Synthesis, characterization and catalytic, cytotoxic and antimicrobial activities of two novel cyclotriphosphazene-based multisite ligands and their Ru(II) complexes

Article abstract of DOI:10.1002/aoc.3328

Two novel cyclotriphosphazene ligands (2 and 3) bearing 3-oxypyridine groups and their corresponding Ru(II) complexes (4 and 5) were synthesized and their structures were characterized using Fourier transform infrared, ¹H NMR and ³¹P

Full text of DOI:10.1002/aoc.3328

Full paper
Received: 8 March 2015
Revised: 26 March 2015
Accepted: 11 April 2015
Published online in Wiley Online Library: 15 June 2015
(wileyonlinelibrary.com) DOI 10.1002/aoc.3328
Synthesis, characterization and catalytic,
cytotoxic and antimicrobial activities of two
novel cyclotriphosphazene-based multisite
ligands and their Ru(II) complexes
Diğdem Erdener Çıralıa*, Zafer Uyarb*, İsmail Koyuncuc
and Nurcihan Hacıoğlud
Two novel cyclotriphosphazene ligands (2 and 3) bearing 3-oxypyridine groups and their corresponding Ru(II) complexes (4 and 5)
were synthesized and their structures were characterized using Fourier transform infrared, 1H NMR and 31P NMR spectroscopic
data and elemental analysis. The Ru(II) complexes were used as catalysts for catalytic transfer hydrogenation of p-substituted
acetophenone derivatives in the presence of KOH. Additionally, the cytotoxic activities of compounds 2–5 were evaluated against
PC3 (human prostate cancer), DLD-1 (human colorectal cancer), HeLa (human cervical cancer) and PNT1A (normal human prostate)
cell lines. Finally the antimicrobial activities of compounds 2–5 were evaluated against a panel of Gram-positive and Gram-
negative bacteria and yeast cultures. The complexes showed efficient catalytic activity towards transfer hydrogenation of
acetophenone derivatives, especially those bearing electron-withdrawing substituents on the para-position of the aryl ring. The
compounds were found to have moderate to high cytotoxic and antimicrobial activities, and Ru(II) complexation enhanced both
cytotoxic and antimicrobial activities in comparison with the parent compounds. Copyright © 2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Keywords: cyclotriphosphazene; Ru(II) complex; transfer hydrogenation; cytotoxicity; antimicrobial activity
Having more than 200 types, each with different causes, symp-
toms and treatments, cancer is one of the major causes of global
Introduction
The use of cyclophosphazenes as ligands to transition metals has
been attracting the attention of researchers due to their reactive pe-
riphery and robust skeleton which provide a perfect platform to build
various types of multisite coordination ligands. Their versatile struc-
ture and coordination ability allow the construction of new macrocy-
clic ligands. Pyridyloxycyclophosphazenes are multisite coordination
ligands where the coordinating pyridine ligand is attached to the
phosphorus atom of the cyclophosphazene ring.[1–5] When these li-
gands are combined with transition metals or main group metals, a
number of different complexes, which exhibit a rich diversity and po-
tential chemical and biological activities, can be readily prepared.
Ru(II) metal-based catalytic conversion of primary and secondary
alcohols into their corresponding aldehydes and ketones is an
essential reaction in organic synthesis.[6–9] Much research has been
aimed at the design of good Ru(II) catalysts for the transfer
hydrogenation of ketones.[10–15] To the best of our knowledge, no
study has so far dealt with the catalytic activities of Ru(II) complexes
of cyclotriphosphazene ligands in transfer hydrogenation of
ketones. Recently, we have shown that pyridyloxy-substituted
phosphazene derivatives and their Ru(II) complexes have good
catalytic activities.[16,17] In the study reported here, we synthesized
two novel cyclotriphosphazene ligands (2 and 3) containing 3-
oxypyridine groups and their corresponding Ru(II) complexes (4
and 5) and evaluated the catalytic activity of the complexes in
transfer hydrogenation of ketones.
deaths. According to the World Health Organization, cancer caused
7.6 million deaths in 2008[18] and the death toll reached 8.2 million
in 2012.[19] Despite rapid technological and medical developments
to fight cancer, the number of deaths caused by cancer keeps
increasing each year. Even though various chemotherapeutics are
employed in the treatment of cancer patients, almost all of these
drugs cause severe side effects and their intended effectiveness
either fails or greatly diminishes as a consequence of continued
*
Correspondence to: Digdem Erdener Çirali, Çanakkale Onsekiz Mart University,
Faculty of Science and Arts, Department of Chemistry, Çanakkale, Turkey.
E-mail: digdem_erdener@hotmail.com Zafer Uyar, Harran University, Faculty of
Science and Arts, Department of Chemistry, Şanlıurfa, Turkey. E-mail: zaferuyar@
gmail.com
a Çanakkale Onsekiz Mart University, Faculty of Science and Arts, Department of
Chemistry, Çanakkale, Turkey
b Harran University, Faculty of Science and Arts, Department of Chemistry,
Şanlıurfa, Turkey
c
Harran University, Faculty of Science and Arts, Department of Biology, Şanlıurfa,
Turkey
d Çanakkale Onsekiz Mart University, Faculty of Science and Arts, Department of
Biology, Çanakkale, Turkey
Appl. Organometal. Chem. 2015, 29, 536–542
Copyright © 2015 John Wiley & Sons, Ltd.

Cyclotriphosphazene-based multisite ligands and their Ru(II) complexes
administration of the drug. This keeps the door open for the
development of new drugs with improved efficiency and
minimum side effects. Cyclotriphosphazene derivatives have long
been attracting attention as potential anti-cancer agents.[20–23]
Cyclotriphosphazenes are a unique group of inorganic ring systems
and their chemical, physical and biological properties vary depen-
ding on the substituents they bear. With this fact in mind, we also
investigated the cytotoxic activities of the cyclotriphosphazene
ligands 2 and 3 and their Ru(II) complexes 4 and 5 against PC3
(human prostate cancer), DLD-1 (human colorectal cancer), HeLa
(human cervical cancer) and PNT1A (normal human prostate) cell
lines. Finally the antimicrobial activities of compounds 2–5 were
evaluated against a panel of Gram-positive and Gram-negative
bacteria and yeast cultures.
Experimental
Materials and methods
Scheme 1. Synthesis of ligands 2 and 3 and Ru(II) complexes 4 and 5.
All chemicals for the synthesis and the solvents used were of analy-
tical grade quality from commercial sources and were used without
further purification.
1.3 mmol) and Cs2CO3 (0.85 g, 2.61 mmol). After stirring under ar-
gon atmosphere for 24 h, evaporation of the solvent gave a white
solid, which was purified using TLC on a silica plate. Ligand 3 was
separated as a pure white solid (Scheme 1), using a 5:1 ratio of
CHCl3–THF as eluent.
FT-IR spectra were recorded as pressed KBr discs, using a
PerkinElmer FTIR 1000 series spectrophotometer in the range
400–4000 cmꢀ1. Melting points were determined with an Electro
Thermal IA 9100 apparatus using a capillary tube. The 1H NMR
spectra were recorded in DMSO with tetramethylsilane as inter-
nal reference using a Bruker AVANCE spectrometer at 300 MHz.
The 31P NMR spectra were recorded with an Agilent spectrome-
ter at 202.5 MHz. Column chromatography was performed on
silica gel (60-mesh, Merck). TLC was carried out on Merck
0.2mm silica gel 60 F254 analytical aluminium plates. Elemental
analyses of the compounds were carried out using a LECO
CHNS-932 analyser.
Yield 0.385g (78%). IR (KBr, νmax, cmꢀ1): ν(P¼N) 1173–1200, ν(P–
O–C) 1072, ν(C¼C) 1719, ν(Ar–H) 3065. 1H NMR (400 MHz,
DMSO-d6, δ, ppm): 8.41 (d, J = 4.7Hz, 2H, H-4), 7.96 (d, J = 8.1 Hz,
2H, H-2), 7.70–7.61 (m, 6H, H-10, H-3), 7.52 (dd, J1 = 8.0 Hz,
J2 = 7.6Hz, 4H, H-8), 7.43 (dd, J1 = 7.6Hz, J2 = 7.6 Hz, 4H, H-9), 7.15
(d, J = 8.00 Hz, 4H, H-7). 13C NMR (DMSO-d6, δ, ppm): 147.4 (C-6),
147.1 (C-1), 143.5 (C-4), 143.3 (C-5), 131.3 (C-11), 130.8 (C-10),
130.5 (C-8), 128.1 (C-7), 127.3 (C-3), 125.2 (C-9), 122.0 (C-2). 31P
NMR (DMSO-d6, δ, ppm): 24.38 (d, P(–O2C12H8)), 11.92 (t, P(–
OC5H3ClN-3)2). Anal. Calcd for C36H28N5O6P3 (%): C, 53.70; H,
2.92; N, 9.21. Found (%): C, 53.52; H, 2.87; N, 9.25.
Synthesis and characterization
Synthesis of spiro-N3P3[(O2C12H8)2(OC5H4N-3)2 (2)
General procedure for synthesis of Ru(II) complexes
To a solution of [N3P3Cl2(O2C12H8)2] (1; 0.518 g, 1.115 mmol) in ace-
tone (80 ml) were added 3-hydroxypyridine (0.285 g, 3 mmol) and
Cs2CO3 (2.1g, 6.5mmol). After stirring under argon atmosphere
for 24h, evaporation of the solvent gave a white solid, which was
purified using TLC on a silica plate. Ligand 2 was separated as a
pure white solid (Scheme 1) using a 5:1 ratio of CHCl3–THF as
eluent.
Complexes 4 and 5 were prepared according to the following gen-
eral method. [RuCl2(p-cymene)]2 (0.05 mmol) in 5 ml of THF was
added to the ligand (0.05 mmol) in 10 ml of THF. The reaction mix-
ture was stirred at room temperature for 12 h. After evaporation of
THF, the resulting residue was washed with diethyl ether (20 ml)
and recrystallized from MeOH.
Compound 4. Yield 0.045 g (65%). IR (KBr, νmax, cmꢀ1): ν(P¼N)
Yield 0.632 g (82%). IR (KBr, νmax, cmꢀ1): ν(P¼N) 1231–1167, ν(P–
O–C) 1091, ν(C¼C) 1604, ν(Ar–H) 3061. 1H NMR (400 MHz, DMSO-d6,
δ, ppm): 8.63 (s, 2H, H-5), 8.56 (d, J =4.7 Hz, 2H, H-4), 7.82 (d,
J =8.4 Hz, 2H, H-2), 7.66 (d, J = 7.6Hz, 4H, H-10), 7.61 (dd,
J1 = 8.4 Hz, J2 = 4.7Hz, 2H, H-3), 7.52 (dd, J1 =8.0 Hz, J2 = 7.6Hz,
4H, H-8), 7.43 (dd, J1 = 7.6Hz, J2 = 7.6 Hz, 4H, H-9), 7.17 (d,
J =8.0 Hz. 4H, H-7). 13C NMR (DMSO-d6, δ, ppm): 147.5 (C-6),
147.0 (C-1), 143.1 (C-4), 139.6 (C-5), 130.8 (C-10), 130.5 (C-8),
129.2 (C-11), 128.2 (C-7), 127.3 (C-3), 125.4 (C-9), 122.0 (C-2). 31P
NMR (DMSO-d6, δ, ppm): 24.90 (d, P(–O2C12H8)2), 12.00 (t, P(–
OC5H4N-3)2). Anal. Calcd for C34H24N5O6P3 (%): C, 59.05; H, 3.50;
N, 10.13. Found (%): C, 58.85; H, 3.62; N, 10.18.
1
1219–1166, ν(P–O–C) 1092, ν(C¼C) 1770, ν(Ar–H) 3051. H NMR
(400MHz, DMSO-d6, δ, ppm): 8.63 (s, 2H, H-5), 8.57 (d, J = 4.6Hz,
2H, H-4), 7.82 (d, J = 8.4Hz, 2H, H-2), 7.66 (d, J = 7.6Hz, 4H, H-10),
7.61 (dd, J1 = 8.4Hz, J2 = 4.6Hz, 2H, H-3), 7.52 (dd, J1 = 8.0Hz,
J2 = 7.6Hz, 4H, H-8), 7.43 (dd, J1 = 7.6 Hz, J2 = 7.6Hz, 4H, H-9), 7.17
(d, J = 8.0 Hz. 4H, H-7), 5.78 (dd, J1 = 17.0 Hz, J2 = 6.2Hz, 8H, H-14,H-
15), 2.81 (m, 2H, H-17), 2.07 (s, 6H, H-12), 1.17 (d, J = 6.9 Hz, 12H,
H-18). 13C NMR (DMSO-d6, δ, ppm): 147.5 (C-6), 147.0 (C-1), 143.1
(C-4), 130.8 (C-10), 130.5 (C-8), 129.2 (C-11), 128.2 (C-7), 127.2 (C-3),
126.5 (C-5), 125.4 (C-9), 122.0 (C-2), 106.8 (C-16), 100.5 (C-13), 86.8
(C-14), 8519 (C-15), 30.4 (C-17), 22.0 (C-18), 18.3 (C-12). 31P-NMR
(DMSO-d6, δ, ppm): 24.87 (d, P(–O2C12H8)2), 11.95 (t, P(–OC5H4N-3)
2). Anal. Calcd for C56H56 Cl4N5O6P3Ru2 (%): C, 50.50; H, 4.24; N,
5.26. Found (%): C, 50.55; H, 4.36; N, 5.15.
Synthesis of spiro-N3P3[(O2C12H8)2(OC5H3ClN-3)2 (3)
To a solution of [N3P3Cl2(O2C12H8)2 ] (1; 0.3g, 0.65 mmol) in acetone
(80 ml) were added 2-chloro-3-hydroxypyridine (0.1675g,
Compound 5. Yield 0.040 g (56%). IR (KBr, νmax, cmꢀ1): ν(P¼N)
1
1224–1172, ν(P–O–C) 1094, ν(C¼C) 1704, ν(Ar–H) 3058. H NMR
Appl. Organometal. Chem. 2015, 29, 536–542
Copyright © 2015 John Wiley & Sons, Ltd.
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D. Erdener Çıralı et al.
(400 MHz, DMSO-d6, δ, ppm): 8.41 (d, J= 4.5 Hz, 2H, H-4), 7.96 (d,
J =8.1 Hz, 2H, H-2), 7.67 (m, 6H, H-3, H-10), 7.52 (dd, J1 = 8.0Hz,
J2 = 7.6 Hz, 4H, H-8), 7.43 (dd, J1 = 7.6Hz, J2 = 7.6 Hz, 4H, H-9), 7.15
(d, J = 8.0 Hz, 4H, H-7), 5.78 (dd, J1 = 17.0 Hz, J2 = 6.2 Hz, 8H,
H-14,H-15), 2.81 (m, 2H, H-17), 2.06 (s, 6H, H-12),1.17 (d, J = 6.9Hz,
12H, H-18). 13C NMR (DMSO-d6, δ, ppm): 147.4 (C-6), 147.2 (C-1),
143.4 (C-4), 143.2 (C-5), 131.4 (C-11), 130.8 (C-10), 130.5 (C-8),
128.1 (C-7), 127.3 (C-3), 125.2 (C-9), 121.9 (C-2), 106.8 (C-16), 100.5
(C-13), 86.8 (C-14), 85.9 (C-15), 30.4 (C-17), 21.9 (C-18), 18.3
(C-12). 31P NMR (DMSO-d6, δ, ppm): 24.32 (d, P(–O2C12H8)),
11.87 (t, P(–OC5H3ClN-3)2). Anal. Calcd for C56H54 Cl6N5O6P3Ru2
(%): C, 48.01; H, 3.89; N, 5.00. Found (%): C, 48.10; H, 3.85; N, 5.04.
growth by 50% under the experimental conditions and is the aver-
age from at least three independent measurements that were re-
producible and statistically significant. The IC50 values were
reported at 95% confidence intervals ( 95% CI). This analysis
was performed with Graph Pad Prism (San Diego, CA, USA).
Antibacterial assay
Microorganisms
Antimicrobial activities of compounds 2–5 were evaluated against
Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Bacillus
cereus ATCC 7064, Listeria monocytogenes ATCC 15313, Micrococcus
luteus La 2971), Gram-negative bacteria (Escherichia coli ATCC
11230, Klebsiella pneumoniae UC57, Pseudomonas aeruginosa
ATCC 27853, Proteus vulgaris ATCC 8427, Enterobacter aerogenes
ATCC 13048) and yeast cultures (Candida albicans ATCC 10231,
Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra DSM 70403)
using both disc diffusion and dilution methods. Lyophilized pure
strains of bacteria and yeast were obtained from the Basic and
Industrial Microbiology Research Laboratory in Canakkale Onsekiz
Mart University, Faculty of Science and Arts, Department of Biology.
General method for transfer hydrogenation of ketones
A mixture of Ru(II) complex 4 or 5 (0.085 mmol), acetophenone
derivative (0.85 mmol) and KOH (3.4 mmol) was refluxed at 80°C
in 2-propanol (4ml) for 2 h. The reaction was monitored using gas
chromatography. At the end of this time, the mixture was cooled
to room temperature and diluted with diethyl ether (5 ml) and
filtered from a mini-column filled with silicagel. The yields were
related to the residual unreacted acetophenone.
Agar disc diffusion method
In vitro cytotoxic activity
Antimicrobial susceptibility testing was performed using the disc
diffusion method according to the protocol described by the Clini-
cal and Laboratory Standards Institute.[24] Fresh stock solutions of
compounds 2–5 were prepared in DMSO (2mg mlꢀ1). To ensure
that the solvent had no effect on bacterial growth, a control test
was performed with test medium supplemented with DMSO using
the same procedure as used in the experiments. The bacteria were
incubated at 30°C for 24 h in nutrient broth. The yeasts were incu-
bated in malt extract broth for 48h. Sterile antibiotic discs (6mm,
Schleicher & Schull no.2668, Germany) were impregnated with
20μl of solutions. After that the discs were placed on the agar
inoculated with test microorganisms and then the plates were
incubated at 35°C for 24h for bacteria and at 25°C for 72 h for yeast.
All experiments were done in triplicate and the average was taken
as the final reading. The antibacterial and antifungal activities of
compounds 2–5 were compared with known antibiotics: ampicillin,
cefotaxime, tetracycline, nystatin, ketoconazole and clotrimazole.
Cell cultures
The human prostate cancer (PC3), colorectal adenocarcinoma
(DLD-1), cervix carcinoma (HeLa) and normal prostate epithelium
(PNT1A) cells were obtained from American Type Culture Collection
(ATCC Manassas) and propagated as recommended in DMEM/F12
and RPMI-1640 medium supplemented with 5% foetal bovine se-
rum, L-glutamine (2 mM), penicillin (100 U mlꢀ1) and streptomycin
(100 mg mlꢀ1) in humidified atmosphere with 5% CO2 at 37°C. Cells
were harvested using 0.25% trypsin (Hyclone) when they were
70–80% confluent in culture.
Cytotoxicity assay and determination of IC50
Cellular viability upon exposure to compounds 2–5 was deter-
mined using the colorimetric 3-(4,5-dimethylthiazole-2-yl)-2,5-bi-
phenyl tetrazolium bromide (MTT) assay. This assay is based on
the conversion of yellow MTT into purple formazan crystals by
living cells, which determines mitochondrial activity. The total mito-
chondrial activity is related to the number of viable cells. Basically,
PC3, DLD-1, HeLa and PNT1A cells (1× 105 cells per well) were
seeded in 96-well microtitre plates (Nunc, Denmark). The cultured
cells were exposed to different concentrations of compounds 2–5
for 24 h and then washed once with phosphate-buffered saline. Af-
ter that, 100 μl of serum-free medium containing 5 mg mlꢀ1 of MTT
(Sigma, Missouri) was added to each well. After incubation for 4 h,
the supernatant was removed and the formazan crystals obtained
were dissolved in 100μl of DMSO (Sigma). The mixture was stirred
for 20 min on a microtitre plate shaker and the absorbance was
read at 570 nm. Cell viability was expressed as the percentage of
untreated cells that served as the control group and was desig-
nated as 100%.
Dilution method
Minimum inhibitory concentrations (MICs) of the ligands and their
Ru(II) complexes against the test microorganisms were determined
using a standard method.[25] Briefly, tested bacteria were activated
in nutrient broth after incubation at 30°C for 24 h while the yeasts
were activated in malt extract broth for 48h. The compounds were
dissolved in DMSO and then diluted using Müller–Hinton broth.
Two-fold serial concentrations of the compounds were employed
to determine the MIC ranging from 200 to 1.56 μg mlꢀ1. In each
case triplicate tests were performed and the average was taken as
the final reading. MICs of compounds 2–5 were compared with
those of gentamycin antibiotic and nystatin antifungal drug.
Cytotoxicity was expressed as mean percentage increase relative
to the unexposed control standard deviation. Control values were
set at 0% cytotoxicity. Cytotoxicity data (where appropriate) were
fitted to a sigmoidal curve and a four-parameter logistic model
was used to calculate the IC50, which is the concentration of mate-
rial causing 50% inhibition in comparison to the untreated controls.
The mean IC50 is the concentration of material that reduces cell
Results and discussion
Synthesis and characterization
Compound 1 was synthesized by adapting a known synthetic
procedure.[26] Compounds 2 and 3 were prepared by the reaction
of 1 with 3-hydroxypyridine and 2-chloro-3-hydroxypyridine,
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Copyright © 2015 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2015, 29, 536–542

Cyclotriphosphazene-based multisite ligands and their Ru(II) complexes
respectively. Ru(II) complexes 4 and 5 were obtained from the reac-
tion of ligands 2 and -3 with [RuCl2(p-cymene)]2 in THF. All com-
pounds were characterized using 1H NMR, 31P NMR and FT-IR
spectroscopies, giving results that are consistent with their pro-
posed compositions.
Under these optimum conditions, the catalytic activity of com-
pounds 4 and 5 was investigated and the results are given in Table 1.
Complex 5, which has a chloro substituent on the pyridyl ring, shows
lower conversions in comparison with complex 4. The difference in
the catalytic activities of these complexes can be explained on the
basis of the electron densities on the metal centre. The presence
of the electron-withdrawing group of chloride on the aromatic ring
of compound 5 possibly decreases the electron density on its metal
centre and in turn the transfer hydrogenation rate.[9]
The complexes exhibit more efficient catalytic activity towards
transfer hydrogenation of acetophenone derivatives that have
electron-withdrawing substituents, chloride and bromide, at the
para position compared to acetophenone derivatives that have
electron-donating groups, methoxy and methyl, instead. Introduc-
tion of multiple methyl groups at the para and ortho positions de-
creases the catalytic activity even more, even though they are
weak electron-donating groups. This is because the introduction
of electron-withdrawing substituents to the para position of the
aryl ring of the ketone decreases the electron density on the C¼O
bond and this leads to an improved catalytic activity giving rise to
easier hydrogenation. Introduction of electron-donating groups
has just the opposite effect.
The absorption bands assignable to the stretching of –P¼N– and
P–O–C are, respectively, 1231–1167 and 1091 cmꢀ1 for 2; 1173–
1200 and 1072 cmꢀ1 for 3. Also, the characteristic –P¼N– and P–
O–C bands are seen, respectively, at 1219–1166 and 1092 cmꢀ1
for complex 3; 1224–1172 and 1094 cmꢀ1 for complex 4.
According to 1H NMR spectra, the aromatic proton signals of li-
gands are observed in the region 8.63–7.17 ppm for 2; 8.41–
1
7.15 ppm for 3. In the H NMR spectra of complexes, the signals
due to the methyl protons relating to p-cymene are easily distin-
guishable and they are observed at 2.07 ppm for 4 and 2.06 ppm
for 5 as singlets. In addition, –CH protons of isopropyl groups are
observed at 2.81 ppm for both 4 and 5.
The 31P NMR spectra of ligands 2 and 3 and complexes 4 and 5
are of the AB2 type. In the 31P NMR spectra of the ligands, the spiro
cyclic phosphorus atoms (P(O2C12H8)) resonate at 24.90 and
24.38 ppm (doublet) while signals of other phosphorus atoms are
seen at 12.00 and 11.92 ppm (triplet). The Ru(II) complexes show
triplets at 11.95 ppm (4) and 11.87 ppm (5) and doublets at
24.87 ppm (4) and 24.32ppm (5).
In vitro cytotoxicity studies
Cytotoxic activity of the newly synthesized compounds 2–5 against
PC3, DLD-1 and HeLa tumour cells and healthy PNT1A cells was de-
termined using MTT assay. Normal PNT1A cells were used as a con-
trol group. All test cells were exposed to compounds 2–5 at
concentrations of 10, 25, 50, 100 and 200 μM for 24 h. The results
were analysed using cell viability curves in a concentration range
Catalytic studies
To determine the most suitable reaction conditions for catalytic
transfer hydrogenation, we first examined the influence of time
and various bases on the reactivity of acetophenone (0.85mmol)
in the presence of compound 4 (0.0085 mmol) as catalyst at 82°C.
The base variation experiments were performed using a catalyst-
to-substrate ratio of 1:400 in 4 ml of 2-propanol with Cs2CO3,
NaHCO3, K2CO3, NaOH and KOH bases. In addition, optimization
studies showed that better activities were obtained with a base-
to-ketone ratio of 4:1. The conversion rate results after 1 and 2 h
are shown in Fig. 1. When the reaction is done without any base,
no reaction is observed even after 12h. The best results are ob-
tained with KOH for 2 h. The question about whether ligand-free
moieties contribute to the catalysis is eliminated by carrying out
the reaction with only [RuCl2(p-cymene)]2 as catalyst under the
same conditions: a conversion rate of 20% is observed.
Table 1. Catalytic activity for transfer hydrogenation of ketones
catalysed by Ru(II) complexesa
Entry
Catalyst
R
Conversion (%)
1
4
5
4
5
4
5
4
5
4
5
4
5
4
5
4
5
4
5
H
H
86
81
93
91
91
87
72
69
89
83
35
31
10
5
2
3
p-NO2
p-NO2
p-CN
4
5
6
p-CN
7
p-Br
8
p-Br
9
p-Cl
10
11
12
13
14
15
16
17
18
p-Cl
p-Me
p-Me
p-OMe
p-OMe
p-OH
p-OH
2,4,6-Me
2,4,6-Me
7
<5
<5
<5
aReaction
conditions:
acetophenone
(0.85 mmol),
catalyst
Figure 1. Base variation study. (Experimental conditions: acetophenone
(0.85 mmol), catalyst 4 (0.0085 mmol), base (3.4 mmol), i-PrOH (4 ml),
reflux, under argon atmosphere at 82°C.
(0.0085 mmol), KOH (3.4 mmol), i-PrOH (4 ml), 2 h, reflux, under argon
atmosphere at 82°C.
Appl. Organometal. Chem. 2015, 29, 536–542
Copyright © 2015 John Wiley & Sons, Ltd.
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D. Erdener Çıralı et al.
from 0 to 200 μM. The relationship between cell viability percent-
age and drug concentration was plotted (Fig. 2) to determine the
growth inhibition curve for each cell line.
The results show that exposure to the compounds decreases the
cell viability for all tested cells in a dose-dependent manner. A com-
parison of the cell viability results reveals that Ru(II) complexes 4
and 5 show better cytotoxic effects than their corresponding
metal-free cyclotriphosphazene ligands 2 and 3 in general, but
there is not a specific pattern. In other words, cytotoxic response
to compounds differs from cell to cell and does not follow a certain
trend. Compound 4 shows the highest antiproliferative effect
against PC3 tumour cells and decreases cell viability by 47% at
200 μM, while compound 5 has the highest cytotoxic activity
against DLD-1 cancer cell line and causes 42% cell viability decrease
at 200 μM. Other compounds show around 30% reduction in cell vi-
ability for both PC3 and DLD-1 cells at 200 μM. Compounds 2 and 3
cause 16 and 35% cell viability reduction in HeLa cells, respectively.
Ruthenium complexation seems to increase the cytotoxic effect of
compounds 2 and 3 and a significant decrease in the number of vi-
able cells is observed against HeLa cells with compounds 4 and 5,
reaching 46 and 48% reduction, respectively. Taken together, the
data indicate that compounds 4 and 5 show the highest cytotoxic
activity against the tested cancer cells. However, compound 5 also
shows severe toxic effects on normal PNT1A cells. A 59% reduction
in the number of viable PNT1A cells is observed upon treatment
with compound 5. This significant decrease in viable cells can be
contrasted with the much lower 25% decrease in cell viability upon
treatment with compound 4. The non-toxic nature of compound 4
to host cells may render it a candidate for potential drugs.
In the literature, it has been reported that some cyclotri-
phosphazene derivatives are not very effective against HeLa
cells,[27] and our results for cyclotriphosphazene ligands 2 and 3
also support that finding. However, Ru(II) complexes of these
ligands (4 and 5) increase the effectiveness of their parent com-
pounds (2 and 3) at inhibiting proliferation of HeLa cells by up to
almost three times. This effect may be explained in view of the che-
lation theory described by Tweedy[28] and the cell permeability con-
cept described by Overtone.[29] According to Tweedy, the polarity
of the metal ion will be lowered on complexation because its posi-
tive charge will be shared with donor groups. Moreover, it will in-
crease the delocalization of π-electrons over the entire chelate
ring. Consequently, this enhances the lipophilicity of the
Table 2. In vitro antimicrobial activities of 2–5 and standard
antimicrobialsa according to disc diffusion assay
Microorganism
Inhibition zone (mm)b
AM CT TE NY KE CL
2
3
4
5
Bacteria
M. luteus
12 13 13 12 30 34 20
16 13 16 14 15 14 26
13 16 14 14 15 16 30
13 11 12 11 18 20 24
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
S. aureus
K. pneumoniae
P. vulgaris
L. monocytogenes 10 10 10 10 14 14 28
E. coli
12 12 12 15 14 12 25
10 10 13 11 14 14 22
B. cereus
Fungi
R. rubra
C. albicans
K. fragilis
10
9
9
10
9
9
—
—
—
—
—
—
—
—
—
23 22 24
20 22 16
16 15 18
12
10
18 13 13 15
aAM, ampicillin 10 μg; CT, cefotaxime 30 μg; TE, tetracycline 30 μg; NY,
nystatin 100 μg; KE, ketaconazole 20 μg; CL, clotrimazole 10 μg.
bIncludes filter paper disc diameter (6 mm).
Figure 2. Plot of viable cells at various concentrations of 2, 3, 4 and 5
against (a) PC3, (b) DLD-1 and (c) HeLa tumour cells and (d) PNT1A normal
cell line.
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Copyright © 2015 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2015, 29, 536–542

Cyclotriphosphazene-based multisite ligands and their Ru(II) complexes
complexes. Overtone’s concept of cell permeability states that
entry of any molecule into a cell is governed by its lipophilicity
because the lipid membrane surrounding the cell favours the
passage of materials that are soluble in lipids. Thus, the
increased lipophilicity upon complexation enhances the penetration
of the complexes into cells and blocks the metal binding sites
of receptors.
to the partial sharing of positive charge with donor group and pos-
sible π-electron delocalization over the whole ring. This increased li-
pophilicity of the metal chelate amplifies its permeation through
lipid layers of bacterial membranes and blocks the metal binding
sites in the enzymes of microorganisms. The complexes also disturb
the respiration process of the cell and thus block the synthesis of
proteins, which restricts further growth of organisms.[30] The varia-
tion in the effectiveness of different compounds against different
organisms depends either on the impermeability of the cells of
the microbes or differences in ribosomes of microbial cells.[31]
These results suggest that the compounds which exhibit good
antimicrobial activities can be further developed for application as
effective antimicrobial agents.
Antimicrobial screening
The results for in vitro antimicrobial activities of compounds 2–5 to-
gether with inhibition zones of standard drugs are summarized in
Table 2. Disc diffusion assay results depend not only on the poten-
tial antimicrobial potency but also on the diffusion ability of the
components. If the compounds do not diffuse well in the polar agar
medium, poor results might be obtained due to the low diameter of
inhibition even if the compounds are actually good antimicrobials.
Disc diffusion is good for qualitative analysis but may not be so ap-
plicable for evaluating the potency of large compounds and com-
plexes which diffuse slowly into the culture medium. Therefore,
we also determined MIC values of compounds 2–5 and standard
drugs (Table 3). The antimicrobial data reveal that the ligands and
Ru complexes show moderate to high antimicrobial activities
against all the tested microorganisms.
The ligands and complexes show higher antibacterial activity
against M. luteus and S. aureus than the commercial antibiotic
gentamycin. Compounds exhibit about the same antibacterial ac-
tivity against K. pneumoniae and moderate activity against the
other bacteria in comparison with the standard drug. The results
also show that Ru(II) complexes have generally better antimicrobial
activities than metal-free ligands. This trend can especially be dis-
cernible against fungal strains. This can be attributed to the Tweedy
chelation theory of reduced metal polarity upon complexation due
Conclusions
Two novel cyclotriphosphazene ligands (2 and 3) and their ruthe-
nium(II) complexes (4 and 5) were prepared and characterized
using spectral and analytical methods, and their catalytic, cytotoxic
and antimicrobial activities were investigated. Catalytic activity
studies of Ru(II) complexes 4 and 5 in catalytic transfer hydrogena-
tion of para-substituted acetophenone derivatives in the presence
of KOH revealed that complex 4 showed better catalytic activity
than complex 5. Presence of a chloro substituent on the pyridyl ring
most likely reduced the electron density on the metal centre of
complex 5 and caused a lower transfer hydrogenation rate
compared to complex 4. Ruthenium complexation also enhanced
cytotoxic and antimicrobial activities of metal-free ligands, and
complexes 4 and 5 showed higher cytotoxic and antimicrobial ac-
tivities compared to their parent ligands (2 and 3). This enhancing
effect is most likely due to the increased lipophilicity of the
complexes resulting from the dissipation of positive charge on
the metal by donor groups. However, unlike complex 4, complex
5 showed detrimental effects on healthy PNT1A cells. The non-
toxicity of compound 4 to host cells make it a candidate for
potential chemotherapeutics considering that most of the drugs
used today also impair healthy cells along with tumour cells.
Table 3. Minimum inhibitory concentrations (MICs) of compounds
2–5 and standard drugsa
Acknowledgments
We are grateful to the Scientific and Technical Research Council of
Turkey (TUBITAK) for the financial support of this work, grant
number 112T584.
References
[1] E. W. Ainscough, A. M. Brodie, C. V. Depree, J. Chem. Soc. Dalton Trans.
1999, 23, 4123.
[2] E. W. Ainscough, A. M. Brodie, C. V. Depree, B. Moubaraki, K. S. Murray,
C. A. Otter, Dalton Trans. 2005, 20, 3337.
[3] E. W. Ainscough, A. M. Brodie, C. V. Depree, G. B. Jameson, C. A. Otter,
Inorg. Chem. 2005, 44, 7325.
[4] E. W. Ainscough, A. M. Brodie, C. V. Depree, C. A. Otter, Polyhedron 2006,
25, 2341.
[5] V. Chandrasekhar, B. M. Pandian, R. Azhakar, Polyhedron 2008, 27, 255.
[6] L. Wang, Q. Yang, H. Chen, R.-X. Li, Inorg. Chem. Commun. 2011, 14,
1884.
[7] S. Gunnaz, N. Ozdemir, S. Dayan, O. Dayan, B. Cetinkaya,
Organometallics 2011, 30, 4165.
[8] D. Pandiarajan, R. Ramesh, J. Organometal. Chem. 2013, 723, 26.
[9] J.-I. Ito, H. Nishiyama, Tetrahedron Lett. 2014, 55, 3133.
[10] J.-X. Gao, T. Ikariya, R. Noyori, Organometallics 1996, 15, 1087.
[11] A. M. Hayes, D. J. Morris, G. J. Clarkson, M. Wills, J. Am. Chem. Soc. 2005,
127, 7318.
aGEN, gentamycin; NY, nystatin. Light red, low activity; yellow, moderate
activity; green, high activity.
Appl. Organometal. Chem. 2015, 29, 536–542
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

D. Erdener Çıralı et al.
[12] W. Baratta, E. Herdtweck, K. Siega, M. Toniutti, P. Rigo, Organometallics
2005, 24, 1660.
[13] A. Çetin, O. Dayan, Chin. J. Chem. 2009, 5, 978.
[25] E. H. Lennette, A. Balows, W. J. Hausler, H. J. Shadomy (Eds), Manual of
Clinical Microbiology, 4th edn, American Society for Microbiology,
Washington, DC, 1985, pp. 1149.
[14] J. E. D. Martins, D. J. Morris, B. Tripathi, M. Wills, J. Organometal. Chem.
[26] G. A. Carriedo, L. Fernández-Catuxo, F. J. Garcia Alonso, P. Gómez-Elipe,
2008, 693, 3527.
P. A. González, Macromolecules 1996, 29, 5320.
[15] H. Cheng, R. Liu, J. Hao, Q. Wang, Y. Yu, S. Cai, F. Zhao, Appl.
Organometal. Chem. 2010, 24, 763.
[27] B. R. Patil, S. S. Machakanur, R. S. Hunoor, D. S. Badiger, K. B. Gudasi,
S. W. A. Bligh, Der Pharma Chemica 2011, 3, 377.
[16] D. E. Çıralı, O. Dayan, N. Özdemir, N. Hacıoglu, Polyhedron 2015, 88, 170.
[17] D. E. Çıralı, O. Dayan, Phosphorus Sulfur Silicon Relat. Elem. in press.
doi:10.1080/10426507.2014.966190).
[18] American Cancer Society, Global Cancer Facts & Figures, 2nd edn,
American Cancer Society, Atlanta, GA, 2011.
[28] B. G. Tweedy, Phytopathology 1964, 55, 910.
[29] A. Kleinzeller, Curr. Topics Membr. 1999, 48, 1–22.
[30] Y. Vaghasia, R. Nair, M. Soni, S. Baluja, S. Chanda, J. Serb. Chem. Soc.
2004, 69, 991.
[31] P. G. Lawrence, P. L. Harold, O. G. Francis, Antibiot. Chemother. 1980, 1597.
[19] J. Ferlay, I. Soerjomataram, M. Ervik, R. Dikshit, S. Eser, C. Mathers,
M. Rebelo, D. M. Parkin, D. Forman, F. Bray, GLOBOCAN 2012.
Available from: http://globocan.iarc.fr (accessed 8 February 2015).
[20] J. O. Bovin, J. Galy, J. F. Labarre, F. Sournies, J. Mol. Struct. 1978, 49, 421.
[21] J. L. Sassus, M. Graffeuil, P. Castera, J. F. Labarre, Inorg. Chim. Acta 1985,
108, 23.
[22] M. Siwy, D. Sek, B. Kaczmarczyk, I. Jaroszewicz, A. Nasulewicz,
M. Pelczynska, D. Nevozhay, A. Opolski, J. Med. Chem. 2006, 49, 806.
[23] K. Brandt, R. Kruszynski, T. J. Bartczak, I. P. Czomperlik, Inorg. Chim. Acta
2001, 322, 138.
Supporting information
Additional supporting information may be found in the online ver-
sion of this article at the publisher’s web-site.
[24] National Committee for Clinical Laboratory Standards, Performance
standards for antimicrobial disk susceptibility tests: approved standard,
NCCLS Publication M2-A5, Villanova, PA, 1993.
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