Novel ruthenium and palladium complexes as potential anticancer molecules on SCLC and NSCLC cell lines

Article abstract of DOI:10.1007/s11696-020-01129-x

Lung cancer is one of the major causes of cancer-related deaths in the world. Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, and small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Proper therapies for SCLC have not yet been developed. However, new molecules have been designed and big innovation in treating SCLC has been achieved. Platinum-based antitumor drugs like cisplatin and carboplatin have several disadvantages including side effects, cisplatin-resistant tumors and limited solubility in aqueous media. Thus, two novel chiral aminoalcohol-based bis(phosphinite) ligands containing (η⁶-p-cymene)-Ru(II)-phosphinite and bis(phosphinite)–Pd(II) complexes were synthesized and evaluated for anticancer activity. In this study, the results showed that complex 1 has the strongest cytotoxic effects on SCLC and NSCLC cell lines. On the other hand, cisplatin, ruthenium and palladium complexes are capable to induce apoptosis. Especially, complexes 1 and 2 can induce apoptosis for both SCLC and NSCLC. When compared to the qRT-PCR and TUNEL results, we obtained a significant correlation between apoptotic index and p21, Bax gene expressions. This work revealed the potential of the synthesized complexes as anticancer agents with cytotoxic and pro-apoptotic activity as leading compounds for further anticancer researches.

Full text of DOI:10.1007/s11696-020-01129-x

Chemical Papers
https://doi.org/10.1007/s11696-020-01129-x
ORIGINAL PAPER
Novel ruthenium and palladium complexes as potential anticancer
molecules on SCLC and NSCLC cell lines
1
2
3
1
4
1
5
Onur Tokgun · Duygu Elma Karakas · Semih Tan · Ege Rıza Karagür · Behcet İnal · Hakan Akca · Feyyaz Durap ·
5
5
Akın Baysal · Murat Aydemir
Received: 1 October 2019 / Accepted: 5 March 2020
Institute of Chemistry, Slovak Academy of Sciences 2020
©
Abstract
Lung cancer is one of the major causes of cancer-related deaths in the world. Non-small-cell lung cancer (NSCLC) is the
most common type of lung cancer, and small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Proper
therapies for SCLC have not yet been developed. However, new molecules have been designed and big innovation in treating
SCLC has been achieved. Platinum-based antitumor drugs like cisplatin and carboplatin have several disadvantages includ-
ing side eꢀects, cisplatin-resistant tumors and limited solubility in aqueous media. Thus, two novel chiral aminoalcohol-
6
based bis(phosphinite) ligands containing (η -p-cymene)-Ru(II)-phosphinite and bis(phosphinite)–Pd(II) complexes were
synthesized and evaluated for anticancer activity. In this study, the results showed that complex 1 has the strongest cytotoxic
eꢀects on SCLC and NSCLC cell lines. On the other hand, cisplatin, ruthenium and palladium complexes are capable to
induce apoptosis. Especially, complexes 1 and 2 can induce apoptosis for both SCLC and NSCLC. When compared to the
qRT-PCR and TUNEL results, we obtained a signiꢁcant correlation between apoptotic index and p21, Bax gene expressions.
This work revealed the potential of the synthesized complexes as anticancer agents with cytotoxic and pro-apoptotic activity
as leading compounds for further anticancer researches.
Keywords SCLC · NSCLC · Phosphinite · Palladium · Ruthenium · p21 · Bax
Introduction
Transition metal complexes may play a variety of important
roles in many biological processes (Patel et al. 2013) such as
cell division and gene expression, as well as carcinogenesis
(
Heydari et al. 2017). Among the synthesized compounds,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11696-020-01129-x) contains
supplementary material, which is available to authorized users.
organometallic- and inorganic-based compounds are very
useful molecules. Organophosphorus compounds, especially
phosphines (Gümgüm et al. 2007; Durap et al. 2008), ami-
nophosphines (Gümgüm et al. 2006; Biricik et al. 2007a),
phosphinites (Karakas et al. 2018; Al-Bayati et al. 2018),
etc., are excellent versatile organometallic ligands that are
easily prepared and form a variety of complexes with vari-
ous transition metals including platinum (Durap et al. 2008),
ruthenium (Karakas et al. 2018; Ok et al. 2014), palladium
*
Onur Tokgun
otokgun@pau.edu.tr
1
2
3
4
5
Faculty of Medicine, Department of Medical Genetics,
Pamukkale University, 20160 Denizli, Turkey
Science and Technology Application and Research Center,
Siirt University, 56100 Siirt, Turkey
(
Deepthi et al. 2012; Karakas et al. 2016), gold (Lazarevic
Department of Histology and Embryology, Faculty
et al. 2017) and copper (Biricik et al. 2007b) metal centers.
In the literature, most studies showed that organometallic
and inorganic compounds have potential anticancer eꢀects
of Medicine, Pamukkale University, 20160 Denizli, Turkey
Department of Agricultural Biotechnology, Agriculture
Faculty, Siirt University, 56100 Siirt, Turkey
(
Mjos and Orvig 2014; Demkowicz et al. 2016; Wani et al.
Department of Chemistry, Science Faculty, Dicle University,
2
1280 Diyarbakir, Turkey
Vol.:(0
1
123456789)
3

Chemical Papers
2
016) Their anticancer activity is currently a hot topic of
approximately 5–10% (Fiorentino et al. 2016). Reducing
power, radical scavenging, DNA binding, antibacterial activ-
ity and DNA cleavage activity of ionic liquid-based Ru(II)-
phosphinite complexes were reported (Meriç et al. 2019).
However, to the best of our knowledge, there are no study
on the use of chiral aminoalcohol-based bis(phosphinite)
ligands and their transition metal complexes in anticancer
activity. Herein, for the ꢁrst time, synthesis and poten-
tial anticancer properties of chiral aminoalcohol-based
cancer research.
In the last decades, ruthenium(II) complexes bearing
chelating phosphine and phosphinite ligands have been
received much attention due to their application in the ꢁeld
of homogeneous catalysis (Lindner et al. 2003; Arauj et al.
2
005; Baysal et al. 2017). These compounds have also been
successfully tested as anticancer, antibacterial and antioxi-
dant agents. Biological properties of Ru ion similar to Fe
mimicking in binding to a variety of serum proteins such
as transferrin and albumin (Deshpande and Kumbhar 2005;
Richert and Budzisz 2010). Ruthenium complexes generally
show low systemic toxicity and antitumor activity. Chemi-
cal properties of Ru complexes under physiological con-
ditions are dependent on the two oxidation states (+2 and
6
bis(phosphinite) ligands containing (η -p-cymene)-Ru(II)-
phosphinite and bis(phosphinite)—Pd(II) complexes were
explored.
Experimental
+
3) (Lazarevic et al. 2017). Therefore, organometallic Ru
complexes have been considered to be an alternative to plat-
inum and the organometallic half-sandwich ruthenium(II)
Materials
6
complexes in type of [(η -arene)RuL] (arene: p-cymene,
Bis(phosphinite) ligands 1 and 2 (Karakas et al. 2016),
bis(phosphinite) palladium(II) complexes (Karakas et al.
2016) and bis(phosphinite) ruthenium(II) complexes (Kara-
kas et al. 2018) were prepared according to procedures in our
previous works. Synthesis of bis(phosphinite) palladium(II)
complexes (1 and 2) and bis(phosphinite) ruthenium(II)
complexes (3 and 4) were carried out under dry argon
atmosphere using Schlenk line technique. All solvents were
benzene, etc.) have been extensively studied in recent years
(
Debreczeni et al. 2006; Liu et al. 2006; Pettinari et al. 2014;
Ellahioui et al. 2019).
Pd(II) and Pt(II) complexes share similar properties like
structural and thermodynamic analogy (Ruiz et al. 2008a;
Rocha et al. 2010). In recent years, newly synthesized pal-
ladium complexes have been a very popular ꢁeld of research
due to their similarity to traditional platinum-based anti-
cancer molecules (Moghadama et al. 2016). There is a huge
tendency to study the antitumor activity of Pd(II) complexes
on many cancer cell lines (Miklásová et al. 2012; Ruiz et al.
3
1
1
dried and distilled under argon prior to use. P-{ H} NMR
1
13
(162.0 MHz), H (400.1 MHz) and C (100.6 MHz) spectra
were evaluated by a Bruker AV400 spectrometer, with tetra-
1
3
methylsilane (TMS) as an internal reference for C NMR
1
2
008b). In addition, palladium has higher lability than plati-
and H NMR or 85% H PO as an external reference for
3
4
3
1
1
num analogs. Bidentate ligands like amino acids that are pre-
sent in biological systems have been utilized to synthesize
palladium anticancer complexes (Anzellotti et al. 2006). In
order to obtain eꢀective Pd(II) complexes, stabilization of
the N–Pd interaction with the amine carrier ligand is neces-
sary to decrease the excessive reactivity. Usage of sterically
hindered amines increases the lipophilicity of the complexes,
thus making easier for complexes to enter the cells (Fanelli
et al. 2016). Especially, phosphine-based palladium(II)
complexes have been studied for their valuable bioactivities
P-{ H} NMR.
Synthesis of bis(phosphinite) ligands 1 and 2
(2R)-1-[benzyl({[6-({benzyl[(2R)-2-hydroxypropyl]amino}
methyl)pyridin-2-yl]methyl}) amino]propan-2-ol and (2R)-
1-[benzyl({[3-({benzyl[(2R)-2-hydroxypropyl]amino}
methyl) phenyl]methyl})amino]propan-2-ol (1.5 mmol) were
dissolved in dry toluene (20 mL) under an argon atmos-
phere. Then, trimethylamine (Et N) (3 mmol) and mono-
3
(
Priyarega et al. 2011; Shabbir et al. 2017). The cytotoxicity
chlorophosphine (ClPPh ) (3 mmol) were added dropwise
2
of the palladium(II) complexes was comparable to that of
platinum-based drugs; it is mediated by apoptosis. Accord-
ing to the literature, Pd(II) complexes can be potential can-
didates for anticancer therapy (Fanelli et al. 2016).
into this mixture and stirred at room temperature for 1 h. The
white precipitate (triethylammonium chloride) was ꢁltered
under argon, and the remaining part was dried in vacuo to
produce a white viscous oily compound.
Lung cancer is one of the major causes of cancer-related
deaths in the world. Non-small-cell lung cancer (NSCLC) is
the most common type of lung cancer and represents about
approximately 80–85% of lung cancers (James et al. 2018).
Small-cell lung cancer (SCLC) is the most aggressive sub-
type of lung cancer, and the overall survival at 5 years is
Ligand 1
3
1
1
1
P-{ H} NMR (CDCl , δ, ppm): 106.86 (s, O-P-(C6H5)2);
3
H-NMR (δ ppm, CDCl ): 1.26 (d, 2H, J = 6.20 Hz,
3
–CHCH ); 2.56 (br, 2H, –CHCH (a)); 2.80 (br, 2H, –CHCH
2
3
2
1
3

Chemical Papers
(
(
b)); 3.63–3.75 (m, 4H, –NCH Ph + –NCH ); 4.11–4.15
protons), 3.87 (broad, 2H, –CHCH3), 3.21–3.29 (m,
8H, –NCH Ph + –NCH ), 2.37–2.42 (m, 4H, –CHCH ),
2
2
m, 2H, –CHCH ); 7.20–7.54 (m, 33H, aromatic protons
3
2
2
2
1
3
13
of ligand); C-NMR (δ ppm, CDCl ): 20.85 (–CHCH );
0.84–0.90 (m, 6H, –CHCH ). C NMR (CDCl , δ, ppm):
3
3
3 3
5
9.45 (–NCH Ph + –NCH Py); 75.34 (–CHCH ); 60.86
138.02, 138.25 (i-carbons), 127.24, 127.86, 128.37,
128.60, 128.73, 130.98, 131.69, 131.86, 132.07, 132.42,
2
2
3
(
–CHCH ); 120.96, 126.88, 128.19, 128.21, 128.45, 128.97,
2
1
1
29.07, 130.00, 130.25, 130.52, 130.69 (aromatic carbons);
132.72, 133.12 (aromatic carbons), 74.84 (–CHCH ), 59.51
3
36.50, 142.61, 158.94 (i-carbons).
(–CHCH ), 57.96 (NCH Ph+–NCH ), 19.47 (–CHCH ).
2
2
2
3
6
Ligand 2
Synthesis of (η ‑p‑cymene)‑Ru(II)‑phosphinite
complexes (3 and 4)
3
1
1
1
P-{ H}- NMR (δ ppm, CDCl ): 108.05 (s, O-P-(C6H5)2);
3
6
H-NMR (δ ppm, CDCl ): 1.24–1.30 (m, 6H, –CHCH );
Bisphosphinite (1.5 mmol) and [Ru(η -p-cymene)(µ-CI)Cl]
3
3
2
2
–
.54–2.58 (m, 2H, –CHCH (a)); 2.72–2.76 (m, 2H,
(1.5 mmol) were dissolved in 30 mL of dichloromethane
under argon atmosphere and stirred for 1 h at room tempera-
ture. Final product concentrated to ca. 2 ml under reduced
pressure. Addition of 15 ml petroleum ether caused precipi-
tation of a tile-red solid. The supernatant was removed and
the solid was washed with hexane/diethyl ether (1:1) and
then dried in vacuum to obtain ruthenium(II) complexes
(complexes 3–4) as a ꢁnal product.
2
CHCH (b)); 3.57–3.63 (m, 4H, –NCH Ph+–NCH ); 4.20
2
2
2
(
br, 2H, –CHCH ); 7.23–7.50 (m, 34H, aromatic protons of
3
1
3
ligand); C-NMR (δ ppm, CDCl ): 139.24, 139.42, 142.65
3
(
i-carbons of phenyl); 126.80, 127.57, 128.14, 128.20,
1
1
–
2
28.32, 128.43, 128.95, 129.42, 130.06, 130.25, 130.34,
30.58 (aromatic carbons of phenyl); 75.53 (d, J=23.1 Hz,
CHCH ); 60.50 (–CHCH ); 59.08 (–NCH Ph+–NCH );
3
2
2
2
0.92 (–CHCH ).
3
Complex 3
Synthesis of dichlorobis(phosphinite)Pd(II)
complexes (1 and 2)
3
1
1
P-{ H}- NMR (δ ppm, CDCl ): 110.32 (s, O-P-
3
1
(
C6H5)2); H NMR (δ, ppm, CDCl ): 1.05–1.13 (m, 18H,
3
Bisphosphinite (1.5 mmol) and Pd(cod)Cl (1.5 mmol)
–CH(CH ) p-cymene+ –CHCH ); 1.83 (s, 6H, –CH
2
3 2
3
3
were dissolved in 30 mL of dichloromethane under an argon
atmosphere. The reaction mixture was stirred for 1 h at room
temperature. The solution was concentrated to ca. 2 ml and
adding 15 ml petroleum ether caused precipitation of the
dark yellow solid. The supernatant was removed and the
solid was washed with hexane/diethyl ether (1:1) and then
dried by vacuum to obtain bis(phosphinite) palladium(II)
complexes (complexes 1–2) as a ꢁnal product.
p-cymene); 2.33–2.40 (m, 2H, –NCH Ph(b)); 2.56–2.72
2
(m, 4H, –NCH Ph(a) + –CH(CH ) p-cymene); 3.27 (d,
2
3 2
2H, J =13.7 Hz, –NCH (b)); 3.40 (d, 2H, J =14.5 Hz,
2
–CHCH (b)); 3.56 (d, 2H, J=13.72 Hz, –NCH (a)); 3.62
2
2
(d, 2H, J=14.5 Hz, –CHCH (a)); 4.63 (br, 2H, –CHCH );
2
3
5.15 (br, 6H, aromatic protons of p-cymene); 5.25 (br, 2H,
aromatic protons of p-cymene); 7.25–7.901 (m, 33H, aro-
1
3
matic protons); C NMR (δ ppm, CDCl ): 17.43 (–CH of
3
3
p-cymene); 20.46 (–CHCH ); 21.77, 21.95 (–CH(CH ) of
3
3 2
Complex 1
p-cymene); 29.97 (–CH(CH ) of p-cymene); 59.22–59.63
3 2
(
–NCH + NCH Ph); 60.75 (–CHCH ); 74.36 (–CHCH );
2 2 2 2
3
1
1
P-{ H} NMR (CDCl , δ, ppm): 108.22 (s, O-P-
87.41, 88.14, 89.56, 90.60 (aromatic carbons of p-cymene);
97.45, 111,23 (i-carbons of p-cymene); 121.00, 126.76,
127.65, 127.77, 128.06, 128.98, 130.83, 132,84, 132.95,
133.11, (aromatic carbons); 137.630, 138.12, 139.40, 158.98
3
1
(
C6H5)2); H NMR (CDCl , δ, ppm): 1.28 (m, 6H,
3
–
–
CHCH ); 2.50 (broad, 4H, –CHCH ); 3.11–3.82 (m, 8H,
3
2
NCH Ph + –NCH ); 4.10 (m, 2H, –CHCH ); 7.22–7.54
2
2
3
1
3
+
(
m, 33H, aromatic protons); C NMR (CDCl , δ, ppm):
(i-carbons); m/z: 1415.25 [M–H ] C H N O P Ru Cl
3
71 81
3
2
2
2
4
1
9.84 (–CHCH ); 59.52 (–NCH Ph + –NCH Py); 74,93
(MA: 1414.23).
3
2
2
(
–CHCH ); 60.35 (–CHCH ); 120.62, 126.75, 127.40,
3 2
1
1
27.46, 127.75, 128.43, 128.96, 131.78, 131.99, 132.45,
32.85, 133.40 (aromatic carbons); 136.23, 140.60, 159.78
Complex 4
3
1
1
(
i-carbons).
P-{ H}- NMR (δ ppm, CDCl ): 110.24 (s, O-P-
3
1
(
C6H5)2); H NMR (δ, ppm, CDCl ): 0.98–1.05 (m, 18H,
3
Complex 2
–CH(CH ) of p-cymene+–CHCH ); 1.77 (s, 6H, –CH of
3
2
3
3
p-cymene); 2.32 (d, 2H, J=8.8 Hz, –CHCH (b)); 2.44 (d,
2
3
1
1
1
P-{ H} NMR (CDCl , δ, ppm): 103.76 (s, O-P-(C6H5)2);
2H, J=8.7 Hz, –CHCH (a)); 2.56 (br, 2H, –CH(CH ) of
3
2
3 2
H NMR (CDCl , δ, ppm): 7.20–7.58 (m, 34H, aromatic
p-cymene); 3.14 (d, 4H, J=13.4 Hz, –NCH Ph); 3.50 (d, 4H,
3
2
1
3

Chemical Papers
J=13.5 Hz, –NCH ); 5.08–5.30 (m, 8H, aromatic protons of
the end of the incubation, treated and untreated cell lines
were washed with ice-cold phosphate-buffered saline
(PBS), and then, total RNA from the cell lines was isolated
as described previously (Tokgun et al. 2015). Quantitative
real-time qRT-PCR analysis performed by using SYBR
green (Qiagen, Germany) reagent and Corbett Rotorgene
(Qiagen, Germany) instrument according to the manu-
facturer’s protocol. The relative mRNA expression levels
were calculated using the comparative Ct method. Expres-
sion levels of Bax and p21 were evaluated. The transcript
level of the ß-actin gene was used as the endogenous refer-
ence. The results were assessed with delta delta CT for-
2
p-cymene); 4.55 (br, 2H, –CHCH ); 7.06–7.85 (m, 34H, aro-
3
1
3
matic protons); C NMR (δ ppm, CDCl ): 17.41 (–CH of
3
3
p-cymene); 20.49 (–CHCH ); 21.97, 21.70 (–CH(CH ) of
3
3 2
p-cymene); 30.08 (–CH(CH ) of p-cymene); 59.24–59.00
3
2
(
–NCH + NCH Ph + –CHCH ); 74.40 (–CHCH ); 87.22,
2 2 2 3
8
1
1
8.15, 89.59, 91.00 (aromatic carbons of p-cymene); 97.32,
10,91 (i-carbons of p-cymene); 126.68, 127.39, 127.66,
27.79, 128.03, 128.85, 129.31, 130.78, 130,86, 132.94,
(
aromatic carbons); 137.35, 137.89, 139.43, 139.62 (i-car-
+
bons); m/z: 1413.20 [M-H ] C H N O P Ru Cl (MA:
7
2
82
2
2
2
2
4
1
413.36).
−
ΔCT
mulas (2
).
Cell culture and cell proliferation assay
NSCLC and SCLC cell lines were obtained from Dr. Jun
YOKOTA (IGTP, Barcelona). NSCLC cell line H1975,
HCC78 and SCLC cell line H209, N417 were cultured in
RPMI 1640 supplemented with 10% fetal bovine serum
and 1% penicillin–streptomycin in a humidified of 5%
Statistical analysis
All experiments were repeated independently to conꢁrm the
results by performing in three replicates. The Student’s t
test is used to calculate a p value based on the diꢀerence in
the mean values between two groups of data and p ≤ 0.05
considered to be statistically signiꢁcant.
CO at 37 °C. Cell proliferation was determined by using a
2
CellTiter-Glo kit (Promega, USA) which is a homogenous
method to determine the number of viable cells. Cells were
counted with an automated cell counter, seeded in 96 well
3
plates 3 × 10 cell/well. Diꢀerent concentrations (1, 10,
3
0, 50, 75, 100, 200 µg/mL) of ruthenium and palladium
Results and discussion
complexes were added per well. On the other hand, to com-
pare the eꢀect of synthesized complexes to the cisplatin we
treated cells with diꢀerent concentrations (0.1, 0.5, 1, 10, 30,
Synthesis of bis(phosphinite) ligands and their
complexes
5
0, 100 µg/mL) of cisplatin (CAS 15663-27-1). Afterward,
cells were cultured in 96 well plates for 48 h. IC values
Coordination chemistry studies of the P-donor ligands have
become center of attention through their unique applications
in bioinorganic chemistry (Dong et al. 2007; Appelt et al.
2017). Recently, the preparation of ionic liquid-based Ru(II)-
phosphinite (James et al. 2018) and phosphine containing
heterobimetallic ruthenium(II) complexes and their applica-
tions were reported (Appelt et al. 2017). Attracting features
of this type of compound prompted us to focus on chiral
aminoalcohol-based bis(phosphinite) ligands (Fig. 1). In the
present study, two bis(phosphinite) palladium(II) complexes
5
0
were calculated by regression analysis.
Apoptosis assay
To evaluate the potential apoptotic eꢀects of the synthesized
molecules on H1975, HCC78, H209 and N417 cells, cell
lines were treated with cisplatin, ruthenium and palladium
complexes and survival rates were evaluated based on their
IC values. Cell lines were incubated in a humidiꢁed of 5%
5
0
6
CO at 37 °C at 24 h. At the end of the incubation period,
(1–2) and half-sandwich (η –p-cymene)-Ru(II)-phosphinite
2
in situ Apoptosis Detection Kit (Millipore, USA) was used to
detect apoptotic cells in situ by the indirect TUNEL method.
The results of TUNEL assay were analyzed by using ꢂuo-
rescence microscopy.
complexes (3–4) were prepared using the diꢀerent synthetic
strategies as shown in Fig. 2. These bis(phosphinite) ligands
and their Ru(II) and Pd(II) complexes were originally syn-
thesized by our research group (Karakas et al. 2016, 2018).
Pd(cod)Cl was used in the preparation of bis(phosphinite)
2
Real‑time polymerase chain reaction
palladium(II) complexes (1–2) through the replacement
of cod (1,5-cyclooctadiene) by the phosphinites (Fig. 2).
Complexes 1–2 were isolated in good yields as bright yel-
low solids. Air stable red compounds (3–4) occur in high
Quantitative real-time polymerase chain reaction (qRT-
PCR) analysis was applied to determine apoptosis and
proliferation-dependent mRNA expression changes in
ruthenium and palladium complexes treated for 48 h. At
yields by the reaction of [Ru(p-cymene) (µ-Cl)Cl] with one
2
equivalent of bis(phosphinite) ligands in CH Cl at room
2
2
1
3

Chemical Papers
Fig. 1 Synthesis of bis(phosphinite) ligand 1 and 2
6
Fig. 2 Synthesis of bis(phosphinite)Pd(II) complexes (1, 2) and (η -p-cymene)-Ru(II)-phosphinite complexes (3–4)
1
3

Chemical Papers
Fig. 3 Cytotoxic eꢀects of the complexes on NSCLC cells. Cell death was determined by the luminometric method. The Student’s t test
(
*p<0.001) was used to evaluate the signiꢁcance of cytotoxic eꢀects of the complexes
Fig. 4 Cytotoxic eꢀects of the complexes on SCLC cells. Cell death was determined by the luminometric method. The Student’s t test
*p<0.001) was used to evaluate the signiꢁcance of cytotoxic eꢀects of the complexes
(
temperature (Fig. 2). Both complexes have high melting
points and are highly soluble in MeCN, MeOH, CH Cl ,
Table 1 IC50 values for each molecule for each cell line
2
2
NSCLC
H1975
SCLC
N417
CHCl , DMF and DMSO, but exhibit very poor solubility
3
HCC78
H209
in water. The bis(phosphinite)-Ru(II) and Pd(II) complexes
are stable for a month in ambient air and moisture. The com-
plexes were characterized by nuclear magnetic resonance
Complex 1 10.43± 0.85 19.85± 1.54 8.17± 1.43 6.52± 1.24
Complex 2 15.17± 0.75 48.48± 4.96 12.15± 3.53 22.21± 6.94
Complex 3 55.38± 4.12 52.38± 3.92 53.65± 3.02 44.97± 2.52
(
NMR) spectroscopy.
Complex 4 66.67± 3.84 50.85± 2.98
136± 8.8
51.6± 3.74
The cytotoxic eꢀect of the bis(phosphinite)Pd(II)
6
(
1–2) and (η –p‑cymene)‑Ru(II)‑phosphinite
complexes (3–4) on lung cancer cells
Afterward, the half-maximal inhibitory concentration
(
IC ) has been calculated by using regression analysis. We
50
Luminometric CellTiter-Glo analysis was performed to
determine the speciꢁc cytotoxic activity of each complex.
NSCLC and SCLC cells were incubated with diꢀerent con-
centrations (1, 10, 30, 50, 75, 100, 200 µg/mL) of ruthe-
nium and palladium complexes for 48 h (Figs. 3 and 4,
respectively).
obtained that complex 1 has the strongest cytotoxic eꢀects
on both SCLC and NSCLC cell lines, while complex 4 has
the lowest cytotoxic eꢀects on both cell lines.
1
3

Chemical Papers
Table 2 IC50 values for cisplatin for each cell line
oxidation states are available for ruthenium species in physi-
ological solution. These two oxidation states are of impor-
tant for their biological activity. In both oxidation states, Ru
has good aꢃnity for sulfur and nitrogen ligands and coordi-
nated with octahedral geometry. Most of the Ru compounds
have a low level of toxicity, and some of the Ru complexes
have the capacity to be selective elimination of cancer cells.
Therefore, ruthenium has been considered to be a strong
alternative to platinum complexes (Deshpande and Kumbhar
NSCLC
H1975
SCLC
H1975
HCC78
HCC78
Cisplatin
16.72± 0.98 1.87± 0.24 14.23± 1.01 0.89± 0.05
When the results of cytotoxic eꢀects of the ruthenium
and palladium complexes were compared, it was observed
that palladium has more cytotoxic activity than ruthenium
2
005). On the other hand, in the literature, there are some
reports which mentioned that palladium complexes are inter-
calated with DNA, thereby exerting a signiꢁcant eꢀect on
in vitro antitumor activity (Farhangian et al. 2017). In the
literature, recent studies show that ruthenium complexes are
more cytotoxic than palladium ones (Onar et al. 2019). How-
ever, we thought that in our study these diꢀerences come
from the diꢀerences of the side groups of molecules that
designed in our study.
(
Table 1).
On the other hand, the cytotoxicity results clearly indicate
that complexes 1–3 are more cytotoxic than complexes 2–4
in dose-dependent manner. The cytotoxic activity of each
complex are also compared with cisplatin via treating cells
with diꢀerent concentrations of cisplatin. Depending on the
CellTiter-Glo results, we calculated IC50 values for cisplatin
and we obtained that cisplatin is an eꢀective molecule, espe-
cially against to complexes 1 and 2 in H209 and HCC78 cell
lines (Table 2). These results revealed that complexes 1 and
2
are more eꢀective proliferation inhibitors for fast grow-
ing cells such as H1975 and N417. This study has shown
for the ꢁrst time that ruthenium and palladium complexes
could induce apoptosis in both human small- and non-small-
cell lung carcinoma cell lines. Among the anticancer agents
containing metals, ruthenium and palladium complexes have
become the most promising therapeutic molecules. Recent
studies in the literature showed that ruthenium polypyridyl
and ruthenium(II) salicylate complexes could inhibit can-
cer cell proliferation (Chen et al. 2019; Jiang et al. 2019).
5
6
Ru(III) (d , paramagnetic) and Ru(II) (d , diamagnetic)
Fig. 6 Complexes induce apoptosis in SCLC cell lines. Apoptotic
cells were analyzed by terminal transferase dUTP Kit (Millipore) sys-
tem. The Student’s t test (*p<0.001) was used to evaluate the signiꢁ-
cance of the TUNEL results
Fig. 5 Complexes induce apoptosis in NSCLC cell lines. Apoptotic
cells were analyzed by terminal transferase dUTP Kit (Millipore) sys-
tem. The Student’s t test (*p<0.001) was used to evaluate the signiꢁ-
cance of the TUNEL results
Fig. 7 Cisplatin induce apoptosis in lung cancer cell lines. Apoptotic
cells were analyzed by terminal transferase dUTP Kit (Millipore) sys-
tem. The Student’s t test (*p<0.001) was used to evaluate the signiꢁ-
cance of the TUNEL results
1
3

Chemical Papers
Fig. 8 Complexes induce p21 and bax mRNA expressions. The Student’s t test (*p<0.001) was used to evaluate the signiꢁcance of the qRT-
PCR results
Bis(phosphinite)Pd(II) (1–2)
at lower concentrations. Previous works have shown that
ruthenium complexes are more cytotoxic than palladium
complexes in general. However, it should be remembered
that this eꢀect can change depending on the additional side
groups. These ꢁndings obviously demonstrate that four
complexes are very promising antitumor agents and sup-
port further investigations concerning its molecular targets
in lung cancers.
6
and (η ‑p‑cymene)‑Ru(II)‑phosphinite complexes
(3–4) induce apoptosis on lung cancer cell lines
To obtain the cell death mechanism after treatment with
ruthenium and palladium complexes and to determine apop-
totic cell index, we performed the indirect TUNEL method.
DNA fragmentation of apoptotic cells has been detected
by using the TUNEL assay. The TUNEL assay has been
the most widely used for identifying apoptotic cells in situ.
Our results indicate that cisplatin, ruthenium and palladium
complexes are able to induce apoptosis in each cell line.
Especially, complexes 1 and 2 can induce apoptosis for both
SCLC and NSCLC (Figs. 5, 6 and 7, respectively).
When the eꢀects of Pd and Ru side groups were com-
pared, we obtained that Pd has stronger eꢀect on induction
of apoptosis.
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