Schiff base triphenylphosphine palladium (II) complexes: Synthesis, structural elucidation, electrochemical and biological evaluation

Article abstract of DOI:10.1016/j.molstruc.2016.04.003

The complexes N-(2-oxidophenyl)salicylideneiminatotriphenylphosphine palladium(II) (1) and N-(2-sulfidophenyl)salicylideneiminato triphenylphosphine palladium(II) (2) of tridentate Schiff bases derived from salicylaldehyde and an amino- or thiophenol, have been synthesized and characterized by various spectroscopic, analytical and electro-analytical techniques. X-ray single crystal analysis of complex 1 has revealed its square planar geometry. The thermal analysis has shown the absence of coordinated water and final degradation product is PdO. The alkaline phosphatase studies have indicated that enzymatic activity is concentration dependent which is inversely proportional to the concentration of the compounds. The biological assays (brine shrimp cytotoxicity, DPPH) have reflected their biologically active and mild antioxidant nature. However, results of DNA protection assay have shown that they possess moderate protective activity against hydroxyl free radicals (OH). The voltammetric studies ascertain two-electron reduction of the compounds through purely diffusion controlled process and reveal intercalative mode of drug DNA interactions.

Full text of DOI:10.1016/j.molstruc.2016.04.003

Accepted Manuscript
Schiff base triphenylphosphine palladium (II) complexes: synthesis, structural
elucidation, electrochemical and biological evaluation
Muhammad Shabbir, Zareen Akhter, Iqbal Ahmad, Safeer Ahmed, Maryam Shafiq,
Bushra Mirza, Vickie McKee, Khurram Shahzad Munawar, Ahmad Raza Ashraf
PII:
S0022-2860(16)30312-X
10.1016/j.molstruc.2016.04.003
MOLSTR 22417
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 2 January 2016
Revised Date: 2 April 2016
Accepted Date: 2 April 2016
Please cite this article as: M. Shabbir, Z. Akhter, I. Ahmad, S. Ahmed, M. Shafiq, B. Mirza, V. McKee,
K.S. Munawar, A.R. Ashraf, Schiff base triphenylphosphine palladium (II) complexes: synthesis,
structural elucidation, electrochemical and biological evaluation, Journal of Molecular Structure (2016),
doi: 10.1016/j.molstruc.2016.04.003.
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Graphical abstract:

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Schiff base triphenylphosphine palladium (II) complexes: synthesis, structural elucidation
, electrochemical and biological evaluation
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Muhammad Shabbir , Zareen Akhter a,*, Iqbal Ahmad , Safeer Ahmed , Maryam Shafiq ,
Bushra Mirza b, Vickie McKee c, Khurram Shahzad Munawar a, Ahmad Raza Ashraf a
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a Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
b Department of Biochemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
c School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.
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* Corresponding author. Email: zareenakhter@yahoo.com (Z. Akhter)
Telephone +92 051 90642111; fax: +92 051 90642224
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Abstract
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The complexes N-(2-oxidophenyl)salicylideneiminatotriphenylphosphine palladium(II)(1) and
N-(2-sulfidophenyl)salicylideneiminato triphenylphosphine palladium(II)(2) of tridentate Schiff
bases derived from salicylaldehyde and an amino- or thiophenol, have been synthesized and
characterized by various spectroscopic, analytical and electro-analytical techniques. X-ray single
crystal analysis of complex 1 has revealed its square planar geometry. The thermal analysis has
shown the absence of coordinated water and final degradation product is PdO. The alkaline
phosphatase studies have indicated that enzymatic activity is concentration dependent which is
inversely proportional to the concentration of the compounds. The biological assays (brine
shrimp cytotoxicity, DPPH) have reflected their biologically active and mild antioxidant nature.
However, results of DNA protection assay have shown that they possess moderate protective
activity against hydroxyl free radicals (.OH). The voltammetric studies ascertain two-electron
reduction of the compounds through purely diffusion controlled process and reveal intercalative
mode of drug DNA interactions.
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Key Words: Palladium (II) complexes; structural elucidation; Drug-DNA intercalation
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1. Introduction
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Schiff bases, the condensation products of primary amines with either aldehydes or ketones, are
versatile compounds capable of coordinating with metals through imino nitrogen and another
donor atom (O&O/S) [1]. Such ligands having functional groups like -OH and/or -SH present
ortho to the azomethine group form five or six membered stable chelates. The metal complexes
having O, N, S donor Schiff bases possess unusual configuration, structural lability and are
sensitive to molecular environment. The environment around the metal center (coordination
geometry, number of coordinating ligands and their donor groups) is the key factor for metallo
proteins in order to carry out specific physiological functions. S-ligated complexes may mimic
the ligation of certain biomolecules in proteins. The presence of oxygen moiety can enhance
antitumor activity of compounds. Similarly, antibacterial and antifungal activities of the
complexes appear due to the chelating behavior of the ligands with most of the metal ions
coordinated through N and S donor atoms [2].
Schiff base transition metal complexes are widely used for organic synthesis, drug design and
[3, 4]
catalysis
.The redox behavior of palladium is dominated by electrochemical conversions
between palladium(0), palladium(II), and palladium(IV) species. Palladium(I) complexes are
rarely found due to their instability and disproportionation whereas Palladium(II) complexes are
comparatively stable [5,6]. The interest in the electrochemical investigation of palladium and
platinum based complexes has emerged in last few years because of their higher cytotoxic
activity and less side effects in comparison to the established drug molecules like Cisplatin.
Palladium(II) complexes have been found to be potential inhibitors of enzyme alkaline
phosphatase (ALP). They interact with the negatively charged functional groups of ALP that are
necessary for regulating the structural and catalytic activities of enzymes [7]. Alkaline
phosphatases (EC 3.1), catalyze the transfer of phosphate groups to water (hydrolysis) or alcohol
(transphosphorylation) using a wide variety of phosphomonoesters [8, 9]. Palladium complexes
with nitrogen and sulfur containing ligands are known to exhibit antibacterial and antitumor
activity. They play a vital role in controlling gene expression, inhibiting cell replication and
hence are used as valuable drugs. They show various modes of interactions, for example
electrostatic, intercalative and groove binding that can be monitored by cyclic voltammetry, UV–
Vis., fluorescence, Raman and NMR spectroscopies [10-12]. However, problem associated with
such complexes is their ready dissociation in solution leading to very reactive species that are
unable to reach their pharmacological targets such as DNA. This rapid aquation and formation of
very reactive species could be overcome if palladium(II) complexes are stabilized by bulky
ligands such as triphenylphosphine [13]
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Inspired by the wide applications of such compounds in various fields, palladium(II)
triphenylphosphine complexes (1&2), containing N-(2-oxidophenyl)salicylideneiminate) (H2L1)
and N-(2-sulfidophenyl)salicylideneiminate) (H2L2) Schiff bases were synthesized. Contrary to
[14, 15]
their previous studies
we have explored them for enzyme inhibition towards ALP,
biological (brine shrimp cytotoxicity, potato disc antitumor and hydroxyl free radical (•OH)
scavenging assays) and voltammetric studies to investigate their DNA binding mode.
2. Experimental
2.1 Materials and methods
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All chemicals were of reagent grade and solvents were purified by standard techniques and
drying methods [16]. Triphenylphosphine, palladium(II) chloride and ALP buffer solution were
used as received from Aldrich. [PdCl2(PPh3)2] was prepared by mixing anhydrous PdCl2
dissolved in acidified methanol and triphenylphosphine in acetone with constant stirring for an
hour in molar ratio 2:1 [17]. The Schiff bases H2L1 and H2L2 were synthesized by condensation
reactions of salicylaldehyde with 2-aminophenol and 2-aminobenezenethiol in ethanol following
the reported method [18]. Elemental analysis was carried out on a CHNS 932 (Leco-USA)
elemental analyzer. Melting points were determined, using a MPD Mitamura Riken Kogyo
(Japan) electrothermal melting point apparatus. FTIR spectra were recorded on a
Thermoscientific (USA) Nicolet 6700 spectrometer, number of resolutions 4 cm-1, the scan's
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numbers16, detector DTGS-KBr and the frequency range of 4000-400 cm-1. H NMR (300
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MHz), C NMR (75 MHz) and P NMR (122 MHz) spectra were recorded on a Bruker NMR
Spectrometer in deuterated chloroform using tetramethylsilane as internal reference.
Thermogravimetric analyses of the complexes was performed on TGA/SDTA 851e Mettler
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Toledo at a heating rate of 20 C /min over a temperature range of 40-800 C under nitrogen
flow.
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A single crystal of complex 1 was obtained by the slow evaporation from the ethanol mother
liquor at room temperature. X-ray data were collected at 150(2) K on a Bruker Apex II CCD
diffractometer using MoKα radiation (λ = 0.71073Å). The structure was solved by direct methods
and refined on F2 using all the reflections [19]. All the non-hydrogen atoms were refined using
anisotropic atomic displacement parameters and hydrogen atoms were inserted at calculated
positions using a riding model. There was a disorder in the structure arising from a 180° rotation
of the ligand (positions of O1 and O2 exchanged) and this also affected the position of the metal
atom. This was modeled for the Pd ion and the “linker” atoms C7 and N1 but no attempt was
made to model the resultant alight disorder of the phenolate groups (Fig. 1). The relative
occupancy of the two sites refined to 82:18. Parameters for data collection and refinement are
summarized in Table 1.
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Activity of the enzyme ALP was determined at room temperature by following the increasing
absorbance at 405 nm due to para-nitrophenol formed by the hydrolysis of the substrate para-
nitrophenyl phosphate (pNPP). In this method, 100 µL of ALP was added to 5 mL of an assay
system containing pNPP (2 mM) in 0.01 M Na2CO3-NaHCO3 buffer (pH=10.1) containing
different concentrations of palladium(II) complexes [20,21]
.
The biologically active nature of compounds was assessed by brine shrimp cytotoxic assay.
Seawater (34 g/L) was used for hatching of brine shrimp (Artemia salina) eggs (Ocean Star Inc.,
USA) under constant aeration at room temperature. After 24 h of hatching, shrimps were
gathered via pipette from lightened side of the dish and were transferred to marked vials.Ten
shrimps were transferred to each vial using a Pasteur pipette and the volume was raised up to 5
mL with artificial seawater having four different concentrations 200, 66.6, 22.2 and 7.4 µg mL -1
for each compound. Three replicates were prepared for each concentration.Vials were kept at
room temperature under illumination for 24 h and then survivors were counted by the aid of 3×
magnifying glass. Percentage death was calculated via Abbott’s formula and the LD50 value was
calculated by using Finney (1971) software [22]. Antitumor activity was screened by using potato
disc antitumor assay [23]. Agrobacterium tumefacians, refreshed AT-10 culture was used to
prepare inoculum with three different concentrations (1000, 100, 10 &1µg mL-1). Surface
sterilized potatoes were then loaded with 50 µL of inoculum. DMSO was used as negative
control while positive control was doxorubicin. Tumor inhibition ability of compounds was
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calculated by following formula, (Percentage inhibition = 100 – average number of tumours of
sample/ average number of tumours of negative control × 100). Antioxidant activity of the
compounds (1&2) was evaluated by DPPH free radical scavenging assay [24] employing ascorbic
acid and DMSO as positive and negative controls respectively. Five different concentrations of
both compounds (200, 66.6, 22.2, 7.4, 2.7 µg mL-1) were tested in triplicate. The reaction
mixture was incubated for 30 min at 37oC and absorbance at 515 nm was determined by
spectrophotometer. DNA damaging activity was assessed by inhibition of hydroxyl (.OH) free
radical induced DNA protection assay [25]. Compounds were analyzed at four different
concentrations (1000, 100, 10 and 1µg mL-1). Reaction mixture of 15 µL (5 µL compound, 3 µL
FeSO4, 4 µL H2O2 and 3 µL plasmid) was prepared, using DMSO as positive control. For
negative control 3 µL of plasmid and 12 µL of phosphate buffer were taken. Damaging effect of
compound on DNA (control compound) was checked in the absence of H2O2. Reaction mixture
along with controls was incubated for an hour in dark at 37oC and then it was subjected to gel
electrophoresis after staining with ethidium bromide. Ladder consisting of 1 kb was employed as
marker. Gel was then analyzed via Gel- Doc (Bio Rad) software.
Voltammetric (cyclic, differential pulse and square wave voltammetry) experiments were
performed employing Eco Chemie Autolab PGSTAT 302 potentiostat/galvanostat (Utrecht, The
Netherlands) using software version GPES 4.9. All the voltammetric experiments were
performed with conventional three-electrode cell with Ag/AgCl as a reference, home-made thin
platinum wire as a counter and a glassy carbon electrode (GCE) with surface area of 0.09 cm2 as
a working electrode. Prior to use the active surface of GCE was polished with 0.25 m alumina
paste on a nylon buffing pad, followed by washing with distilled water and finally rinsing with
DMSO. Tetrabutylammonium tetrafluoroborate (TBATFB) was used as supporting electrolyte.
For compound-DNA interaction studies solutions of the compounds were prepared in aqueous-
DMSO mixture (2:8, v:v) and subjected to CV analysis at a scan rate of 100 mVs-1 with
progressive addition of DNA.
2.2 General Procedure for the Synthesis of Pd (II) Complexes
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Complexes 1 and 2 were prepared by refluxing equimolar ethanolic solutions of Schiff base
(H2L1 or H2L2) and PdCl2(PPh3)2 in 250 mL two neck flask under nitrogen for 24 h. The red solid
was filtered off, washed with ethanol, dried in air and stored in vacuum desiccator (Scheme 1).
H
H
N
C
C
N
Cl
Cl
PPh3
PPh3
Pd
Pd
Y
O
PPh3
OH HY
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where PPh3 = triphenylphosphine, Y = O for 1 ,S for 2
Scheme 1: Synthesis of complexes1and 2.
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2.2.1 N-(2-oxidophenyl)salicylideneiminato triphenylphosphiepalladium(II)(1)
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Yield: 83%. mp 254 oC, υmax/ cm-1 1595 (-CH=N), 1338 (C - O), 545 (Pd-O), 412 (Pd-N) δH 7.9-
6.6 (m, 23 H, Ar–H), 8.8 (s, H, –CH= N–). δC 169.0 (C–O),163.5 (C=N),147.6 (C–P), 138.1-
114.5(Ar-C), δP 22.8. Calc. for C31H24NO2 PPd: C 64.2, H 4.1, N 2.4.Found: C 64.3, H 4.1, N
2.3%.
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2.2.2 N-(2-sulfidophenyl)salicylideneiminato triphenylphosphine palladium(II)(2)
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Yield: 79%. mp 242 oC, υmax/ cm-1 1612 (-CH=N), 1327 (C - O), 744 (C-S), 538 (Pd-O),408 (Pd-
N) δH 7.7–6.5 (m, 23H, Ar–H), 8.7 (s, 1H,–CH= N–). δC 169.4 (C–O),159.9 (C= N), 145.3 (C–
P), 135.1-117.9 (Ar-C), δP 23.3. Calc for C31H24NOS PPd: C 62.4, H, 4.0, N 2.4, S 5.4.Found: C
62.4, H, 4.0, N 2.4, S 5.3%.
3 Results and Discussion
3.1 Synthesis and characterization
The complexes 1 and 2 were prepared from equimolar ethanolic solutions of Schiff base (H2L1 or
H2L2) and PdCl2(PPh3)2.The synthesized complexes(1&2) were characterized by various spectral
techniques(FTIR, 1H NMR, 13C NMR, 13P NMR) and single crystal X-ray analysis.
All the characteristic bands observed for the complexes in FTIR spectra are in close agreement
with their structures. The bands at 1595, 1612 ν(C= N), 1338, 1327 ν(C–O) vibrations are for 1
and 2 respectively. The C=N bond vibrations are shifted to lower frequency due to reduction in
electron density which shows coordination of the ligand with palladium. For complex 2 a band at
744 was shown due to ν(C–S) vibration [26, 27]. The bands at 545 and 538 cm−1 in the complexes
(1 & 2) were assigned to ν (Pd–O) vibrations. The bands observed at 412 and 408cm−1 were due
to ν (Pd–N) mode [15]. Further, the compounds showed strong bands near 1425, 1108, and 659
cm−1characteristic of triphenylphoshine indicating the coordination of this ligand with palladium
metal [28] .The FTIR spectra of complexes (1 & 2) is presented in Fig. S1.
In the 13C NMR spectra of the complexes, azomethine carbon resonances were observed at 163.5
and 159.9 ppm. The resonances at 169.0(C–O), 147.6 (C–P) and 169.4 (C–O), 145.3 (C–P) ppm,
respectively, were observed for both the complexes. The signals at 138.1-114.5 and 135.1-117.9
ppm were due to the aromatic rings of Schiff bases and triphenylphosphine for the complexes 1
and 2, respectively as revealed in Fig. S3.
31P NMR spectra exhibited singlets at 22.8 and 23.3 ppm suggesting coordinated
triphenylphosphine in each case [29] as displayed in Fig. S4.
3.2 X-ray structure analysis of complex 1
A perspective diagram of neutral complex Pd(L1)PPh3 (1) is shown in Fig. 1. Data collection and
refinement parameters are given in Table 1 while selected bond lengths and angles are given in
Table 2.The palladium ion has approximate square planar coordination (rms deviation from the
PdO2NP mean plane, 0.22Å) bound to the phenolate and imine donors of L1 and to the PPh3
ligand. The two Pd – O distances are distinctly different, presumably reflecting the different
chelate ring size. There is a disorder in the structure arising from a 180° rotation of the ligand
(positions of O1 and O2 exchanged) and this also affects the position of the metal atom. The
disorder was modeled for the palladium ion and the “linker” atoms C7 and N1 but no attempt
was made to model the resultant slight disorder of the phenolate groups. The relative occupancy
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of the two sites refined to 82:18.The complexes are arranged into interdigitated, π-stacked
columns.
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Fig.1. Perspective view of Pd(L1)PPh3 (1). Ellipsoids are drawn at the 50% probability level.
White bonds and primed labels show the minor (18%) component of the disorder.
The principal π interaction is between the imine of one L1 ligand and neighboring C1-C6 and
C8-C13 phenol rings (under symmetry operations 1-x, 1-y, -z and -1x, -y, -x respectively); the
imine centroid - ring centroid distances are 3.340 and 3.665Å (Fig. 2).
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Fig. 2. Packing diagram for 1 showing imine-phenol π-stacking. Purple dots indicate the centroid
of the imine; green and red dots indicate the centroids of the C1-C6 and C8-C13 phenolate rings
respectively.
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The only significant link between stacks is an (aromatic) C-H ... π interaction between C34-H34
to the centroid of the phenyl ring containing C41 (H34−centriod 2.594 Å under symmetry
operation x, y-1, z (Fig.S5).
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Table 1. Data collection and refinement parameters.
C31H24NO2PPd
V = 2486.4 (3) Å3
Z = 4
Mr = 579.88
Monoclinic, P21/n
a = 15.4856 (12) Å
b = 9.0872 (7) Å
Mo Kα radiation, λ = 0.71073 Å
µ = 0.84 mm-1
T = 150 K
c = 18.3498 (14) Å
β = 105.655 (1)°
0.37 × 0.23 × 0.05 mm
Bruker APEX 2 CCD diffractometer
6196 independent reflections
Absorption correction: multi-scan
5310 reflections with I > 2σ(I)
SADABS v2012/1, Sheldrick, G.M., (2012)
T
min = 0.339, Tmax = 0.431
Rint = 0.031
measured reflections
R[F2 > 2σ(F2)] = 0.037
wR(F2) = 0.085
S = 1.05
24586
5 restraints
H-atom parameters constrained
∆〉max = 0.78 e Å-3
∆〉min = -0.62 e Å-3
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6196 reflections
parameters
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Table 2. Selected bond lengths [Å] and angles [°] for 1 (major component)
Pd1—O1
2.019 (3)
Pd1—N1
2.006 (4)
Pd1—O2
1.948 (3)
Pd1—P1
2.2980 (13)
91.55 (7)
174.48 (12)
89.85 (9)
O2—Pd1—N1
O2—Pd1—O1
N1—Pd1—O1
83.66 (12)
176.57 (10)
95.08 (11)
O2—Pd1—P1
N1—Pd1—P1
O1—Pd1—P1
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3.3 Thermal analysis
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Thermal analysis was carried out in an inert atmosphere of argon at a heating rate of 20 C/min
over a temperature range of 25-800 C to check the presence of coordinated water molecules.
The weight loss for each complex was calculated within corresponding temperature range. The
decomposition of both the complexes occurred in a single step starting above 140 C, showing
absence of coordinated water
(found/calc. 79.59/79.82 %) leaving behind the residue, PdO, (found/calc. 20.40/20.17 %).
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[30]
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and completed at 706 C with the loss of organic moiety
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3.4 Electrochemical studies
Electrochemical behavior of the complexes was investigated by employing advanced
electrochemistry techniques namely cyclic voltammetry (CV), differential pulse voltammetry
(DPV) and square wave voltammetry (SWV). The objective of these studies was to assess the
redox mechanism of the complexes, both qualitatively and quantitatively, the former tells about
the characteristic redox potential and the nature of electron transfer (ET) process while latter
gives the number of such ET processes and the electrons involved in each step. The kinetics
across the electrode solution interface was also investigated while determining the diffusion
coefficient value which in turn is applied to compare the ET process of complex alone and in
presence of DNA- a medicinal application through in vitro study.
3.4.1 Cyclic Voltammetry
The CV of 2 mM each of 1 and 2 in their argon saturated aqueous/DMSO solution (2:8, v: v)
containing 0.1M tetrabutylammonium tetrafluoroborate as a supporting electrolyte was
performed at glassy carbon electrode first in the potential range of -1.5 V to +1.5 V at scan rate
of 100 mVs-1 at 25o C (Fig. 3). During anodic scan no well-defined oxidation signal (except a
hump) was observed for both the compounds while scanning cathodically a reduction peak
centered at Epc = -1.34 V (vs. Ag/AgCl) for 1 and for 2 two reduction signals centered at Epc = -
1.40 V and -1.26 V (vs. Ag/AgCl) were observed represented as 1c and 2c, respectively. Hence
further experiments were performed in potential window of -1.6 to -0.5 V. The absence of
oxidation wave clearly indicates the highly reactive nature of the reduced form of the complexes
and thus an electron transfer followed by a chemical reaction i.e. EC mechanism was followed.
The higher reduction values (beyond -1.0V) can be correlated to the lower electron affinity of the
electrophore having electron donating ligands of PPh3. Relatively easier reduction in 2 can be
ascribed to the less electronegative character of S(2.58) in comparison to 1(3.44). The very weak
anodic signal, as a hump around -1.3V, particularly in complex 2, indicates the outer
reorganization phenomenon commonly present in metal complexes [31]. The appearance of two
explicit reduction peaks in complex 2 and one reduction peak in complex 1 could be attributed to
the presence of less electronegative S in complex 2 which not only facilitates the first reduction
(anion formation) but also allows the dianion formation. It is notable that there is no structural
difference other than replacement of oxygen in complex 1 by sulphur in complex 2. Thus there is
high probability of intramolecular rearrangement that makes the PPh3 more available to induce e-
donating character in presence of less electronegative S in comparison to that of more
electronegative O.
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Fig. 3. Cyclic voltammograms of (2 mM each) 1 (A) and 2 (B) at GCE (0.09 cm2) in aqueous-
DMSO (2:8, v: v) solution containing 0.1M TBATFB at scan rate of 100 mVs-1 at 25oC.
Assignment of number of electrons involved in the reduction step was confirmed by DPV
discussed later. The reduction process involving two electrons might be attributed to the
formation of a palladium(0) center with coordination number between 2 and 4 (or to an
equilibrium mixture of several palladium(0) species) [32]. There are many examples of stable
Pd(0) complexes available in literature, the most relevant is [Pd(diars)2]0 (diars = 1,2-
bis(dimethylarsino)benzene), which is sufficiently stable to be isolated in the solid form [33]. The
absence of an oxidation peak during anodic scan for both the complexes indicates that the
complexes undergo apparently irreversible reduction.
To further investigate the redox behavior of complexes CV studies were performed at different
potential scan rates ranging from 20 mVs-1 to 140 m Vs-1 which are shown in Fig. S6.
By increasing the scan rate, a shift in cathodic peak potential towards more negative potential
was observed for both the complexes establishing irreversible reductive behavior. It was
observed that cathodic peak 2 of complex 2 disappeared at higher scan rate (≥ 1000mVs-1).
For irreversible processes peak current is given by Randles-Sevcik equation given below [34]
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ip = (2.99 x 105)n(αn)1/2AC*(Doν) 1/2
(1)
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Where ip is peak current, n is number of electrons involved in the redox process, α is the charge
transfer coefficient, A is the surface area of working electrode, C* is the bulk concentration of
analyte, v is the potential scan rate and Do is the diffusion coefficient.
It was observed that with the increase of scan rate the cathodic peak current increases linearly
following the equation 1 for both of the complexes. Plots of cathodic peak currents (ipc) vs. v1/2
(Fig. S7) give a straight line with slope values of 5.91 × 10-6 and 3.47×10-6 for 1 and 2,
respectively. By substituting the values of α as 0.5 (supposed), n as 2 (calculated from DPV), A
and C*in the slope (Slope = (2.99 x 105) n (αn) 1/2AC*Do1/2) of equation 1, the values of diffusion
coefficient were calculated to be 3.02 × 10-9 cm2s-1 and 1.04 × 10-9cm2s-1 which reveal that the
electrochemical reduction of 1 and 2 is diffusion controlled.
3.4.2 Differential pulse voltammetry
DPV is an excellent technique to determine the number of electrons involved in the electron
transfer process. In DPV the number of electrons involved in the electrochemical process can be
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calculated from the value of peak width at half of the maximum height by employing equation
[35]
314
mV
(2)
315
316
317
318
319
320
321
Where W1/2 is the peak width at half height, n is the number of electrons involved in
electrochemical process and F is the Faraday constant.
The DPVs of 1 and 2 (2 mM each) in their argon saturated aqueous-DMSO (2:8, v: v) solutions
containing 0.1M TBATFB as a supporting electrolyte at GCE are presented in Fig. 4 at 25o C.
The values of W1/2 are 62 mV and 60 mV (peak 1) for 1 and 2, respectively. These values are
close to the theoretical value of 45 mV for a redox process involving two electrons. Hence the
two electron redox process is proposed for the electrochemical reduction of the complexes.
322
323
Fig. 4. DP voltammograms of 2 mM complex 1(A) and 2mM complex2 (B) in aqueous-DMSO
324
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330
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332
333
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336
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340
(2:8, v: v) solution containing 0.1M TBATFB as a supporting electrolyte at GCE at 25o C.
3.4.3 Square wave voltammetry
The advantages of square wave voltammetry (SWV) on DPV are greater speed of analysis, lower
consumption of electroactive species and reduced problems with the poisoning of electrode
surface [36]. Further SWV study is the best criterion to diagnose the reversibility of redox process.
The reversibility of the obtained peaks for the complexes was evaluated by plotting the forward
and backward components of the total current obtained in 2mM solution of the complexes.
SWVs of 1 and 2 recorded at 25o C are shown in Fig. S8. It is clear that the reduction process of
both the complexes are quasi-reversible instead of completely irreversible as apparently observed
in case of CV. The steric hindrance of triphenylphosphine group may be one of the reasons to
cause electrochemical irreversibility; as geometrical influence affects the electron transfer
process both in terms of its thermodynamic feasibility and kinetics. Moreover, the observed
broad redox waves complement the inference of structural rearrangements (both of inner and
outer sphere) as conferred above. This can be further validated by computational studies such as
DFT calculations.
341
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344
3.5 Enzymatic activity
The synthesized Schiff base ligands and their corresponding complexes (1 & 2) were screened
for the study of inhibitory effects on ALP. Both ligands were found to be inactive but
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palladium(II) complexes inhibit the enzyme in a concentration dependent manner (Fig. S9). It
was observed that enzyme loses its more than 80% of activity at the concentration of 0.73 mM.
The Pd may replace Zn or Mg of the enzyme and hence the enzyme fails to bind with the
substrate. This affect is similar to earlier studies that more than 50% inhibition of the enzyme
was observed when the concentration of Pd complex was 0.25 mM [37]
350
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3.6 Biological studies
3.6.1 Brine shrimp cytotoxic assay
Brine shrimp cytotoxic assay was carried out in order to prescreen biologically active nature of
the compounds. Results were compared with standard drug doxorubicin (LD50 25.2 µg mL-1) and
it was observed that the compound 1 showed promising cytotoxic potential with LD50 value of
64.2 µg mL-1. Results indicate that the compounds were toxic even at low dose which is
consistent with the study of 1-indanone thiosemicarbazones coordinated to palladium (II) [38]
.
3.6.2 Potato disc antitumor assay
Cytotoxic potential of compounds is predictive of their antitumor potential. The data revealed
that complex 1 had promising antitumor activity as compared with doxorubicin showing IC50
value 24.9 µg mL-1 while 2 did not show any antitumor activity.
Table 3. Results of cytotoxicity, potato disc antitumor, DPPH free radical scavenging
antioxidant and inhibition of hydroxyl free radical induced DNA damage assays
complex Cytotoxic
activity
antitumor activity
IC50
% scavenging of DPPH at different IC50
conc.(µg mL-1)
DNA protection activity at
different conc.(µg mL-1)
(µg mL-1)
(µg mL-1)
LD50
value
Percentage inhibition ±
SD(µg mL-1)
(µg mL-1)
1000 100 10
1
200
66.6 22.2 7.4
42.2 26.4 1.0
2.7
0
1000 100 10
1
64.2
69
36
80
62
20
69
39 27 18.5
nil
40 37 24.9
76
90
116.9
-
-
+
+
++ ++
++ ++
1
2
724.8
6
0
40.3 14.7 10.4 5.57 101.8
Standard 25.2
82.4 76.4 58.6 39.2 19.9 20.2
367
368
Doxorubicin was used as standard drug for cytotoxic and antitumour activity while ascorbic acid
for % scavenging of DPPH (-) = no protection. (+) = slight protection. (++) = good protection.
[39]
369
370
371
372
373
374
These results are consistent with the findings of Butour et al
that most of the palladium
compounds displayed antitumor activity. Moreover the presence of oxygen moiety (mostly at
ortho position in ring structure) can also enhance antitumor activity of palladium compounds [40]
.
This might seems to be the reason for non antitumoral activity of 2 which contains sulphur.
3.6.3 DPPH free radical scavenging antioxidant assay
375
376
DPPH free radical scavenging activity of complexes was determined spectrophotometrically by
measuring absorbance at 515 nm. Both the compounds show mild antioxidant potential with IC50
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values of 116.9 and 101.8 µg mL-1 (Table 3) by using ascorbic acid (IC50 value = 20.2 µg mL-1)
as standard and these are in agreement as already reported [41]. This antioxidant potential may be
due to ligand associated electronic factors, ligand complexation with palladium and extended π
configuration of aromatic ligands [42]
.
3.6.4 DNA protection assay
Inhibition of hydroxyl (•OH) free radical induced DNA damage assay was performed in vitro in
order to check the antioxidant and prooxidant behaviors of compounds at four different
concentrations (1 ꢀg/mL, 10 ꢀg/mL, 100 ꢀg/mL and 1000 ꢀg/mL) listed in Table 3. It was
observed that complex 1 and 2 showed no protection at 1000 µg mL-1 while DNA protection
activity was observed for rest of tested concentrations (Fig. 5).
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401
402
403
404
405
406
Fig. 5. Effect of compounds 1 and 2 on pBR322 plasmid DNA[L=1Kb ladder , P=pBR322
plasmid, , X = Plasmid DNA + FeSO4 + H2O2, 1(C+P) =Plasmid + phosphate buffer + 1000 µg
mL-1 of 1, C1 =Plasmid + phosphate buffer + 1000 µgmL-1 of 1 + FeSO4 + H2O2,C2=Plasmid +
phosphate buffer +100 µg mL-1 of 1 + FeSO4 + H2O2, C3 = Plasmid + phosphate buffer + 10 µg
mL-1 of 1 + FeSO4 + H2O2, C4=Plasmid + phosphate buffer + 1 µg mL-1 of 1 + FeSO4 + H2O2,
2(C+P)=Plasmid + phosphate buffer + 1000 µg mL-1of 2, C5 =Plasmid + phosphate buffer +
1000 µg mL-1of 2 + FeSO4+H2O2, C6 = Plasmid + phosphate buffer +100 µg mL-1 of 2 + FeSO4
+ H2O2, C7 = Plasmid + phosphate buffer + 10 µg mL-1 of 2 + FeSO4 + H2O2, C8 = Plasmid +
phosphate buffer + 1 µg mL-1 of 2 + FeSO4 + H2O2].
3.7 Drug DNA Interaction Study
The cyclic voltammetric method was employed to study the interaction of palladium complexes
with the DNA. The amounts of 21, 41.2, 61.2, 81, 100, 119 and 137 µM DNA were added into
the 2 mM solution each of 1 and 2 separately and corresponding CV response was recorded at
glassy carbon electrode under the same conditions as were performed for pure complexes. It was
noted that the cathodic peak currents for both the complexes decreased substantially along with a
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positive shift of reduction peak potentials on DNA addition (Fig. 6 & Table 4). The regular
anodic shift accompanying the decline in peak current is noticeably showing the decrease in the
concentration of free drug molecules (Pd complex used) while rest of the drug intercalates with
the added DNA molecules [43]
.
411
412
413
414
Fig. 6. Cyclic voltammograms of 2 mM each of 1 (A) and 2(B) (▬) without DNA and (▬) in the
presence of 41.2 µM DNA on GCE at 100 mVs-1 in aqueous-DMSO (2:8, v: v).
415
416
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418
419
420
421
422
423
424
425
To further evaluate and quantify the interaction of complexes with DNA, CV studies were
executed at different potential scan rates. The relationship of cathodic peak current with square
root of scan rate (v1/2) for both the complexes was found to be linear in the presence of DNA
(drug-DNA adduct) (Fig. S10) and directed that the redox process of complexes persisted mainly
diffusion controlled as observed for pure complexes. However, the slope of the linear line
declined substantially in presence of DNA as shown in Fig. S10. The diffusion coefficient values
of these adducts were considerably smaller than that of free complexes, showing that DNA-
bound complexes diffused more slowly as compared to free complexes (Table 4). The observed
behavior could be ascribed to the diffusion of an equilibrium mixture of the free and DNA-bound
complexes to the surface of working electrode [44]. To promote the stated argument, binding
constant values were evaluated by employing the following equation [45]
426
1/ [DNA] = {K (1-A) / (1-I/I0)} – K
(3)
427
428
429
430
431
Where A is proportionality constant and K is the binding constant calculated from the intercept
of the plots of equation 3 as shown in Fig. S11. The K values given in Table 4, define the
binding strength of a complex to the DNA molecule. The value of binding constant of complex 2
is close to (1.74 × 104 M–1) the typical intercalator lumazine-DNA complex [46].The binding site
size in terms of concentration of base pairs was calculated by employing equation 4 [47,48]
.
432
Cb/Cf = K {[DNA]/2s}
(4)
433
434
Where ‘s’ is the size of binding site, Cf and Cb are the concentrations of free and DNA-bound
species, respectively. The Cb/Cf can be given by equation 5.
435
Cb/Cf = (Io − I)/I
(5)
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437
438
439
440
441
The values of binding site size (s) were determined from the slope of plots of Cb/Cf vs. [DNA]
(Fig. S12) and are presented in Table 4. The values of binding site size show that the complex 2
includes ≈ 1 base pair of the DNA and thus has stronger interaction than 1 as depicted from the
values of binding constant for the complexes. Relatively a bit stronger interaction of complex 2
was as per expectation due to its easy reduction and thus more susceptible for interaction through
intercalation into staked base pairs of with DNA molecules.
442
443
Table 4. The drug-DNA interaction electrochemical parameters of complexes on glassy carbon
electrode vs. Ag/AgCl in aqueous DMSO (2: 8, v: v) solution at 100 m Vs-1 scan rate at 25oC
In the absence of DNA
In the presence of DNA
Complex
Eo (V)
-1.39
-1.45
Do × 109(cm2s-1) Eo (V) Do×1010 (cm2s-1) K ×10-4(M-1)
s(bp)
0.27
0.64
3.02
1.04
-1.36
-1.44
3.89
5.42
0.38
1.02
1
2
444
445
4 Conclusion
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
Palladium
oxidophenyl)salicylideneiminate)
(II)
complexes
derived
and
from
the
condensation
of
N-(2-
with
N-(2-sulfidophenyl)salicylideneiminate)
salicyaldehyde were synthesized and characterized by physio-chemical, spectroscopic and
electrochemical methods. The results suggested the coordination of the ligand to the metal
through O, N, S donors and attachment of one coordinated triphenylphosphine with palladium.
Structure of complex 1 was determined by X-crystallography which is stabilized by
intermolecular π-π stacking between the ligand and by various C-H....π interactions. Based on
the spectroscopic studies and single crystal analysis, the new complexes possess square planar
geometry. Thermal analysis has shown that decomposition of both the complexes occurred in a
single step above 140 C, verifying that no water was physically or chemically bonded with the
complexes. A rapid weight loss above 140 C indicated the decomposition of coordinated
ligands. The broad band above 706 C is resulted the formation of stable PdO.The enzymatic
activity exhibited the inactivation of the enzymes by increasing the concentration of the
compounds. These complexes demonstrated cytotoxic and antitumor activity due to which they
can be used for therapeutic purposes. They unveiled quasi-reversible redox behavior on glassy
carbon electrode involving two-electron reduction of palladium(II) to palladium(0). Diffusion
coefficients of the complexes have divulged that the electron transfer process is diffusion
controlled. Voltammetric and biological studies explored DNA damaging nature of these
compounds suggesting their anticancerous potential. Although our work is preliminary but in
future it can serve as a ground to study their mechanistic action as well as structure activity
relationship.
o
o
o
467
Acknowledgements
468
469
The authors are highly grateful to Chemistry & Biochemistry Departments Quaid-I-Azam
University Islamabad, Pakistan, Chemistry Department and School of Chemical Sciences,
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Dublin City University, Glasnevin, Dublin 9, Ireland for providing technical support and
laboratory facilities.
472
473
Disclosure Statement
Authors have no financial interest or benefit from the direct applications of this research work.
474
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Highlights
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Synthesis and characterization of palladium(II)complexes.
The enzymatic activity declines by increasing concentration of the compounds.
Biological assays (brine shrimp cytotoxicity, DPPH) reflect mild antioxidant nature.
Voltammetric studies ascertained two-electron reduction.
Electrochemical findings illustrate intercalative mode of drug DNA interactions.