

THAKOR ET AL.
13 of 15
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hydrolysis rate of palladium complexes in the biological
atmosphere and thus improve their biological activities.
All compounds show noticeable antibacterial activity
against five different microorganisms. Compounds with
more electronegative fluorine substituted derivative give
significant antimicrobial activity. Cytotoxicity studies
using brine shrimp and S. pombe have been carried
out and result suggest that fluorine substituted moiety
is better cytotoxic agent than rest of five complexes.
The results of absorption titration as well as viscosity
measurement study suggest that all complexes bind to
HS DNA via intercalative mode of binding. The binding
constant of complexes are comparable to cisplatin and
some reported complexes, while better than trans
platin. The results of molecular docking and Gibb's free
energy corroborated to both above studies. Also, all the
thermodynamic parameters suggest the intercalative
mode of complex to DNA binding. Fluorescence study
provides additional evidence and suggest that metal
complexes intercalate in between the stacks of DNA
base pairs. Thus all the complexes bind to DNA via
intercalative mode of binding and chelation helps to
improve the binding strength of ligands to DNA.
Complexes can efficiently cleave the plasmid DNA in
absence of any external agents and this leads to the
conclusion that hydrolytic cleavage mechanism involves
in this study. Complexes 5c and 5d are compatible
with cisplatin.
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[18] N. R. Filipovic, S. Bjelogrlic, A. Marinkovic, T. Z. Verbic, I. N.
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Authors are thankful to Head, Department of Chemistry,
Sardar Patel University, India for making it convenient to
work in laboratory, Dhanji P. Rajani, microcare labora-
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G.C., New Delhi for providing financial assistance of
UGC BSR grant No. C/2013/BSR/Chemistry/59 and
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Bhupesh S. Bhatt http://orcid.org/0000-0003-0693-3226
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