Synthesis, characterization, structural-activity relationship and biomolecular interaction studies of heteroleptic Pd(II) complexes with acetyl pyridine scaffold

Article abstract of DOI:10.1016/j.molstruc.2020.128802

A series of substituted hydrazinyl pyridine based Schiff base ligands (L¹–L⁶) and corresponding palladium(II) complexes were synthesized and characterized by conductivity measurement, ¹H NMR, ¹³C NMR and liquid

Full text of DOI:10.1016/j.molstruc.2020.128802

Journal Pre-proof
Synthesis, characterization, structural-activity relationship and biomolecular
interaction studies of heteroleptic Pd(II) complexes with acetyl pyridine scaffold
Nikita J. Patel, Bhupesh S. Bhatt, Pankajkumar A. Vekariya, Foram U. Vaidya,
Chandramani Pathak, Juhi Pandya, Mohan N. Patel
PII:
S0022-2860(20)31127-3
DOI:
https://doi.org/10.1016/j.molstruc.2020.128802
MOLSTR 128802
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 April 2020
Revised Date: 18 June 2020
Accepted Date: 30 June 2020
Please cite this article as: N.J. Patel, B.S. Bhatt, P.A. Vekariya, F.U. Vaidya, C. Pathak, J. Pandya, M.N.
Patel, Synthesis, characterization, structural-activity relationship and biomolecular interaction studies of
heteroleptic Pd(II) complexes with acetyl pyridine scaffold, Journal of Molecular Structure (2020), doi:
https://doi.org/10.1016/j.molstruc.2020.128802.
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Nikita J. Patel: Conceptualization, Methodology, Software, Formal analysis, Validation,
Investigation, Resources, Writing- Original draft Bhupesh S. Bhatt: Software, Validation,
Conceptualization, Writing- Review & editing, Resources Pankajkumar A. Vekariya:
Software, Data Curation Foram U. Vaidya: Investigation, Data Curation Chandramani
Pathak: Resources Juhi Pandya: Investigation, Data Curation Mohan N. Patel: Writing-
Original draft, Writing- Review & editing, Supervision

Synthesis, characterization, structural-activity relationship and
biomolecular interaction studies of heteroleptic Pd(II) complexes with
acetyl pyridine scaffold
Nikita J. Patel1, Bhupesh S. Bhatt1, Pankajkumar A. Vekariya1, Foram U.
Vaidya2, Chandramani Pathak2, Juhi Pandya3 and Mohan N. Patel*1
1Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar–388 120, Gujarat (INDIA)
Phone number: (+912692) 226856*218
Corresponding address: jeenen@gmail.com
2Department of Cell Biology, School of Biological Sciences and Biotechnology,
Indian Institute of Advanced Research, Koba Institutional Area,
Gandhinagar-382007, Gujarat (INDIA)
3Department of Biosciences, Sardar Patel University,
Vallabh Vidyanagar, Gujarat, (INDIA)
Abstract
A series of substituted hydrazinyl pyridine based Schiff base ligands (L1-L6) and
corresponding palladium(II)
complexes were synthesized and characterized by
1
conductivity measurement, H NMR, 13C NMR and liquid chromatography mass
spectrometry (LC-MS). Synthesized compounds were screened against various biological
activities. UV-Vis spectroscopy, fluorescence spectroscopy, viscosity measurement, and
molecular docking studies were used to determine the binding mode between complexes
and HS-DNA, which suggest intercalation mode of DNA binding. The protein binding
study of complexes was evaluated by UV-visible spectroscopy. Antibacterial activity of
the complexes was screened against two Gram (+ve) and three Gram (-ve) bacteria and the
result shows that all complexes are more effective against microorganisms than their
respective ligands. The cytotoxicity of the synthesized compounds was tested against brine
shrimp and S. pombe cell. The LC50 values of the ligand and complexes are found in the

range of 9.24-14.12 µg/mL and 5.68-7.94 µg/mL, respectively. The DNA cleavage
activity of metal complexes was performed using agarose gel electrophoresis. All
compounds were tested for anticancer activity against human colorectal (HCT-116) cancer
cell lines. The result shows that all the compounds exhibited effective cytotoxicity against
the tested cell line.
Keywords: Mixed-ligand Pd(II) complexes, SAR, Protein binding, Cell line study
1. Introduction
The success of cisplatin in cancer treatment led the foundation of metal-organic,
organometallic complexes, and inorganic compounds in modern medicinal chemistry [1-
7]. Various factors are responsible for the increasing incidence of cancer, of which
modernization of our society is a major one. In cancer treatment, resistance to drugs is one
of the most significant problems [8, 9]. Several platinum drugs are used for the treatment
of cancer, but the side effects connected with the usage of these drugs considerably limit
their application [10]. Thus, transition metal like titanium, iron, osmium, rhodium, iridium,
palladium, and coinage metal ions have been considered as alternatives to platinum based
metallodrugs due to the cytotoxic properties of some of their compounds [11].
Effective biological activity of palladium(II) and platinum(II) complexes have attracted
significant attention in the research area [9, 12]. The interest in platinum(II) and
palladium(II) complexes containing S, S-, N, N- and O, S-donor have increased in
nowadays with the desire to synthesize antitumor drugs having reduced toxicity than
cisplatin and its analogues [13]. Anticancer agents have resulted in the development of a
large number of palladium(II) complexes with nitrogen donor ligands, such as various
alkyl and aryl substituted amines and imines, pyridine and pyrimidine derivatives, in
which some of exhibit effective antitumor properties both in vivo and in vitro [14].
Schiff base ligands are mostly applied in coordination chemistry and it can coordinate
with a large number of 3d, 4d, 5d transition metals [15]. Palladium is an important metal in
medicinal research due to significant similarities in the coordination chemistry with
platinum [16]. The initial way for cancer therapy will be metallodrugs started in the
interaction between DNA and coordination complexes. These include three main

interactions between DNA and metal complexes: intercalation, groove binding, and
electrostatic interaction [17, 18].
In continuation of our earlier work [19], six new hydrazinyl pyridine based Schiff base
and their mixed-ligand palladium(II) complexes were synthesized and characterized.
Synthesized compounds were screened against various biological activities.
2. Experimental section
2.1 Chemicals and materials
Commercial grade solvents and reagents were used without further purification. Sodium
tetrachloropalladate (II) was purchased from S.D. fine chem. Ltd (SDFCL). 2-Acetyl
pyridine, 2-hydrazinopyridine, 2-chlorobenzaldehyde, 2-bromobenzaldehyde, 4- chloro
benzaldehyde, 4-bromo benzaldehyde, 4-flourobenzaldehyde, 4- methylbenzaldehyde,
Hearring sperm HS-DNA and bovine serum albumin (BSA) were purchased from sigma
aldrich chemical co. (India). Nutrient Broth (NB), agarose, ethidium bromide (EtBr), tris-
acetate-EDTA (TAE) and bromophenol blue were purchased from Himedia (India). The
solvents used for spectral measurements were of spectroscopic grade from aldrich.
2.2 Physical measurements
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Brucker Avance
400 spectrometer at room temperature using deuterated dimethyl sulphoxide (DMSO-d6)
solvent. IR spectra were recorded on a FT-IR ABB Bomen MB-3000 spectrophotometer.
The melting points were determined on the thermocal10 melting point apparatus (Analab
Scientific PVT.LTD, India) in open capillaries. Electronic spectra were recorded with UV-
160A UV-Vis spectrophotometer, Shimadzu, Kyoto (Japan). Conductance measurements
were carried out using conductivity meter model number E-660A. Model Euro Vector EA
3000 and Perkin Elmer 240 elemental analyzer were used for C, H, N- elemental analysis
of ligand and complexes, respectively. Electron Spray ionization (ESI) mass spectra were
obtained using thermo scientific mass spectrometer (USA). The structures were optimized
by quantum mechanics methods. Optimization was performed using the Becke’s three
parameter hybrid exchange functional (B3) and the Lee–Yang–Parr correlation functional
(LYP) (B3LYP) with the Gaussian 09 program package[20]. Calculations were done using

an Intel CORE i3, 2.40 GHz based machine running MS Windows 8.1, 32 bit, x64 based
processor as the operating system.
2.3 Synthesis of substituted hydrazinyl pyridine based ligands (L1-L6)
A solution of substituted aldehyde (2a-2f, 1 mmol) was stirred in methanol at room
temperature with 2- hydrazinyl pyridine (1a, 1 mmol) in the presence of the catalytic
amount of acetic acid. The reaction was completed after 1 h, which was confirmed by
TLC. The white precipitates of the desired product were obtained, which were filtered. The
purity of product was checked on the TLC plate in ethyl acetate: n-hexane system (30:70).
Scheme 1. Reaction scheme for mixed-ligands palladium complexes (I-VI).
2.3.1 2-(2-(4-Chlorobenzylidene)hydrazinyl)pyridine (L1)
This ligand (L1) was prepared using 2- hydrazinyl pyridine (1a) (0.01 mmol) and 4- chloro
benzaldehyde (2a) (0.01 mmol). Color: White, Yield: 81%, mol. wt.: 231.68 g/mol, m.p.:
275 - 278 °C, Anal. Calc. (%) for C12H10CIN3: C, 62.21; H, 4.35; N, 18.14; Found (%): C,
62.03; H, 4.54; N, 17.99. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.92 (1H, s, -N-H), 8.17-
8.16 (1H, m, H6), 7.74 (1H, s, H2’), 7.67-7.61 (3H, m, H4’,8’,4), 7.40- 7.36 (3H, m, H5’,7’,5),
6.84-6.80 (1H, m, H3). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 156. 6 (C2, Cqut), 147.4

(C2’, -CH), 138.2 (C6, -CH.), 137.7 (C4’,8’, -CH.), 134.5 (C6’, Cquat.), 133.5 (C3’, Cquat), 128.9
(C5’,7’, -CH.), 127.5 (C4, -CH), 116.0 (C5, -CH), 107.6 (C3, -CH). [Total signal observed =
10: signal of Cqut = 3, signal of -CH = 7)].
2.3.2 2-(2-(4-Bromobenzylidene)hydrazinyl)pyridine (L2)
This ligand (L2) was prepared using 2- hydrazinyl pyridine (1a) (0.01 mmol) and 4- bromo
benzaldehyde (2b) (0.01 mmol). Color: White, Yield: 83%, mol. wt.: 276.14 g/mol, m.p.:
268 – 271 °C, Anal. Calc. (%) for C12H10BrN3: C, 52.20; H, 3.65; N, 15.22; Found (%): C,
52.16; H, 3.54; N, 15.39. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.62 (1H, s, -N-H), 8.16
(1H, d, J = 4.4 Hz, H6), 7.72 (1H, s, H2’), 7.67-7.62 (2H, m, H4’,8’), 7.58-7.52 (3H, m,
H4,5’,7’), 7.38 (1H, d, J= 8.4 Hz, H3), 6.82 (1H, dd, J1 = 5.2, J2 = 7.2 Hz, H5). 13C NMR (100
MHz, DMSO-d6) δ/ppm: 157.4 (C2, Cqut), 147.6 (C2’, -CH), 142.4 (C6’, -Cquat), 138.2 (C6, -
CH.), 137.7 (C4, -CH.), 131.8 (C5’,7’, -CH), 127.8 (C4’,8’, -CH.), 122.6 (C3’, Cquat), 116.1 (C5,
-CH), 107.6 (C3, -CH). [Total signal observed = 10: signal of Cqut = 3, signal of -CH = 7)].
2.3.3 2-(2-(4-Flourobenzylidene)hydrazinyl)pyridine (L3)
This ligand (L3) was prepared using 2- hydrazinyl pyridine (1a) (0.01 mmol) and 4- flouro
benzaldehyde (2c) (0.01 mmol). Color: White, Yield: 86%, mol. wt.: 215.23 g/mol, m.p.:
249 – 251 °C, Chemical formula: C12H10FN3. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.69
(1H, s, -N-H), 8.16 (1H, s, H2’), 7.77 (2H, d, J = 12.0 Hz, H4’,8’), 7.67-7.62 (2H, m, H4,6),
7.38 (2H, d, J = 8.0 Hz, H5’,7’), 7.12-7.07 (1H, m, H3), 6.79 (1H, dd, J1 = 3.2, J2 = 8.0 Hz,
H5). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 154.4 (C2, Cqut), 147.6 (C6, -CH), 138.1 (C2’, -
CH.), 137.8 (C4, -CH.), 134.2 (C6’, Cquat.), 128.1 (C4’,8’ -CH), 123.4 (C3’, Cquat.), 115.9 (C5, -
CH), 115.6 (C5’,7’, -CH), 107.5 (C3, -CH). [Total signal observed = 10: signal of Cqut = 3,
signal of -CH = 7)].
2.3.4 2-(2-(4-Mehtylbenzylidene)hydrazinyl)pyridine (L4)
This ligand (L4) was prepared using 2- hydrazinyl pyridine (1a) (0.01 mmol) and 4- methyl
benzaldehyde (2d) (0.01 mmol). Color: White, Yield: 80%, mol. wt.: 211.27 g/mol, m.p.:
243 – 245 °C, Anal. Calc. (%) for C13H13N3: C, 73.91; H, 6.20; N, 19.89; Found (%): C,
74.06; H, 6.54; N, 19.99. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 8.60 (1H, s, -N-H), 8.17-
8.15 (1H, m, H6), 7.76 (1H, s, H2’), 7.65-7.58 (3H, m, H4’,8’,4), 7.39 (1H, d, J = 8.4 Hz, H3),
13
7.22 (2H, d, J= 8.0 Hz, H5’,7’), 6.81-6.79 (1H, m, H5), 2.40 (3H, s, -CH3). C NMR (100
MHz, DMSO-d6) δ/ppm: 156.8 (C2, Cqut), 147.6 (C6, -CH), 139.3 (C2’, -CH.), 139.0 (C6’,
Cquat.), 138.1 (C4, -CH.), 132.2 (C3’, Cquat), 129.4 (C5’,7’, -CH.), 126.4 (C4’,8’, -CH), 115.6

(C5, -CH), 107.5 (C3, -CH), 21.4 (-CH3, -Methyl). [Total signal observed = 11: signal of
Cqut = 3, signal of –CH and CH3 = 8)].
2.3.5 2-(2-(2-Chlorobenzylidene)hydrazinyl)pyridine (L5)
This ligand (L5) was prepared using 2- hydrazinyl pyridine (1a) (0.01 mmol) and 2- chloro
benzaldehyde (2e) (0.01 mmol). Color: White, Yield: 89%, mol. wt.: 231.68 g/mol, m.p.:
269 – 272 °C, Anal. Calc. (%) for C12H10CIN3: C, 62.21; H, 4.35; N, 18.14; Found (%): C,
62.16; H, 4.54; N, 18.09. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 9.56 (1H, s, -N-H), 8.23
(1H, s, H2’), 8.09 (2H, dd, J1 = 1.6, J2 = 7.6, H6, 8’), 7.67-7.63 (1H, m, H5’), 7.43-7.37 (2H,
m, H6’,7’), 7.31 (1H, t, J = 7.2 Hz, H4), 7.26 (1H, dd, J1 = 5.6, J2 = 7.6 Hz, H5), 6.84 (1H, t,
J = 5.6 Hz, H3). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 157.7 (C2, Cqut), 147.6 (C2’, -CH),
142.8 (C3’, Cquat.), 138.1 (C6, -CH.), 135.4 (C4, -CH.), 130.0 (C5’, -CH), 129.6 (C6’, -CH.),
126.9 (C7’, -CH), 126.6 (C8’, -CH), 120.2 (C4’, -Cquat), 116.1 (C5, -CH), 107.6 (C3, -CH).
[Total signal observed = 12: signal of Cqut = 3, signal of -CH = 9)].
S
2.3.6 2-(2-(2-Bromobenzylidene)hydrazinyl)pyridine (L6)
This ligand (L6) was prepared 2- hydrazinyl pyridine (1a) (0.01 mmol) and 2- bromo
benzaldehyde (2f) (0.01 mmol). Color: White, Yield: 87%, mol. wt.: 276.14 g/mol, m.p.:
254 – 257 °C, Anal. Calc. (%) for C12H10BrN3: C, 52.20; H, 3.65; N, 15.22; Found (%): C,
52.10; H, 3.59; N, 15.49. 1H NMR (400 MHz, DMSO-d6) δ/ppm: 9.14 (1H, s, -N-H), 8.22
(1H, dd, J1 = 0.8, J2 = 4.8 Hz, H4), 8.18 (1H, s, H2’), 7.67- 7.63 (1H, m, H5), 7.61-7.56 (1H,
m, H6), 7.42-7.32 (4H, m, H5’,6’,7’,8’), 7.21- 7.18 (1H, m, H3). 13C NMR (100 MHz, DMSO-
d6) δ/ppm: 153.6 (C2, Cqut), 147.6 (C2’, -CH), 144.0 (C3’, Cquat.), 138.2 (C6, -CH.), 137.8 (C4,
-CH.), 133.1 (C6’, -CH), 129.9 (C5’, -CH.), 127.5 (C8’, -CH), 127.1 (C7’, -CH), 123.1 (C4’,
Cquat), 116.1 (C5, -CH), 107.6 (C3, -CH). [Total signal observed = 12: signal of Cqut = 3,
signal of -CH = 9].
2.4 General method for synthesis of mixed-ligands palladium(II) complexes (I-VI)
The methanolic solution of substituted hydrazinyl pyridine based Schiff base (L1-L6)
(0.0001 M) was drop wise added to a methanolic solution of Na2PdCl4 (0.0001 M). The
mixture was warmed for 10 minutes, followed by the addition of 2-acetylpyridine (0.0001
M). The resulted solution was refluxed at 60 °C. After 30 min, the stirring was carried out
at room temperature for 12-16 h. The obtained product was washed with diethyl ether and
hexane and dried to get yellow powder.

2.4.1 Palladium(II) complex (I) [Pd(L1)2-acpy]Cl2
This complex was prepared using ligand (L1) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 86%,
mol. wt.: 530.24 g/mol, m.p.: > 300 °C Anal. Calc. (%) for C19H17Cl3N4OPd: C, 43.05; H,
1
3.23; N, 10.57; Pd, 20.07; Found (%): C, 43.50; H, 3.56; N, 10.23; Pd, 20.78. H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.44 (1H, s, -N-H), 8.17 (1H, d, J = 5.6 Hz, H2’’), 8.01
(1H, s, H2’), 7.83 (2H, t, J = 8.0 Hz, H3’’,4’’), 7.93 (2H, d, J =5.2 Hz, H3,6), 7.47 (2H, d, J =
4.8 Hz, H5’,7’), 7.26-7.23 (2H, m, H4,5), 7.17 (1H, d, J = 6.8 Hz, H5’’), 6.97 (2H, t, H4’,8’),
3.44 (3H, s, -Methyl). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 173.2 (>C=O, Cquat), 157.3
(C6’’, Cqut), 155.4 (C2, Cquat), 147.4 (C2’, -CH.), 146.0 (C2’’,4’’, -CH.), 141.3 (C4, -CH.), 138.4
(C3’, Cquat), 133.2 (C6, -CH.), 130.0 (C5’,7’, -CH), 127.5 (C4’,8’, -CH), 124.9 (C5’’, -CH), 122.1
(C6’, Cquat), 116.8 (C3’’, -CH), 114.4 (C5, -CH), 108.5 (C3, -CH), 33.5 (Methyl, -CH3).
[Total signal observed = 16: signal of Cqut = 5, signal of -CH and CH3= 11)].
2.4.2 Palladium(II) complex (II) [Pd(L2)2-acpy]Cl2
This complex was prepared using ligand (L2) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 82%,
mol. wt.: 574.96 g/mol, m.p.: > 300 °C Anal. Calc. (%) for C19H17BrCl2N4OPd: C, 39.72;
1
H, 2.98; N, 9.57; Pd, 18.52; Found (%): C, 39.06; H, 2.52; N, 9.99; Pd, 18.43. H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.60 (1H, s, -N-H), 7.91 (1H, d, J = 4.4 Hz, H2’’), 7.82
(2H, t, J = 8.0 Hz, H3’’,4’’), 7.74 (1H, s, H2’), 7.29 (2H, d, J = 1.6 Hz, H3,6), 7.16 (2H, dd, J1
= 1.6, J2 = 8.0 Hz, H5’,7’), 7.11 (1H, d, J = 8.0 Hz, H5’’), 6.96 (2H, t, J = 6.0 Hz, H4, 5), 6.87
13
(2H, d, J = 8.4 Hz, H4’,8’), 3.45 (3H, s, -Methyl). C NMR (100 MHz, DMSO-d6) δ/ppm:
175.7 (>C=O, Cquat), 157.9 (C6’’, Cqut), 155.8 (C2, Cquat), 150.4 (C2’, -CH.), 149.6 (C3’,
Cquat.), 146.0 (C2’’,4’’, -CH.), 141.2 (C6, -CH), 136.2 (C5’,7’, -CH.), 131.8 (C4’,8’, -CH), 128.0
(C3’’, -CH), 127.2 (C5’’, -CH), 121.7 (C6’, Cquat), 120.0 (C4, -CH), 116.6 (C5, -CH), 108.4
(C3, -CH), 35.2 (Methyl, -CH3). [Total signal observed = 16: signal of Cqut = 5, signal of -
CH and CH3= 11)].
2.4.3 Palladium(II) complex (III) [Pd(L3)2-acpy]Cl2

This complex was prepared using ligand (L3) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 85%,
mol. Wt.: 513.69 g/mol, m.p.: > 300 °C, Anal. Calc. (%) for C19H17Cl2FN4OPd. Pd, 20.72;
1
Found (%): Pd, 20.86. H NMR (400 MHz, DMSO-d6) δ/ppm: 12.42 (1H, s, -N-H), 8.00
(1H, d, J = 4.4 Hz, H2’’), 7.92 (1H, d, J = 5.2 Hz, H6), 7.81 (2H, t, J = 7.6 Hz, H3’’,4’’), 7.74
(1H, s, H2’), 7.45 (2H, t, J = 8.8 Hz, H4,5), 7.24 (1H, d, J = 6.0 Hz, H3), 6.96- 6.90 (4H, m,
H4’,5’,7’,8’), 6.76 (1H, d, J = 6.0 Hz, H5’’), 3.74 (3H, s, -Methyl). 13C NMR (100 MHz,
DMSO-d6) δ/ppm: 178.4 (>C=O, Cquat), 158.4 (C6’’, Cquat), 156.8 (C2, Cquat), 150.3 (C2’’,4’’
-
CH.), 146.0 (C2’, -CH.), 141.1 (C6, -CH.), 139.3 (C3’, Cquat), 132.8 (C4’,8’, -CH.), 128.1 (C6’,
Cquat), 121.1 (C4, -CH), 120.9 (C5’’, -CH), 116.3 (C5’,7’, -CH), 110.9 (C3’’, -CH), 110.7 (C5, -
CH), 108.4 (C3, -CH), 34.9 (Methyl, -CH3). [Total signal observed = 16: signal of Cquat
5, signal of -CH and CH3= 11)].
=
2.4.4 Palladium(II) complex (IV) [Pd(L4)2-acpy]Cl2
This complex was prepared using ligand (L4) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 89%,
mol. wt.: 509.73 g/mol, m.p.: > 300 °C, Anal. Calc. (%) for C20H20Cl2N4OPd: C, 47.13; H,
1
3.96; N, 10.99; Pd, 20.88; Found (%): C, 47.16; H, 3.54; N, 10.19; Pd, 20.93. H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.34 (1H, s, -N-H), 7.90 (2H, d, J = 5.6 Hz, H2’’,5’’), 7.78
(2H, t, J = 8.0 Hz, H3’’,4’’), 7.67 (1H, s, H2’), 7.42 (1H, d, J = 8.4 Hz, H6), 7.04 (2H, t, J =
6.4 Hz, H4,5), 6.93 (2H, d, J = 6.4 Hz, H4’,8’), 6.84 (2H, d, J = 8.8 Hz, H5’,7’), 6.73 (1H, d, J
= 7.6 Hz, H3), 3.52 (3H, s, -Methyl( acetyl moiety)), 2.18 (3H, s, Methyl). 13C NMR (100
MHz, DMSO-d6) δ/ppm: 177.0 (>C=O, Cquat), 157.8 (C6’’, Cqut), 155.3 (C2, Cquat), 151.0
(C2’, -CH.), 145.8 (C3’, Cquat.), 140.8 (C2’,’4’’, -CH.), 138.0 (C4, -CH), 135.4 (C6’, -Cquat.),
130.1 (C6, -CH), 129.7 (C5’,7’, -CH), 126.2 (C4’,8’, -CH), 124.8 (C3’’, -CH), 120.0 (C5’’, -CH),
116.2 (C5, -CH), 108.2 (C3, -CH), 33.2 (Methyl (acetyl moiety), -CH3), 21.5 (Methyl, -
CH3). [Total signal observed = 17: signal of Cqut = 5, signal of -CH and CH3= 12)].
2.4.5 Palladium(II) complex (V) [Pd(L5)2-acpy]Cl2
This complex was prepared using ligand (L5) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 86%,
mol. wt.: 530.14 g/mol, m.p.: > 300 °C, Anal. Calc. (%) for C19H17Cl3N4OPd: C, 43.05; H,
1
3.25; N, 10.57; Pd, 20.07; Found (%): C, 43.32; H, 3.84; N, 10.89; Pd, 20.83. H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.54 (1H, s, -N-H), 8.17 (1H, d, J = 5.2 Hz, H2’’), 7.93

(2H, d, J = 4.8 Hz, H3,6), 7.83 (1H, s, H2’), 7.76 (2H, t, J = 8.0 Hz, H3’’,4’’), 7.47 (2H, d, J =
3.2 Hz, H5’,8’), 7.28 (2H, t, H4,5), 7.17 (1H, d, J = 6.0 Hz, H5’’), 6.97 (2H, t, J = 6.0 Hz,
H6’,7’), 3.43 (3H, s, -Methyl). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 176.4 (>C=O,
Cquat), 156.3 (C6’’, Cqut), 154.5 (C2, Cquat), 147.4 (C2’, -CH.), 146.0 (C2’’,4’’, -CH.), 141.3 (C4,
-CH.), 139.1 (C3’, Cquat), 133.2 (C6, -CH.), 130.0 (C5’,6’,8’, -CH), 127.5 (C7’, -CH), 124.9
(C3’’,5’’, -CH), 122.3 (C4’, Cquat), 116.9 (C5, -CH), 108.5 (C3, -CH), 33.8 (Methyl, -CH3).
[Total signal observed = 15: signal of Cqut = 5, signal of -CH and CH3= 10)].
2.4.6 Palladium(II) complex (VI) [Pd(L6)2-acpy]Cl2
This complex was prepared using ligand (L6) (0.01 mmol), 2- acetyl pyridine (0.01 mmol)
and Na2PdCl4 salt (0.01 mmol) as described in general process. Color: yellow, Yield: 86%,
mol. wt.: 574.60 g/mol, m.p.: > 300 °C, Anal. Calc. (%) for C19H17BrCl2N4OPd: C, 39.72;
1
H, 2.98; N, 9.75; Pd, 18.52; Found (%): C, 39.07; H, 2.13; N, 9.97; Pd, 18.78. H NMR
(400 MHz, DMSO-d6) δ/ppm: 12.54 (1H, s, -N-H), 7.93 (1H, d, J = 5.2 Hz, H2’’), 7.84
(2H, t, J = 8.0 Hz, H3’’,4’’), 7.79 (1H, s, H2’), 7.22 (2H, d, J = 7.2 Hz, H3,6), 7.11 (2H, d, J =
8.0 Hz, H5’,8’), 6.99 (2H, t, J = 6.4 Hz, H4,5), 6.91 (1H, d, J = 8.4 Hz, H5’’), 6.84 (2H, t, J =
8.0 Hz, H6’,7’), 3.52 (3H, s, -Methyl). 13C NMR (100 MHz, DMSO-d6) δ/ppm: 176.6
(>C=O, Cquat), 158.5 (C6’’, Cqut), 156.8 (C2, Cquat), 151.3 (C2’, -CH.), 150.1 (C3’, Cquat),
146.2 (C2’’,4’’, -CH.), 142.1 (C4, -CH.), 138.5 (C6, -CH.), 133.8 (C5’,8’, -CH), 128.0 (C6’,7’,
-
CH), 127.8 (C5’’, -CH), 122.5 (C4’, Cquat), 121.4 (C3’’, -CH), 113.2 (C5, -CH), 105.2 (C3, -
CH), 34.4 (Methyl, -CH3). [Total signal observed = 16: signal of Cqut = 5, signal of -CH
and CH3= 11)].
2.5 Biological screening of compound
2.5.1 Antibacterial activity by broth dilution method
The minimum inhibitory concentration (MIC) is defined as the lowest concentration of
bacterial agents that prevent the growth of bacteria (bacteriostatic). The in vitro
antibacterial activity of hydrazinyl pyridine based Schiff bases (L1-L6) and their metal
complexes (I-VI) were carried out against the two Gram(+ve) and three Gram(-ve) bacteria
using broth dilution method. All the bacteria were grown in 5 mL sterile nutrient or Luria
broth solution and incubated overnight at 37 °C. Different concentrations of the
compounds were injected with cultured bacteria. The next day, effects were checked to
determine at what level the MIC value is established [21, 22].

2.5.2 Evaluation of in vitro cytotoxicity
Brine shrimp lethality bioassay is a simple, reliable and convenient method used for the
evaluation of bioactivity of compounds and it extends for preliminary assessment of
toxicity. The cytotoxicity was reported in terms of lethal concentration (LC50).
Commercial sea salt was used for the preparation of artificial sea water (5%) for hatching
brine shrimp eggs. The incubation at 26-30 °C for 48 h results into free nauplii from
eggshells, which were collected using a light source and used for the screening. Ten active
shrimps were added in each test tube and volume was adjusted to 2500 µL per test tube to
obtain different concentrations. The surviving shrimp were counted after 24 h and lethal
concentration LC50 was assessed and percentage mortality (% M) of nauplii was plotted
against the log of the concentration of compounds [23, 24].
2.5.3 Cellular level cytotoxicity against S. pombe cells
The cellular level bioassay was carried out using S. pombe cells. S. pombe is harmless,
rapidly growing eukaryotes that are popular as model systems to understand basic
biological processes. In vivo cytotoxicity experiment was performed according to
previously published literature [25].
2.5.4 Cell line study
The human colorectal cancer cell line HCT-116 was purchased from the national centre for
cell science (NCCS), Pune, Maharashtra, India. The cell cultures were maintained under
controlled conditions in a humidified atmosphere with 5% CO2 at 37°C in culture flasks.
The cells were grown in complete RPMI-1640 medium supplemented with 10% FBS and
1% PSN, an antibiotics mixture (Life technologies, USA). Exponentially growing HCT-
116 cells were exposed to compounds and vehicle (1% DMSO) for 24 h. therefore, the
cells were washed with DPBS and incubated with MTT solution for 4 h in dark at 37°C.
After incubation, the MTT was removed and DMSO was added to each well. The
absorbance was taken at 570 nm with a reference wavelength of 650 nm by using a
multimode microplate reader.
2.6 DNA interaction studies
2.6.1 Electronic absorption spectra

The interaction of complexes with HS-DNA was investigated using UV-visible
spectroscopy. The stock solutions of metal complexes were prepared using DMSO and
diluted with phosphate buffer (pH = 7.2) to the required concentration for whole
experiments. Absorption titration experiments were performed by maintaining a constant
concentration of the complexes and HS-DNA concentration (0-100 µM) with varying
volume (0-500 µL). The UV-visible spectra of the complexes in the presence of HS-DNA
were scanned against phosphate buffer solution in the range from 200 to 800 nm
wavelength [26].
2.6.2 Hydrodynamic volume measurement
Viscosity measurement was used for further clarification of the DNA binding interaction
of the metal complexes. In this experiment, the HS-DNA solution (200 µM) was mixed
with an increasing amount of complexes and the relative viscosity of these solutions was
measured using Ubbelohde viscometer at 25.0 ± 0.5 °C. The data were obtained as (ƞ
/ƞ0)1/3 vs. [complex/HS-DNA], where ƞ0 and ƞ are the viscosity of HS-DNA in the absence
and presence of the metal complexes, respectively. Viscosity values were calculated from
the observed flow time of DNA containing solution (t) corrected for buffer alone (t0), ƞ α
(t-t0) [27, 28].
2.6.3 EB-displacement study
Fluorescence emission studies were carried out according to the previously reported
literature [29].
2.6.4 Molecular docking study
The docking study of Pd(II) complex with DNA was performed using the Hex 8.0
software. The binding position and bound conformation of the macromolecule with
complexes and approximate calculation of their interactions were analyzed with the hex
software. Docking needs data bank for the search target with proper PDB format. These
tools provide the information about the interaction between DNA and complexes. DNA
sequence d(ACCGACGTCGGT)2 (1BNA) of the structure was used for interaction study
and obtained from the protein data bank [30, 31].
2.6.5 DNA cleavage via agarose gel electrophoresis

Agarose gel electrophoresis experiment was carried out to monitor the cleavage of DNA
by synthesized complexes. The experimental procedure was performed according to the
reported procedure [25].
2.7 Protein binding study
The Serum albumin(SA)-binding study was performed by UV-visible electronic absorption
titration using BSA in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2) solution. In this
study, titration of BSA with mixed-ligand Pd(II) complexes was carried out. The
absorption titration spectra of BSA (15 µM) and the solution of mixed-ligand Pd(II)
complexes at 0, 8, 16, 24, 32, 40 µM concentration were measured in the range of 200-800
nm. In each fraction, after the addition of metal complexes, solution was incubated for 7
min., and the absorption spectra were overlaid [32].
3. Result and discussion
3.1 1H NMR spectra
1
The H NMR spectra of the Schiff base ligands (L1-L6) and their mixed-ligand
palladium(II) complexes (I-VI) were obtained in solvent DMSO-d6 as shown in
supplementary material 1 and their physiochemical data of the compounds are summarized
in the experimental section. In the aromatic region, ten protons are observed for ligands
(L1-L6) and fourteen protons are observed for palladium(II) complexes (I-VI). In Schiff
base ligands (L1-L6), all aromatic protons appear in the range of 8.23 – 6.79 δ ppm while
for Pd(II) complexes, appeared in the range of 8.17 – 6.73 δ ppm. The NMR signal for
imine (-N-H) group of ligands and complexes are appeared at around 9.56 – 8.60 δ ppm
and 12.60 – 12.34 δ ppm, respectively. The signal shift in the 1H NMR spectra suggests the
involvement of pyridine ring nitrogen as a coordinating atom.
3.2 13C NMR spectra
The 13C NMR spectra of the synthesized Schiff base ligands (L1-L6) and mixed-ligand
palladium(II) complexes (I-VI) are shown in supplementary material 2 and their
physicochemical data are represented in the experimental section. The peak of the methyl
group in the ligand (L4) and complex (IV) is observed at 21.43 ppm and 21.47 ppm,
respectively. The heteroleptic Pd(II) complexes (I-VI) containing 2-acetylpyridine

bidentate ligands having >C=O peak appeared in the range of 173.0-177.0 ppm. In ligands,
the peak of the C2 and Cquat are observed in the range of 153.6-157.7 ppm. In complexes,
the peaks of C2, and Cquat are shifted to the downfield region at 156.3-158.4 ppm.
3.3 Mass spectra
The mass spectra and fragmentation pattern of hydrazinyl pyridine based ligands (L1-L6)
are shown in supplementary material 3. The mass spectra of complex (I) shows a
molecular ion peak [M-2Cl]+ at 459.0 m/z and [M-2Cl+2]+ at 461.3 m/z. The peak at
338.10 m/z is due to the loss of 2-acetyl pyridine moiety from the complex. The peak at
233.90 m/z is due to the loss of palladium metal ion. The ESI-MS spectrum and possible
mass fragmentation pattern of complex (I) are shown in supplementary material 4.
3.4 Electronic spectra, magnetic behaviour, conductance measurement, and structure
optimization
The electronic spectra of mixed-ligand Pd(II) complexes were recorded in DMSO. The
electronic spectra of palladium(II) complexes show absorption peak at around 273-277 nm
(MLCT) and 451-456 nm (d-d transition), which suggest square planar geometry of the
complexes. The magnetic moment measurement of all the synthesized mixed-ligand Pd(II)
complexes is found to be zero, which is comparable to theoretical spin only value of
square planar complexes indicating diamagnetic nature and d8-low spin, with dsp2
hybridization. The observed molar conductance (25-40 Ω-1 cm2 mol-1) in 10-3 DMSO
solution of the complexes is consistent with the electrolytic nature of the complexes and
presence of two chlorine ion as a counter ion outside the coordination sphere [33, 34]. The
structure of complexes was optimized by Gaussian 09 program software to get information
about the relative position of ligands in the complexes. The optimized structures
(Supplementary material 5) clearly show that the oxygen atom of the acetylpyridine ligand
is trans to the pyridine group of the benzylidene hydrazinyl pyridine ligand. The pyridine
rings of both the ligands are cis to each other.
3.5 Biological screening of compounds
3.5.1 In vitro antibacterial activity
The persistence and growth of antibiotic resistance in bacterial pathogens represent a
public health concern. Antibacterial resistance may manipulate a biological cost, thereby

reducing the fitness of resistant strains, which can restrict the development of antibacterial
resistant bacteria. Increased antibacterial resistance is the grounds of severe infections,
complications, and increased mortality. Some newly developed antibiotics have lost their
efficacy against various bacterial infections within a few years after their debut. Metal
coordination can alter the mechanism of drug activity, the antibacterial activity of the
newly synthesized compound was tested against two Gram(+ve) negative and three
Gram(-ve) bacterial strain using the broth dilution method. The results of the antibacterial
study are represented in Fig. 1 and the data of the compounds are represented in
supplementary material 6.
Figure 1. Effect of different concentrations of hydrazinyl pyridine based Schiff base (L1-
L6) and mixed-ligand Pd(II) complexes on Gram(+ve) and Gram(-ve) bacteria.
MIC values of hydrazinyl based Schiff base and their corresponding mixed-ligand
palladium(II) complexes are in the range of 150-215 µM and 30-85 µM respectively,
which is comparable to previously reported palladium(II) complexes [35]. Complex VI has
antibacterial activity against all tested bacterial strains with MIC value in the range of 30-
45 µM. Compound VI has more potent activity with MIC value 30 µM against B. subtilis,
40 µM against P. aeruginosa. The complex V is most potent against E. coli with MIC
value 30 µM. From this result, we can conclude that the metal ion upon chelation enhances
the bacterial activity than the hydrazinyl based ligands. Tweedy’s chelation theory predicts

that chelation reduces the polarity of the metal ions mainly because of partial sharing of its
positive charge with donor group and possible electron delocalization over the entire ring.
Therefore, increases in the lipophilic character of the chelates, favouring their permeation
through the lipid layer of the bacterial membrane [36].
3.5.2 Brine shrimp lethality bioassay
In recent years, the brine shrimp lethality bioassay procedure of bioactive compounds has
gained great attention to evaluate cytotoxicity as well as pharmacological activity of the
compounds. The mortality rate of brine shrimp nauplii was found to increase with increase
in the concentration of the compounds. The main advantages of brine shrimp lethality
bioassay .are economical, inexpensive, short time generation than other cytotoxicity, and
more reliable method. The LC50 data was calculated by the plot of percentage mortality of
nauplii against the log of the concentration of the compounds, which is shown in Fig. 2,
and data are represented in supplementary material 7. The LC50 values of the hydrazinyl
Schiff base and their corresponding mixed ligand palladium(II) complexes are observed in
the range of 9.24-14.12 µg/mL and 5.68-7.94 µg/mL, respectively, which are comparable
with the previously reported Pd(II) complexes (8.31-16.80 µg/mL) [37]. From the data
recorded, the palladium(II) complexes show good activity than ligands. The order of
potency of compounds is cisplatin > V > VI > III > I > II > IV > L3 > L5 > L6 > L1 > L2 >
L4 > transplatin.
Figure 2. LC50 values of hydrazinyl based ligands and their corresponding mixed-ligand
Pd(II) complexes, cisplatin and transplatin in µg/mL

3.5.3 In vivo cytotoxicity against S. pombe cells
The cell viability assay is designed to measure activities attributable to cellular
maintenance and survival. The current most useful cytotoxicity biomarkers are constitutive
conserved and relatively stable, high- abundance enzymes released into the culture
medium following the loss of membrane integrity. The sixth model eukaryotic organism,
Schizosaccharomyces pombe, was used in the study of the cellular response to DNA
damage. The trypan blue dye used for the detection of S. pombe cells. Live cells or tissues
with intact cell membranes are not colored. Since cells are very selective in the compounds
that pass through the membrane in a viable cell trypan blue is not absorbed. Dead cells
appear as a distinctive blue color under the microscope. Cellular level in vivo cytotoxicity
against S. pombe cells was carried out using synthesized compounds, cisplatin, and
transplatin. The toxicity was found to change with the different substituents present in the
compounds. Complexes VI and V are found more toxic than other complexes against S.
pombe cells. After 17 h treatment, S. pombe cells were killed due to the toxic nature of the
compounds. Percentage viability graph and data of in vivo cellular level cytotoxicity
against S. pombe are represented in Fig. 3 and supplementary material 8, respectively.
Figure 3. Cytotoxicity in terms of % cell viability of compounds at different
concentrations.

3.5.4 Cell line study
For the investigation of potential synergetic cytotoxic effects, we evaluated cell anti-
proliferation activity of Pd(II) complexes. Colorectal cancer is one of the most common
malignancies worldwide [38] and hence the cytotoxicity of complexes I-VI was evaluated
by MTT assay against human HCT-116 cancer cell line with cisplatin as positive controls.
The IC50 value of the Pd(II) complexes (II, III, IV) exhibit some inhibitory effect on HCT-
116 cancer cell line, while the palladium(II) complexes (I, V, VI) are found to be
practically ineffective. Complexes (IV and V) are the most cytotoxic effect against human
colorectal carcinoma HCT-116 cancer cell line compared to other complexes. The IC50
value of the palladium(II) complexes are found in increasing order of IV (51 µg/mL) > V
(132 µg/mL) > II (138 µg/mL) > I (165 µg/mL) > VI (272 µg/mL) > III (283 µg/mL) >
DMSO. Furthermore, the IC50 value of the mixed-ligand palladium(II) complex IV is lower
than to standard drug carboplatin, but remaining complexes have higher IC50 value than
standard drugs cisplatin (15 µg/mL), carboplatin (64.97 µg/mL) and oxaliplatin (25.74
µg/mL) [39]. The graph and values are represented in Fig. 4 and supplementary material 9,
respectively.
Figure 4. Effect of synthesized mixed-ligand palladium(II) complexes on the proliferation
of HCT-116 cells and determination of IC50 value of 10, 25, 50, 100, 250, 500, 1000
µg/mL Pd(II) complexes (I-VI), control (100% DMSO), and vehicle control (1% DMSO).
Error bar represents standard deviation of three replicates.

3.5.5 General structure-activity relationship (SAR)
Different substituted benzyl rings on 2-hydrazino pyridine are widely affected the results
of biological activities (Fig. 5). The complexes (I, II, IV) containing chloro (-CI), bromo (-
Br), and methyl (-CH3) substituent at para position on benzyl ring increase the cytotoxicity
in brine shrimp lethality bioassay. The para position having fluoro (complex III) and ortho
position having –CI and –Br (complex V and VI) substituent on benzyl ring decrease the
LC50 value in brine shrimp assay. All the complexes have higher antibacterial activity
against B. substilis except complex- IV, which has an electron releasing group attached to
the ancillary ligand. Complexes (I and III) have potent antibacterial activity against B.
substilis and E. coli. Complexes (I, II, and IV) have less percentage viability and
complexes (III, V, VI) have higher percentage viability in in vivo cytotoxicity against S.
pombe cells.
Figure 5. General structural-activity relationship of mixed ligand Pd(II) complexes for
evaluation of LC50 value by brine shrimp lethality bioassay, antibacterial activity against
two Gram(+ve) and three Gram(-ve), and in vitro cellular level bioassay against S. pombe
cell.

3.6 DNA binding studies
3.6.1 Absorption titration study
The absorption titration method is one of the most useful tools for the investigation of the
mode of interaction of synthesized compounds with HS-DNA. The absorption spectrum of
hydrazinyl based Schiff base and their corresponding Pd(II) complexes at the constant
concentration (125 µM) in the absence and presence of HS-DNA (0-500 µL) with
different concentration is shown in Fig. 6. As shown in Fig. 6, when titrated by HS-DNA,
the complexes presented significant hypochromism effect around 240-265 nm and
accompanied the slightly red shifts in the absorbance maxima [40]. These spectral
characteristics suggest that the complex may interact with HS-DNA through the
intercalation mode. After the compounds intercalate to the base pairs of DNA, the π*
orbital of the intercalated compounds could couple with π orbital of the base pairs, hence
decreasing the π-π* transition energies, thus resulting in hypochromism. The hydrazinyl
based Pd(II) complexes showed more hypochromicity than the ligands, indicating that the
binding strength of the Pd(II) complexes is higher than the free ligands.
To quantitatively evaluate the binding affinity of the complexes with increasing DNA
concentration, the intrinsic binding constant (Kb) was determined by monitoring the
changes in absorbance at about 267-280 nm using the following equation.
[DNA]/ɛa-ɛf = [DNA]/ (ɛb-ɛf)+ 1/Kb (ɛb-ɛf)
where, ɛa, ɛf, and ɛb are the apparent extinction coefficients Aobs/[complex], the extinction
coefficient for the free complexes, and the extinction coefficient for the complexes in the
fully bound form, respectively. [DNA] is the concentration of DNA in base pairs, and Kb is
the intrinsic binding constant determined from the slop to intercept ratio of a plot of
[DNA]/ɛa-ɛf versus [DNA]. The Kb value of hydrazinyl based ligands and mixed-ligand
Pd(II) complexes are found in the range of 0.10 - 0.83 × 105 M-1 and 1.10 - 1.89 × 105 M-1
respectively, which is comparable to the standard drug cisplatin, i.e. 5.51 × 104 M-1 and
transplatin i.e. 1.75 × 104 M-1 and lower than classical intercalator EB, i.e. 7 × 107 M-1.
The binding strength of metal complexes with DNA is comparable to the reported
complexes [22]. From the data, we can note that the chelation of ligand to metal enhances
the binding affinity of the ligands. Percentage hypochromicity was calculated by [(Afree-
Abound)/Afree × 100]. Hypochromism of hydrazinyl based ligands (L1-L6) and Pd(II)
complexes (I-VI) are 9.20- 32.6% and 11.0-30.9%, respectively. The Gibb’s free energies
of the synthesized compounds are observed in the range of -22.81 to -30.07 kJmol-1. The

binding constant (Kb) values of metal complexes with HS-DNA are found in increasing
order of V > IV > VI > III >I > II. The binding constant (Kb), percentage
hypochromism(%H) and Gibb’s free energy data of the ligands (L1-L6), and metal
complexes are represented in Table 1. All the ligands and metal complexes graph are
represented in supplementary material 10.
Table 1. Binding
constant (Kb) and
Compounds Kb
%H ∆G°
(kJmol-1)
Compounds Kb
%H ∆G°
(kJmol-1)
(M1)×105
(M-1)× 105
L1
L2
L3
L4
L5
L6
0.83
0.42
0.52
0.20
0.17
0.10
32.6 -28.06
27.3 -26.37
13.1 -26.90
9.90 -24.53
24.5 -24.13
23.3 -22.81
I
1.37
1.10
1.63
1.84
1.87
1.81
11.0 -29.30
28.8 -28.76
26.8 -29.73
31.9 -30.03
26.0 -30.07
30.9 -29.99
II
III
IV
V
VI
Gibb’s free energy and percentage hypochromism (%H) of ligands (L1-L6) and Pd(II)
complexes (I-VI).

Figure 6. Absorption titration spectra of mixed ligand Pd(II) complex (II) (125 µM) with
increasing concentration of HS-DNA (0-500 µM) in phosphate buffer (Na2HPO4/
NaH2PO4).
3.6.2 Fluorescence study
Ethidium bromide (EB) displacement is a useful method to examine the reactivity of
chemical and biological systems it gives information about binding mechanisms and
provides clues for the nature of binding. EB emits intense fluorescent light in the presence
of DNA due to strong intercalation between the adjacent DNA base pairs. The
intercalation of Pd(II) complexes with HS-DNA was monitored by studying the quenching
the fluorescence of HS-DNA with increasing concentration of Pd(II) complexes. With the
gradual increase of the concentration of complexes, the fluorescence intensity of DNA-
EtBr gradually decreased as shown in Fig. 7. The displacement of EB by complexes at 610
nm suggests the intercalative mode of complex to DNA binding. The observed quenching
of DNA-EB fluorescence by the complexes suggest that the complexes can significantly
displace ethidium bromide from the DNA-EB system, thus illuminating the intercalation
mode of binding [41, 42]. Ethidium bromide displacement data of Pd(II) complexes and
graph are represented in supplementary material 11. This shows a dramatic increase in the
fluorescence intensity of the EB-DNA by adding the Pd(II) complexes. The quenching plot
illustrates that the fluorescence quenching of EB bound to DNA by the palladium (II)
complexes is in linear agreement with the stern-Volmer relationship.
I0/I = 1 + Ksv[Q]
where, I0 and I represent the fluorescence intensities in the absence and presence of
quencher, respectively. Ksv is a linear stern volmer quenching constant and [Q] is the
concentration of quencher.
The Ksv value calculated from the plot of I0/I versus [complex] is shown in Figure 7. The
stern-Volmer quenching constant (Ksv), change in standard Gibb’s free energy (∆G°), and
binding constant (Kf) of palladium (II) coordination compounds are shown in Table 2,
respectively. The Kf values calculated from the Scatchard plots indicate that the complex
V displays the higher binding ability and the order of binding strength of the complexes to
HS-DNA is V > IV > VI > III > I > II, which is the same as observed in the UV titration
study.

Table 2. Linear stern volmer quenching constant (Ksv, M-1), number of binding sites (n),
associative binding constant (Kf, M-1) of mixed ligand Pd(II) complexes with HS-DNA.
Figure 7. Fluorescence emission spectra of EtBr binds to HS-DNA in the absence and
presence of compounds.
3.6.3 Effect of HS-DNA on hydrodynamic volume measurement
Hydrodyna
Complexes
Ksv (M-1)
5.3 × 103
9.1 × 103
7.7 × 103
6.0 × 103
4.4 × 103
6.7 × 103
Binding sites (n)
1.138
Kf (M-1)
2.1 × 104
0.6 × 104
3.5 × 104
5.5 × 103
5.0 × 104
6.8 × 103
mic volume
measureme
nts, such as
viscosity of
HS-DNA is
critical test
for
I
II
0.969
III
IV
V
1.148
1.209
1.222
VI
1.208
evaluation of binding mode in solution in the absence of crystallographic structural data.
Classical intercalation mode should result in lengthening the DNA helix as base pairs are
separated to accommodate the binding ligand or the non-classical intercalation could bend
or kink the DNA helix, thereby decreasing its length and viscosity [35]. From the viscosity

measurements, it was observed that there is an increase in the relative viscosity of the HS-
DNA solution by increasing the concentration of complexes given in Fig. 8. The values of
relative viscosity, (ƞ/ƞ0)1/3 (ƞ0 and ƞ are the specific viscosity contributions of DNA in the
absence and the presence of the complexes) were plotted against 1/R (R =
[DNA]/[complex]). In the viscosity curve, the results reveal that the increasing
concentration of metal complexes increases the viscosity of HS-DNA, which provide
strong evidence for intercalation mode of binding [43]. Thus, we may deduce that the
mentioned complex can be considered as a DNA intercalator.
Figure 8. Effect of an increasing amount of mixed ligand Pd(II) complexes on the relative
viscosity of HS-DNA at 27 °C, [DNA] = 200 µM. The results are the triplicate
experiments carried out under identical conditions.
3.6.4 Computational study
Molecular docking is an attractive scaffold to understand drug biomolecular interactions
for the rational drug design. The information about the docking technique can be employed
to suggest the binding energy, stability of complexes, and to predict the tentative binding
parameters of the ligand-receptor complex. The molecular docking results exhibit the
ligands and their corresponding Pd(II) complexes approach the DNA cleavage site forming
stable complexes through the intercalation mode of binding. The relative binding energy of

the docked structures of ligands is -210.14 (L1), -211.27 (L2), -195.52 (L3), -205.43 (L4), -
204.72 (L5), -196.05 (L6) kJ mol-1and complexes are -257.48 (I), -259.71 (II), -258.08 (III),
-244.63 (IV), -254.68 (V), -241.37 (VI) kJ mol-1. The representative docked structure is
shown in Fig. 9 and other structures are shown in supplementary material 12.
Figure 9. Molecular docking study of mixed ligand Pd(II) complexes (II and V) with DNA
helix (PDB ID: 1BNA).
3.6.5 Photochemical analysis of DNA nuclease activity

Figure 10. DNA damage of the S. pombe cells by the treatment of complex (II and V) in
the range of (20-100 µM).
The electrophoresis experiment was performed to monitor the outcome of DNA nuclease
activity against stimulated S. pombe cell to see whether the complexes have any
consequence on the degradation of DNA or not. The study of DNA cleavage efficiency of
the molecule was examined by agarose gel electrophoresis. The treated and untreated S.
pombe cells were separated from DNA for DNA degradation analysis of S. pombe cells.
The damage caused by complex on DNA can be seen as DNA smearing due to the toxic
nature of the compound, whereas control cell DNA appeared normal as an intact band.
Smearing of DNA in Pd(II) complexes concluded that the damage occurs because of the
toxic nature of the compounds (Fig. 10). The results suggest complexes as a good
genotoxic agent, data of DNA cleavage of all Pd(II) complexes is shown in supplementary
material 13.
3.7 Protein binding study

Figure 11. UV-absorption spectra of BSA (15 µM) in the presence different concentration
of complexes (0- 40 µM).
UV- vis absorption spectroscopy is a common method to explore the structural changes of
protein and investigate protein complex formation. Static and dynamic quenching can be
distinguished by UV-absorption spectroscopy, which is done by the absorption spectra of
BSA in the presence of complexes. The UV absorption spectra of BSA in the presence and
absence of complexes revealed that the absorption intensity of BSA was enhanced as the
concentration of the compounds increased, and there was a little blue shift for all
compounds [44, 45]. Fig. 11 indicates that upon adding the complexes, BSA skeleton
absorption intensity decrease in the range of 278-283 nm and blue shift appear due to the
perturbation of the secondary structure of the protein. Pd(II) complexes absorption titration
graph with BSA is represented in supplementary material 14.
4. Conclusion
The novel mixed-ligand Pd(II) complexes(I-VI) were synthesized by the complexation of
hydrazinyl pyridine based substituted Schiff base (L1-L6), 2-acetyl pyridine with Na2PdCl4.
1
Spectroscopic characterization (UV-vis, H NMR, 13C NMR, and mass) supported the
formation of the complexes. The Pd(II) complexes having square planar geometry,
diamagnetic nature, and d8-low spin configuration with dsp2 hybridization. The structure

optimization of complexes suggest that pyridine rings of both the ligands are cis to each
other. The UV-visible spectral titration for protein binding study suggests perturbation in
the secondary structure of protein influenced by Pd(II) complexes. The UV-visible,
viscosity, fluorescence titration and molecular docking studies of Pd(II) complexes reveal
that the interaction of synthesized complexes with HS-DNA is specifically through
intercalation type of binding. The synthesized Pd(II) complexes have potent antimicrobial
activity against Gram(+ve) and Gram(-ve) bacterial species than the Schiff bases. All the
mixed-ligand Pd(II) complexes exhibit good in vitro cytotoxic activity and cellular level
cytotoxicity.
ACKNOWLEDGMENT
The authors are thankful to the Head, Department of Chemistry, Sardar Patel University,
for providing necessary research facilities; DST-PURSE Sardar Patel University, for LC-
MS analysis; CAS-Phase-II and UGC-CPEPA program, Sardar Patel University for
providing instrumental and chemicals facilities.
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