New phosphorus ylide palladacyclic: Synthesis, characterization, X-Ray crystal structure, biomolecular interaction studies, molecular docking and in vitro cytotoxicity evaluations

Article abstract of DOI:10.1016/j.jorganchem.2018.09.018

The ylide-phosphonium salt [PPh₃CH₂C(O)CH₂Cl]⁺Cl^? was reacted with Pd(OAc)₂ to give the chloro-bridged dinuclear complex [Pd{C(H)PPh₃C(O)CH₂Cl}(μ-Cl)(OAc)]₂, which experienced bridge cleavage reactions with triphenylphosphine (PPh₃) and pyridine (Py), and to prepare the new orthometallated complexes [Pd{(C,C)–C₆H₄PPh₂C(H)C(O)CH₂Cl}L]Cl, [L = PPh₃ (1) and Py (2)]. The complexes were identified and characterized using various techniques. X-ray crystallography was used to determine the crystal structure of 1, which revealed the presence of an orthometallated C₆H₄-PPh₂ unit. CT-DNA binding interaction of the synthesis compounds was tested by fluorescence spectroscopy, UV–Vis absorption spectroscopy, and the viscometric titration method. The analysis of the obtained data indicated that the Pd complexes could bind to DNA via groove binding by the partial intercalation mode. The emission titration of bovine serum albumin (BSA) with two Pd complexes showed a static process for the fluorescence quenching mechanism of BSA. In addition, the results of competitive binding by Eosin-Y, Ibuprofen and Digoxin site markers revealed that the complexes were bound to the site I of BSA. The donor (BSA) - acceptor (Pd complexes) distance was calculated using fluorescence resonance energy transfer (FRET). Notably, molecular docking studies were used for the determination of DNA and BSA-Pd (II) complexes binding. Finally, the two complexes exhibited significant in vitro cytotoxicity against human leukemic T cell (Jurkat) and chronic myelogenous leukemia (K562) cancer cell lines using MTT([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] colorimetric. In cell cycle analysis conducted on Jurkat and K562 cells treated with ligand and Pd complexes, a decrease in DNA cell content and shift in the main population of cells toward the subG1 phase were observed.

Full text of DOI:10.1016/j.jorganchem.2018.09.018

Accepted Manuscript
New phosphorus ylide palladacyclic: Synthesis, characterization, X-Ray crystal
structure, biomolecular interaction studies, molecular docking and in vitro cytotoxicity
evaluations
Kazem Karami, Mahzad Rahimi, Mostafa Zakariazadeh, Orhan Buyukgungor, Zahra
Amirghofran
PII:
S0022-328X(18)30542-4
10.1016/j.jorganchem.2018.09.018
JOM 20575
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 1 July 2018
Revised Date: 12 September 2018
Accepted Date: 21 September 2018
Please cite this article as: K. Karami, M. Rahimi, M. Zakariazadeh, O. Buyukgungor, Z. Amirghofran,
New phosphorus ylide palladacyclic: Synthesis, characterization, X-Ray crystal structure, biomolecular
interaction studies, molecular docking and in vitro cytotoxicity evaluations, Journal of Organometallic
Chemistry (2018), doi: https://doi.org/10.1016/j.jorganchem.2018.09.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New phosphorus ylide palladacyclic: Synthesis, characterization, X-
Ray crystal structure, biomolecular interaction studies, molecular
docking and in vitro cytotoxicity evaluations
a*
a
b
c
d
Kazem Karami , Mahzad Rahimi , Mostafa Zakariazadeh , Orhan Buyukgungor , Zahra Amirghofran
a
Department of Chemistry, Isfahan University of Technology, Isfahan, 84156/83111, I.R. Iran
Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran
Faculty of Arts and Sciences, Department of Physics , Ondokuz, Mayıs University, 55139 Samsun
b
c
d Immunology Department, Autoimmune Diseases Research center, Shiraz University of Medical Sciences, Shiraz 71454,
Iran
Abstract
+
-
The ylide-phosphonium salt [PPh CH C(O)CH Cl] Cl was reacted with Pd(OAc) to give
3
2
2
2
the chloro-bridged dinuclear complex [Pd{C(H)PPh C(O)CH Cl}(µ-Cl)(OAc)] , which
3
2
2
experienced bridge cleavage reactions with triphenylphosphine (PPh ) and pyridine (Py), and
3
to prepare the new orthometallated complexes [Pd{(C,C)-C H PPh C(H)C(O)CH Cl}L]Cl,
6
4
2
2
[
L=PPh (1) and Py (2)]. The complexes were identified and characterized using various
3
techniques. X-ray crystallography was used to determine the crystal structure of 1, which
revealed the presence of an orthometallated C H -PPh unit. CT-DNA binding interaction of
6
4
2
the synthesis compounds was tested by fluorescence spectroscopy, UV-Vis absorption
spectroscopy, and the viscometric titration method. The analysis of the obtained data
indicated that the Pd complexes could bind to DNA via groove binding by the partial
intercalation mode. The emission titration of bovine serum albumin (BSA) with two Pd
complexes showed a static process for the fluorescence quenching mechanism of BSA. In
addition, the results of competitive binding by Eosin-Y, Ibuprofen and Digoxin site markers
revealed that the complexes were bound to the site I of BSA. The donor (BSA) - acceptor (Pd
complexes) distance was calculated using fluorescence resonance energy transfer (FRET).
Notably, molecular docking studies were used for the determination of DNA and BSA-Pd (II)
complexes binding. Finally, the two complexes exhibited significant in vitro cytotoxicity

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against human leukemic T cell (Jurkat) and chronic myelogenous leukemia (K562) cancer
cell lines using MTT([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide]
colorimetric. In cell cycle analysis conducted on Jurkat and K562 cells treated with ligand
and Pd complexes, a decrease in DNA cell content and shift in the main population of cells
toward the subG1 phase were observed.
Keyword: Orthometallated complexes; BSA binding; CT-DNA binding; Anticancer activity;
Molecular docking
1
. Introduction
Phosphorus ylides are useful ligands in many synthesis due to their ambidentate characters
[
1,2]. The reactivity and coordination chemistry of them are important in organometallic
chemistry and their application in synthetic chemistry has been well recorded [3,4]. Recently,
orthometallation of these ligands has been reported by many researchers because of
regioselectivity at Ph rings of the phosphine, showing that the orthopalladated complexes
derived from CH activation at Ph rings belong to the R′ or R″ substituents of the ylidic carbon
[
5,6]. Because of some problems associated with platinum-based anticancer drugs, such as
limited solubility, limited spectrum of tumors, ototoxicity and neurotoxicity, significant
attempts are being made to improve the effectiveness of them [7]. Thus, various new
complexes have been synthesized in order to overcome those disadvantages. The complexes
of ylide palladacyclic have received special attention due to their potential application in
organic synthesis [5]. Ylide palladacyclic is a heterogeneous cyclic organic compound;
palladium is the core of a specific structure stabilized by intramolecular connections [8].
Recently, the preparation of a wide variety of new complexes with medicinal properties such
as anticancer activity and their biomolecular interaction studies has been reported [9,10].

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Among metal-based complexes, palladium complexes are interesting anticancer drugs
[
11,12]. Anticancer properties of ylide palladacyclic have been specified due to their
cytotoxity assessment [13]. The initial way for cancer therapy will be metallodrugs started in
the interaction between DNA and coordination complexes [14]. There are three important
styles of interactions between DNA and metal complexes; these include intercalation, groove
binding and electrostatic interaction [15].
The most abundant proteins in blood plasma are serum albumins, whose major physiological
function is to carry various ligands to a particular target organ [16]. Bovine Serum Albumin
(BSA) was chosen because of the advantages of low price and accessibility and the similarity
of the tertiary structure to the human serum albumin (HSA). Moreover, three sites of
albumins are the specific binding sites for pharmaceutical metals [17-19].
In our work, synthesis of two new phosphorus ylide palladacyclic complexes was done using
triphenylphosphine (PPh ) and pyridine (Py) , something not reported before; then it was
3
characterized by Elemental analysis, spectroscopic methods (UV-vis, FT-IR and NMR), and
single crystal X-ray structure analysis. Additionally, biological application of them has been
reported. Because of medical significance, low charge, easy accessibility, natural
fluorescence emission, and the structural similarity of bovine serum albumin (BSA) with
human serum albumin (HSA), it was utilized as the protein modeling for drug delivery
[
20,21]. The BSA binding capacity and site selective binding were monitored by UV
absorption and fluorescence spectroscopy in the absence and presence of the complexes. In
addition, the complex-DNA interaction was verified by UV visible absorption and
fluorescence emission spectroscopy, molecular docking, and the viscometery method.
Furthermore, the molecular docking technique was performed on BSA. Additionally, the
prepared complexes exhibited antitumor activity and cytotoxic activity against human
leukemic T cell (Jurkat) and chronic myelogenous leukemia (K562) cancer cell lines, as

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evaluated
by
the
MTT
([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide]assay.
2
2
. Experimental
.1. Materials
All solvents and starting materials were obtained from Sigma Aldrich or Alfa Aesar
and used without further purification. Methylene blue (MB), Calf thymus DNA (CT‐DNA),
and BSA were supplied by Sigma Aldrich. Stock solutions of BSA and CT-DNA were
prepared by dissolving in a Tris buffer (NaCl (50 mM), Tris–HCl (5 mM) at the pH of 7.3).
The UV absorbance ratio (A /A ) of the solutions of CT-DNA was over 1.8, showing the
2
60
280
purity of DNA [22]. The CT‐DNA concentration was standardized by the UV-Vis absorption
-1
-1
method, using its molar absorption coefficient (ε = 6600 M cm ) [23,24]. Distilled
deionized water as the solvent and dimethylsulfoxide (DMSO) as the co‐solvent were used
for the preparation of the stock solution of the complex. The final DMSO concentration never
exceeded 0.13% (v/v). Jurkat (human leukemic T cell line) chronic myelogenous leukemia
(K562) was bought from the National Cell Bank of Pasteur Institute, Tehran, Iran.
2
.2. Measurements
Infrared spectra were recorded on a FT-IR JASCO 680-PLUS spectrometer using KBr
pellets. UV-visible spectra were recorded with a JASCO 7580 UV-Vis-NIR
spectrophotometer. Cary Eclipse fluorescence spectrophotometer was the secondhand for
1
fluorescence emission. A 500 MHz Bruker spectrometer operating at 500.13MHz for H and
3
1
31
1
1
61.97MHz for P was referenced to H PO (85%) for P{ H}-NMR spectra, which were
3 4
obtained in CDCl . For viscometric titrations, a cannon Fenske routine viscometer was used
3
in a thermostatic water bath at 298 °K. Cells were cultivated in the Dulbecco’s Modified

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Eagle Medium (DMEM) supplemented in the activated fetal bovine serum (FBS). 5 mM L-
-1
-1
glutamine, 100 µg.ml streptomycin, and 100 U.ml penicillin were used. Afterward, the cell
lines were grown in a humidified atmosphere containing 5% CO at 37 °C. Elemental
2
analyses were performed on a Leco, CHNS-932 apparatus. The Tm spectra were recorded on
a Varian BioCary-100 UV–Vis spectrophotometer using a 1 cm path length cell. CD spectra
were recorded on an Aviv Circular Dichroism Spectrometer, the model 215 (USA), using a
cylindrical cuvette with 0.1 cm path length.
2
1
.3. Synthesis of compounds
. Synthesis of [Pd(ClCH COCH(PPh )L)]Cl, L= PPh (1)
2
3
3
First, the phosphorus ylide, [PPh CH C(O)CH Cl][Cl], was prepared according to our
3
2
2
previous study [1]. Then, palladium (II) acetate (0.25 mmol, 0.053 g was added to a solution
of mono-phosphonium salt [PPh CH C(O)CH Cl][Cl] (0.5 mmol, 0.194 g) in 15 mL of
3
2
2
methanol and the resulting solution was stirred at room temperature for 24 h. The resulting
solution was concentrated by reducing the solvent in vacuum to 2 mL. N-hexane (15 mL) was
then added to separate [Pd(ClCH COCHPPh )(µ-Cl)]
2
as an orange solid washed with
2
3
diethyl ether, collected and air-dried. After that, PPh (0.2 mmol, 0.052 g) was added to a
3
solution of [Pd(ClCH COCHPPh )(µ-Cl)] (0.1 mmol, 0.098 g) in dichloromethane (15 mL).
2
3
2
The resulting mixture was stirred at room temperature for 12 h. The suspension formed was
filtered off, washed with diethyl ether, and dried to give 5 as a yellow powder.
-1
-1
-1
1
Yield: 85%; IR (KBr, cm ): ν(C=O): 1663 cm , ν (P-C) = 650-750 cm . H NMR
(
400.13MHz, DMSO-d , ppm): δ 4.5 - 4.7 (d, 2H, CH ), δ 4.9 (t, 1H, CH), δ 7.2-7.9 (m, 29H,
6
2
1
3
1
1
aromatic), C{ H} NMR (DMSO-d , ppm): δ = 37.3 (d, J = 56 Hz), 49.24 (Caliphatic),
6
pc
1
1
19.04, 121.16, 121.26, 122.34, 123.43, 123.62, 124.25, 125.17, 126.05, 126.35, 126.64,
27.37, 128.63, 129.14, 129.46, 129.72, 131.06, 131.27, 131.60, 131.72, 132.05, 132.22,

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1
1
33.39, 133.45, 135.54, 135.66, 135.78, 135.83, 137.14, 137.20, 138.38, 138.80, 141.72,
31
1
46.46, 150.67, 153.30 (Caromatic), 194.92 (s, CO), P { H} NMR (DMSO-d , ppm): δ ~ 17
6
(
s, 1P, CHP), 31 (s, 1P, PdP), Elemental Anal. Calcd. For C H Cl OP Pd: C, 62.01; H,
39 32 2 2
4
.11%. Found: C, 61.92; H, 3.98%.
2
. Synthesis of [Pd(ClCH COCH(PPh )L)]Cl, L=Py (2)
2
3
Pyridine (0.2 mmol, 0.016 g) was added to a solution of [Pd(ClCH COCHPPh )(µ-Cl)]
2
2
3
(0.1 mmol, 0.098 g) in dichloromethane (15 mL). The resulting mixture was stirred at room
temperature for 12 h. The suspension formed was filtered off, washed with diethyl ether,
collected, and dried to give a brown powder.
-1
-1
-1
-
Yield: 80%; IR (KBr, cm ): ν (C=O):1668 cm , ν (C-N) = 1602 cm , ν (PC) = 650-750 cm
1
1
.
H NMR (400.13MHz, DMSO-d , ppm): δ 4.3 - 4.4 (d, 2H, CH ), δ 4.5 (t, 1H, CH), δ 7 -
6
2
1
3
1
1
8
.1 (m, 19H, aromatic), C{ H} NMR (DMSO-d , ppm): δ = 37.5 (d, J = 57 Hz), 49.35
6 pc
(Caliphatic), 119.15, 121.44, 124.32, 125.64, 126.07, 127.37, 129.42, 129.65, 130.05, 130.87,
1
1
32.75, 133.17, 133.55, 135.46, 135.79, 138.30, 140.64, 141.77, 142.09, 144.18, 145.38,
3
1
1
51.21, 154.56 (Caromatic), 195.44 (s, CO), P { H} NMR (DMSO-d , ppm): δ ~ 17 (s, 1P,
6
CHP), Elemental Anal. Calc. For C H Cl ONPPd: C, 55.02; H, 4.02; N, 2.05%. Found: C,
2
6
22
2
5
4.94; H, 3.82; N, 1.96%.
2
.4. X‐ray crystallography
X‐ray diffraction data collection was carried out at 296 K using the X-scan
technique and the X-RED32, developed by Stoe & Cie, 2002 package. The single crystal
structure was solved by direct methods and refined by employing SHELXL-2016/6
(Sheldrick, 2016) program and using the ORTEP crystallographic software package. Table S1
shows the corresponding crystallographic data.

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2
.5. Studies of DNA binding
For DNA-binding studies, first, a stock solution of CT-DNA was prepared in the Tris-
HCl buffer (5 mM Tris-HCl, 50 mM NaCl at pH 7.2) and stored at 4 °C. Then electronic
-1
-1
absorption spectra were recorded in the 200-400 nm range using Ɛ260 = 6600 M cm [25];
this was done by increasing 20 µl of CT‐DNA to the complexes 1 and 2 solutions in the Tris
buffer at room temperature. Each sample was retained for 5 min to reach equilibrium before
recording the UV–visible spectrum by electronic absorption measurement and fluorescence
quenching experiments. In addition, the stock solution of CT-DNA was prepared in the Tris-
HCl buffer (5 mM Tris-HCl, 50 mM NaCl at pH 7.2) and stored at 4 °C. Concentrated stock
solutions of Pd complexes were prepared by dissolving them in DMSO and diluted with the
buffer to develop the vital suitable concentrations in all experiments. The final DMSO
concentration did not exceed 0.13% v/v and our complexes displayed good stability, as tested
by electronic absorption spectrometry. All DNA-binding studies were done at room
temperature by electronic absorption spectroscopy and fluorescence spectroscopy
measurement. The test was done in the case of artificial model and differentiated with the real
system biological environment of blood. Also, in our complexes, the ylide ligand could do
the penetration phenomenon in the cell membrane because of its hydrophobic nature due to
hydrolysis, but Pd remained in the aqueous solution [26].
2
.6. Studies of protein binding
The fluorescence quenching of the tryptophan remainder of BSA, via the Pd
complexes as a quencher, with variable complex/BSA molar ratios was proved in the range of
85-485 nm, at room temperature, and in the Tris buffer at 343 nm. In addition, titration of
BSA and the marker compounds with the complexes was done to investigate the complex-
2

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BSA interaction by the existing BSA site markers, site I (Eosin-Y), site II (Ibuprofen), and
site III (Digoxin).
2
.7. Molecular docking of complexes with bio-macromolecules
Molecular docking simulation was performed in this study in order to survey the non-
covalent interactions between the new synthesized complexes with BSA and DNA. Also, the
binding affinity and the binding region of these complexes with bio-macromolecules was
considered by the docking procedure. Free binding energy (∆G) between complexes and
macromolecules was calculated via molecular docking simulation. To prepare the complexes
structures as ligands, the three dimensional structure of the pph3 complex (complex 1) was
obtained by converting the CIF file to PDB file using Open Babel 2.3.2 software [27]. The
chemical structure of pyridine containing complex (complex 2) was prepared using the Gauss
View 5.0 software. Molecular energy optimization of this complex structure was performed
using Gaussian 09 software (Quantum Chemistry package), by employing the density
functional theory (DFT) method with Becke's three-parameter hybrid functional (B3LYP)
through the 3-21G basis set. Crystallographic structures of BSA and DNA as the receptors
were selected from the RCSB protein data bank (http://www.pdb.org). The BSA (PDB ID:
4
F5S) with 2.47Å resolution and DNA (PDB ID: 2GVR) with the 1.65Å resolution of the
self-complementary dodecamer 5'-d(CGCGAATTCGCG)-3' were chosen for docking
simulation. Excessive molecules such as hetero atoms and waters were removed. Free
molecular docking packages, Auto Dock 4.2.6 and Auto Dock Tools 1.5.6, were used in this
study for docking simulation [28,29].
The PDB formats of complexes and bio-macromolecules were modified by computing polar
hydrogens, Gasteiger and Kollman charges. At first, the blind docking approach was

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employed to probe the possible binding sites with the highest binding energy affinity of
complexes to the three dimensional structure of BSA and DNA [30]. Finally, focus docking
was done on the best binding site. The grid boxes dimensions size at the grid points in x*y*z
3
directions were set to 60*60*60 Å with 0.375 Å grid spacing for both bio-macromolecules.
The Lamarckian genetic algorithm method with the assigned 100 numbers run of genetic
algorithm has been mentioned [29]. All parameters default values in Auto Dock Tools
software were adjusted in this docking study.
2
.8. Cytotoxicity assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay was
used to calculate the cytotoxic activity of the complexes 1 and 2 against Jurkat and K562
cancer cell lines. This test was carried out using a fixed concentration of tumor cells that had
been scattered in the wells of a 96-well plate with variable concentrations of the synthesized
complexes (0–100 mmol dm_3) and incubated for 48 h. (37°C, 5% CO 95%air humidified).
2
,
-1
Next, 20 µl of the MTT solution (5 mg.ml in phosphate buffer solutions) was added to each
0
well and the plates were incubated for 4 h at 37 C. Afterwards, 100 µl of the medium
including MTT was quietly substituted by dimethyl sulfoxide (DMSO) and pipetted to
dissolve any formazan crystal wrought. Finally, after the complete dissolution of the dye, the
optical density (OD) was measured at 560 nm by means of an enzyme-linked immunosorbent
assay (ELISA) plate reader (Bio-Tek’s ELx808, USA). The ratio inhibition of the cells was
measured as follows: % Inhibition= 100 - [(test OD/non-treated OD) × 100]. Non-treated
cultures in all treatments consisted of only the DMSO solvent at a concentration identical to
those in the test wells. IC (value of 50% maximal inhibitory concentration) was determined
50
using the non-linear regression of concentration response curves [31,32].

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2
.9. Cell cycle analysis by flow cytometry
The effects of ylide ligand and complexes 1 and 2 on cell cycle changes in Jurkat and
K562 cells were studied by propidium iodide staining using flow cytometry. Cells at
6
concentration of 1×10 /ml/well were seeded in 24-well plates in triplicate and treated with the
compounds at concentrations equal to their IC values calculated from the results of MTT
5
0
°
cytotoxicity assay. After 48 h of culture at 37 C in a CO incubator, the cells were harvested
2
and then permeable with 1ml hypotonic solution containing sodium citrate (1%) and triton X-
1
00 (0.1%). Then propidium iodide (Sigma Aldrich, Steinheim, Germany) at final
°
concentration of 50 µg/ml was added. After 2 h incubation at 4 C, cells were examined for
the intensity of fluorescent dye by flow cytometry (Becton Dickinson, San Diego, CA) in
FL2 channel. The results were analyzed by FlowJo software version 7 (TreeStar Inc,
Ashland, OR). Cells treated with cisplatin were considered as positive control and those
treated only with DMSO (untreated) considered as negative control.
3
3
. Results and discussion
.1. Synthesis and spectroscopic characterization
In this work, the reaction of mono phosphonium salt (A) (scheme 1) with
palladium(II) acetate in a 2:1 ratio in methanol and at room temperature for 24 h resulted in
the chloro-bridged dinuclear complex [Pd{C(H)PPh C(O)CH Cl}(µ-Cl)(OAc)] (B) (scheme
3
2
2
1
) [1]. The treatment of PPh and Py with (B) (2 : 1 M ratio) in dichloromethane at room
3
temperature for 12 h yielded yellow solids soluble in the DMSO solvent; their stoichiometry
corresponded to cationic orthometalated derivatives [Pd(ClCH COCH(PPh )PPh )]Cl
2
3
3
(
complex 1) and [Pd(ClCH COCH(PPh )Py)]Cl (complex 2) in good yields. So, complexes
2 3
were characterized using FT-IR and NMR spectroscopy methods. The crystal structure of the

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complex 1 was addressed by X-ray crystallography studies (Figure 1). The spectroscopic data
of 2 was in accordance with the similar structure. The IR spectrum of the complexes 1 and 2
-1
showed strong bands at 1630-1670 cm that were assigned to ν (C=O) stretching bands. The
C-bonding of the ylide-phosphonium salt could be concluded from the IR spectrum, since a
-1
decrease in the C=O stretch was observed [33,34]. Furthermore, three bands at 650-750 cm
were due to ν (P–C), lower than the parent phosphonium salt 1[35] because of the
elimination of electron density in the P–C bond, confirming the coordination of the ligand to
Pd through the carbon, not through oxygen; this was confirmed for the complex 1 by X-ray
diffraction. Additionally, the IR spectra of the complex 2 showed ν (C=N) as a strong band at
-1
-1
1
602 cm . Also, the strong bands at 1580 and 1335 cm were due to asymmetric and
symmetric ν (COO) stretching bands, respectively, which were consistent with monodentate
-1
acetate, and the band at 470 cm was due to ν (Pd-OAc) [36].
1
The H NMR spectra of 1 and 2 showed signals for the PC(H) at 4.5-4.9 ppm, which were
shifted up field in comparison with the parent phosphonium salt (5.5), similar to other C-
coordinated phosphonium ylide complexes [34]. Furthermore, the aromatic protons were
observed in the ~7-8 ppm range and signals for the protons of CH were assigned at 4.3-4.7
2
3
1
ppm. In P {1H} NMR spectra of 1 and 2, signals due to PC(H) groups were 17 ppm and at
3 ppm for extra phosphonium salt, so we purified to remove it. Also, the signal due to PdP
for the complex 1 was assigned at 31 ppm.
2
Scheme 1
3
.2. Molecular structure of the complex
Single X‐ray crystallography was utilized to study the synthesized complexes. The
ORTEP view of the complex 1 is displayed in Figure 1. Table 1 represents the selected bond

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distances and angles. The data showed that the complex 1 was crystallized in the monoclinic
space group, with four molecules within the unit cell. Distortion from the regular square
planar geometry was indicated by the bond angles around the Pd center. The Pd_Caryl bond
distance (2.001(4) Å) was within the range generally reported for C,C-orthopalladated
complexes [37,38].
Bond lengths and angles at Pd were slightly distorted (C1—Pd) 2.001(4), (P2—Pd)
2
.3108(12), (Cl1—Pd1) 2.3828(12), (C37—Pd1) 2.144(4), (P2—Pd1—Cl1) 94.91(5), (C1—
Pd1—C37) 85.44(16). The stabilized resonance construction for the parent ylide-
phosphonium was destroyed because of complexation; therefore, the C1-C2 bond lengths
(1.408(5) Å) were slightly longer than the corresponding distance found in similar non
complexed phosphoranes (1.407(8)Å, suggesting that this bond has been relatively week.
Although the C38-O1 bond lengths (1.216(6)) Å were shorter than those observed in a similar
ligand (1.256(2) Å) [39], they showed that the C-bonding of the ligand kept the charge
density at C and discontinued the conjugation.
Figure 1
Table 1
3
.3. Stability of complex in solution
The electronic spectra of the complexes displayed a band around 250 nm which could
be attributed to the n-π* electron transition of triphenylphosphine groups in the structure of
complexes. The stability of the compounds in the DMSO solution was tested by electronic
absorption (S2). No changes were observed in absorbance after incubating the synthesized

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complexes in DMSO with increasing time to 10 days; this confirmed the stability of the
complexes and the preservation in solution.
3
3
.4. DNA-binding studies
.4.1. Electronic absorption investigations
One of the common, sensitive, reproducible and simple techniques to study the
binding mode of DNA with metal complexes is electronic absorption spectrometry [40]. The
electronic absorption spectra of the complexes were recorded for a stable concentration of the
-5
free metal complexes [6×10 M] by increasing CT‐DNA; the results are shown in Figure 2,
which represents the mode of interactions [41]. The absorption band was around 250 nm for
the complexes, which could be assigned to the intra ligand transition [42]. Thus, the titration
data obtained by adding DNA to the complexes showed the decrease of absorbency with no
shift of the absorption peak in the UV-vis spectrum, thereby confirming that the
complexes/CT-DNA interactions were not intercalated. To determine the binding power of
the Pd complexes along with CT-DNA, the intrinsic binding constant, K , was calculated via
b
the Wolfe-Shimmer equation (1): [43]
[
DNA]/(ɛ - ɛ ) = [DNA]/(ɛ -ɛ ) + 1/K (ɛ - ɛ )
(1)
a
f
b
f
b
b
f
,
where [DNA] is the DNA concentration, and ɛ , ɛ and ɛ correspond to A /[complex], the
a
f
b
obs
destruction coefficients for the free complex, and the entirely attached form of the complex,
respectively. A plan of [DNA]/(ɛ − ɛ ) vs. [DNA]gave a slope and an intercept fit equal to
a
f
1
/(ɛ − ɛ ) and 1/K (ɛ − ɛ ) (Figure 2). The intrinsic binding constant (K ) could be obtained
b f b b f b
from the ratio of the slope to the intercept [44]. The calculated K value of the complexes was
b
6
-1
6
-1
about 4.9 × 10 M for the complex 1 and 2.3× 10 M for the complex 2 at 298 K,
indicating the high binding ability of the complexes with CT-DNA. DNA has different
hydrogen bonding sites in its grooves. Therefore, the ylidic ligand would encourage the
intercalation of the phenyl group in the groove mode. Free energy (∆G) of compound-DNA

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complexes could be achieved from the amounts of binding constant (K ) through the van’t
b
Hoff equation (2): [45]
∆G = - RT lnK
(2)
Binding constants are typical of the stability of compound-DNA complexes, while free
energy shows the spontaneity/non-spontaneity of the compound-DNA binding. The free
-1
-1
energy of the complexes 1 and 2 was -9.13 Kcalmol and -8.7 Kcalmol , respectively, and
the negative values showed the spontaneity of the compound-DNA interaction.
Figure 2
3
.4.2. Fluorescence emission titration
To study whether the type of the binding of the Pd complexes to DNA was the groove
or intercalative mode, the comparative methylene blue (MB) displacement assay was used.
The emission intensity of MB was quenched by the addition of CT‐DNA [46]. This emission
quenching process also indicated the alterations in the excited state electronic structure as a
result of the electronic interactions in the MB-DNA complexes [47]. Figure 3 shows that the
fluorescence intensity of MB-DNA was increased by adding the complexes. This revealed
that the complex was bound to the base pairs of DNA [48]. The Fluorescence intensity of the
MB probe molecule ([DNA]/[MB] = 10) at 680 nm was slightly increased by adding the two
complexes 1 and 2 to DNA; this established that the DNA/Pd complexes interaction was in
the partial intercalative mode. Moreover, from the plot of F /F vs. [complex], the apparent
0
DNA binding constant (K ) was calculated using the following equation: [49]
app
KMB [MB] = Kapp [complex]
(3)
,
where KMB is the DNA binding constant of MB, [MB] is the concentration of MB, and
[
complex] is the concentration of the complex at 50% quenching of the DNA-bound
5
methylene blue fluorescence emission intensity. The value of Kapp was
1.2×10 and
5
–1
1
.04×10 M for the complexes 1 and 2, respectively, which was slightly less than the

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7
–1
binding constant of the classical intercalators and metallointercalators (10 M ) [50],
revealing that the compounds were bound to DNA in the groove binding mode.
Figure 3
3
.4.3. CD spectroscopy
Circular dichroic (CD) studies are useful in identifying the changes in the morphology
of DNA. CD spectra of DNA showed two characteristic bands; the first one at 245nm was
related to the helicity of the right handed B-form and the second one at 260-285 nm was due
to base stacking [51]. This method could be useful in monitoring the changes in the DNA
structure in the presence of the synthesized complexes as drugs in solution, resulting in some
changes in the intensity and wavelength of its CD spectra [52]. Thus, less or no perturbation
was displayed by the simple groove binding and the electrostatic interaction of small
molecules with DNA; on the other hand, increasing the intensities of both bands
demonstrated that the stabilization of the right handed B conformation of CT-DNA could be
improved by intercalation, as observed in the classical intercalator methylene blue [53,54].
The CD spectra of DNA in the absence and presence of complexes were recorded in the
0
range of 230–330 nm at 25 C using a bandwidth of 1 nm, a step interval of 1 nm, an average
time of 1 second, and the resolution of 1 nm. For the complexes 1 and 2, the CD spectra of
DNA exhibited changes in intensity by the addition of complexes, without any changes in the
wavelength (Fig. 4). The less decrease of the negative bands and the decrease of the positive
bands demonstrated that the two complexes were bound to the DNA via groove mechanism
with the partial intercalative mode in the DNA-base pairs [55-57].
Figure 4

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3
.4.4. Thermal denaturation studies
DNA denaturation studies can be used to distinguish the nature of the interaction of
the complexes with DNA via the groove or intercalation mode. The melting temperature
Tm) is related to the stability of the double helical structure of DNA [58], which is raised
(
sharply in the presence of metallo-intercalators. The small shift in the Tm value of CT‐
DNA could propose non‐classical modes such as partial intercalation, groove binding,
and/or surface binding modes [59,60]. The melting curves of CT‐DNA in the absence and
presence of the complexes are shown in Figure 5. The melting temperature of CT‐DNA was
found to be 82 °C in the buffer. After the addition of the Pd(II) complexes, Tm was
increased to 86.16 and 85.22 °C for the complexes 1 and 2, respectively. Since increasing
Tm in 4-12 °C range was due to the intercalation of the compounds into DNA base pairs and
this range for non-intercalative binding mode was 0-4 °C, this amount for our complexes
showed the possibility of the partial intercalation between the CT‐DNA and the two
complexes [60].
Figure 5
3
.4.5. Viscosity measurement
The DNA length (L/L ) and relative viscosity (η/η ) are measured by L/L = (η/η )
0
0 1/3
,
0
0
where η and L are the specific viscosity and the molecular length of DNA at a gained
0
complex/DNA ratio, respectively; also, η and L are the corresponding values for the alone
0
DNA [61]. For an intercalated molecule, a hypothetical rise in the amount of DNA viscosity
was considered because of the insertion between the DNA base pairs and the lengthening of
the DNA double helix [62].
Additionally, the bounded molecules via the groove mechanism made minor or no effect on
the viscosity of the DNA solution [63]. However, the viscosity of CT-DNA was not seriously

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affected through the addition of the two complexes 1 and 2 (S3). This presentation suggested
that the Pd complexes might be bound to DNA upon groove binding with a slight
intercalative style.
3
.5. Protein binding experiments
Serum albumins are well known for binding to small aromatic groups mainly
containing part of the protein in blood plasma; they play a significant part in drug delivery.
Thus, to identify the possible binding interaction between BSA and complexes, emission
titration and absorption experiments were used at the ambient temperature.
3
.5.1. Fluorescence spectroscopy studies
Tryptophan emission quenching experiments were used to study the interaction of the
complex with the protein; f tyrosine fluorescence is ionized, it has been totally quenched and
phenylalanine has a very low quantum yield [64]. The effect of the Pd complexes on the BSA
fluorescence emission can be seen in Figure 6. The intensities of the fluorescence emission of
BSA were decreasedregularly with the addition of the two complexes 1, 2, which established
the interaction between the complex and BSA. Stern–Volmer equation (4) has also been used
in the data analysis of the fluorescence quenching and the quenching ability of the
complexes: [65]
F /F = 1 + k τ [Q] = 1 + K [Q]
(4)
0
q
0
SV
,
where F and F are the fluorescence powers in the absence and presence of the complexes as
0
the quencher, respectively, [Q] is the concentration of the quencher, K is the Stern–Volmer
sv
quenching constant, τ is the average life time of the fluorophore without quencher, and k is
0
q
6
-1
the bimolecular quenching rate constant. In this research, the value of K was 1.58×10 M
sv

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6
-1
and 1.56×10 M for Pd complexes 1 and 2. Since the value of τ for tryptophan
0
-8
fluorescence is 2× 10 s [63], the quenching rate constant, k , can be measured via the
q
following equation:
k = K / τ
0
(5)
q
SV
1
2
12
-1 -1
The obtained values for k were 79×10 and 78×10 M S for the complexes 1 and 2
q
respectively. Also, the highest concentration of k for diverse quenchers with the biopolymer
q
1
0
-1 -1
is 2×10 M S [66]. Therefore, the amount calculated was greater than k of the scatter
q
procedure. This exhibited that a static quenching mechanism was operative [67].
Figure 6
3
.5.2. Determination of the binding constant (K ) and the number of binding sites (n) on
b
BSA
The number of binding sites and the binding constant for the static quenching
interaction can be calculated according to the following equation: [68]
Log (F - F)/F = logK + n log [Q]
(6)
0
b
,
where K is the binding constant of the complex with protein and n is the average number of
b
the binding sites per albumin molecule. The plot of log [(F − F)/F] vs. Log [Q] is exposed in
0
Figure 6. K and n can be determined from the slope of such a plot. The values of K and n at
b
b
6
-1
6
-1
2
98 K were 1.58×10 M and 0.99 for the complex 1 and 1.56×10 M and 0.98 for the
complex 2. The values of n point were such that there was only one class of binding site for
the two Pd complexes on BSA.
3
.5.3 Determination of site selective binding

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BSA crystal structure comprises three distinct drug binding domains (sites I, II, and
III) [69]. Sites I, II and III in serum albumin are suggested to correspond to Eosin-Y,
Ibuprofen and Digoxin binding sites, respectively [70]. The experiments, due to competition,
were done through Eosin-Y, Ibuprofen and Digoxin to achieve the drug binding sites. In this
-6
-6
test, the BSA/site marker concentration proportion was 1:1 (6 × 10 M: 6 × 10 M). Figure 7
shows that by the addition of the site specific probe into the BSA solution, the fluorescence
intensity was significantly decreased. Thus, Eq. (6) was used for the calculation of the
binding constants (Table 2). So, in the presence of Eosin-Y probe, the binding constant of the
complexes 1 and 2 was decreased. The results pointed to the competition of Eosin-Y and
Ibuprofen with the complexes. Thus, the complexes 1 and 2 could bind to the site I and the
site II of serum albumin, respectively.
Figure 7
Table 2
3
3
.6. Molecular docking
.6.1. Molecular docking study of the complexes with BSA
The lowest free binding energy (∆G) calculated for the complex 1 and the complex 2 to
-
1
-1
BSA was -7.19 Kcalmol and -10.68 Kcalmol in docking simulation, respectively. In order
to study the fluorophore residue (Tryptophan) quenching in the presence of complexes , the
distance between the best docked conformer of the complex 1 and the complex 2 and
Trp134 and Trp213 as a fluorophore residue of bovine serum albumin was measured. The
length between the complex 1 and Trp134 was 19.7Å and that between the complex 1 and
Trp213 was 22.5Å (Fig.8A). Also, these distances for the complex 2 were 27.0Å and 20.0Å
for Trp134 and Trp213, respectively (Fig.9A). The obtained distances for both complexes

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were less than 80 Å. In order to do the quenching of protein fluorescence emission, this
distance was enough in the presence of the ligand (in this study, the complex 1 and the
complex 2). Docking simulation confirmed our findings by experimental quenching studies
[
71,72]. For the better study of the interacted residues with the new synthesized complexes,
all docking conformations between the complexes and BSA were examined. Residues
interacted with complex 1 included: Thr2, His3, His9, Arg10, Lys12, Asp13, Asp236,
Lys239, Asp254, Asp255, Ala257, Asp258, Leu259 and Lys261. In Fig.8B, 8C and 8D, the
interaction of the complex 1 with BSA is represented by three and two-dimensional modes,
respectively. AutoDookTools has the ability to present the non-covalent interactions such as
hydrogen bond, π–π stacking and π-cation interactions between the receptor residues and the
ligand from the output file of the docking procedure. The receptor residues and the ligand
must have close contacts with each other for these types of interactions. The amino functional
group of Arg10 from BSA participated in hydrogen bond formation via the methoxy
functional group of the complex 1. The measured distance between the hydrogen atom and
the oxygen atom in hydrogen bonding was 2.1Å (Fig.8B). Due to presence of aromatic rings
in the complex 1 molecular structure, the formation of other non-covalent interactions,
especially π-cation interaction and π–π stacking interaction by the complex 1 occurred.
Lys261 and His3 are the residues with which the complex 1 have π-cation and π–π stacking
interaction, respectively. The distances between these residues and the aromatic ring of the
complex 1 that participated in the interactions were 3.6Å and 5.8Å for the π-cation
interaction and the π–π stacking interaction, respectively (Fig.8B and Fig.8C). According to
the best docking conformer results, the complex 1 had hydrophobic interactions with His3,
His9, Lys12, Asp13, Asp254, Asp258, and Lys261. (Fig.8D (spoked arcs)).
Complex 2 and BSA interactions were analyzed for all docking conformations. The
residues participated in this reaction were found. The residues that interacted with the

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complex 2 included: Glu100, Leu103, Ser104, Ile202, Gln203, Lys242, Cys245, and His246.
Interactions of the complex 2 with BSA are demonstrated by the three and two- dimensional
mode in Fig.9B and Fig.9C, respectively. According to the best docking conformer results,
the complex 2 had hydrophobic interactions with Leu103, Ser104, Ile202, Gln203, Lys242,
Cys245, and His246 (Fig.9C (spoked arcs)). Pyridine heterocyclic ring in the complex 2
structure facilitated the formation of the hydrogen bond, where the hydrogen bond was
formed between the oxygen (OE1) of Glu100 and the nitrogen of pyridine (Fig. 9C (green
dashed line)). Also, the Pd atom of the ligand was interacted (external bond) with the side-
chain of Glu100 by oxygen (OE1) (Fig. 9C (purple line)). The ability of the external bond
formation as well as hydrogen bonds due to the nitrogen of the pyridine ring by the complex
2
showed the high affinity of the complex 2 to BSA rather than the complex 1 in a molecular
docking study.
Figure 8
Figure 9
3
.6.2. Molecular docking study of the complexes with DNA
The calculated lowest free binding energy (∆G) of the molecular docking simulation
-1
-1
of the complex 1 and the complex 2 with DNA was -7.07 Kcalmol and -12.50 Kcalmol ,
respectively. The interaction between DNA and both synthesized complexes was of the
groove type, as determined from docking results analyses (Fig.10A and Fig.11A). The other
docked conformers revealed the partial intercalation mode, supporting the experimental
absorption spectroscopy findings in this study. Analyzing f all conformations from docking
between DNA and the complex 1 identified the main nucleotides participating in the

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interaction with the complex. These nucleotides included: DG2, DC3, DG4, DA5, DA6,
DT7, DT8, DG16, DA17, DA18, DT19, and DT20. Hydrogen bonding was formed between
the methoxy functional group of the complex 1 and the exocyclic amino groups of DG4,
DA5, DA6, DG16, DA17, and DA18 nucleotides. The measured distance between the donor
(N) and acceptor (O) atoms in hydrogen bonding between DG4 and the complex 1 was 2.8Å
(Fig.10B). Purines nucleotides played a major role in hydrogen bonding formation with the
complex 1. The presence of aromatic rings caused the formation of the non-covalent π–π
stacking interaction between the nucleotides aromatic rings and the phenyl groups of the
complex 1. The nucleotides taking part in non-covalent π–π stacking interactions were DG4,
DA5, DA6, DG16, DA17, and DA18 (Fig.10B and Fig.10C). The three-dimensional
interaction between the complex 1 and DNA is shown in these figures. Based on these
figures, the length of the non-covalent π–π stacking interaction between the complex 1 and
DA5 and DA6 was 4.8Å and 5.4Å, respectively (Fig.10B). In addition, the π–π stacking
interaction distance between DA17 and DA18 and the complex 1 was 4.5Å and 5.2Å,
respectively (Fig.10C). The two dimensional representation of the complex 1 and DNA
interaction is demonstrated in Fig.10D. According to this figure, DA17, DA18 and DT19 had
the hydrophobic interaction with the complex 1 (spoked arcs in Fig.10D).
All conformers from the molecular docking study between the complex 2 and DNA
indicated that main nucleotides with which the complex 2 had interacted. They were DT8,
DC9, DG10, DC11, DG12, DA17, DA18, DT19, and DT20 nucleotides. In Fig11.B, the
three-dimensional interaction between the best docked conformer of the complex 2 and DNA
is indicated. The two-dimensional representation of the interaction between the complex 2
and DNA is depicted in Fig.11C. The complex 2 had hydrophobic interaction with DT8,
DC9, DA18, DT19, and DT20 nucleotides (spoked arcs in Fig.11C). Like the interaction of
the complex 2 with BSA, the complex 2 could form hydrogen bond via the nitrogen of the

ACCEPTED MANUSCRIPT
pyridine ring with the oxygen (OP1) of DG10 (Fig. 11C ( the green dashed line)), and the
palladium atom could form the external bond with the oxygen of DG10 (Fig. 11C (purple
line)).
Totally, the external bond formation via palladium atom, van der Waals interactions, and
strong hydrogen bonds formation by the nitrogen of the pyridine ring caused the complex 2 to
have high affinity binding for both bio-macromolecules in docking studies. The outstanding
tendency of the complex 2 to DNA proposed the usage of this palladium as a new synthesized
complex serving as an anticancer. All non-covalent interactions (hydrogen bonding, π-cation
and π–π stacking interactions) distances between BSA and DNA with the complex 1 are
shown in the three- dimensional scheme in Fig 8 (B,C) and Fig 10 (B,C), and in Table 3.
Figure 10
Figure 11
Table 3
3
.7. Selective cancer cytotoxic activity
Cytotoxic activity of the ylide ligand and the complexes 1 and 2 was evaluated against
human leukemic T cell (Jurkat) and chronic myelogenous leukemia (K562) cell lines. The
viability of the treated cell lines was determined after 48 hours via a standard bioassay, by the
MTT-dye reduction method. The cytotoxic activity of cisplatin as the standard reference was
evaluated for the comparison purposes.
As shown in Figure 12, both the ligand and the complexes 1 and 2 could dose-dependently
decrease the viability of Jurkat and K562 cell lines at the evaluated doses. The complex 1 at
the doses ranged from 1 to 100 µM, exerting 10% – 91% and 10.5% - 95% toxicity against
K562 and Jurkat cancer cells, respectively. Additionally, the complex 2 at the doses ranged

ACCEPTED MANUSCRIPT
from -1 to 100 µM, exerting 20% – 98% and 2.5% - 95% toxicity against K562 and Jurkat
cancer cells, respectively. The ligand at the same concentrations was found to exert 2.2% -
9
8% cytotoxic effect against the K562 cell line, while showed 3.2% – 93 % cytotoxicity
against the Jurkat cell line. The cytotoxicity data, expressed as IC values, are presented in
5
0
(
Table 4). IC (µM) values of cisplatin, as the reference anticancer compound and a standard
50
drug on K562 and Jurkat cells, are shown; the results for the ligand and complexes in
comparison with the reference were noticeable. The results implicated that the ligand and the
two complexes 1 and 2 could highly and significantly exert a cytotoxic effect against K562
and Jurkat cancer cell lines. According to the IC values, the cytotoxicity of 1 against K562
5
0
cells was more than 2, probably due to the presence of the triphenylphosphine moiety in its
molecular structure, could improve the cytotoxic activity [73]. Also, the IC value of ligand
50
in that cells is near to cis-platin, but for complexes 1 and 2 these values had been better. No
considerable difference in cytotoxicity of the compounds against Jurkat cells was detected,
suggesting that Complexes 1 and 2 may do not increase the cytotoxicity of the ligand against
this cell line, or the difference between the biological nature and origin of the tumor cell lines
has resulted in their different sensitivity to the compounds. The results have been supported
by the other in vitro studies reporting the cytotoxic activity of the ylide complexes against
cancer cell lines [74,75].
Figure 1
Table 4
3
.8. Effects of compounds on cell cycle
In order to determine the effects of ligand and Pd complexes on cell cycle progression, Jurkat
and K562 cell lines were cultured in the presence of the compounds for 48 h and then were

ACCEPTED MANUSCRIPT
stained with propidium iodide to be analyzed for DNA content by flow cytometry. As shown
in Figure 13, the ligand and two complexes 1 and 2 have significantly induced apoptosis in
treated cells. Cisplatin as positive control induced accumulation of 42.7±6.5% of Jurkat and
4
1.2±8% of K562 cells in subG1 phase (apoptotic cells) compared to the negative controls
<4.1%). The percentages of compound-treated Jurkat cells underwent apoptosis were
1.1±3.9 (ligand), 44.9±5.8 (complex 1) and 55.2±6.2 (complex 2). Similarly, treatment of
K562 cells with the compounds significantly shifted the main population of cells toward the
subG1 phase The complexes simultanously with inducing accumulation of cells in subG1
phase, have efficiently decreased the percentage of Jurkat cells (27.3±2.3%) and K562 cells
27.4±8.2%) in G0-G1 phase as compared to that in negative control (≈51-55%) suggesting
(
4
.
(
an induction of subG1 arrest in the cells. A representative example of flow cytometry
analysis of cell cycle distribution in treated Jurkat and K562 cells is shown in Figure 14.
Thus, the effects of ylide and Pd complexes on cell cycle changes were determined by the
propidium iodide staining using flow cytometry. Propidium iodide is a fluorescent dye with
the ability to enter the cells and bind DNA. Evaluation of cell cycle phases in treated cells
showed that the ligand and Pd complexes have decreased DNA content by inducing apoptosis
and accumulation of cells in subG1 phase. The percentage of subG1 cells and decreased
number of G0-G1cells treated with Pd complexes were comparable to those treated with
cisplatin. Reduction in the number of G0-G1 cells suggested their ability to induce subG1
arrest.
Figure 2 Figure 3
3
.9. Energy transfer between Pd complexes and BSA

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A non-destructive spectroscopic procedure for monitoring the proximity and the
relevant angular orientation of fluorophores is fluorescence resonance energy transfer (FRET)
[
76]. It has been used to measure the donor-acceptor distance in macromolecular and
biological systems. In addition, the overlap between the excitation band of the acceptor and
the donor is significant and the distance between them is within 2–8 nm [77]. The efficiency
of energy transfer (E) then depends on the distance, R , between the acceptor and the donor,
0
as shown by the equation (7): [78]
6
6
6
E = 1- F/F = (R ) / (R + r )
(7)
0
0
0
,
where r is the distance between the donor and acceptor, and R is the critical energy transfer
0
distance at which 50% of the energy is transferred to the acceptor; it is defined by the
following equation: [79]
6
-25
2
-4
R = 8.8 × 10 κ N ФJ
(8)
0
2
2
In Eq. (8), κ is the orientation space factor and κ = 2/3 for the random orientation is as in the
unsolidified solution. N is the ordinary deflected medium index in the range of wavelength
with the significant spectra, Φ is the fluorescence quantum output of the donor, and J is the
effect of the spectral overlay between the emission spectrum of the donor and the absorption
spectrum of the acceptor (Figure 15) which could be obtained from the following equation:
[
80]
4
J = [∑F(λ)ε(λ)λ ∆λ] / [∑F(λ)∆λ]
(9)
F (λ) is the modified fluorescence intensity of the donor in the wavelength array λ to λ +∆λ,
and ε (λ) is the extermination constant of the acceptor at λ. In the present case, n = 1.336 and
-15
-14
3
-1
Ф= 0.15 [81]. Pursuant to equations (7)–(9), J=3.66×10 , 1.13×10 cm L mol , R = 2.15,
0
2
.6 nm, E=0.11, 0.12 and r=2.06, 2.01 nm for the complexes 1 and 2, respectively. The mean
distance between the donor and the acceptor was on the 2–8 nm range and 0.5R <r < 1.5R ,
0
0

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thereby indicating that the energy transfer from BSA to the Pd complexes might have
occurred with high probability.
Figure 4
4
. Conclusion
Two new orthometallated complexes were promoted under mild conditions by the
reaction of [Pd{C(H)PPh C(O)CH Cl}(µ-Cl)(OAc)] with PPh and Py as the chelating
3
2
2
3
ligands, yielding the two mononuclear orthometallated complexes 1 and 2. Single X-ray
crystallography method was applied to characterize the structure of the complex 1, showing a
monoclinic geometry for the Pd atom. The DNA binding properties of the prepared
complexes were checked out using fluorescence spectroscopy, electronic absorption, and
viscometry measurements. The complex-CT-DNA interaction for the two complex 1 and 2
was via groove binding with the partial intercalation, according to the results. The quenching
of BSA emission through the complex was of the static form, as shown by the reactivity
toward BSA. Site marker competitive analysis showed that the two Pd complexes could bind
to the BSA molecule in one site. The molecular docking studies also supported the above
results and successfully verified the interaction mode of the complex with DNA and BSA.
Cytotoxic activity of the Pd complexes against K562 and Jurkat cell lines demonstrated the
potent effect against K562 and Jurkat cancer cell lines and confirmed the anti-tumor activity
of Pd compounds. Cell cycle analysis showed that the complexes have significantly increased
the proportion of subG1 cells and effectively reduced G0-G1 cells implying the effect of
compounds on cell cycle arrest by decreasing cell DNA content and inducing cell death. The
binding distances with BSA based on the Foster’s theory illustrated that the energy transfer
could occur from BSA to the ylide complexes.

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Acknowledgements
This research was funded by Isfahan University of Technology (IUT).
Crystallography was provided by the Faculty of Arts and Sciences, Department of Physics,
Ondokuz Mayrs University.
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