Novel nickel(II), palladium(II), and platinum(II) complexes having a pyrrolyl-iminophosphine (PNN) pincer: Synthesis, crystal structures, and cytotoxic activity

Article abstract of DOI:10.1016/j.jinorgbio.2020.111015

A pyrrolyl-iminophosphine (PNNH) which would act as a potential terdentate ligand has been prepared by Schiff base reaction. Complexes [M(PNN)X] (M = Ni; X = Cl (1), Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl (5)) were prepared. The title complexes were characterized by various spectroscopic (IR, ¹H, ¹³C, and ³¹P NMR) and elemental analyses. The molecular structures of 1, 2, and 5 have been established by single-crystal X-ray crystallography, demonstrating a distorted square planar geometry comprising two 5-membered metallacyclic rings. Complexes 1 and 2 were found to crystallize in the orthorhombic while complex 5 crystallizes in the monoclinic. Cytotoxicities of the complexes along with PNNH were evaluated against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines by sulforhodamine B assay. Notably, the palladium(II) complex (2) shows the highest activity. Apoptosis activity along with the caspase inhibitor Z-VAD (Z-Val-Ala-Asp-fluoromethyl ketone) assay of 2 and 5 against A549 and HCT15 cancer cell lines were investigated to learn a mechanistic pathway for the observed cytotoxicity, practically eliminating an apoptotic cell-death route. Complexes 2 and 5 were studied to DNA cleavage assay and molecular docking simulation. The DNA (pcDNA3.0) cleavage experiment evaluates complex 5 interacting with DNA, more effectively, in comparison to complex 2. Molecular docking simulation of 2 and 5 toward DNA and GRP78 (glucose-regulated protein 78) was performed to predict binding sites of ligand-receptors and a plausible mechanistic aspect of metallodrug-action.

Full text of DOI:10.1016/j.jinorgbio.2020.111015

Journal of Inorganic Biochemistry 205 (2020) 111015
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel nickel(II), palladium(II), and platinum(II) complexes having a
pyrrolyl-iminophosphine (PNN) pincer: Synthesis, crystal structures, and
cytotoxic activity
a
a
b
b
c
Youngwon Kim , Jinwook Lee , You-Hwa Son , Sang-Un Choi , Mahboob Alam ,
a,⁎
Soonheum Park
a
b
c
Department of Advanced Materials Chemistry, Dongguk University, 123 Dongdae-ro, Gyeongju 780-714, Republic of Korea
Center for Drug Discovery Technology, Korea Research Institute of Chemical Technology, 141 Gajeongro, Daejeon 34114, Republic of Korea
Division of Chemistry and Biotechnology, Dongguk University, 123 Dongdae-ro, Gyeongju 780-714, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
A pyrrolyl-iminophosphine (PNNH) which would act as a potential terdentate ligand has been prepared by Schiff
PNN-pincer complex
X-ray crystal structure
Cytotoxicity
base reaction. Complexes [M(PNN)X] (M = Ni; X = Cl (1), Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl (5))
1
13
31
were prepared. The title complexes were characterized by various spectroscopic (IR, H, C, and P NMR) and
elemental analyses. The molecular structures of 1, 2, and 5 have been established by single-crystal X-ray
crystallography, demonstrating a distorted square planar geometry comprising two 5-membered metallacyclic
rings. Complexes 1 and 2 were found to crystallize in the orthorhombic while complex 5 crystallizes in the
monoclinic. Cytotoxicities of the complexes along with PNNH were evaluated against A549 (lung), SK-OV-3
Apoptosis
DNA cleavage
Molecular docking simulation
(
ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines by sulforhodamine B assay. Notably, the
palladium(II) complex (2) shows the highest activity. Apoptosis activity along with the caspase inhibitor Z-VAD
(
Z-Val-Ala-Asp-fluoromethyl ketone) assay of 2 and 5 against A549 and HCT15 cancer cell lines were in-
vestigated to learn a mechanistic pathway for the observed cytotoxicity, practically eliminating an apoptotic cell-
death route. Complexes 2 and 5 were studied to DNA cleavage assay and molecular docking simulation. The DNA
(
pcDNA3.0) cleavage experiment evaluates complex 5 interacting with DNA, more effectively, in comparison to
complex 2. Molecular docking simulation of 2 and 5 toward DNA and GRP78 (glucose-regulated protein 78) was
performed to predict binding sites of ligand-receptors and a plausible mechanistic aspect of metallodrug-action.
1
. Introduction
monodentate ligands as supporting ligands showed lower anticancer
activity than their platinum analogs [9–12]. This suggests that the
The discovery of cisplatin as a potent antitumor agent initiated the
palladium complexes do not maintain their structure until they reach
the pharmacological target in biological fluids because of the rapid li-
field of pharmaceutical metallodrugs [1]. Despite its high antitumor
activity, however, cisplatin has side effects, such as toxicity to normal
cells and drug resistance. To solve these problems, cisplatin derivatives
have been developed [2–6], and most of the transition metals have been
studied in the pharmaceutical field [7]. The development of new an-
ticancer drugs is needed to reduce the side effects and improve effi-
ciency.
5
gand-exchange kinetics, which is ~10 times faster than that of the
platinum analogs [13,14]. As a way to overcome this high lability,
chelate ligands have been used to produce thermodynamically stable
and kinetically inert palladium complexes. Palladium complexes with
chelate ligands showed high anticancer activity and occasionally higher
activity than their platinum analogs [15,16]. The interpretation is that
the chelation of metals exhibits high thermodynamic stability, as in the
case of palladium and platinum. On the other hand, chelates of pla-
tinum are too kinetically inert, whereas the analogs of palladium are
kinetically labile. In some cases, these properties of palladium com-
plexes with an appropriate chelating ligand are more suitable for
The efficacy of metallodrugs is based on the central metal and li-
gand [8]. Among the transition metals, palladium, which has similar
properties to platinum in terms of coordination chemistry, has attracted
considerable attention as an alternative to platinum in the development
of new anticancer agents. On the other hand, palladium complexes with
⁎
Corresponding author.
E-mail address: shpark@dongguk.ac.kr (S. Park).
https://doi.org/10.1016/j.jinorgbio.2020.111015
Received 17 December 2019; Received in revised form 23 January 2020; Accepted 27 January 2020
Available online 30 January 2020
0162-0134/ © 2020 Elsevier Inc. All rights reserved.

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
interacting with cancer cells and exhibiting high anticancer activity
as a terdentate pincer-type ligand were evaluated their cytotoxic ac-
tivities against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and
HCT15 (colon) human cancer cell lines. Among the title complexes, the
palladium(II) and platinum(II) derivatives exhibited excellent activity
toward all the cancer cell lines tested. Apoptosis assay in the presence of
the complexes, along with a caspase inhibitor Z-VAD, was explored to
discuss a cell-death route. DNA cleavage assays of the complexes as well
as molecular docking simulation with DNA and GRP78 suggested that
the observed cytotoxicities are likely involved different cell-death
pathways, depending on the nature of the central metal in the com-
plexes.
[
17].
Potential terdentate ligands have been developed to prepare pincer-
type complexes that have chelating ability to form stable complexes
with various transition metal ions [18–24]. Pincer complexes have been
studied in many fields [25–27] and demonstrated efficacy in the
pharmaceutical field, such as antibacterial [28], antifungal [29], anti-
oxidant [30], and anticancer agents [31–34]. In some cases, although
the ligand in itself is active against microorganisms, the incorporation
of a transition metal frequently enhances the biological activity, and
often decreases the cytotoxicity of both the metal ion and ligand
[
35–40]. Pincer complexes with various transition metals such as va-
nadium, iron, nickel, copper, ruthenium, palladium, osmium, platinum,
and gold have been studied [41–49]. In particular, pincer complexes of
iron, copper, ruthenium, palladium, osmium, platinum and gold
showed significantly higher anticancer activity than cisplatin as a po-
sitive control. The anticancer activities of the reported pincer com-
plexes against various cancer cell lines, such as AGS (gastric), NCI-H460
2
. Experimental
2.1. General methods and materials
All preparations of air-sensitive compounds were carried out on a
standard Schlenk line under high purity nitrogen atmosphere. Absolute
ethanol was supplied from Hayman Chemical Company and used
without purification. Dichloromethane and diethyl ether were used
(
(
lung), HCT116 (colorectal), HeLa (cervical), Hep G2 (liver), MCF7
breast), PC3 (prostate), and A172 (glioma), varied according to the
central metal ions and ligand frameworks along with structural varia-
tions [42,44–49].
after storing over molecular sieves (4 Å) under
N
2
.
2-
(
Diphenylphosphino)ethylamine from Alfa Aesar, pyrrole-2-carbalde-
hyde from Aldrich, and triethylamine from Junsei Chemical were pur-
chased and used as supplied. Anhydrous NiCl was from Aldrich and
dried at 120 °C in a ventilated oven. PdCl and K PtCl were supplied by
the Pressure Chemical Company and used as received. All other re-
agents were acquired from various commercial companies. Pd(COD)X
X = Cl, Br; COD = 1,5-cyclooctadiene) [58] and Pt(SEt )Cl [59] were
prepared according to the literature methods.
The antitumor activity of transition metal complexes can be mani-
fested by various mechanisms. The binding of a metal complex to nu-
cleic acid in DNA is one of the most common causes of its anticancer
activity, a typical example of cisplatin. Intracellular aquation of the
chloride ligand can generate the active species of cisplatin, forming
adducts to the DNA. This binding of cisplatin causes DNA cleavage,
damage, and torsion that trigger a cell death mechanism [50,51].
Transition metal complexes can also act as pro-oxidants to produce
reactive oxygen species (ROS), such as hydroxyl free radicals, which
cause DNA damage (both mitochondria and nuclear) [52–54]. Damage
to the mitochondria by ROS discharges mitochondrial cytochrome-C,
encouraging the intrinsic death pathway [55].
2
2
2
4
2
(
2
2
2.2. Physical measurements
IR spectra were recorded on a Bruker (Tensor 37) FT-IR spectro-
1
13
1
31
1
meter, as pressed KBr pellets. The H, C{ H}, and P{ H} NMR
A recent study showed that a palladium(II) complex with O-donor
chelate ligands exhibited anticancer activity in another pathway from
the DNA interactions of platinum complexes [56]. This phenomenon
induced by the palladium complex is referred to as endoplasmic re-
ticulum (ER) stress, which has been demonstrated to be a cytosolic
target and a proapoptotic pathway, being described as the over-
expression of glucose-regulated protein 78 (GRP78). In addition,
overexpression of GRP78 confronts resistance to chemotherapeutic
agents in various cancer cell lines [57].
1
spectra were measured on a Varian Gemini 2000 spectrometer ( H
1
3
1
31
1
(
199.975 MHz), C{ H} (50,288 MHz), P{ H} (80.950 MHz)) in
1 13 1
CDCl
3
at ambient temperature. The chemical shifts for H and C{ H}
Si) and
, respectively. For P{ H} NMR spec-
troscopy, the chemical shifts were measured in parts per million re-
NMR are reported in parts per million (δ) relative to TMS (Me
4
1
3
31
1
natural contents of C in CDCl
3
lative to external 85% H PO in a sealed capillary. Elemental analyses
3
4
were performed at Korea Basic Science Institute in Seoul, Korea.
Biological study of the title compounds on antitumor activity, apoptosis
activity, and DNA cleavage were carried out at Korea Research Institute
of Chemical Technology in Daejeon, Korea.
In this study, a series of novel nickel-triad complexes (Fig. 1) con-
taining a pyrrolyl-iminophosphine (PNN = PPh
2
2
(CH )
2
N=CHC
4
3
H N)
2
.3. Synthesis
.3.1. PPh (CH
2
2
2
)
2
N=CHC
4
H NH (PNNH)
3
Absolute ethanol (30 mL) was bubbled with nitrogen gas for 15 min
to eliminate dissolved oxygen. Under nitrogen atmosphere, pyrrole-2-
carbaldehyde (0.415 g, 4.36 mmol) was dissolved in bubbled absolute
ethanol. To this solution, 2-(diphenylphosphino)ethylamine (1.00 g,
4
.36 mmol) was added. The mixture was stirred for 3 h at ambient
temperature. White powders of compound PPh
PNNH) were precipitated during the course of the reaction. The pre-
cipitates were filtered under vacuum, washed with cold ethanol, and
2
(CH
2
)
2
N=CHC
4
H NH
3
(
−
1
dried in vacuo. Yield 1.22 g (91%). IR: ν(NH) = 3153 cm
,
−1
1
3
ν(N=C) = 1639 cm
.
H NMR (CDCl
3
): δ 2.41 t (2H, NCH
2
;
J
J
3
2
(
(
HH) = 7.7 Hz), δ 3.65 dt (2H, PCH
2
;
J(HH) = 8.3 Hz,
PH) = 7.6 Hz), δ 6.21 t (1H, pyrrole), δ 6.43 (1H, pyrrole), δ 6.83 (1H,
3
1
1
pyrrole), δ 7.3–7.5 m (10H, phenyl). P{ H} NMR (CDCl
3
): δ −19.1 s.
M = Ni, Pd, Pt; X = halide
1
3
1
C{ H} NMR (CDCl
3
): δ 151.74 s (imine, C=N). Anal. Calcd for
Fig. 1. Target complexes of the nickel-triad having a PNN pincer as anticancer
C
19
H
19
N P: C, 74.49; H, 6.25; N, 9.14. Found: C, 74.15; H, 5.98; N,
2
agents.
8
.84.
2

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
2
.3.2. Ni(PPh
Absolute ethanol (30 mL) was bubbled with nitrogen gas for 15 min.
Under nitrogen atmosphere, NiCl (21 mg, 0.163 mmol),
PPh (CH N=CHC NH (PNNH) (50 mg, 0.163 mmol), and trie-
2
(CH
2
)
2
N=CHC
4
H
3
N)Cl, Ni(PNN)Cl (1)
0.112 mmol), PNNH (34.3 mg, 0.112 mmol), and triethylamine
(750 mg, 7.46 mmol) in dichloromethane (15 mL) gave complex 5 as
−
1 1
2
yellow solids. Yield 44.4 mg (74%). IR: ν(N=C) = 1570 cm . H NMR
3
3
2
2
)
2
4
H
3
(CDCl
3
): δ 2.61 dt (2H, NCH
2
;
J(HH) = 6.8 Hz, J(PH) = 10 Hz), δ
3
2
thylamine (25 mg, 0.247 mmol) were added into the bubbled absolute
ethanol. The reaction mixture was stirred for 3 h at 80 °C, resulting in a
reddish solution. The volume of solution was reduced to ca. 5 mL under
high vacuum. Diethyl ether (30 mL) was added to the concentrated
solution, resulting in red precipitates. The precipitates were filtered
under vacuum, washed with distilled water, cold ethanol and diethyl
ether, and then dried in vacuo. An analytically pure compound of 1 can
be obtained by column chromatography (a short glass-column,
3.68 dt (2H, PCH
2
;
J(HH) = 6.4 Hz, J(PH) = 23 Hz), δ 6.37 s (1H,
pyrrole), δ 6.81 s (1H, pyrrole), δ 7.34 s (1H, pyrrole), δ 7.5–8.0 m
3
1
1
1
(10H, phenyl). P{ H} NMR (CDCl
3
): δ 23.3 t ( J(PtP) = 3584 Hz).
Anal. Calcd for C19
H
18
N ClPPt: C, 42.59; H, 3.39; N, 5.23. Found: C,
2
42.23; H, 3.41; N, 4.98.
2.4. Biological studies
2.4.1. Cytotoxic activity
0
.7 × 15 cm) on silica gel (ca. 2 cm) with CH
2
Cl eluent to give red
2
−1
solids from n-hexane. Yield 51.4 mg (79%). IR: ν(N=C) = 1574 cm
.
All the cell line using in this study, that is the human non-small cell
lung cancer cell lines A549, ovarian cancer cell line SK-OV-3, skin
cancer cell line SK-MEL-2 and colorectal cancer cell line HCT15 were
1
3
3
H
NMR (CDCl
3
):
δ
2.28 dt (2H, NCH
2
;
;
J(HH)
=
7
Hz,
J
J
3
2
(
(
(
PH) = 9.8 Hz), δ 3.30 dt (2H, PCH
2
J(HH) = 6.8 Hz,
PH) = 22.6 Hz), δ 6.17 s (1H, pyrrole), δ 6.66 d (1H, pyrrole), δ 7.02 s
maintained using RPMI1640 (RPMI = Roswell Park Memorial
3
1
1
1H, pyrrole), δ 7.4–8.1 m (10H, phenyl). P{ H} NMR (CDCl
3
): δ
Institute) cell growth medium (Gibco, Carlsbad, CA), supplemented
with 5% fetal bovine serum (FBS) (Gibco), and grown at 37 °C in a
3
4.3 s. Anal. Calcd for C19 ClPNi: C, 57.13; H, 4.54; N, 7.01.
H N
18 2
Found: C, 57.05; H, 4.62; N, 6.87.
humidified atmosphere containing 5% CO . The cytotoxicity of the
2
compounds against cultured human tumor cell lines was evaluated by
the sulforhodamine B (SRB) method [60]. Each tumor cell line was
inoculated over standard 96-well flat-bottom microplates and then in-
2
.3.3. Pd(PPh
A similar procedure as for complex 1, using Pd(COD)Cl
.175 mmol), PNNH (53.6 mg, 0.175 mmol), and NEt (27.3 mg,
2
(CH
2
)
2
N=CHC
4
H
3
N)Cl, Pd(PNN)Cl (2)
2
(50 mg,
0
0
3
cubated for 24 h at 37 °C in a humidified atmosphere of 5% CO . The
2
.269 mmol) gave complex 2 (yellow solids). Yield 63 mg (80%). IR:
attached cells were then incubated with the serially diluted each sam-
ples. After continuous exposure to the compounds for 72 h, the culture
medium was removed from each well and the cells were fixed with 10%
cold trichloroacetic acid at 4 °C for 1 h. After washing with tap water,
the cells were stained with 0.4% SRB dye and incubated for 30 min at
room temperature. The cells were washed again and then solubilized
with 10 mM unbuffered Tris base solution (pH 10.5). The absorbance
was measured spectrophotometrically at 520 nm with a microtiter plate
reader. Each experiment was conducted in triplicate. The IC50 values of
compounds were calculated by the nonlinear regression analysis.
−
1
1
3
3
ν(N=C) = 1574 cm
.
H NMR (CDCl
3
): δ 2.66 dt (2H, NCH
2
;
J
J
3
(
(
HH) = 6.7 Hz, J(PH) = 10.6 Hz), δ 3.64 dt (2H, PCH
2
;
2
HH) = 6.5 Hz, J(PH) = 26.8 Hz), δ 6.24 s (1H, pyrrole), δ 6.74 s (1H,
3
1
1
pyrrole), δ 7.32 s (1H, pyrrole), δ 7.4–8.0 m (10H, phenyl). P{ H}
1
3
1
NMR (CDCl
C=N). Anal. Calcd for C19
Found: C, 50.50; H, 4.02; N, 6.03.
3
): δ 46.8 s. C{ H} NMR (CDCl ): δ 158.71 s (imine,
3
H
18
N
2
ClPPd: C, 51.02; H, 4.05; N, 6.26.
2
.3.4. Pd(PPh
2
(CH
2
)
2
N=CHC
4
H
3
N)Br, Pd(PNN)Br (3)
A similar procedure as for complex 1, using Pd(COD)Br (61 mg,
(27.3 mg,
.269 mmol) in dichloromethane (10 mL) gave complex 3 (orange so-
2
0
.163 mmol), PNNH (50 mg, 0.163 mmol), and NEt
3
2.4.2. Apoptosis activity
0
The induced apoptosis ability of complexes 2 and 5 was evaluated in
A549 (lung) cancer cells by flow cytometry with annexin V-FITC/PI
(fluorescein isothiocyanate/propidium iodide) staining. The A549 cells
−
1 1
lids). Yield 68 mg (85%). IR: ν(N=C) = 1575 cm . H NMR (CDCl
3
):
3
3
δ 2.67 dt (2H, NCH
2
; J(HH) = 6.8 Hz, J(PH) = 9.8 Hz), δ 3.63 dt (2H,
3
2
6
PCH
2
;
J(HH) = 6.5 Hz, J(PH) = 27 Hz), δ 6.22 s (1H, pyrrole), δ
were seeded in a 6-well plate 1 × 10 per well. After 24 h, various
6
.76 s (1H, pyrrole), δ 7.42 s (1H, pyrrole), δ 7.5–8.0 m (10H, phenyl).
concentrations (0, 0.1, 0.3, and 1 μM) of complexes 2 and 5 were
3
1
1
13
1
P{ H} NMR (CDCl
3
): δ 49.2 s. C{ H} NMR (CDCl
3
): δ 158.58 s
added. After 48 h, cells were washed with cold phosphate-buffered
6
(
imine, C=N). Anal. Calcd for C19
H
18
N
2
BrPPd: C, 46.42; H, 3.69; N,
saline (PBS), mixed in 1× binding buffer at a concentration of 1 × 10
5
5
2
0
.70. Found: C, 46.72; H, 3.75; N, 5.55.
cell/mL. 100 μL of the solution (1 × 10 cells) was transferred to a 5 mL
culture tube. The cells were incubated with 5 μL of Annexin V-FITC and
5 μL of PI at room temperature for 15 min in the dark. 400 μL of 1×
binding buffer was added to each tube. The samples were analyzed by
flow cytometer within 1 h.
.3.5. Pd(PPh
2
(CH
2
)
2
N=CHC
4
H N)I, Pd(PNN)I (4)
3
Under nitrogen atmosphere, a mixture of complex 3 (10 mg,
.02 mmol) and sodium iodide (60.85 mg, 0.4 mmol) was dissolved in
acetone (10 mL) and stirred for 3 h at ambient temperature. Removal of
all volatiles from the solution under high vacuum gave red-yellow re-
sidues, which were washed with distilled water and cold ethanol. The
resulting residues were extracted with dichloromethane (10 mL) to give
a reddish brown solution. The volume of solution was reduced to ca.
2.4.3. Caspase inhibitor Z-VAD experiment
HCT15 (colon) cancer cells were inoculated over 96-well cell cul-
ture plates, and then incubated for 24 h at 37 °C in a humidified at-
mosphere of 5% CO . Various concentrations (0.3, 1, 3, and 10 μM) of
2
5
mL and poured diethyl ether (30 mL) to give reddish brown powders.
complexes 2 and 5 were added. After 4 h, the culture solution was re-
moved from each well and the cells were washed 3 times with phos-
phate-buffered saline (PBS). The culture solution containing Z-VAD
(30 μM) was added and incubated for 48 h. The cells were photo-
graphed.
The resulting precipitate was filtered under vacuum, washed with dis-
tilled water, cold ethanol and diethyl ether, and then dried in vacuo.
−
1 1
Yield 8 mg (73%). IR: ν(N=C) = 1576 cm . H NMR (CDCl ): δ 2.72
3
3
3
dt (2H, NCH
2
;
J(HH) = 6.6 Hz, J(PH) = 10.2 Hz), δ 3.61 dt (2H,
3
2
PCH
2
;
J(HH) = 6.4 Hz, J(PH) = 26.4 Hz), δ 6.18 s (1H, pyrrole), δ
3
1
1
6
.76 s (1H, pyrrole), δ 7.5–8.0 m (10H, phenyl). P{ H} NMR (CDCl
3
):
): δ 158.42 s (imine, C=N). Anal. Calcd
IPPd: C, 42.37; H, 3.37; N, 5.20. Found: C, 42.02; H, 3.43;
2.4.4. DNA cleavage experiment
1
3
1
δ 51.9 s. C{ H} NMR (CDCl
for C19
N, 4.88.
3
DNA cleavage assays of complexes 2 and 5 with plasmid DNA
(pcDNA3.0) were performed by agarose gel electrophoresis. Plasmid
DNA aliquots (50 μg/mL) were incubated in TE buffer (Tris-EDTA;
H
18 2
N
1
0 mM Tris-HCl, 1.0 mM EDTA) with 10 μM of complexes 2 and 5 at
2.3.6. Pt(PPh
2
(CH
2
)
2
N=CHC
4
H
3
N)Cl, Pt(PNN)Cl (5)
25 °C for 24 h. After incubation, the aliquots were subjected to elec-
trophoresis on 1.5% agarose gel in a TAE buffer (Tris Acetate-EDTA;
A similar procedure as for complex 1, using Pt(SEt
2
)Cl (50 mg,
2
3

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Scheme 1. Synthesis of pyrrole-iminophosphine PPh
2
(CH
2
)
2
N=CHC
4
H NH (PNNH).
3
4
0 mM Trisacetate, 2.0 mM EDTA). The gel was stained with ethidium
been prepared via Schiff base reaction from pyrrole-2-carbaldehyde and
2-(diphenylphosphino)ethylamine (Scheme 1). PNNH was isolated as
white powders in good yields up to 91%. A single resonance at δ −19.1
bromide before migration. The bands were imaged using a Gel Doc EZ
system (Bio-Rad, USA).
3
1
1
in the P{ H} NMR spectrum of PNNH ruled out any contamination of
phosphine oxide from the reaction. The new complexes M(PNN)X
2.5. X-ray crystallographic analyses
(
M = Ni; X = Cl (1), M = Pd; X = Cl (2), Br (3), I (4), M = Pt; X = Cl
The X-ray diffraction data for complexes (1, 2, 5) were collected
(5), PNN = PPh
2
(CH
2
)
2
N=CHC
4
H N) have been prepared by reacting
3
with a Bruker SMART CCD diffractometer equipped with a graphite-
monochromated Mo Kα (λ = 0.71073 Å) radiation source and a ni-
trogen cold stream (−100 °C), at the Western Seoul Center of Korea
Basic Science Institute in Seoul, Korea. Data collection and integration
were performed with SMART (Bruker, 2000) and SAINT-Plus (Bruker,
the ligand with (COD)PdCl
2
, (COD)PdBr
2
, NiCl
2
, and (Et
S)
2 2
PtCl , re-
2
spectively (Scheme 2). Palladium(II) complex Pd(PNN)I (4) has been
metathetically prepared from the reaction of complex 3 and NaI (ex-
cess) in acetone (Scheme 3). The title complexes 1–5 were character-
1
31
ized by various spectroscopic methods (IR, H, and P NMR) including
satisfactory microanalytical data (C, H, N). The molecular structures of
complexes 1, 2, and 5 have been determined by single crystal x-ray
crystallography (vide infra: Section 3.2).
2
001) [61]. Absorption correction was performed by multi-scan method
implemented in SADABS [62]. The structure was solved using direct
2
methods and refined using full-matrix least-squares on F using
SHELXTL [63]. All the non-hydrogen atoms were refined aniso-
tropically, and hydrogen atoms were added to their geometrically ideal
positions. Crystallographic data for complexes 1 (Ni), 2 (Pd), and 5 (Pt)
have been deposited with the Cambridge Crystallographic Data Centre
as CCDC 1970178, 1970184, and 1970185, respectively.
In the IR spectrum of the ligand PNNH, the stretching frequency of
−1
−1
the ν(NeH) and the ν(C=N) shows at 3153 cm
and 1639 cm
,
respectively. These two absorption peaks in the ligand are anticipated
to be removed (the ν(NeH)) and shifted (the ν(C=N)) upon co-
ordination to metal ion. Complexation of PNNH with the metal ions
3
affords η -P,N,N pincer-type complexes consisting of two 5-membered
2.6. Molecular docking simulation
metallacyclic rings (P-N(imino)-N(pyrrolyl) pincer, see Fig. 1 and
Scheme 2). In the IR spectra of all the title complexes, the absence of
NeH absorption band clearly indicates that the NeH proton in the
pyrrole moiety has been removed to be a terdentate ligand, pyrrolyl-
imino phosphine. The characteristic ν(C=N) band in the complexes is
also shifted to a lower wave number (red shift), compared to that of the
free ligand, due to π-back donation of electron density from metal ion
to the π* orbital of imine group. The ν(C=N) bands in all complexes
Molecular docking simulation was performed using a series of
software packages such as Mercury, MGLtools (ADT) and Discovery
Studio. The crystal data of DNA and protein were retrieved from the
Protein Data Bank. The water and associated heteromolecules to the
retrieved DNA named 12-mer DNA (PDB code = 1aio) [64] and protein
named GRP78 (glucose-regulated protein 78, PDB code = 3ldl) [57]
were removed and Gasteiger charges were applied by Autodock Tools
−
1
were observed at nearby 1570 cm
(Table S1), indicative of N-co-
(
ADT) [65] before performing docking simulation. The PDB format of
ordination of the imine group to the metal ions.
1
complexes 2 and 5 were generated by converting CIF files of complexes
using Mercury software. The docking site for DNA and protein acting as
a receptor was defined by the size of the grid box set at 60 × 60 × 60
in the x, y, and z directions with a grid spacing. 0.375 Å. Docking was
executed employing the Lamarckian genetic algorithm with default
parameters and the best pose was chosen on the basis of the binding
energy (Kcal/mol). Visualization of the best pose and its interactions
was displayed using Discovery Studio.
In the H NMR spectrum of PNNH, the resonances of ethylene
protons (CH
2
-CH ) displayed at 3.65 ppm and 2.41 ppm with a quartet
2
and a triplet split-pattern, respectively. Thus the quartet resonance
(overlapping with the doublet of the triplet) is due to the one CH
2
1
protons coupling with phosphorous. In the H NMR spectra of all the
metal complexes, the resonance peaks corresponding to the two CH
2
protons in the ethylene group (NCH
2
-CH
2
2
P) showed their increased
3
2
coupling constants (δ NCH
2
( J(PH)), δ PCH
( J(PH))) with splitting of
3
1
1
double of triplet, respectively (Table S1). In the P{ H} NMR spectrum
3
1
1
of PNNH, a single peak resonates at −19.1 ppm. The P{ H} NMR
spectra of all the complexes showed their respective single resonance
shifted to the downfield, considerably, compared to that of the free li-
gand. This significant shift to the downfield is partly attributed to the
3
. Results and discussion
3.1. Synthesis and characterization
3
1
ring formation in the complex, what is called ring shifts [75]. The
P
Complexes with labile ligands lead to dissociation of complexes by
1
hydrolysis and/or reaction with a nucleophile in the body. These
complications can be overcome using a chelate ligand [14,66,67]. A
PNN pincer-type ligand is able to form stable complexes via chelation of
P,N,N donors with metal ion. Metal complexes with phosphine ligands
exhibit high stability even in vivo conditions [68]. In addition, a number
of antitumor activities of metal complexes with phosphine ligands have
been reported [69–74].
{ H} NMR spectrum of the platinum complex (5) shows a single re-
1
sonance at 23.34 ppm, flanked with platinum satellites ( J
(
PtP) = 3584 Hz). These phenomena such as the absence of NeH ab-
sorption peak, the lower shift of the imine absorption peak in the IR
1
spectra, the increased coupling constant values in the H NMR, and
3
1
1
downfield shift of phosphorous resonance peak in the P{ H} NMR
indicated that the ligand coordinated to the metal ion in a chelate
mode. The IR and NMR data are summarized in Table S1.
A pyrrole-iminophosphine PPh
2
(CH
2
)
2
N=CHC
4
3
H NH (PNNH) has
4

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Scheme 2. Synthesis of M(PNN)X (M = Ni, Pd, Pt; PNN = PPh
2
(CH
2
)
2
N=CHC
4
H N).
3
Scheme 3. Synthesis of Pd(PNN)I (4).
Table 1
Crystal data and structure refinement for 1, 2, and 5.
Compound
Ni(PNN)Cl (1)
Pd(PNN)Cl (2)
Pt(PNN)Cl (5)
Empirical formula
Formula weight
Temperature
C
19
399.48
H
18ClN
2
PNi
C
19
447.17
H
18ClN
2
PPd
C
19
535.86
H
2
18ClN PPt
200(2) K
200(2) K
223(2) K
Crystal system
Space group
Orthorhombic
Orthorhombic
Monoclinic
P2
1
2
1
2
1
P2
2
1 1
2
1
1
P2 /c
Unit cell dimensions
a = 11.3120(7) Å
b = 12.1276(7) Å
c = 12.9257(8) Å
α = β = γ = 90°
a = 11.3150(9) Å
a = 14.8417(16) Å
b = 12.1391(9) Å
b = 10.9952(11) Å
c = 13.0906(10) Å
α = β = γ = 90°
c = 11.1886(11) Å
α = γ = 90°, β = 99.921(4)°
3
3
3
Volume
1773.24(19) Å
1798.0(2) Å
1798.5(3) Å
Z
4
4
4
3
1
3
3
−1
Density (calculated)
Absorption coefficient
F(000)
1.496 Mg/m
1.652 Mg/m
1.979 Mg/m
−
−1
1.337 mm
824
1.272 mm
8.041 mm
896
1024
3
3
3
Crystal size
0.18 × 0.10 × 0.04 mm
2.30 to 28.29°.
12,586
0.12 × 0.08 × 0.03 mm
2.29 to 28.29°.
0.190 × 0.160 × 0.060 mm
Theta range for data collection
Reflections collected
Independent reflections
Completeness to theta = 28.29°
Data/restraints/parameters
2.617 to 28.321°.
52,371
13,011
4334 [R(int) = 0.0293]
99.5%
4431 [R(int) = 0.0387]
99.7%
4469 [R(int) = 0.0496]
99.9%
4334/0/217
4431/0/217
4469/0/217
2
Goodness-of-fit on F
1.143
1.143
1.070
Final R indices [I > 2sigma(I)]
R indices (all data)
R1 = 0.0322, wR2 = 0.0640
R1 = 0.0486, wR2 = 0.0839
−0.007(15)
R1 = 0.0354, wR2 = 0.0631
R1 = 0.0593, wR2 = 0.0931
−0.02(5)
R1 = 0.0181, wR2 = 0.0395
R1 = 0.0240, wR2 = 0.0429
Absolute structure parameter
Largest diff. peak and hole
−
3
−3
0.895 and −0.613 e·Å−3
0.460 and −0.530 e·Å
1.050 and −0.814 e·Å
5

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Fig. 2. ORTEP representation of Ni(PNN)Cl (1) with 50% probability ellipsoids.
All hydrogens were omitted for clarity.
Fig. 4. ORTEP representation of Pt(PNN)Cl (5) with 50% probability ellipsoids.
All hydrogens were omitted for clarity.
Microanalytical data for PNNH and complexes (1–5) are all in good
agreement with calculated values (Table S2).
3.2. Single crystals X-ray diffraction studies
The molecular structures of complexes 1, 2, and 5 have been es-
tablished by single crystal x-ray diffraction. Single crystals suitable for
an x-ray diffraction study were grown by slow diffusion of n-hexane
into a dichloromethane solution of the respective complex. Crystal data
and structure refinements for 1, 2, and 5 are listed in Table 1. The
molecular structures of 1, 2, and 5, shown in Figs. 2, 3, and 4,
Table 2
Selected bond lengths (Å), bond angles (°), and torsion angles (°) for complexes
1
, 2, and 5.
1
2
5
M-N(1)
1.908(2)
1.880(3)
2.1550(9)
2.1694(8)
1.292(4)
1.339(4)
1.383(4)
83.50(11)
85.48(9)
94.47(3)
97.15(8)
173.74(9)
167.41(8)
43.4(3)
2.062(4)
2.044(2)
1.994(2)
2.2142(7)
2.3099(7)
1.299(4)
1.345(4)
1.386(4)
79.93(9)
85.40(7)
99.62(3)
95.22(7)
173.79(7)
165.03(7)
−40.3(3)
M-N(2)
2.006(5)
M-P(1)
2.2280(15)
2.3080(15)
1.296(7)
M-Cl(1)
N(2)-C(5)
N(1)-C(1)
1.331(7)
N(1)-C(4)
1.397(7)
N(2)-M-N(1)
N(2)-M-P(1)
P(1)-M-Cl(1)
N(1)-M-Cl(1)
N(2)-M-Cl(1)
N(1)-M-P(1)
N(2)-C(6)-C(7)-P(1)
80.78(19)
84.19(14)
96.94(6)
98.42(14)
175.57(15)
164.19(14)
45.4(5)
Fig. 3. ORTEP representation of Pd(PNN)Cl (2) with 50% probability ellipsoids.
All hydrogens were omitted for clarity.
6

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Fig. 5. The relative bond length of the Pd-N(imine) due
to the trans-positioned Cl and PPh , respectively, to the
3
imino ligand. (a) Complex 2 (Pd-N(imine) = 2.006 Å),
and (b) for Pd(NN)(PPh )(Me), (Pd-N(imine) = 2.121 Å)
79].
3
[
Fig. 6. Resonance forms of pyrrolide-imine ligand and its complex [81].
Table 3
Cytotoxic activity of cisplatin, PNNH, and compound 1–5 against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin), and HCT15 (colon) human cancer cell lines.
Compound
IC50 (μM) ± SDa
A549
SK-OV-3
SM-MEL-2
HCT15
Cisplatin
0.17 ± 0.087
> 30
1.55 ± 0.329
> 30
0.19 ± 0.14
> 30
0.68 ± 0.95
> 30
PNNH
1
2
3
4
5
> 30
> 30
> 30
> 30
0.22 ± 0.21
0.36 ± 0.41
3.90 ± 3.59
2.56 ± 2.13
1.23 ± 0.700
1.20 ± 0.618
10.02 ± 6.940
4.97 ± 2.58
0.53 ± 0.53
0.76 ± 0.62
10.48 ± 5.065
2.52 ± 1.48
0.14 ± 0.088
0.18 ± 0.14
4.26 ± 3.64
2.00 ± 1.66
a
SD is standard deviation of the value.
respectively, illustrate a distorted square planar geometry comprising
been reported; the Ti-N(pyrrole) is shorter than the Ti-N(imine) [78].
The difference from these two bond lengths observed in pyrrolide-imine
based complexes is ascribed mainly to the trans-influence. A pyrrolide-
two 5-membered metallacyclic rings via P,N,N-chelation of a pincer-
type pyrrolyliminophosphine (P,N,N = PPh
2
(CH
2
)
2
N=CHC
4
H N) li-
3
gand. Crystals of complexes 1 and 2 are isomorphic, differing for the
imine based palladium(II) complex Pd(NN)(PPh )(Me) in which the
3
2
+
2+
ionic radii of Ni and Pd in the square planar complexes of 63 and
phosphine and the methyl ligand coordinated to the corresponding
7
8 pm, respectively, that alter the bond lengths and angles of atoms
trans-position of the N of the imine and the pyrrole, respectively, has
directly associated with the central metal atoms. The bond lengths and
angles for these isomorphic complexes are presented in Table 2 for
comparison.
been reported (Fig. 5(b)) [79]. In Pd(NN)(PPh )(Me), the bond length
3
of the Pd-N(imine) is 2.121 Å which is longer than that of the Pd-N
(imine) (2.006 Å) in complex 2. In complexes 1 and 5, nickel(II) and
platinum(II) analogs, a similar trend in the relative bond lengths of the
M-N(imine) and the M-N(pyrrolyl) have been observed as for those of
the palladium complex 2 (see Table 2). The respective bond length of
M-N(imine), M-N(pyrrole), and M-P in the complexes are in the order of
1 < 5 < 2. This sequence corresponds to an order of nickel (63 pm),
platinum (74 pm), and palladium (78 pm) in the ionic radius of the
square planar of metal 2+ oxidation state [80]. On the other hand, the
M-Cl bond lengths are in the order of 1 < 2 < 5. This can be at-
tributed to the elongated N(2)-C(5) (imine) bond length observed in
In complex 2, bond angles along with torsion angles exhibit a slight
distorted square-planar geometry mainly arising from the formation of
two five-membered palladacyclic rings [76]. The bond length of Pd(1)-
N(1) and Pd-N(2) in complex 2 is 2.062(4) and 2.006(5) Å, respectively.
The Pd(1)-N(2)(imine) is shorter than the Pd(1)-N(1)(pyrrolyl). This
result is consistent with precedents of nickel(II) and palladium com-
plexes bearing an imino-pyrrolyl ligand. The M-N(imine) bond length is
shorter than the M-N(pyrrolyl) [77]. However an opposite tendency in
those bond lengths in a pyrrolide-imine based titanium complex has
7

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Fig. 7. Apoptosis of A549 (lung) cancer cells with complex 2. Percentage of cells in each quadrant are given as live (lower left), early apoptotic (lower right), late
apoptotic (upper right), necrotic cells (upper left).
complex 5. An increasing electron density on the central metal leads to
strong π-back donation to the imine group, which may weaken the C]
N imine bond followed by the M-N (imine) bond stronger, resulting in
elongation of the M-Cl bond length.
bond lengths (Å), angles (°), and torsion angles (°) for complexes 1, 2, 5
are listed in Table 2.
3.3. Biological studies
In the pyrrolyl ring of complex 2, the CeC bond lengths of C(1)-
C(2), C(2)-C(3), and C(3)-C(4) are nearly all the same of 1.39 Å within
the standard deviations. But the CeN bond lengths are quite different
each other as of the N(1)-C(1) (1.331 Å) and the N(1)-C(4) (1.397 Å).
Han et al. suggested that the difference in the bond lengths of the N(1)-
C(1) and the N(1)-C(5) in the pyrrole group of a pyrrolide-imine based
complex is due to electron delocalization in N(1)-C(4)-C(5)-N(2) [77].
A theoretical study on density functional theory (DFT) for a pyrrole-
imine aluminum complex reported by Huang et al. revealed that re-
sonance of the pyrrole-imine ligand in the complex occurs across the
entire range from the pyrrole ring to imine group (see Fig. 6) [81]. In
this study for complex 2, the structural phenomena for delocalization of
the electrons in the pyrrole ring including the imino group, judged from
the related bond lengths, is consistent with precedents. Structural si-
milarity of complexes 1 and 5 is analogous to complex 2. The selected
3
.3.1. Cytotoxic activity
The cytotoxic activities of PNNH and the title complexes were
evaluated against A549 (lung), SK-OV-3 (ovarian), SM-MEL-2 (skin),
and HCT15 (colon) human cancer cell lines by using SRB (sulforhoda-
mine-B) assay. Cisplatin was used as a positive control. Cytotoxicity
data are summarized in Table 3.
As shown in Table 3, PNNH and nickel(II) complex (1) exhibited
negligible activity with IC50 values of > 30 μM. However, the palla-
dium complexes (2–4) and the platinum complex (5) were found to be
highly active toward all the tested cancer cell lines. Among those, the
chloropalladium complex Pd(PNN)Cl (2) showed the highest cytotoxi-
city against all the cancer cell lines. For HCT15 and SK-OV-3 cell lines,
IC50 values of the palladium complex (2) are even low in comparison
with those of cisplatin. On the other hand, the palladium complex (2) is
8

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Fig. 8. Apoptosis of A549 (lung) cancer cells with complex 5. Percentage of cells in each quadrant are given as live (lower left), early apoptotic (lower right), late
apoptotic (upper right), necrotic cells (upper left).
nearly comparable with cisplatin in IC50 values for A549 and SM-MEL-2
cell lines. Comparing cytotoxic activities of the palladium halides (2–4),
the chloro and the bromo derivatives (2, 3) showed relatively high
activity than the iodo derivative 4. This consequence can be explained
by high lability of the iodide ligand than the chloride or the bromide in
the complex, likely resulting in a less active palladium species. Fur-
thermore, the palladium chloride 2 shows cytotoxicity of 4 times higher
than that of the platinum chloride 5 against all the cancer cell lines
tested. Considering significantly different cytotoxic activities resulted
from the chloro derivatives of the nickel triad having a PNN pincer (1,
detecting the Annexin V binding to PS. The DNA-binding fluorescent
dye propidium iodide (PI) cannot penetrate membranes of live cells.
However, PI can stain DNA, owing to loss of cell membranes as apop-
tosis progresses, enabling confirmation of late apoptosis or necrosis
[87]. A549 (lung) cancer cells were treated with various concentrations
(0, 0.1, 0.3, 1.0 μM) of complexes 2 and 5 for 48 h. Annexin V and PI
binding to apoptotic cells were analyzed by flow cytometry to evaluate
the apoptotic effects of complexes 2 and 5.
As shown in Fig. 7, treatment with 0.1 μM of complex 2 resulted in
15% of early apoptotic and 2% of late apoptotic cells. However, the
observed percentages of early and late apoptotic cells were practically
comparable as concentration of complex 2 increases (0.3 and 1.0 μM).
The apoptosis results of complex 5 showed 4%, 7%, and 9% in early
apoptotic cells at concentration of 0.1, 0.3, and 1.0 μM of the complex,
respectively, as shown in Fig. 8. Apoptotic activity was slightly in-
creased in proportion to the concentration of the complex but was less
than complex 2. These results demonstrate that an apoptotic cell-death
pathway is not considerably responsible for the observed cytotoxic
activity of the complexes, likely comprising other cell-death pathways.
2
, 5), the employed central metal is to be critical in efficacy of antic-
ancer metallodrugs [82–84].
3
.3.2. Apoptosis assay
Apoptosis, commonly referred to a programmed cell death, con-
tributes to the homeostasis of multicellular organisms by eliminating
aged, damaged, and/or abnormal cells. Apoptotic activity has been
studied as a factor of anticancer activity of pharmaceuticals [85,86].
Apoptosis begins when phosphatidylserine (PS) inside a plasmic
membrane is expressed, externally. Early apoptosis can be confirmed by
9

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
3
.3.3. Z-VAD assay
Apoptosis is mainly expressed by means of activated caspase
through intrinsic and extrinsic pathways. An intrinsic pathway is trig-
gered by loss of mitochondrial membranes, releasing cytochrome C
from mitochondria, which activates caspase [88]. On the other hand, an
extrinsic pathway involves both TRADD (TNF receptor-associated death
domain) and FADD (Fas-associated death domain) arising from acti-
vation of TNF (tumor necrosis factor) receptor family in membrane.
FADD activates caspase, followed by apoptosis [89]. Z-VAD inhibits
apoptosis induced by caspase activation, confirming cytotoxicity of the
complexes attributable to caspase stimulus [90]. Thus, Z-VAD assay was
performed to learn whether the observed cytotoxic activities of com-
plexes 2 and 5 involve an apoptotic pathway induced by caspase acti-
vation.
Figs. 9 and 10 are the photographs of colon cancer cells untreated
(
a) and treated (b) with Z-VAD after treatment with each of various
concentrations of complexes 2 and 5. In Fig. 9, the cells treated with 0.3
and 1.0 μM of complex 2 shows a similar amount of living cells, which
was treated (a) or untreated (b) with 30 μM of Z-VAD. Moreover, the
cells treated with 3.0 and 10 μM of complex 2 displays most of the
cancer cells dead whether treated or untreated with Z-VAD. The results
with complex 5 also shows that the cytotoxic activity is independent on
treatment with caspase-inhibitor Z-VAD as similar to that of complex 2
Fig. 9. Image of HCT 15 (colon) cancer cells untreated (a) and treated (b) with
(
Fig. 10).
3
0 μM of Z-VAD after treatment with various concentrations of complex 2.
These results suggest that complexes 2 and 5 exhibit cytotoxic ac-
tivity through other pathways rather than apoptosis. Apoptosis and
necroptosis can be expressed through activation pathways of the TNF
receptor family. Activation of TNF receptor by means of TNFα-signals
releases TRADD which recruits RIPK1 (receptor-interacting protein ki-
nase 1). In case, caspase is inhibited, RIPK1 associates with RIPK3 to
form necrosomes. The necrosome facilitates phosphorylation of MLKL
(
mixed lineage kinase domain like pseudokinase), which can express
necroptosis, as a caspase-independent pathway, leading to cell-death
[
91]. Consequently, the results of Z-VAD assay may imply that cyto-
toxicity of complexes 2 and 5 likely involves a pathway of necroptosis,
which is another programmed cell-death pathway occurring apoptosis
suppressed.
3
.3.4. DNA cleavage
Relative interaction of the chloro complexes of palladium(II) (2)
and platinum(II) (5) with DNA has been investigated by monitoring the
matching bands of a supercoiled DNA (Form I), a nicked DNA (Form II),
and a linear DNA (Form III) resulted from a Plasmid DNA (pcDNA 3.0)
after treatment with the respective metal complex.
The experimental result from DNA cleavage measured by agarose
gel electrophoresis shows detached bands in the order of Form II, Form
III, and Form I from top to bottom. DNA cleavage ability of complexes is
proportional to an increasing amount of the resulting Form II and Form
III. Fig. 11 shows a photograph for the bands from electrophoresis of
DNA treated with 10 μM of the respective complex 2 and 5 for 48 h. The
Fig. 10. Image of HCT 15 (colon) cancer cells untreated (a) and treated (b) with
0 μM of Z-VAD after treatment with various concentrations of complex 5.
3
Fig. 11. Cleavage of pcDNA3.0 by complexes 2 and 5 (Control = pcDNA 3.0 (untreated), Form I: supercoiled DNA, Form II: nicked DNA, Form III: linear DNA).
10

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Fig. 12. Interactions of complexes 2 and 5 with receptors: 2 with DNA (a), 2 with GRP78 (b), 5 with DNA (c), and 5 with GRP78 (d).
result shows that the amount of Form I decreased and those of Form II
and Form III increased by treatment of the platinum complex (5). On
the other hand, the Form II fragments slightly increased by treatment of
the palladium analog (2) compared to the control. This result demon-
strates that the platinum complex interacts with plasmid DNA more
effectively than the palladium analog does. Consequently, the platinum
11

Y. Kim, et al.
Journal of Inorganic Biochemistry 205 (2020) 111015
Table 4
(molecular docking simulation) and DNA cleavage experiment may
imply that the palladium species likely proceeds in a different pathway
from the platinum in the observed cytotoxicity, i.e. the palladium pre-
fers interacting with proteins rather than DNA, while the platinum does
the other way. In the observed cytotoxicity, the platinum complex
shows less active than the palladium analog against all the cancer cell
lines (Table 3). The thermodynamic data resulted from molecular
docking simulation of complexes 2 and 5 are summarized in Table 4.
Thermodynamic data resulted from molecular docking simulation of complexes
2
and 5 toward DNA and GRP78.
DNA
GRP78
ΔGa
K
b
ΔGa
K
i
b
i
2
5
−5.86
−5.94
50.8
44.4
−7.63
−7.44
2.56
3.53
ΔG and K
i
are the free energy of binding and inhibition constant, respectively.
4. Conclusions
a
kcal/mol.
b
μM.
In the present study, novel nickel(II), palladium(II), and platinum
(
II) complexes having a PNN-pincer have been synthesized and char-
1 31 1
complex likely involves a DNA interaction pathway while the palladium
analog does an alternative, considering from the observed cytotoxic
activity of the two complexes against the cancer cell lines.
acterized by various spectroscopic methods (IR, H and P{ H} NMR)
and elemental analysis. The molecular structures of M(PNN)X (M = Ni;
X = Cl (1), M = Pd; X = Cl (2), and M = Pt; X = Cl (5),
2
PNN = PPh (CH
2
)
2
N=CHC
4
3
H N) were determined by single crystal x-
3.4. Molecular docking simulation
ray crystallography. The cytotoxicity of the synthesized metal com-
plexes against human cancer cell lines (A549, SK-OV-3, SM-MEL-2, and
HCT15) was evaluated in a comparative manner. The general trends
were found as follows: 2 (PdeCl) > 3 (PdeBr) > 4 (PdeI) and 2
(PdeCl) > 5 (PteCl) ⋙ 1 (NieCl). In addition, the study of apoptotic
activity and caspase inhibitor (Z-VAD) experiments, with complexes 2
and 5 against A549 and HCT15 cancer cells, demonstrates that an
apoptosis pathway would not be fully accountable for the observed
cytotoxicity, probably involving other pathways. The DNA cleavage
experiment demonstrates that the platinum complex (5) interacts with a
plasmid DNA more effectively than the palladium analog (2) does.
These results imply that the palladium analog likely involves an alter-
native pathway in the observed cytotoxicity. In order to investigate the
interaction of complexes with proteins and DNA, molecular docking
studies were performed, showing that docking energy-based anti-cancer
activities of 2 and 5 are consistent with experimental results.
The synthesized metal complexes were studied to determine the
binding affinity of the crystal structure of human GRP78 (PDB 3LDL)
and the crystal structure of double-stranded DNA (PDB 1AIO) in order
to discover the interaction between the studied complexes and the re-
ceptor, and for lead optimization using molecular docking study. To
find out how complexes interact with active sites of receptor and the
difference between palladium and platinum complexes with the same
ligand, complexes 2 and 5 were simulated with DNA and GRP78.
As shown in Fig. 12(a), there are various types of non-covalent in-
teractions of complex 2 with DNA. The π-electrons of DA17 and DC18
aryl rings in DNA interact with the ethylene protons in the PNN ligand
of the complex via π-alkyl interaction. The π-electrons of the pyrrolyl
moiety in the complex interact with the O-anion and the C5 of DC9, and
with the C2 of DT8 in DNA via π-anion(O) and π-sigma interaction,
respectively. Fig. 12(b) shows interactions of complex 2 with GRP78,
revealing that there are similar non-covalent interactions such as the π-
alkyl and the π-sigma along with hydrogen bonding of π-HN (ASN389).
Detailed interaction types and distances obtained from the molecular
docking simulation of complex 2 with DNA and GRP78 are shown in
Table S3.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Similarly, Fig. 12 shows molecular interactions resulted from mo-
lecular docking simulation of the platinum(II) complex 5 with the same
receptors of DNA (c) and GRP78 (d), elucidating that there are com-
parable covalent interactions such as π-alkyl, π-sigma, and π-hydrogen,
as observed from the interactions of the palladium analog 2. In addition
to these types of interactions, intermolecular π-π interactions have been
observed from associates of phenyl ring moieties in the complex with
DC28 and DG47 in DNA (π-π stacked), and with TYR396 in GRP78 (π-π
T-shaped) as well (Table S4).
Acknowledgments
This work was supported by the Dongguk University Research Fund
of 2019.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2020.111015.
The thermodynamic values of ΔG (free energy of binding) and K
inhibition constant) obtained from molecular docking simulation of
complex 2 with DNA and GRP78, respectively, are of −5.86 and
7.63 kcal/mol for ΔG, and are of 50.8 and 2.56 μM for K (Table 4).
for complex 5 with the receptors is of
5.94 kcal/mol along with 44.4 μM (toward DNA), and is of
i
(
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