Inorganica Chimica Acta 547 (2023) 121368

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Inorganica Chimica Acta

journal homepage: www.elsevier.com/locate/ica

Spectral studies, crystal structures, DNA binding, and anticancer potentials

of Pd(II) complexes with iminophosphine ligands: Experimental and
computational methods
Mahsa Pooyan a, Zahra Shariatinia a, *, Fahimeh Mohammadpanah b, Khodayar Gholivand b, *,
Peter C. Junk c, Zhifang Guo c, Mohammad Satari d, Vahid Noroozi Charandabi e
a
Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic), P.O.Box:15875-4413, Tehran, Iran
b
Department of Chemistry, Faculty of Science, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran
c
College of Science & Engineering, James Cook University, Townsville, Queensland, 4811, Australia
d
Department of Biology, Faculty of Sciences, Malayer University, Malayer, Iran
e
Kimia Andish Laboratory, KAGI Company, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Herein, we studied the synthesis and the biological properties of palladium(II) complexes derived from imino­
Iminophosphine ligands phosphine ligands DPPB-3-hydroxybenzohydrazone (DPPB-HBH), DPPB-4-phenylthiosemicarbazone (DPPB-
Molecular docking PTSC), and DPPB-thiosemicarbazone (DPPB-TSC) (DPPB = 2-(diphenylphosphino)benzaldehyde) using theo­
Palladium complexes
retical and experimental methods. The molecular structures of [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]
DFT calculations
DNA binding
Cl2⋅2CH3CN, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)] were characterized by various spectroscopic tech­
Cytotoxicity niques and X-ray crystallographic for [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]Cl2⋅2CH3CN, and [PdCl(DPPB-
PTSC)], confirming the distorted square-planar geometry for the palladium atoms. Complexes with the gen­
eral formula [PdCl(Ligand)x] (x = 1) were employed to evaluate biological activities such as cytotoxicity against
the MDA-MB-231 breast cancer cells and DNA binding ability. The results revealed the high cytotoxicity of the
palladium complexes compared to the reference drug cisplatin (IC50 = 24.59 µg/mL), as well as their moderate
binding ability to DNA through electrostatic interactions and groove binding. It was also found that [PdCl(DPPB-
HBH)]Cl has the highest activity among others (IC50 = 9.3 µg/mL). Their interaction mode with DNA was
investigated using molecular docking and NCI calculations, which showed complete agreement with the
experimental results. Furthermore, quantum chemistry calculations were used to analyze the structure–activity
relationship and introduce reactive sites, which determined the cause of the difference in the reactivity of the
samples.

1. Introduction
Despite significant advances in medical science, cancer is still
considered the most common disease in the world [1]. Organoplatinum
drugs such as cisplatin, oxaliplatin, and carboplatin are among the most
widely employed anticancer drugs, but due to side effects such as renal
and hepatotoxicity, dose limitation, and drug resistance, their long-term
treatment is not recommended [2–4]. Hence much research is being
done in the field of introducing non-platinum complexes as alternative
drugs to overcome these limitations. Palladium complexes are a group of
these candidates that have attracted the attention of researchers due to
their similar coordination chemistry with platinum complexes in terms
of structure and biology so that they cover a wide range of biological
applications such as anticancer, antibacterial, and antifungal activities
[5–9]. Palladium complexes are much more reactive and sensitive to
hydrolysis than their platinum counterparts. However, their low cost,
high solubility, and greater tendency to interact with DNA prompted
researchers to improve the properties of the complexes and increase
their stability by choosing appropriate ligands [5,10–13]. Iminophos­
phines are a group of polyfunctional chelating ligands that contain both
hard and soft donor atoms in their structure [14–16]. These ligands
stabilize the construction of the complex by forming chelate rings
around the central metal due to weak π-accepting and strong σ-donating
characteristics and also increase the reactivity of the complex by

* Corresponding authors.
E-mail addresses: shariati@aut.ac.ir (Z. Shariatinia), gholi_kh@modares.ac.ir (K. Gholivand).

https://doi.org/10.1016/j.ica.2022.121368
Received 18 September 2022; Received in revised form 23 December 2022; Accepted 23 December 2022
Available online 28 December 2022
0020-1693/© 2022 Elsevier B.V. All rights reserved.
M. Pooyan et al.
creating asymmetry in the metal orbitals [17,18].
Hydrazones and thiosemicarbazones derived from 2-(diphenyl­
phosphino)benzaldehyde are a group of iminophosphine ligands that
contain PNO, and PNS donor atoms, respectively. These compounds
show different biological activities depending on the coordinated metal
ion and coordination geometry and the number of donor atoms [19–22].
In addition, their metal complexes are effective on a wide range of
cancer cells, and some of them exhibit similar and even higher cyto­
toxicity than the reference drug cisplatin [19,23–25]. Although the
biological properties of palladium complexes of these derivatives have
been investigated in a limited way, the results indicate their significant
antifungal and anticancer activities [5,21,22,26–29].
Considering the background presented on the anticancer potential of
palladium complexes and the interesting coordination behavior of the
iminophosphines, in this paper, we investigate the structural properties
and biological activity of palladium complexes derived from the
condensation reactions of 2-(diphenylphosphino)benzaldehyde with 3-
hydroxybenzohydrazide, 4-phenylthiosemicarbazide, and thio­
semicarbazide. The structures of the molecules were characterized using
various spectroscopic techniques and X-ray crystallography. Given that
the main target of most anticancer drugs is DNA, the tendency of the
candidate palladium complexes to interact with DNA was assessed by
fluorescence and UV–vis spectroscopic methods. Also, their cytotoxicity
was evaluated against triple-negative breast cancer cells (MDA-MB-
231). Furthermore, to create a logical relationship between structure
and activity and to gain insight into the reason for the difference in the
biological potential of compounds, DFT and MEP studies were also
conducted.
2. Experimental
2.1. Materials and methods
All compounds used in this research were > 95 % pure because they
were provided by Sigma-Aldrich and Merck Companies. Materials used
in biological tests such as DNA, MTT (3–(4,5–dimethyl thiazole–2–yl)–
2,5–diphenyltetrazolium bromide), ethidium bromide (EB) were ob­
tained from Sigma Corporation, but FBS (fetal bovine serum) and high
glucose DMEM medium from GiBCO Company (Grand Island, NY, USA).
Cell Bank at Pasteur Institute of Iran (Tehran) provided the MDA-MB-
231 cell line.
Electrothermal apparatus was utilized to determine melting points.
The spectrophotometer Perkin–Elmer (Lambda 25) and Nicolet 510P
spectrometer were applied to take UV–Vis and FT- IR spectra, respec­
tively. Bruker Avance DRX (300.13 MHz for 1H NMR spectra and 121.49
MHz for 31P NMR spectra) was used to acquire NMR spectra in d6–DMSO
using 85 % H3PO4 and tetramethylsilane as standards for 31P- and 1H
NMR spectra, respectively.
2.2. Synthesis procedures of ligands
2.2.1. Synthesis of DPPB-HBH
A mixture of 0.43 mmol (0.127 g) of 2-(diphenylphosphino) benz­
aldehyde within ethanol (20 mL) and some droplets of glacial acetic acid
were poured into the ethanolic solution (20 mL) of 3-hydroxybenzohy­
drazide (0.43 mmol, 0.065 g) at room temperature. After refluxing for
8 h, a white solid was obtained through slow evaporation of the solvent,
which was subsequently separated and washed with diethyl ether.
(2-(diphenylphosphino) benzaldehyde 3-hydroxybenzohydrazone)
(DPPB-HBH)
(C6H5)2P(o-C6H4-CH = N-NH-C(O)(m-C6H4OH)) (DPPB-HBH). M.p.
130 ◦ C. FT-IR (cm− 1): ν(O–H)asym = 3403w, br, ν(O–H)sym = 3225w, br
ν(N–H) = 3058w, br, ν(C–H)arom. = 2971w, 2928w ν(C– – O) = 1645s,
1
ν(C– N) = 1588s, ν(C P) = 1443m. H NMR (300.13 MHz, ppm); δ=:
– –
11.58 (s, 1H, N–H), 11.93 (s, 1H, O–H), 9.22 (d, 1H, CH = N),
6.53–7.64 (m, aromatic hydrogens). 31P(1H) and 31P NMR (121.49
2
Inorganica Chimica Acta 547 (2023) 121368
MHz); δ = –16.65 ppm.
2.2.2. Synthesis of DPPB-PTSC
A solution of 2-(diphenylphosphino) benzaldehyde (0.43 mmol)
dissolved in 25 mL of hot methanol with a few droplets of glacial acetic
acid was poured into a hot methanolic solution of 4-phenylthiosemicar­
bazide (0.43 mmol). After refluxing for 18 h, finally, slow solvent
evaporation yielded a yellow powder, which was rinsed by diethyl ether
after extraction from the remaining solvent.
(2-(diphenylphosphino) benzaldehyde 4-phenylthiosemicarbazone)
(DPPB-PTSC)
(C6H5)2P(o-C6H4-CH = N-NH-C(S)-NHC6H5) (DPPB-PTSC). M.p.
206 ◦ C. FT-IR (cm− 1): ν(N–H)ph = 3443w, br, 3302w, br, ν(N–H)hyd =
3135w, br, ν(C–H)arom. = 2990w, ν(C– – N) = 1541s, ν(C–P) = 1438m
ν(C–– S) = 1190s. 1H NMR (300.13 MHz, ppm); δ=: 11.93 (s, 1H, N–H),
9.89 (s, 1H, NH-C6H5), 8.77 (d, 1H, JP-H = 4.67, CH = N), 6.80–8.32 (m,
aromatic hydrogens). 31P(1H) and 31P NMR (121.49 MHz); δ = –14.17
ppm.
2.2.3. Synthesis of DPPB-TSC
Synthesis of DPPB-TSC was accomplished according to the literature
method [15].
(2-(diphenylphosphino) benzaldehyde thiosemicarbazone) (DPPB-
TSC)
(C6H5)2P(o-C6H4-CH = N-NH-C(S)–NH2) (DPPB-TSC). M.p. 225 ◦ C.
FT-IR (cm− 1): ν(NH2)asym = 3442w, br, ν(NH2)sym = 3306w, br, ν(N–H) =
3166w, br, ν(C–H)arom. = 3016w, ν(C– – N) = 1574s, ν(C–P) = 1436m,
– S) = 1218s. 1H NMR (300.13 MHz, ppm); δ=: 11.51 (s, 1H,
1366s, ν(C–
N–H), 8.64 (d, 1H, JP-H = 4.61, CH = N), 8.17–8.14 (q, 2H, NH2) 6.74
–7.68 (m, 14H, Ph-P). 31P(1H) and 31P NMR (121.49 MHz); δ = –14.26
ppm.
2.3. Synthesis procedure of complexes
A solution of [PdCl2(COD)] (0.0125 mmol for [PdCl(DPPB-HBH)]Cl
and [Pd(DPPB-HBH)2]Cl2⋅2CH3CN, 0.06 mmol for [PdCl(DPPB-PTSC)]
and [PdCl(DPPB-TSC)]) in acetonitrile (10 mL) was poured into aceto­
nitrile solutions (20 mL) of ligands in 1:1 (for [PdCl(DPPB-PTSC)] and
[PdCl(DPPB-TSC)]) and 1:2 M ratios of metal to ligand (for [PdCl(DPPB-
HBH)]Cl and [Pd(DPPB-HBH)2]Cl2⋅2CH3CN). Then, these solutions
were placed under ambient conditions for several days. Finally, slow
solvent evaporation obtained suitable crystals for X-ray crystallography
determination.
2.3.1. [PdCl(DPPB-HBH)]Cl
Yield = 45 % (yellow color). M.p. 250 ◦ C. FT-IR (cm− 1): ν(O–H)asym
= 3427w, br, ν(O–H)sym = 3122w, br ν(N–H) = 3059w, br, ν(C–H)arom. =
2929w, 2861w, ν(C– – N) = 1582s, ν(C–P) = 1437m, ν(C– – O) = 1648s. 1H
NMR (300.13 MHz, ppm); δ=: 9.08 (s, 1H, N–H), 9.99 (s, 1H, O–H),
8.45 (d, 1H, CH = N), 6.98–7.70 (m, aromatic hydrogens). 31P (1H) and
31
P NMR (121.49 MHz); δ = 35.30 ppm.
2.3.2. [Pd(DPPB-HBH)2]Cl2⋅2CH3CN
Red; yield = 15 %. M.p. 265 ◦ C. 1H NMR (300.13 MHz, ppm); δ=:
9.09 (d, 1H, O–H), 8.49 (d, 1H, CH = N), 7.72–6.91 (m, 36Harom, Ph).
31
P (1H) and 31P NMR (121.49 MHz); δ = 33.32 ppm.
2.3.3. [PdCl(DPPB-PTSC)]
M.p. 286 ◦ C. FT-IR (cm− 1): = ν(N–H)ph = 3405w, br, 3262w, br,
ν(N–H)hyd = 3049w, br, ν(C–H)arom. = 2924w, ν(C– – N) = 1490s,
ν(C–P) = 1430m ν(C– – S) = 1180s. 1H NMR (300.13 MHz, ppm); δ=:
8.67 (s, 1H, N–H), 8.54 (d, 1H, NH-C6H5), 8.46 (d, 1H, CH = N),
7.09–7.69 (m, aromatic protons).
2.3.4. [PdCl(DPPB-TSC)]
M.p. 290 ◦ C. FT-IR (cm− 1): ν(NH2)asym = 3400w, br, ν(NH2)sym =
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

3281w, br, ν(N–H) = 3145w, br, ν(C–H)arom. = 2925w, ν(C–– N) = 1470s, Table 1
ν(C–P) = 1394m, ν(C– – S) = 1154s. 1H NMR (300.13 MHz, ppm); δ=: Crystal data and structural refinement parameters of complexes. [Pd(DPPB-
9.16 (s, 1H, N–H), 7.66 (m, 2H, NH2), 8.36 (d, 1H, CH = N), 6.04–7.97 HBH)2]Cl2•2CH3CN, [PdCl(DPPB-HBH)]Cl, and [PdCl(DPPB-PTSC)].
(m, 14H, Ph-P). 31P (1H) and 31P NMR (121.49 MHz); δ = 22.54 ppm. Compound [Pd(DPPB-HBH)2] [PdCl(DPPB-HBH)] [PdCl(DPPB-
Cl2•2CH3CN Cl PTSC)]
2.4. X-ray crystallographic details Empirical C56H48Cl2N6O4P2Pd C26H21Cl2N2O2PPd C26H22ClN3PPdS
formula
Formula 1108.24 601.72 581.34
Single crystals of complexes [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-
weight
HBH)2]Cl2⋅2CH3CN, and [PdCl(DPPB-PTSC)] were dipped into a viscous T (K) 100(2) 100(2) 100(2)
hydrocarbon oil and mounted onto a loop to determine their structures Crystal system, Monoclinic, C2/c Triclinic, P-1 Monoclinic, P21/
using Australian Synchrotron over MX1 macromolecular beamlines at space group c
100 K and λ = 0.71073 Å. All diffractions were indexed and integrated a(Å) 22.326(5) 9.5020(19) 9.5520(19)
17.221(3) 15.332(3) 13.663(3)
by the XDS software suite [30]. SHELXT was applied to solve data
b(Å)
C(Å) 15.132(3) 17.398(4) 18.321(4)
refining against F2 via the full-matrix least-squares method with α 90 74.67(3) 90
SHELXL-2018 in Olex2 [31,32]. The riding model theoretically deter­ β 118.39(3) 87.18(3) 100.11(3)
mined the positions of all H-atoms. The CCDC numbers of Pd(II) com­ γ 90 86.34(3) 90
plexes are 1,580,485 (for [PdCl(DPPB-HBH)]Cl), 1,580,470 (for [Pd V 5118(2) 2438.0(9) 2353.9(8)
Z 4 4 4
(DPPB-HBH)2]Cl2⋅2CH3CN), and 1,580,469 (for [PdCl(DPPB-PTSC)]). Dcalc (Mg/ 1.438 1.639 1.640
Also, the crystallographic information is available freely on http m− 3(− |-))
s://www.ccdc.cam.ac.uk/data_request/cif (Cambridge Crystallo­ Absorption 0.583 1.073 1.079
graphic Data Centre). The crystal structure of the complexes and crystal coefficient
(mm− 1)
data and refinement parameters are presented in Fig. 2 and Table 1,
F(000) 2272 1208 1172
respectively. Crystal size 0.11 × 0.05 × 0.02 0.10 × 0.04 × 0.02 0.09 × 0.03 ×
(mm) 0.01
2.5. DNA binding studies θ rang for data 1.9–24.99 1.21–24.99 1.87–24.99
collection (◦ )
Limiting − 26 ≤ h ≤ 26 − 11 ≤ h ≤ 11 − 11 ≤ h ≤ 11
2.5.1. Electronic absorption titration method indices − 20 ≤ k ≤ 20 − 18 ≤ k ≤ 18 − 15 ≤ k ≤ 15
Electronic absorption and fluorescence spectroscopy techniques − 17 ≤ l ≤ 17 − 20 ≤ l ≤ 19 − 21 ≤ l ≤ 21
examined the tendency of Pd (II) complexes to interact with ct-DNA. All Total 30,906 19,591 28,137
reflections
tests were accomplished under ambient conditions within buffer Tris-
Unique 4501 [R(int) = 7895 [R(int) = n/a] 4014[R(int) =
HCl solution composed of NaCl (50 mM in deionized water) + Tris- reflections 0.0605] 0.0351]
HCl (5 mM) at pH 7.2, adjusted by concentrated hydrochloric acid. The (Rint)
ct-DNA standard solution concentration was measured by its absorbance Completeness 99.7 % (θ = 25.00) 91.9 % (θ = 25.00) 96.9 % (θ =
(260 nm) via the Beer-Lambert equation (molar absorption coefficient to θ(%) 25.00)
Data/ 4501/0/324 7895/6/ 616 4014/0/299
was 6600 M− 1cm− 1). This solution was used within three days. The
restraints/
constant 48 µM concentration of each complex ([PdCl(DPPB-HBH)]Cl, parameters
[PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)]) within Tris-HCl buffer/ Refinement Full-matrix least Full-matrix least Full-matrix least
DMSO (less than 1 % final volume) mixture was titrated by different ct- method squares on F2 squares on F2 squares on F2
Goodness-of-fit 1.050 1.064 1.061
DNA concentrations (9.94–170 µM) to perform the electronic absorption
(GOF) on F2
titration assays. Various ct-DNA concentrations were added into each Final R indices R1 = 0.0308, wR2 = R1 = 0.0666, wR2 R1 = 0.0241,
complex solution with a fixed concentration so that the ratio of [DNA]/ [I ˃ 2σ(I)] 0.0807 = 0.1718 wR2 = 0.0644
[complex] = r (r = 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4). At R indices (all R1 = 0.0341, wR2 = R1 = 0.1009, wR2 R1 = 0.0260,
each titration step, an equal amount of DNA was added to the control data) 0.0825 = 0.1928 wR2 = 0.0656
Largest 0.840 and − 0.56 1.66 and –1.34 0.47 and − 0.82
and test solutions to remove the DNA adsorption. All absorption spectra
difference in
were recorded after 7 min of incubation at room temperature. Then, the peak and
Wolfe-Shimer formula (Eq. (1)) was used to estimate Kb binding con­ hole (e Å− 3)
stant [33]. In this equation, εb, εf, εa are related to extinction coefficients
of sample completely bound onto DNA, non-bound complex, and in DNA
presence (Aobsd/[Pd] = εa), respectively. (5 µM EB, 100 µM ct-DNA, buffer Tris-HCl with pH 7.2). Fluorescence
intensity changes of the test solutions were recorded after incubation for
[DNA] [DNA] 1 7 min at room temperature from 500 to 700 nm using 471 nm excitation
= + (1)
|(εa − εf )| |(εb − εf )| Kb |(εb − εf )| radiation. The extent of EB-DNA fluorescence quenching by complexes
was calculated using the Stern–Volmer formula (Eq. (3)) [35], where F
Graphical plots of [DNA]/|(εa–εf)| against [DNA] afford Kb values
and F0 reveal fluorescence intensity in quencher (complex) presence and
which can be obtained using the ratio of the slope (1/|(εb–εf)|) to the
absence, respectively, Ksv exhibits linear Stern-Volmer quenching con­
intercept (1/Kb|(εb–εf)|) of the linear plots. Gibbs free energy values for
stant, [Q] represents complex concentration. Ksv value was estimated
DNA binding were obtained using the following equation (Eq. (2)) [34].
using the slope of F0/F vs [Q] linear plot.
ΔG0b = − RT ln Kb (2)
F0 /F = 1 + Ksv[Q] (3)

2.5.2. Competitive binding studies

Since DNA and synthesized complexes do not show fluorescence
activity in free form and in complex form, to determine the model of
interaction of compounds with ct‑DNA, the ethidium bromide (EB)
fluorophore was used as a fluorescence probe in this spectroscopy
technique. EB-DNA competition experiments were performed by adding
different concentrations (20–200 µM) of complexes to EB-DNA solution
2.6. Cytotoxicity activity evaluation
2.6.1. Cell culture
MDA-MB-231 triple-negative breast cancer (TNBC) cell line was
purchased from the Cell Bank of Pasteur Institute of Iran. MDA-MB-231
cells were cultured in DMEM (high glucose) medium supplemented with

3
M. Pooyan et al.
1 % penicillin–streptomycin (GIBCO) as antibiotics and 10 % fetal
bovine serum (FBS) at 37 ◦ C in a humidified incubator containing 5 %
CO2. After 2–3 days, all culture media were renewed, considering MDA-
MB-231 cellular growth rates.
2.6.2. MTT cytotoxicity assays
Cytotoxicity activities of Pd(II) complexes towards the MDA-MB-231
cell line (a typical TNBC line) were evaluated in vitro using the MTT
assay. MTT is a water-soluble tetrazolium salt that is reduced in viable
cells by the enzyme Succinate dehydrogenase in the mitochondrial
membrane to an insoluble purple formazan product. Since formazan
absorption intensity has a direct relationship with alive cells’ density, it
is possible to measure the cytotoxicity potential of the compounds.
MDA-MB-231 cells were cultured into 96-well plates with 7000 cells/
well density using 100 µl DMEM medium including 10 % FBS, which was
then incubated for 12 h under air containing 5 % CO2 at 37 ◦ C [36].
Subsequently, culture environment was changed by 100 µl of new so­
lution composed of various (2.5, 5, 7.5, 10, 25, 50, 100 µg/mL) com­
plexes concentrations. The drugs were dissolved within DMSO and
diluted with culturing solution to desired concentrations. Each dosage
was poured into wells separately, and this was repeated triple times.
Control was a medium containing 1 % DMSO. After 24 and 48 h incu­
bation, 10 µl of 5 mg/mL MTT dissolved in 90 µl of the medium was
added to each well and incubated for another 4 h. After the desired time,
the medium and unreacted MTT were discarded and 100 µl DMSO was
added to each well to dissolve the formed formazan crystals. Finally,
absorbances of specimens were recorded at 540 nm by BioTek ELx808
microplate reader. For comparison, the anticancer drug cisplatin was
used as a reference drug. IC50 values were calculated to evaluate the
cytotoxicity effects of the Pd(II) complexes using logarithmic plots of
viability percentage against drug concentration, at which IC50 is the
concentration that causes 50 % of cell death. The following equation
determined the cellular viability percentage.
mean OD of the treated cells
Cellular viability percentage =
mean OD of the untreated cells (control)
× 100
2.6.3. Statistical study
Results were extracted as mean ± standard deviation of 3 individual
experimental data. Using one-way ANOVA, statistical analysis was
performed utilizing GraphPad Prism 5 program. The P-value < 0.05
represented a significant value.
2.7. Protocol of molecular docking
Molecular docking simulations were performed using the AutoDock
4.2 program [37]. The structures of all three complexes were optimized
as described in the Computational Methods section. The crystal structure
of the B-DNA dodecamer with the sequence d(CGCGAATTCGCG)2 was
extracted from the Protein Data Bank (PDB ID: 1BNA) [38]. All water
and ligand molecules were removed from the macromolecule before the
molecular docking simulation to prepare the receptor. Then, Gasteiger
charges were calculated for each atom, and non-polar hydrogens were
merged into carbon atoms. In all dockings, the grid box covered the
entire macromolecule with a grid spacing of 0.375 Å. All calculations
were done by the Lamarckian genetic algorithm method in 100 runs
[39]. Other parameters were set based on the software’s default settings.
Results that differ by less than 0.5 Å in positional root–mean–square
deviation (RMSD) were clustered together. The optimized clusters were
ranked based on the lowest binding energy. Finally, the conformation
with the lowest free energy of binding and the highest probability was
selected as the desired conformation for the investigation of in­
teractions. The analysis of the resulting structural files and graphical
visualization of their surfaces were performed by AutoDockTools and
Discovery Studio Visualizer [40]. In addition, the two-dimensional
4
Inorganica Chimica Acta 547 (2023) 121368
representation of the interactions of the docked molecules was depic­
ted using the LIGPLOT program (v.1.4.5) [41]. Also, non-covalent in­
teractions between DNA and complexes were investigated through the
non-covalent interaction (NCI) method [42] using Multiwfn software
[43]. This method, which is known as the reduced density gradient
(RDG) method [44], is one of the popular methods for studying weak
interactions, which can differentiate between attractive (such as
π-stacking) and repulsive interactions [43]. Calculating RDG grid data
for large systems takes a long time, so NCI analysis is performed based
on promolecular density for large systems [45]. Promolecular density in
the term is the electron density which is constructed almost by super­
posing the electron density of the atoms in their free state, known as the
Promolecular approximation. Best docked poses were used to conduct
these studies, and the data obtained from the Multiwfn program were
visualized by VMD (Virtual Molecular Dynamic) software [46].
2.8. Computational methods
All quantum-chemical calculations were performed using density
functional theory (DFT) at the B3LYP (Becke, 3-parameter, Lee­
–Yang–Parr) level [47,48] by the Gaussian 09 program package [49].
Basis sets of LANL2DZ [50] and 6–311 + G** were utilized to describe
Pd atoms and main group elements (such as C, N, O, S, Cl, P, and H
atoms), respectively, in all computations. Also, water solvent effects
were investigated in calculations using the self-consistent reaction field
(SCRF) method based on the polarizable continuum model (PCM)
[51,52]. After the geometry optimization of the complexes, frequency
calculations were performed to confirm that the optimized structures are
true minima. The absence of imaginary vibrational frequencies indi­
cated that the optimized structures correspond to the energy minima.
Natural Bond Orbital (NBO) [53] calculations were also done to extract
the natural atomic charges and analyze the frontier molecular orbitals.
The Chemcraft program (version 1.8) [54] was applied to draw the
frontier molecular orbitals. Also, using the energies of HOMO and LUMO
orbitals, global reactivity descriptors were calculated by the methods
reported in [55]. Studies of electron density distribution, atomic con­
tributions of HOMO and LUMO orbitals, and molecular electrostatic
potential (MEP) were completed using Multiwfn software (version 3.7)
[43], known as the multifunctional wave function analysis program. The
outputs were visualized by the Visual Molecular Dynamics (VMD) pro­
gram [46]. All wave function files were generated by Gaussian 09
package. Fukui functions analysis was employed to determine electro­
philic and nucleophilic sites. Single-point calculations of palladium
complexes in N, N-1, and N + 1 states (N = number of electrons in the
stable situation)were conducted at the same level of theory used for
optimization.
3. Results and discussion
3.1. Synthesis
Hydrazone ligand DPPB-HBH was synthesized through a condensa­
tion reaction between 2-(diphenylphosphino) benzaldehyde with 3-
hydroxybenzohydrazide. Ligands DPPB-PTSC and DPPB-TSC were ob­
tained according to published procedures [15,56]. Complexes [PdCl
(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]Cl2⋅2CH3CN, [PdCl(DPPB-PTSC)],
and [PdCl(DPPB-TSC)] were prepared via the substitution reaction of
PdCl2(COD) (COD = 1,5-cyclooctadiene) with ligands DPPB-HBH,
DPPB-PTSC, and DPPB-TSC. From the reaction of DPPB-HBH with
PdCl2(COD), a mixture of [PdCl(DPPB-HBH)]Cl and [Pd(DPPB-HBH)2]
Cl2⋅2CH3CN complexes was obtained. Of these two molecules, only
[PdCl(DPPB-HBH)]Cl was used to evaluate biological activities due to its
dominant percentage and similar coordination model with [PdCl(DPPB-
PTSC)] and [PdCl(DPPB-TSC)] compounds. In complexes [PdCl(DPPB-
PTSC)] and [PdCl(DPPB-TSC)], the coordination of the ligands to
palladium cation is in a tridentate form through the P, N, and S atoms.
M. Pooyan et al.
While the DPPB-HBH ligand acts as a P, N bidentate and P, N, O tri­
dentate donor ligand in the [Pd(DPPB-HBH)2]Cl2⋅2CH3CN and [PdCl
(DPPB-HBH)]Cl complexes, respectively, see Scheme 1. These com­
plexes are all stable to air and moisture, and their diverse structural
characteristics are investigated with X-ray crystallography analysis and
UV–vis, FT-IR, 31P-NMR, and 1H-NMR, spectroscopy techniques.
3.2. Spectroscopic studies
Comparing FT-IR spectra of complexes and corresponding ligands
provided considerable evidence regarding ligand coordination mode to
central metal. Table 2 summarizes FT-IR and NMR spectroscopy results.
There are three characteristic absorption bands in the ranges of
3145–3049, 1582–1490, and 1437–1394 cm− 1 within FT-IR spectra of
Pd(II) complex samples, which are assigned to stretching frequencies of
N–H, C– – N, C–P groups, respectively [57]. The shifts of the C–
– N and
C P bands by 6–64 cm
– − 1
in the direction of lower wavenumbers
compared to those of the corresponding ligands confirm the participa­
tion of azomethine nitrogen atoms and triphenylphosphine phosphorus
to coordinate metal cation [21]. The stretching vibration of the C– –O
bond in the [PdCl(DPPB-HBH)]Cl complex appears at 1648 cm− 1 [58]. It
is moved to a higher frequency than the free ligand. This shift proves
that the oxygen atom has been coordinated as carbonyl (i.e., the
hydrazonic ligand is connected in the keto-form) [59].
The characteristic bands of C(S) appear at around 1033–1093 cm− 1
and 1098–1154 cm− 1 in thiosemicarbazone complexes. These bands
show the values between stretching modes of single bond C–S (ν(C–S))
and stretching modes of C– – S double bond (ν(C–S)) [60,61] due to the
–
Scheme 1. Chemical structures and synthesis process of Pd(II) complexes.
5
Inorganica Chimica Acta 547 (2023) 121368
delocalization of electron density among the C(S)-N-N– – C fragment,
which leads to partial double bond nature for C–S bonds. It was inferred
earlier from the lack of ν(S–H) vibrations in the range of 2500–2600
cm− 1 but ν(N–H) stretching occurrence within 3049–3145 cm− 1 [62].
Also, UV–vis spectra of Pd(II) samples display-three absorption peaks
within regions 202–220, 251–298, and 390–415 nm, as shown in Fig. S1.
The appeared bands around 202–220 and 258–298 nm correspond to
intra-ligand transitions (π-π* plus n-π* transitions of the aromatic rings
and C– – N groups) [63]. The 390–415 nm bands are assigned to charge
transfer (CT) transitions [62,64]. The UV–vis spectra of ligands and Pd
(II) complexes and their data are given in Figs. S1, S2, and Table S1.
1
H and 31P-NMR spectra of complexes and their corresponding li­
gands are recorded in DMSO‑d6. The 31P NMR spectra of the ligands
show signals in the − 16.65 to − 14.17 ppm range due to the phosphorus
atoms resonance of triphenylphosphine. The peaks are firmly moved
downfield upon complexation, indicating phosphorus atoms are
involved in coordination. 1H NMR spectra of ligands exhibit signals
belonging to the hydrogen atom of N-NH-C = X (X = O or S) group at δ
11.51–11.93 ppm [65], but these signals appear in the regions of δ
8.67–9.16 ppm in the spectra of complexes. These shifts support the
coordination of sulfur and oxygen atoms in corresponding ligands onto
central metal ions [66]. For each Pd(II) complex, a doublet appears near
8.36–8.45 ppm due to the coupling of the azomethine proton to the
phosphorus atom of triphenylphosphine, which is transferred to more
shielded regions compared to those of the free ligands (9.22–8.64 ppm).
Thus, this result confirms that the azomethine N atom has participated
in complexation [59]. Furthermore, in the spectra of the complexes,
there are a series of overlapping multiplets in the range of 6.04–7.97
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Table 2
Some spectroscopic data of Pd(II) complexes.
Name X ν(N–H) ν(C–
–O) ν(C–
–N) ν(C–P) ν(CS) ν(C–H)arom δ(N–H) δ(CH = N) Ref.

DPPB-HBH O 3058w, br 1645s 1588s 1443m – 2971w 11.58 9.22 –

[PdCl(DPPB-HBH)]Cl O 3059 w, br 1648s 1582s 1437m – 2929w 9.08 8.45 –
DPPB-PTSC S 3135w, br – 1541s 1438m 1072w 2990w 11.93 8.77 [56]
[PdCl(DPPB-PTSC)] S 3049w, br – 1490s 1430m 1093w, 1033w 2924w 8.67 8.46 –
DPPB-TSC S 3166w, br – 1534s 1436m 1090w 3016w 11.51 8.64 [15]
[PdCl(DPPB-TSC)] S 3145w, br – 1470s 1435m 1098w, 1154w 2925w 9.16 8.36 [15]

ppm assigned to H atoms in aromatic rings of ligands.
3.3. Crystal structure analysis of Pd(II) complexes
To confirm the coordination structure of the Pd(II) complexes, suit­
able single crystals of [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]
Cl2•2CH3CN and [PdCl(DPPB-PTSC)] were obtained by slow evapora­
tion of their acetonitrile solution. Two different types of crystals were
obtained from the reaction of the DPPB-HBH ligand with PdCl2(COD),
including thin yellow crystals ([PdCl(DPPB-HBH)]Cl) and cubic red
crystals ([Pd(DPPB-HBH)2]Cl2•2CH3CN) (Fig. 1). The type of coordi­
nation structure of every single crystal was determined by X-ray crys­
tallography. Fig. 2 and Table 1 illustrate the ORTEP plots and the
crystallographic information of these complexes, respectively. Also,
Table S2 lists relevant bond lengths and bond angles. Crystallographic
data show that [PdCl(DPPB-PTSC)] and [Pd(DPPB-HBH)2]Cl2•2CH3CN
crystals are crystallized within monoclinic crystal systems with P21/c
and C2/c space groups, whereas single crystals of [PdCl(DPPB-HBH)]Cl
belong to the triclinic system and P-1 space group.
In all structures, the central metal reveals distorted square-planar
coordination geometry. The three coordination positions of the [PdCl
(DPPB-HBH)]Cl and [PdCl(DPPB-PTSC)] complexes are coordinated
by the PNO and PNS donor atoms of the corresponding ligands,
respectively, and the chloride ion occupies the fourth position. In [Pd
(DPPB-HBH)2]Cl2•2CH3CN, four coordination positions are occupied by
the P and N atoms of two iminophosphine ligands (DPPB-HBH), Fig. 2.
According to Table S2, the cis bonding angles around Pd are greater or
less than 90◦ , and both trans ones are tinier than 180◦ . Hence, these
displacements confirm the deviation of the central metal ions from the
ideal square-planar geometry. The C–S bond distance (d = 1.753(2) Å)
lies between the C–S single bond (d = 1.82 Å) and the C– – S double bond
(d = 1.56 Å) in the [PdCl(DPPB-PTSC)] crystal structure [67], which
indicates the CS bond has a partial double bond character. Moreover, the
C–N bond (d = 1.299(3) Å) in this structure is shorter compared to the
classical C–N bond (d = 1.471 Å) [68]. All these shreds of evidence
confirm the delocalization of electron density in the thiosemicarbazone
chain. In the crystal structure of [PdCl(DPPB-HBH)]Cl, the C– – O dis­
tance (d = 1.257(10) and 1.262(9) Å) is close to the classical C– – O bond
(d = 1.264 Å) [68], indicating the iminophosphine ligand is coordinated
in the keto form.
The [PdCl(DPPB-HBH)]Cl crystal structural analysis displays that the
structure contains two molecules and two chloride ions in its asym­
metric unit, as shown in Fig. 2. The DPPB-HBH tridentate ligand coor­
dination to central cation forms one five- plus one six-membered chelate
ring (Cg1 = Pd1-O1-C8-N2-N1, Cg7 = Pd2-O3-C34-N4-N3, Cg2 = Pd1-
P1-C2-C1-C7-N1, and Cg8 = Pd2-P2-C28-C27-C33-N3) around the Pd
(II) centers. In the crystal structure of [PdCl(DPPB-HBH)]Cl, molecules
A and B are connected one by one through N2-H2A⋯Cl4, O4-H4A⋯Cl4,
O2-H2⋯Cl3, and N4-H4B⋯Cl3 hydrogen bonds forming zigzag chains
along the a-axis (Table S3). Adjacent zigzag chains are connected
through CH⋯π interactions (C43-H43⋯Cg4 and C30-H30⋯Cg10) and
create two-dimensional layers in the ab plane (Fig. 3A and Table S4).
Complex [Pd(DPPB-HBH)2]Cl2•2CH3CN contains half of the mole­
cule, acetonitrile solvent, and a chloride ion in its asymmetric unit, and
the other half, together with a solvent molecule and another chloride
ion, are generated by the inversion center. In this complex, the two
DPPB-HBH ligands are attached to the palladium atom as bidentate li­
gands through the phosphorus atoms of triphenylphosphine and the
iminic nitrogen atoms, which form 2 six-member rings with Pd(II) cation
(Fig. 2). In this crystal structure, chloride ions outside the coordination
sphere generate zigzag chains along the c-axis by creating N2-H2A⋯Cl1
and O2-H2⋯Cl1 hydrogen bridges between adjacent molecules (Fig. 3B
and Table S3).
The chains are joined together via CH⋯π connections between the
hydrogens of the phenyl rings of one chain of triphenylphosphine
fragment (H18) and the 4‑hydroxyphenyl rings of another chain. In
addition, other C–H⋯π intermolecular interactions such as C3-
H3⋯Cg6 and π⋯π interactions (Cg4⋯Cg4) between molecules of a
chain stabilize the crystal structure of [Pd(DPPB-HBH)2]Cl2•2CH3CN in
the bc-plane; see Fig. 3B and Table S4.
[PdCl(DPPB-PTSC)] crystal contains one molecule in the asymmetric
unit, and each molecule links to its neighboring molecule through C25-
H25⋯Cl1 (dH25⋯Cl1 = 2.93(10) Å) non-classical hydrogen bonding
interactions; hence, infinite one-dimensional polymeric chains are
extended along the a-axis. The one dimensional chains are inter­
connected together through C17-H17⋯N2, C12-H12⋯Cl1, C6-H6⋯Cl1,
and C19-H19⋯S1 contacts (dH17⋯N2 = 2.61(2), dH12⋯Cl1 = 2.79(8),
dH6⋯Cl1 = 2.82(9), and dH19⋯S1 = 2.84(9) Å) and weak π⋯π
stacking interactions (C23-H23⋯Cg4) to form the 2D network along ab-
plane, see Fig. 3C, Tables S3 and S4.

3.4. DNA binding studies

Fig. 1. Image indicating a mixture of [PdCl(DPPB-HBH)]Cl and [Pd(DPPB- 3.4.1. Electronic absorption titration method
HBH)2]Cl2•2CH3CN crystals at 50X magnification. Before starting the experiment, to measure the stability of the

6
M. Pooyan et al.
Fig. 2. ORTEP diagrams of complexes [Pd(DPPB-HBH)2]Cl2•2CH3CN, [PdCl(DPPB-HBH)]Cl, and [PdCl(DPPB-PTSC)] (The thermal ellipsoids have been shown by
50% probability).
complexes, their absorption spectrum was measured in Tris-HCl buffer
solution containing 1 % DMSO after 24 h. The slight changes observed in
the absorption spectrum of the complexes prove their stability in the
Tris-HCl buffer environment (Fig. S3).
Since DNA is one of the main targets of anticancer medications, the
interaction study of Pd(II) complexes with DNA and the model of their
binding to each other provide valuable information. Consequently, the
interactions between the DNA and synthesized complexes are evaluated
through the electron absorption titration technique by monitoring the
absorbance variations. Fig. 4 depicts changes in absorption spectra for
the [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)] and [PdCl(DPPB-TSC)]
samples in the presence and absence of various ct-DNA dosages. Elec­
tronic spectra of the free complexes illustrate three absorptions within
ranges of 202–220, 251–298 (intra-ligand transitions), and 390–415 nm
(CT transitions). By adding different ct-DNA doses, absorption bands
related to intra-ligand transitions exhibit hyperchromism and blue shift
(Fig. 4). These spectral changes are not significant for the CT bands. The
observed hyperchromic effects with blue-shift suggest effective in­
teractions of Pd(II) complexes with DNA helix structure, which is likely
to be due to the weak interactions with nucleobases in minor and major
grooves of DNA or electrostatic interactions with the phosphate back­
bone of the DNA helix [67,69,70]. The intrinsic binding constants (Kb)
between the complexes and ct-DNA are calculated from the ratio of slope
to the Y-intercept of the linear plots of [DNA]/|(εa-εf)| vs [DNA] (Fig. 4).
Kb values were found to be 2.1 × 104, 0.3 × 104, and 0.9 × 104 M− 1 for
[PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)]
complexes, respectively, which indicates that all three complexes have
good binding affinity to ct-DNA, and [PdCl(DPPB-HBH)]Cl interacts
more strongly with DNA than the other complexes.
Compared to the classical intercalator ethidium bromide (Kb = 1.23
(±0.07) × 105 M− 1) [71] and metallointercalators whose binding con­
stants have been reported in the range of 105 to 107 M− 1 [72–76], Pd (II)
complexes exhibited a lower Kb value. These results indicate that these
complexes can interact with DNA moderately and with less strength than
intercalators [77,78]. Values of intrinsic binding constants are presented
in Table 3. Standard Gibbs free energy (ΔG0b ) for DNA binding is
7
Inorganica Chimica Acta 547 (2023) 121368
obtained to be − 24.66, − 19.84, and − 22.56 KJ.mol− 1 at 25 ◦ C for
compounds [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl
(DPPB-TSC)], respectively, indicating the spontaneous interactions of
the complexes with DNA [34].
3.4.2. Competitive binding using ethidium bromide (EB)
To further elucidate the DNA binding modes of Pd(II) complex
samples, the EB displacing technique was performed by monitoring the
variations of EB-DNA fluorescence intensity. Notably, the EB fluores­
cence probe shows high emission intensity upon coupling with DNA,
which changes with adding another DNA-binding compound [79].
Emission reductions may occur when the compound can replace EB in
the DNA strand, or it can accept excited electrons of the EB intercalator
[80]. The fluorescence spectra of the EB-DNA solution are taken with the
presence and absence of diverse palladium complexes concentrations.
Investigation of the effects of the complexes on the photo­
luminescence intensity shows that with increasing concentrations of
[PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)] the
emission intensity gradually decreases. The observed quenching in­
dicates the interactions of palladium complexes with DNA helix strands,
as seen in Fig. 5. Quenching constant values (Ksv) as a measure of the
tendency of Pd(II) complexes to bind to DNA is calculated as 0.70 × 103,
0.349 × 103, and 0.60 × 103 for [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-
PTSC)], and [PdCl(DPPB-TSC)], respectively, using the Stern–Volmer
equation. The DNA-binding ability follows the order of [PdCl(DPPB-
HBH)]Cl˃ [PdCl(DPPB-TSC)]˃ [PdCl(DPPB-PTSC)]. These observations
confirm the results obtained by absorption titration studies (Table 3).
Based on the Ksv values and the information extracted from the experi­
ments, the quenching can be considered a result of the acceptance of
excited electrons during the photoelectron phenomenon. [PdCl(DPPB-
HBH)]Cl creates the most robust binding to the DNA, among other
structures. It is most likely associated with hydroxyl and carbonyl
moieties in the [PdCl(DPPB-HBH)]Cl structure.
3.5. Cytotoxicity activities
Following outstanding data attained by DNA binding studies, the
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Fig. 3. (A) Crystal packing of [PdCl(DPPB-HBH)]Cl; 2D representation of intermolecular interactions in the ab-plane, showing the molecules are connected through
hydrogen bonding and C–H⋅⋅⋅π interactions along the a-axis and in the direction of the ab-plane. (B) Side view of [Pd(DPPB-HBH)2]Cl2•2CH3CN crystal packing in
the bc-plane. (C) Representation of the 2D layer in [PdCl(DPPB-PTSC)] shows how chains are connected in the ab-plane. In all shapes, non-interacting hydrogens were
removed for simplicity.

cytotoxicity effects of the Pd(II) complexes were evaluated on the pro­ the tendency of molecules to bind to DNA. This agreement suggests that
liferation of MDA-MB-231 cells using the MTT method. For this purpose, the cytotoxic activities of Pd(II) complexes are possibly associated with
various Pd(II) complexes concentrations (100, 50, 25, 10, 7.5, 5.0, 2.5 interactions by DNA.
µg/mL) were added to these breast cancer cells at 24- and 48-h intervals.
IC50 values of tested samples are presented in Table 4, where the low
3.6. Molecular docking studies
values of IC50 indicate their performance at low concentrations. Analysis
of the results reveals that all three Pd(II) complexes substantially inhibit
According to the results of experimental studies and the desired
the growth of cells in a time- and dose-dependent manner, meaning with
abilities of Pd(II) complexes to bind to DNA, to obtain an overview of the
increasing concentration and treatment time, the mean cell viability
interaction modes of complexes with DNA, molecular docking simula­
decreases. As a result, they have a positive effect on inhibiting cell
tion was conducted, which is a powerful graphical tool for better un­
proliferation (Fig. 6).
derstanding non-covalent interactions between drug and
Results also verify that all three complexes have higher cytotoxic
macromolecule.
activities than the cisplatin reference drug (IC50 = 24.59 µg/mL) upon
The geometries of all three molecular structures were fully optimized
incubation for 24 h. This increasing trend of inhibitory potency of
in the water phase by density functional theory (DFT) with B3LYP
compounds continues to be observed with the prolongation of the
method using LANL2DZ and 6–311 + G** basis sets for palladium and
treatment time. However, only [PdCl(DPPB-HBH)]Cl shows higher in­
main group elements, respectively. Also, DNA crystal structure (1BNA.
hibition than the cisplatin (IC50 = 6.24 µg/mL) after incubation for 48 h.
pdb) was obtained from the Protein Data Bank.
According to these interpretations, [PdCl(DPPB-HBH)]Cl has the most
The results show that all the complexes interact well with the DNA
potent anti-proliferative activity among other compounds tested as it
and bind to nucleotide residues in the minor groove of the DNA double
shows IC50 amounts of 9.3 and 4.99 µg/mL after 24 and 48 h, respec­
helix by H-bonds, electrostatic and hydrophobic interactions. Among
tively, against selected cancer cells. It could correspond to the mole­
the docked molecules, [PdCl(DPPB-HBH)]Cl demonstrates the highest
cule’s hydroxyl and carbonyl functional moieties, making it more
binding affinity with a free binding energy of − 8.88 kcal/mol (with Ki
reactive than the two complexes containing C(S) group. In general, the
= 0.310 µM) compared to the other two molecules, which is in good
anticancer potentials of these samples change as [PdCl(DPPB-HBH)]Cl
agreement with the experimental results (Table S5). The most probable
> [PdCl(DPPB-TSC)] > [PdCl(DPPB-PTSC)], which is consistent with
and best binding positions of the complexes in the DNA double-helix are

8
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Fig. 4. Absorption titration spectra of complexes [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)] (48 µM) within Tris-HCl buffer upon both the
presence and absence of different DNA concentrations (9.94–170 µM). Arrows display absorption intensity changes by varying DNA concentrations.

Table 3
Electronic absorption spectroscopic data plus DNA binding parameters of sam­
ples [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)].
Compound λmax (nm) Δλ Kb Ksv ΔG* (KJ.
(nm) (M− 1) (M− 1) mol− 1)
Free Bound

[PdCl(DPPB- 298 258 40 2.1 × 0.70 × –24.66

HBH)]Cl 104 103
[PdCl(DPPB- 251 259 8 0.3 × 0.349 × –19.84
PTSC)] 104 103
[PdCl(DPPB- 290 265 25 0.9 × 0.60 × –22.56
TSC)] 104 103

depicted in Fig. 7.
As shown in Fig. 8, the orientation of [PdCl(DPPB-HBH)]Cl is such
that it fits precisely in parallel between the DNA strands and involves
more residues during the interaction process, thus creating more in­
teractions with the target DNA functional groups. In complexes [PdCl
(DPPB-PTSC)] and [PdCl(DPPB-TSC)], on the other hand, the orienta­
tion is such that part of both molecules is located outside the minor
groove and naturally provides fewer sites for binding (Figs. 7 and 8).
Hence, this evidence verifies the importance of the presence of hydroxyl
and carbonyl groups in increasing the binding potential of [PdCl(DPPB-
HBH)]Cl. Eight interactions stabilize [PdCl(DPPB-HBH)]Cl in the DNA
chains. The two classical hydrogen bonds are composed of the interac­
tion of a hydroxyl oxygen atom with the oxygen atoms of residues DT7
Fig. 5. The EB-DNA emission spectra both in the presence and absence of
and DT19 in chains A and B, respectively (with bond lengths of 2.18 and
various complex concentrations ([PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)],
2.77 Å). Also, two non-classical hydrogen bonds are formed by con­ and [PdCl(DPPB-TSC)]). [DNA] = 100 µM, [EB] = 5 µM, [Complex] =
necting oxygen atoms in hydroxyl moiety to iminic hydrogen with res­ 20–200 µM. Arrows display the emission intensity changes.
idues DA18:C2 and DC9:O2 (Fig. 7).

9
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Table 4 complexes, NCI analysis based on promolecular density was performed

IC50 values obtained from the MTT assay for investigated Pd(II) complexes and using VMD 1.9.3 and Multiwfn 3.7 software.
cisplatin against MDA-MB-231 cells after 24 and 48 h. The best binding mode obtained from molecular docking simulations
Tumor Time IC50 (µg/mL) was used for the NCI studies. Different interactions can be identified in
cell (h)
[PdCl(DPPB- [PdCl(DPPB- [PdCl Cisplatin
various regions according to the obtained color-filled NCI gradient iso­
HBH)]Cl PTSC)] (DPPB- surfaces and considering the scale bar [44,45,81]. As can be seen from
TSC)] Figs. 8 and S5, each interaction is shown in a different color. The red
MDA-MB- 24 9.3 17.47 15.35 24.59 color is due to the steric repulsion in the phenyl and phenol rings of the
231 48 4.99 11.45 9.87 6.24 complexes, and the green and blue colors are due to the presence of van
der Waals and H-bonds contacts among the DNA functional groups and
molecules, respectively. It is clear from the results that in all investigated
On the other hand, DG10 and DC11 residues participate in both complexes, a large surface of NCI gradient isosurface is related to van
electrostatic and hydrophobic interactions with [PdCl(DPPB-HBH)]Cl, der Waals interactions (such as π-stacking) [45,67,81,82], which is in
and in addition, hydrophobic interactions are observed with DA18, complete alignment with the results of docking simulation and experi­
DT19, DC9, DT8, DA17, and DG16 (Fig. S4). Information on bond mental studies.
length, type of interaction, and participating residues are presented in
Table 5. In [PdCl(DPPB-PTSC)] and [PdCl(DPPB-TSC)] complexes, hy­
drophobic intermolecular forces are the leading forces in the in­ 3.7. Computational studies
teractions with DNA double-helix, and fewer residues are involved
within electrostatic and H-bonds contacts. 3.7.1. Quantum chemistry descriptors
To better visualize non-covalent interactions (NCI) such as steric Studies of compounds’ structural and electronic properties provide
effect, van der Waals interactions, and hydrogen bonds in drug-DNA deep insights into the influences of functional groups on reactivity.
Therefore, to make a correlation between the structural properties and

Fig. 6. Cell viability of cells treated by various samples concentrations for 24 h (A) and 48 h (B).

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M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Table 5
Details of the interactions between the docked Pd(II) complexes and the DNA
double helix.
Compound Nucleobase Interaction type Distance
residues (Å)

[PdCl(DPPB- A: DT7:O2 Hydrogen Bond 2.18

HBH)]Cl B: DT19:O2 Hydrogen Bond 2.77
B: DA18:C2 Hydrogen Bond (non- 2.72
classical)
A: DC9:O2 Hydrogen Bond (non- 3.33
classical)
A: DG10:OP1 Electrostatic 4.20
A: DC11:OP1 Electrostatic 3.25
A: DC11:OP1 Electrostatic 5.45
A: DG10:C5′ Hydrophobic π-alkyl 3.82
[PdCl(DPPB- B: DG22:C5′ Hydrogen Bond (non- 3.59
PTSC)] classical)
B: DG22:OP1 Electrostatic 4.09
B: DG22:OP1 Electrostatic 5.47
A: DG4 Hydrophobic π-π T- 4.90
shaped
[PdCl(DPPB- B: DC23:OP1 Hydrogen Bond 2.06
TSC)] B: DG22:OP1 Electrostatic 5.09
B: DC23:OP1 Hydrogen Bond; 2.13
Electrostatic

Fig. 7. Demonstration of the possible interactions between investigated com­

plexes and DNA; The various interactions are distinguished by colored dashes in
which the green and gray colors represent the classical and non-classical
hydrogen bonds, respectively, and the orange color illustrates the electro­
static interactions. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
Fig. 8. NCI plots for the most probable binding modes of [PdCl(DPPB-HBH)]Cl
(A), [PdCl(DPPB-PTSC)] (B), and [PdCl(DPPB-TSC)] (C) complexes to DNA.
Surfaces represent various non-covalent interactions based on the color bar.
the activity of the Pd(II) complexes, the electron density distribution, the
atomic contribution of HOMO and LUMO orbitals, and other quantum
chemical parameters affecting the biological activity like electron af­
finity, ionization potential, softness, hardness, chemical potential,
electrophilicity index, and electronegativity, were selected and
computed by respective standard equations [83]. The DFT optimization
of geometrical structures was performed at the B3LYP level using a
6–311 + G** basis set for O, N, C, H, S, and Cl atoms while the LANL2DZ
basis set for Pd atom within the solvent phase (water).
The reactivity of compounds is due to the charge transfer processes
between electron acceptor and donor groups. It is reasonable to analyze
the compounds’ reactivity differences based on their LUMO and HOMO
energies. Diagrams of frontier molecular orbitals (FMOs) of all three
molecules with the energy level values and energy differences between
them are depicted in Fig. 9. Analysis of FMOs displays that, in all
complexes, the HOMO orbitals are mainly located on the palladium and
the atoms coordinated to it, and the LUMO orbitals are spread over the
hydrazide fragment and phenyl ring attached to the C– – N bond of the
ligands.
Frontier orbitals composition was carried out by the Hirshfeld
method using Multiwfn software. Fig. S6 exhibits the percentage con­
tributions of HOMO and LUMO. As can be seen, the atomic contributions
of the HOMO orbitals in [PdCl(DPPB-PTSC)] and [PdCl(DPPB-TSC)] are
such that the sulfur atoms participate to a greater extent (9.55 %), while
in the [PdCl(DPPB-HBH)]Cl, the oxygen atom contributes less to this
orbital (2.99 %). This situation is reversed for LUMOs, where 2.86 % is
localized on the sulfur atom and 4.29 % on the O atom.
Fig. 10 displays the electron density maps of the molecules. As
evident from Fig. 10 and the values of atomic charges presented in
Table S7, there are more electronic densities around the O atoms of
[PdCl(DPPB-HBH)]Cl than sulfur atoms of [PdCl(DPPB-PTSC)] and
[PdCl(DPPB-TSC)] molecules, which illuminate their abilities to interact
and participate in nucleophilic reactions. Table S6 lists the data related
to the quantum reactivity parameters. Energy gap values provide in­
formation about the chemical reactivity and kinetic stability of com­
pounds that are very close to each other for the complexes tested, and
they are consistent with the energy gap values of biologically active
molecules. Other chemical reactivity parameters were calculated using
LUMO and HOMO levels. The results illustrate that the electron transfer
process in terms of energy in the studied complexes is favorable due to
the positive values of chemical hardness (η) and the negative values of
energy change (ΔE) [84].

11
M. Pooyan et al.
Fig. 9. Schematic representation of the frontier molecular orbitals of [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and [PdCl(DPPB-TSC)] with the energy gap values.
The electrophilicity index (ω) is the most crucial parameter in bio­
logical activity studies. The samples with higher ω have more in­
teractions with biological molecules. According to the ω values, all three
complexes have high electrophilicity and are considered the strongest in
the classification of the electrophilic index [85].
The order of increasing electrophilicity of the complexes is [PdCl
(DPPB-HBH)]Cl > [PdCl(DPPB-PTSC)] > [PdCl(DPPB-TSC)]. It suggests
that there may be more interactions between [PdCl(DPPB-HBH)]Cl and
macromolecules.
Electronegativity (χ ) demonstrates the electron capture capability of
the compound. High or low electronegativity values determine the
complexes’ Lewis acidic or basic properties. Samples with higher χ
participate in the reaction as a Lewis acid. The term -χ defines the
chemical potential of a compound. A more negative chemical potential
in a combination shows that it is more difficult to lose the electron,
whereas it is easier to accept electrons. The given high values of elec­
trophilicity and electronegativity and the negative chemical potential
are considerable. It concludes that these complexes tend to accept
electrons and act as electrophiles in the reaction with macromolecules.
The order of increasing electronegativity is [PdCl(DPPB-HBH)]Cl >
[PdCl(DPPB-PTSC)] > [PdCl(DPPB-TSC)], indicating that [PdCl(DPPB-
HBH)]Cl has a greater affinity for electron acceptance among the
other two complexes. This result entirely approves those attained from
the experimental analyses.
3.7.2. Reactive sites of molecules
Fukui functions and molecular electrostatic potential (MEP) were
investigated to assign reactive sites in molecules. MEP is a computa­
tional method highlighting a molecule’s electrophilic or nucleophilic
areas based on electron density [86]. The MEP schemes of the complexes
are plotted using the DFT approach (6–311 + G**/LANL2DZ basis sets),
in which different colors indicate various values of electrostatic poten­
tials. High electron density positions are displayed in red and are
12
Inorganica Chimica Acta 547 (2023) 121368
suitable for electrophilic attack. The blue, green, and yellow regions are
electron deficient, neutral, and less rich in electrons, respectively.
As can be seen in Fig. 11, the areas around the Pd atoms have the
most negative potential, and the most positive one is related to the
attached hydrogen to the hydrazonic nitrogen, which is the most
desirable site for the nucleophilic attack. Localized surface maxima and
minima in ESPs have been exhibited, respectively, with red and blue
spheres. Comparison between the oxygen and sulfur atoms in the
structures of the complexes reveals that the electrostatic potential of the
oxygen is more negative (the red region) than that of the sulfur atom
(the yellow region), which makes it prone to participate in nucleophilic
reactions. Since the dominant color of the electrostatic potential in Pd
(II) complexes is blue, they can act as electrophilic agents interacting
with macromolecules. The highlighted reactivity sites are clear evidence
of the reactivity of the complexes.
The performance of Fukui calculations of atoms helps to understand
the interactions better. According to the Mulliken population, Fukui
functions provide quantitative data about atomic electrophilicity and
nucleophilicity for a compound [87–89]. The following formula esti­
mates these functions.
f + (→
r ) = qr(N + 1) − qr(N) The nucleophilic attack
f − (→
r ) = qr(N) − qr(N − 1)The electrophilic attack
f 0(→
r ) = (qr(N + 1) − qr(N − 1) )/2 The radical attack
In Fukui functions equations, qr is the calculated atomic charge from
analyzing the Mulliken population at the rth atomic site in neutral (N),
anionic (N + 1), and cationic (N-1) chemical species. The 0, − , and +
signs indicate radical, electrophilic, and nucleophilic attacks, respec­
tively. Full details of Fukui calculations for all atoms of three molecules
are presented in Tables S8–S10 (see the Supplementary data). The dif­
ference between the nucleophilic and electrophilic Fukui functions is
M. Pooyan et al.
Fig. 10. Color-filled maps of the electron density of molecules.
defined as the dual descriptor Δf(r) [90].
Δf (r) = f + (→
r ) − f − (→
r ) Dual descriptor
When Δf(r) < 0, the site is suitable for the electrophilic attack, and
when Δf(r) > 0, the location is fit for nucleophilic attack. In general,
according to the dual descriptor data of atoms, palladium atoms and
iminic carbons can act as nucleophile sites in all three complexes. In
[PdCl(DPPB-HBH)]Cl, the oxygen atom of the carbonyl and hydroxyl
groups and the hydrazonic nitrogen atom can be a good place for elec­
trophilic attack, while H9 is more suitable for nucleophilic attack due to
its binding to the electronegative nitrogen atom (N8). In [PdCl(DPPB-
PTSC)] and [PdCl(DPPB-TSC)] complexes, hydrazonic nitrogen and
sulfur atoms are suitable sites for nucleophilic attack due to the partial
double-bond character of the C–S bond and the conjugation effect of
double bonds.
4. Conclusions
In this work, a series of palladium(II) complexes bearing imino­
phosphine ligands based on benzohydrazone and thiosemicarbazone
derivatives of structural and biological significance were synthesized
and characterized with different spectroscopic techniques. The
13
Inorganica Chimica Acta 547 (2023) 121368
structures of complexes [PdCl(DPPB-HBH)]Cl, [Pd(DPPB-HBH)2]
Cl2•2CH3CN, and [PdCl(DPPB-PTSC)] were determined by X-ray crys­
tallography. Analysis of crystal structures confirmed the distorted
square-planar geometry for the palladium centers, it was also found that
the DPPB-HBH ligand could form two different palladium complexes,
acting as a P, N bidentate and P, N, O tridentate in the [Pd(DPPB-HBH)2]
Cl2•2CH3CN and [PdCl(DPPB-HBH)]Cl complexes, while the DPPB-
PTSC ligand behaves as a tridentate ligand through the P, N, and S
donor atoms in the [PdCl(DPPB-PTSC)]. Among the synthesized com­
pounds, complexes [PdCl(DPPB-HBH)]Cl, [PdCl(DPPB-PTSC)], and
[PdCl(DPPB-TSC)] were employed to evaluate the biological activities
due to their similar coordination configuration. The results of the DNA-
binding studies showed that all three complexes have good binding af­
finity to ct-DNA through electrostatic and groove binding modes. Also,
the cytotoxicity assay of palladium(II) complexes in vitro against MDA-
MB-231 breast cancer cells revealed their high activity compared to
the reference drug cisplatin (IC50 = 24.59 µg/mL). [PdCl(DPPB-HBH)]Cl
exhibited the best antiproliferative activity (IC50 = 9.3 µg/mL) and the
highest DNA binding ability among the tested samples, which could be
due to the presence of carbonyl and hydroxyl moieties and their
participation in the interaction with DNA that was further investigated
by molecular docking and NCI studies. Chemical reactivity descriptors
M. Pooyan et al. Inorganica Chimica Acta 547 (2023) 121368

Fig. 11. Molecular electrostatic maps of complexes ([PdCl(DPPB-HBH)]Cl (A), [PdCl(DPPB-PTSC)] (B), and [PdCl(DPPB-TSC)] (C)) along with their electrostatic
potential values.

were calculated to establish a logical relationship between structure and Vahid Noroozi Charandabi: Investigation, Formal analysis,
activity. Small energy gap values of Pd(II) complexes confirmed their Methodology.
visualization as biologically active molecules. Also, the high electro­
negativity and electrophilicity values of [PdCl(DPPB-HBH)]Cl justified Declaration of Competing Interest
its greater reactivity than others. Possible sites for electrophilic and
nucleophilic attacks were determined using MPE calculations and Fukui The authors declare that they have no known competing financial
functions. Considering the concordance of theoretical and experimental interests or personal relationships that could have appeared to influence
results, it seems that the present study can be effective in the targeted the work reported in this paper.
design of anticancer candidates.
Data availability
CRediT authorship contribution statement
Data will be made available on request.
Mahsa Pooyan: Investigation, Formal analysis, Methodology,
Writing – original draft. Zahra Shariatinia: Supervision, Conceptuali­ Acknowledgment
zation, Validation, Visualization, Project administration, Data curation,
Funding acquisition, Resources, Writing – review & editing. Fahimeh This work was supported by Iran National Science Foundation (INSF)
Mohammadpanah: Investigation, Formal analysis, Methodology. (INSF, Grant No. 99025423), and the authors thank all generous
Khodayar Gholivand: Conceptualization, Validation, Visualization, support.
Project administration. Peter C. Junk: Investigation, Formal analysis,
Methodology. Zhifang Guo: Investigation, Formal analysis, Methodol­
ogy. Mohammad Satari: Investigation, Formal analysis, Methodology.

14
M. Pooyan et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ica.2022.121368.
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