Synthesis, structural and thermal characterizations, dielectric properties and in vitro cytotoxic activities of new 2,2,4,4-tetra(4′-oxy-substituted-chalcone)-6,6-diphenylcyclotriphosphazene derivatives

Article abstract of DOI:10.1007/s00044-017-1810-4

In this study, we aimed to investigate the relationship between the cytotoxic and dielectric properties of newly synthesized 2,2,4,4-tetra(4′-oxy-substituted-chalcone)-6,6-diphenylcyclotriphosphazene derivatives (3–10). Firstly, 2,2,4,4-tetrachloro-6,6-di

Full text of DOI:10.1007/s00044-017-1810-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1810-4
ORIGINAL RESEARCH
Synthesis, structural and thermal characterizations, dielectric
properties and in vitro cytotoxic activities of new 2,2,4,4-tetra(4′-
oxy-substituted-chalcone)-6,6-diphenylcyclotriphosphazene
derivatives
Kenan Koran1 Çiğdem Tekin2 Fatih Biryan1 Suat Tekin3 Süleyman Sandal3
●
●
●
●
●
Ahmet Orhan Görgülü1
Received: 11 November 2016 / Accepted: 16 February 2017
© Springer Science+Business Media New York 2017
Abstract In this study, we aimed to investigate the rela-
tionship between the cytotoxic and dielectric properties of
newly synthesized 2,2,4,4-tetra(4′-oxy-substituted-chalcone)-
6,6-diphenylcyclotriphosphazene derivatives (3–10). Firstly,
2,2,4,4-tetrachloro-6,6-diphenyl cyclotriphosphazene (2) was
obtained through Friedel Crafts alkylation in the presence of
hexachlorocyclotriphosphazene, benzene and triethylamine
and anhydrous AlCl3. The compounds 3–10 were synthe-
sized from the reaction of the hydroxychalcone compounds
(1a–h) with 2 in the presence of K2CO3 and within the
acetone solvent for the first time and their dielectric constant,
dielectric loss factor and ac conductivity of compounds 3–10
were examined through the impedance analyzer as a function
of frequency. The in vitro cytotoxic activities of compounds
3–10 in five different concentrations (1, 5, 25, 50, and 100
µM) were analyzed by colorimetric MTT assay which is
based on reduction of MTT salt by mitochondria of alive
cells over the human ovarian cancer (A2780) and human
prostate cancer (PC-3 and LNCaP) cell lines. The LogIC50
values of 3–10 were calculated by using a Graphpad prism 6
programs on a computer. The obtained results suggests that
the compounds have a powerful cytotoxic activity (especially
A2780, p < 0.05).
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Keywords Chalcone-cyclotriphosphazene Cancer cell
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●
●
lines Dielectric property Cytotoxic activity Dielectric
constant
Introduction
Cyclophosphazenes are versatile inorganic ring systems
owing to the great stability of the phosphorus-nitrogen
backbone and the ability to undergo substitution of side
groups with retention of the ring structure (Gleria and De
Jaeger 2004). The phosphazene compounds included in the
group of phosphorus-nitrogen compounds are also termed as
phosphonitrilic and hydroazophosphorane (Allcock 1972;
Gleria and De Jaeger 2004). The phosphazene derivatives
synthesized as the result of phosphazene reactions are gen-
erally formed as the result of the replacement of the chlorines
existing on the phosphorus atom by an appropriate reagent
(Allen 1992). The obtained derivatives are formed with the
bonding of a nitrogen group (as –NH2/NR) (Allcock et al.
1998; Tumer et al. 2013; Başterzi et al. 2015), an oxygen
group R(Ar)O− (Benson et al. 2013; Koran et al. 2014;
Reynes et al. 2016), a sulfur group R(Ar)S− (Ma et al. 2002),
or through the direct bonding of alkyl/aryl groups with the
phosphazene (Neilson et al. 1987; Uslu et al. 2013). Due to
the reactivity of P-Cl bond within the structures of phos-
phazene compounds, the type of the organic or inorganic
groups bonded as a side-group alter the physical, biological,
and chemical properties of these compounds. The cyclo or
poly phosphazene derivatives containing organic, inorganic
or organometallic side-groups have many potential
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1810-4) contains supplementary material,
which is available to authorized users.
* Kenan Koran
kenan.koran@gmail.com
1
Department of Chemistry Elazığ, Faculty of Science, Firat
University, Elazığ, Turkey
2
Department of Public Health, Faculty of Medicine, Inonu
University, Malatya, Turkey
3
Department of Physiology, Faculty of Medicine, Inonu University,
Malatya, Turkey

Med Chem Res
applications such as dielectric (Koran et al. 2016), liquid
crystalline (Bao et al. 2010), chemosensor (Şenkuytu et al.
2015), littium-ion batteries (Xia et al. 2015), fire-retardant
material (Jiang et al. 2014; Zhao et al. 2016), optic material
(Rojo et al. 2000), antibacterial, and antitumor properties
(Brandt et al. 2001; Siwy et al. 2006; Ozay et al. 2010;
Görgülü et al. 2015; Akbaş et al. 2016).
the presence of hexachlorocyclotriphosphazene (trimer,
HCCP), benzene and triethylamine and anhydrous AlCl3
(McBee et al. 1965). In the second stage, the substituted
hydroxychalcone compounds bearing different side
groups at ortho or para positions (1a–h) were synthesized in
accordance with Claisen–Schmidt Condensation protocol
(Funiss et al. 2004; Modzelewska et al. 2006). 2,2,4,4-
tetra(4′-oxy-substituted-chalcone)-6,6-diphenylcyclotripho-
sphazene (3–10) were synthesized for the first time from the
interactions of 2,2,4,4-tetrachloro-6,6-diphenylcyclotripho-
sphazene (2) with 4.1 equiv. of 4′-hydroxy-substituted-
chalcone analogs (1a–h) in the excess of potassium carbo-
nate and within the acetone solvent. Reactions of cyclo-
triphosphazene compounds followed with thin layer
chromatography and 31P-nuclear magnetic resonance
(NMR) spectroscopy. The general synthesis presentations
for compounds are shown in Scheme 1.
On the other hand, the chalcone compounds, which are
also referred to as 1,3-Diaryl-2-propen-1-ones, are natural
and synthetic compounds and members of flavonoid family.
Chalcone groups are the compounds in which two aromatic
rings are bonded with one another through the three carbon
α,β-unsaturated carbonyl group. Such compounds having a
ketovinyl group have been among the natural compounds
that have gained importance in recent years due to their
various biological activities (Patil et al. 2009; Baggio et al.
2016). Furthermore, the fact that they are found densely in
edible plants has increased the importance of these com-
pounds in cancer researches (Bahekar et al. 2016; I-Monica
et al. 2016). It has been found in the literature that chalcones
show anti-cancer (Rao et al. 2004; Kitawat et al. 2013;
Jayashree et al. 2016), anti-inflammatory (Herencıa et al.
1998), anti-HIV activity (Wu et al. 2003), antihyperglycemic
activity (Damazio et al. 2010), antifungal activities (Boeck
et al. 2005) as well as antioxidant (El-Sayed and Gaber
2015) and antituberculosis effects (Lin et al. 2002). Apart
from the biological effects of chalcone and its derivatives,
they are also used as UV-absorption filters in optic materials
(Mager et al. 1997), in food industry and holographic
recording technologies (Fayed and Awad 2004).
In the literature, there is no performed study as to the
synthesis of 2,2,4,4-tetra(4′-oxy-substituted-chalcone)-6,6-
diphenylcyclotriphosphazene derivatives and their cytotoxic
and dielectric properties. For this reason, new phosphazene
compounds carrying phenyl and chalcone rings were
designed and synthesized. The structures of these com-
pounds were characterized through spectroscopic methods.
The dielectric properties of cyclotriphosphazene com-
pounds, as the function of frequency, were identified by
using the impedance analyzer device. Finally, the in vitro
cytotoxic activities of chalcone-phosphazene compounds
were determined through MTT assay method by using
A2780, PC-3, and LNCaP cell lines. The compounds were
determined to have showed a powerful cytotoxic activities
effect against A2780 cancer cell in particular.
The structures of 1a-h were confirmed through Fourier
1
transform infrared spectroscopy (FT-IR), H and 13C NMR
spectroscopy methods. The structures of compounds 2–10
1
were identified through FT-IR, MS, H, 13C-APT and 31P-
NMR spectroscopy methods. When matrix assisted laser
desorption ionization-time of flight mass spectrometry
spectrums of compounds are examined, it is seen that they
are almost the same as the molecular weights calculated
theoretically. The molecular ion peaks of 3–10 have been
given in the experimental section. For example, the mole-
cular ion peak of compound 7 was displayed at 1255.32
(Supplementary data, Figure S2).
The melting points of 3–10 were determined through
differential scanning calorimetry (DSC) thermograms
(Supplementary data, Figure S1 (A)). The thermal stability
of the compounds as well as the temperatures at which they
begin to get degraded was determined through thermo-
gravimetric analysis (TGA) analysis (Supplementary data,
Figure S1 (B)). More than 50% of the structures of com-
pounds do not get degraded at the temperatures reaching up
to 500 °C. At the temperatures reaching up to 900 °C,
however, almost all the compounds get degraded (Table 1).
In the light of these results, the synthesized compounds are
also seen to be thermally stable.
When FT-IR spectra of 3–10 were examined, the -OH
stretching vibration in the structure of 1a–h was not dis-
played. The –P=N stretching vibrations in the FT-IR
spectrum of compound 2 were observed at 1172 and
1221 cm−1. However, the –P=N stretching vibrations in the
phosphazene compounds were observed to have shifted
towards 1174 and 1206 cm−1. The P-O-Ph stretching
vibrations in the structure of phosphazenes were observed
between 928 and 934 cm−1. In addition, the PCl2 bands are
disappeared. When the FT-IR spectra of the chalcone-
cyclotriphosphazene compounds (3–10) were examined, the
carbonyl stretching vibrations (–C=O) were observed at
Results and discussion
Chemistry
2,2,4,4-Tetrachloro-6,6-diphenylcyclotriphosphazene
(2)
compound was obtained through Friedel Crafts alkylation in

Med Chem Res
O
Scheme 1 Chemical structure
and synthetic pathway of
compounds 1a–h and 2–10
O
EtOH
+
O
HO
% 30 NaOH
( 1a-f )
HO
R1
R1
CH
3
O
O
EtOH
R2
+
( 1 )
R2
% 30 NaOH
HO
( 1g, 1h )
Compounds
R1
1a / 3 1b / 4 1c / 5 1d / 6 1e / 7 1f / 8
2-F,
3-CH3,
2-Cl,
3-Cl,
2-CH3
,
3-F,
1g / 9
1h / 10
R2
N
N
Cl Cl
P
P
N
P
N
N
P
N
P
C6H6 / AlCl3
Cl
P
Cl
Et3N
Cl
Cl
N
N
Cl
Cl
Cl
Cl
HO
R1
( HCCP )
( 2 )
O
O
( 1g, 1h )
( 1a-f )
R2
3
4
HO
2
3
2
3
4
1
2
3
P
1
2
O
7
6
O
N
P
N
P
9
8
P
5
O
O
7
6
O
O
N
N
P
11
10
7
6
9
8
N
5
O
R2
O
O
R2
P
O
11
10
7
6
N
12
O
O
O
R1
R1
O
O
O
R2
R2
( 9 and 10 )
R1
R1
( 3-8 )
1661, 1659, 1662, 1662, 1663, 1662, 1661, and 1663 cm−1
respectively.
,
phosphorus peaks of compound 2 were unobserved at 31P-
NMR spectra of 3–10. For example, the 31P-NMR spectrum
of compound 2 and 7 was given in Figure S3 (Supple-
mentary data).
31P NMR spectra of 3–10 observe the expected AB2 spin
system. As expected, there are one double and triple
including two peaks at 31P NMR spectra of 2–10. In 31P-
NMR spectrum of compound 2, the doublet peaks belong-
ing to two equivalent phosphorus was observed at 18.18
ppm, while the triplet peaks belonging to single phosphorus
were observed at 20.40 ppm. When the chalcone com-
pounds linked to the phosphazene ring, the doublet and
singlet peaks belonging to two equivalent phosphorus and
single phosphorus were shifted towards the range of
6.68–7.10 ppm and 22.10–22.14 ppm, respectively. The
When 1H-NMR spectra of 3–10 were examined, The
1
–OH protons observed at 10.43 and 10.54 ppm in the H-
NMR spectra of 1a-h were not observed at NMR spectra of
1
3–10. The ratio of the protons integral height in the H-
NMR spectra of 3–10 supports the proposed structures. The
locations of the primary, secondary and tertiary carbon
peaks was determined by using 13C-attached proton test
(APT) NMR technique and the carbon peaks also support
1H-NMR and the structure. The results are given in detail in

Med Chem Res
the experimental section. For example, the 1H and 13C-APT
NMR spectra of compound 7 were given in Figs. S4 and S5,
respectively (Supplementary data).
Dielectric behaviors
The dielectric parameters (capacitance values (Cp), dielec-
tric loss factor (DF), and conductivity (Gp)) of compounds
3–10 were measured by using with a Quad Tech 7600
precision LRC Meter impedance analyzer against increasing
frequency (from 100 Hz to 20 kHz). And then the ɛ′, ɛ″, and
σ values for each compound were determined with Eqs. (1,
2, and 3). The dielectric constants, dielectric loss factors and
AC conductivities of the compounds were compared with
one another. All the results and comparisons are given in
Fig. 1 and Table 1.
The dielectric properties of the cyclophosphazene com-
pounds (3–10) were measured as a function of the fre-
quency. The dielectric properties of compounds change
with the frequency. The change in the dielectric properties
of cyclotriphosphazenes is the result of electronic, ionic and
molecular polarizability. These properties are associated
with the physical and chemical structures of compounds. It
is seen that the capacitance value and the dielectric constant
decrease with the increasing frequency, while they remain
constant at high frequencies. The reason for this can be
thought to be the polarization effect. Polarization occurs
since the effect of the dipoles increases as the frequency
increases.
The change graphics along with the frequency,
which pertain to the dielectric constant, dielectric loss
factors and conductivity values of the compounds having
–CH3 (3 and 4), –Cl (5 and 6), and –F (7 and 8) groups in
their ortho and meta position, is given in Fig. 1. Separately,
the comparison related to compounds 9 and 10 having the
pyridine ring instead of the phenyl ring is also given in the
graphics. It was seen that the dielectric constant and
dielectric loss values of the compounds had decreased along
with the increasing frequency and remained unchanged after
some point.
The dielectric constant of compound 5 having chlorine in
its ortho position was found to be higher than that of
compound 7 having fluorine. This is thought to be due to
the fact that the –Cl atoms within the structure of compound
2 increase polarity; hence, the dipole moment increases. On
the other hand, the dielectric constant in pyridine-
substituted compounds 9 and 10 was found to be the
highest (Fig. 1 (a)). Since the presence of a hetero-atom in
the ring can increase polarization, this can be caused the
situation mentioned above. Similar cases were also
observed in dielectric loss and AC conductivity values
(Fig. 1 (b) and (c)). The dielectric constant of compound 8

Med Chem Res
Fig. 1 Variation of dielectric parameters of compounds 3–10 against frequency; Dielectric constants (a), Dielectric loss factors (b), and AC
Conductivity graphics (c)
having fluorine in its meta position proved to be higher than
that of compound 6 having chlorine (Fig. 1 (a)).
(3–10) compounds on the androgen independent (negative)
prostate cancer cell line (PC-3) and on the androgen
dependent (positive) prostate cancer cell line (LNCaP) and
also on the human ovarian cancer cell line (A2780), the %
changes in the cell viability rates that were caused by 1, 5,
25, 50, and 100 μM concentrations of the substance for 24 h
were determined through MTT assay. The LogIC50 values
of the compounds were given in Table 1. The comparisons
of the compounds were made by considering the solvent
control as the basis. According to the results of experiments,
compounds 3–10 were quite effective on A2780 cancer
cells. On the other hand, compounds 3–10 were observed to
be effective on PC-3 and LNCaP cells in general.
The effect of the positions of R groups on the dielectric
constants of the compounds is clearly understood through
the graphic in Fig. 1. For instance, while the dielectric
constant of the compound having fluorine group in its ortho
position is lower than the dielectric constant of the com-
pound having chlorine in its ortho position, this situation is
the opposite in the meta position. This situation remained
unchanged in the compounds carrying the pyridine ring.
The highest dielectric constant was observed in pyridine-
substituted compound 10.
All the comparisons of compounds were made by con-
sidering the control group as the basis. When compounds
containing –CH3 (3), –Cl (5), and –F (7) in the ortho-
position of the phenyl ring are examined in terms of
In vitro cytotoxic activities activity
In order to determine the effective dose of 2,2,4,4-tetra
(substituted-chalcone)-6,6-diphenylcyclotriphosphazene

Med Chem Res
structure activity, compounds 5 and 7 were found to be
quite effective on A2780 cells (p < 0.05, Fig. 2 (a)). Sepa-
rately, it was observed that all the doses of compounds 5
and 7 had reduced the cancer cells. On the other hand, only
100 μM dose of compound 3 was determined to be effec-
tive. When it is the pyridine ring instead of the phenyl ring,
they showed even a little cytotoxic effect on A2780 cell
lines.
Compound 3 were not showed cytotoxic activity against
PC-3 and LNCaP cell lines. Compound 5 were showed
highest cytotoxic activity against PC-3 and LNCaP cell
lines. Compounds 7 were found to be effective on LNCaP
cell lines (p < 0.05, Fig. 2 (b)). When it is the pyridine ring
instead of the phenyl ring, compound 9 showed even a little
cytotoxic effect on PC-3 and LNCaP cell lines (p < 0.05).
When examined in terms of structure activity, the electron-
withdrawing group, –Cl and F, in the ortho position showed
more effect than the electron releasing group (–CH3),
against A2780, PC-3, and LNCaP cell lines (p < 0.05,
Fig. 2).
to be effective on A2780 cell lines (p < 0.05, Fig. 2 (a)).
Separately, it was observed that all the doses of compounds
6 and 25, 50 and 100 µM doses of compounds 4 had
reduced the cancer cells. Compounds 4, 6, 8, and 10 were
usually showed cytotoxic activity on PC-3 and LNCaP cell
lines (p < 0.05, Fig. 2 (b) and (c)). Compound 6 bearing –Cl
group in the meta-position showed more effect than other
compounds against all cancer cell lines. When it is the
pyridine ring instead of the phenyl ring, compound 10
showed cytotoxic effect on A2780 and LNCaP cell lines (p
< 0.05, Fig. 2 (a) and (c)).
The cytotoxic effect of 5 and 6 compounds having Cl in
their ortho- and meta-positions was examined according to
the position of the chlorine atom. They showed a cytotoxic
effect against three cancer cell lines in the cases in which
–Cl atom was in the ortho or meta position (p < 0.05).
However, it was determined that the cytotoxic effect in 5
compound having –Cl in its ortho position was found to be
more effective than in 6 compound having –Cl in its meta
position all the cancer cell lines (A2780, PC-3, and
LNCaP).
When compounds carrying –CH3 (4), –Cl (6), and –F (8)
in the meta-position of the phenyl ring are investigated in
terms of structure activity, compounds 4 and 6 were found
The common results generally observed in three cancer
cells are mentioned below:
Fig. 2 The relative cell viability (%) of A2780 (a), LNCaP (b), and PC-3 (c) cells after a 24-h treatment with all the compounds 3–10. The changes
on the cell viability (%) caused by compounds 3–10 are compared with the control data. Each data point is an average of 10 viability (*p < 0.05)

Med Chem Res
The compounds containing chlorine at ortho and meta
positions are effective on A2780, PC-3, and LNCaP cancer
cells. The best cytotoxic effect in all of the three cell types
was exhibited by chlorine-containing structures. The com-
pound having methyl in its ortho position did not show any
significant effect in the three cell types. However, the
compound 4 having methyl in its meta position showed a
better effect on especially A2780 cell lines. The compound
having fluorine group in its ortho position showed better
effect on all the cells when compared with its localization in
meta position.
pyridinecarboxaldehyde, 3-pyridinecarboxaldehyde, ben-
zene, ethanol, and acetone were supplied from Merck.
Hexachlorocyclotriphosphazene (trimer, HCCP), potassium
carbonate, sodium hydroxide (powder), chloroform-d and
DMSO-d6 for NMR analysis were supplied from Sigma-
Aldrich.
Physical measurement
1
Elemental analysis, Mass spectra, FT-IR, and H, 13C and
31P-NMR spectra were recorded on a LECO 932 CHNS-O
apparatus, a Bruker Daltonics microflex mass spectrometer,
a Perkin Elmer FT-IR spectrometer and a Bruker DPX-400
and, respectively. Thermal characterizations of the
chalcone-phosphazene compounds were determined by
Conclusion
DSC and TGA using a SHIMADZU DSC (10 °C min−1
)
In conclusion, we successfully reported the synthesis of
2,2,4,4-tetra(4′-oxy-substituted-chalcone)-6,6-diphenylcy-
clotriphosphazene derivatives (3–10). The structures of
compounds were identified by using spectroscopy and
thermal analysis techniques (microanalysis, FT-IR, 31P, 1H,
13C-APT NMR, MALDI-TOF MS, DSC, and TGA thermal
analysis) and their the dielectric parameters (dielectric
constant, dielectric loss and AC conductivity) of the com-
pounds were determined through the impedance analyzer as
a function of frequency.
The in vitro cytotoxic activities of compounds 3–10 in
five different concentrations (1, 5, 25, 50, and 100 µM) were
analyzed by colorimetric MTT assay which is based on
reduction of MTT salt by mitochondria of alive cells over
the human ovarian cancer (A2780) and human prostate
cancer (PC-3 and LNCaP) cell lines. The LogIC50 values of
3–10 were calculated by using a Graphpad prism 6 pro-
grams on a computer. The obtained results suggests that the
compounds have cytotoxic activity (especially A2780, p <
0.05). All the doses of the compounds 5 and 7, which are
orto-substituted phosphazenes containing chloro and fluoro
groups on the chalcone ring, are very effective. It can be
said that cytotoxicity effects of 5 and 7 on A2780 cells are
structure and dose-dependent. In the light of the obtained
results, it was studied to be determined with this study that
besides the importance of the organic side-group in cancer
researches, the type of the substituents linked to this side-
group and their ortho or meta position within the structure
change the effect on activity to a considerable degree.
and a TGA-50 thermobalance (20 °C min−1), respectively.
Dielectric measurements against increases frequencies were
detected by using a Quad Tech 7600 precision LRC Meter
impedance analyzer.
General reaction method for the synthesis of chalcones
(1a–h)
4′-Hydroxy-substitutedchalcone compounds were synthe-
sized and purified in accordance with Claisen–Schmidt
Condensation protocol (Scheme 1) (Funiss et al. 2004;
Modzelewska et al. 2006).
3 g (22.02 mmol) 4′-hydroxyacetophenone (1) was dis-
solved in 50 mL absolute ethyl alcohol and was added into
250 mL reaction flask. The reaction medium was set to
0 °C, after which 30% of NaOH solution was added into the
reaction flask. After it was mixed for half an hour, 22.02
mmol of substituted aldehyde was added in drop by drop.
The reagent was mixed at room temperature for 24 h. After
the reaction was stopped, the reagent mixture was pre-
cipitated in acidic water. The solid matter that was pre-
cipitated was filtered and washed with plenty of water.
Afterwards, the substance in question was dissolved once
again in acetone, and after it was precipitated once again
within the water containing sodium bisulphate, it was
washed with plenty of water and was then dried up in a
vacuum-oven. The obtained compounds were recrystallized
in acetone-water mixture, and then compounds 1a–h was
obtained.
Materials and methods
Chemistry
Synthesis of 2,2,4,4-tetrachloro-6,6-diphenylcyclotriphosph
azene (2)
Triethylamine, anhydrous AlCl3, 2-Methylaldehyde, 3-
methylbenzaldehyde, 2-chlorobenzaldehyde, 3-chlorobenzal
deyde, 2-fluorobenzaldehyde, 3-fluorobenzaldehyde, 2-
2,2,4,4-tetrachloro-6,6-diphenylcyclotriphosphazene com-
pound (2) was obtained through Friedel Crafts alkylation
protocol (Scheme 1) (McBee et al. 1965).

Med Chem Res
200 mL benzene was added into a three-necked reaction
flask in argon atmosphere. 26.56 g (0.2 mol) anhydrous
AlCl3 and 7.91 g (0.078 mol) triethylamine was added onto
it. The reaction was heated up and mixed at the boiling
point of benzene for 30 min Following this process, 10 g
(0.029 mol) HCCP was added into it gradually in solid
form, and it became refluxed for 48 h. The reaction was
stopped and left for cooling 48 h later. The solution was
added into 200 mL HCl solution and mixed. Then, the
mixture was put into the separating funnel, and the benzene
extract was removed. 15 mL benzene was added into the
aqueous solution three times, and the extraction was per-
formed. Then, the benzene solutions were combined, and
anhydrous MgSO4 was added into this mixture, after which
it was mixed and filtered. Then it was evaporated by means
of a rotary evaporator until 15 mL of benzene solution was
left in the reaction flask, and then hexane was added into the
solution. The residue that had precipitated was filtered and
separated. The solvent of the remaining was re-evaporated,
and the solid matter that remained within the balloon was
dissolved once again in 5–10 mL benzene, after which it
was recrystallized. A white-colored solid crystal was
obtained. Yield: 7.4 g (60%). M.p.: 93–96 °C. Anal. Calcd.
For C12H10N3Cl4P3: C, 33.44; H, 2.34; N, 9.75. Found: C,
33.48; H, 2.39; N, 9.82. MALDI-MS: m/z calc. 430.96.
Found: 431.97 [M+H]+. FT-IR (KBr, cm−1) ν 3056 and
3069 (C–H aromatic), 1438, 1483, 1590 and 1610 (C=C),
1172 and 1221 (P=N). 31P NMR (400 MHz, CDCl3) δ =
the reaction mixture was filtered. The mixture was pre-
cipitated within 5% of 250 mL NaOH. The precipitated
solid was filtered and washed with plenty of water until it
reached pH~7, after which it was dried up. The dried solid
was dissolved in chloroform, and n-hexane was precipitated
once again, after which it was washed up with ethyl alcohol
three times and was then dried up under the vacuum. The
white solid matter (compound 3) was obtained in pure form.
Yield: 2.15 g (75%). M.p.: 142–143 °C. Anal. Calcd. For
C76H62N3O8P3: C, 73.72; H, 5.05; N, 3.39. Found: C,
73.76; H, 5.09; N, 3.31. MALDI-MS: m/z calc. 1238.24.
Found: 1238.43. FT-IR (KBr, cm−1) ν 3026 and 3058 (C–H
aromatic), 2922 and 2957 (C–H Aliphatic), 1661 (C=O),
1503, 1576 and 1601 (C=C), 1182 and 1206 (P=N), 934
(P-O-Ph). 31P NMR (400 MHz, DMSO-d6) δ=7.05 (d, 2P,
PB), 22.14 (t, 1P, PA). 1H NMR (400 MHz, DMSO-d6) δ =
2.38 (12H, s, H18, –CH3), 7.75 (4H, d, J = 15.6, H10,
–CH=), 7.22–7.46 (30H, m, H3,4, H6, H14–17, Ar–H),
7.93–7.99 (8H, m, H2, Ar–H and H11, =CH–), 8.01–8.03
(8H, d, H7, Ar–H).13C NMR (400 MHz, DMSO-d6) δ =
133.61 C1 (P–C(Ar)), 128.80–128.93 C2 (Ar–CH),
130.26–130.37 C3 (Ar–CH), 130.96 C4 (Ar–CH), 153.97
C5 (Ar–C), 121.56 C6 (Ar–CH), 131.26 C7 (Ar–CH),
135.09 C8 (Ar–C), 188.16 C9 (C=O), 122.78 C10 (–CH=),
141.63 C11 (=CH–), 133.61 C12 (Ar–C), 138.56 C13
(Ar–C), 130.96 C14 (Ar–CH), 130.96 C15 (Ar–CH), 126.83
C16 (Ar–CH), 127.32 C17 (Ar–CH), 19.75 C18 (–CH3).
1
16.18 (d, 2P, PB), 20.40 (t, 1P, PA). H NMR (400 MHz,
CDCl3) δ = 7.52–7.55 (2H, Ar-H4), 7.58–7.60 (4H,
Ar–H3), 7.81–7.87 (4H, Ar–H2). 13C NMR (400 MHz,
CDCl3) δ = 131.95–133.31 C1 (P-C(Ar)), 128.73-128-87
C2 (Ar-CH), 130.60–130.72 C3 (Ar–CH), 132.56–132.59
C4 (Ar–CH).
2,2,4,4-Tetra(4′-oxy-3-methylchalcone)-6,6-diphenylcyclotr
iphosphazene (4)
For the synthesis of compound 4 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1b (3.32 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 24 h. Yield: 2.35 g (82%). M.p.: 127–128 °C. Anal.
Calcd. For C76H62N3O8P3: C, 73.72; H, 5.05; N, 3.39.
Found: C, 73.78; H, 5.11; N, 3.43. MALDI-MS: m/z calc.
1238.24. Found: 1238.32. FT-IR (KBr, cm−1) ν 3029 and
3064 (C–H aromatic), 2864 and 2921 (C–H Aliphatic),
1659 (C=O), 1484, 1501, and 1595 (C=C), 1175 and 1203
(P=N), 933 (P-O-Ph). 31P NMR (400 MHz, DMSO-d6) δ =
General reaction method for the synthesis of 2,2,4,4-tetra
(4′-oxy-substituted-chalcone)-6,6-diphenylcyclotriphosphaz
ene compounds (3–10)
Synthesis and characterization of chalcone substituted
cyclophosphazene compounds (3–10) were prepared along
with a similar procedure. Therefore, detailed procedures for
the synthesis and purification procedure were only given
compound 3.
50 mL of acetone was added into a three-necked reaction
flask of 100 mL, and then 1 g (2.32 mmol) 2,2,4,4-tetra-
chloro-6,6-diphenylcyclotriphosphazene (2), 3.32 g (13.92
mmol) 4′-hydroxy-2-methylchalcone (1a), and 1.92 g
(13.92 mmol) K2CO3 were added onto it in cold condition
(0 °C) in argon atmosphere. After the reaction was mixed at
room temperature for 30 min in argon atmosphere at 0 °C, it
became refluxed for 12 h. After the reaction was stopped,
1
6.82 (d, 2P, PB), 22.10 (t, 1P, PA). H NMR (400 MHz,
DMSO-d6) δ = 3.40 (12H, s, H18, –CH3), 7.24–7.45 (26H,
m, H3,4, H6, H13, H15, H17, Ar–H), 7.62–7.68 (12H, m, H2,
H16, Ar–H, H10, –CH=), 7.81–7.85 (4H, d, J = 15.6 Hz,
H11, =CH–), 8.02–8.04 (8H, d, J = 8.4 Hz, H7, Ar–H).13C
NMR (400 MHz, DMSO-d6) δ = 134.95 C1 (P–C(Ar)),
128.78–128.92 C2 (Ar–CH), 130.24–130.35 C3 (Ar–CH),
131.89 C4 (Ar–CH), 153.98 C5 (Ar–C), 121.48 C6
(Ar–CH), 130.94 C7 (Ar–CH), 135.94 C8 (Ar–C), 188.22
C9 (C=O), 121.88 C10 (–CH=), 144.78 C11 (=CH–),
138.63 C12 (Ar–C), 130.94 C13 (Ar–CH), 138.63 C14

Med Chem Res
(Ar–C), 129.68 C15 (Ar–CH), 126.26 C16 (Ar–CH), 126.75
C17 (Ar–CH), 21.34 C18 (–CH3).
(Ar–CH), 131.12 C7 (Ar–CH), 134.25 C8 (Ar–C), 188.02
C9 (C=O), 123.55 C10 (–CH=), 142.86 C11 (=CH–),
137.28 C12 (Ar–C), 138.43 C13 (Ar–CH), 134.87 C14
(Ar–C), 128.36 C15 (Ar–CH), 131.12 C16 (Ar–CH), 131.12
C17 (Ar–CH).
2,2,4,4-Tetra(4′-oxy-2-chlorochalcone)-6,6-diphenylcyclotr
iphosphazene (5)
For the synthesis of compound 5 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1c (3.6 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 18 h. Yield: 2.63 g (86%). M.p.: 157–158 °C. Anal.
Calcd. For C72H50Cl4N3O8P3: C, 65.52; H, 3.82; N, 3.18.
Found: C, 65.59; H, 3.87; N, 3.24. MALDI-MS: m/z calc.
1319.92. Found: 1320.02. FT-IR (KBr, cm−1) ν 3005 and
3063 (C–H aromatic), 2921 and 2952 (C–H Aliphatic),
1662 (C=O), 1469, 1501, and 1598 (C=C), 1178 and
1203 (P=N), 931 (P-O-Ph). 31P NMR (400 MHz, DMSO-
2,2,4,4-Tetra(4′-oxy-2-fluorochalcone)-6,6-diphenylcyclotr
iphosphazene (7)
For the synthesis of compound 7 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1e (3.37 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 24 h. Yield: 1.75 g (60%). M.p.: 147–148 °C. Anal.
Calcd. For C72H50Cl4N3O8P3: C, 68.96; H, 4.02; N, 3.35.
Found: C, 69.02; H, 4.09; N, 3.39. MALDI-MS: m/z calc.
1254.10. Found: 1255.32. FT-IR (KBr, cm−1) ν 3064 (C–H
aromatic), 2921 and 2952 (C-H Aliphatic), 1663 (C=O),
1504, 1578, and 1602 (C=C), 1180 and 1204 (P=N), 933
(P-O-Ph). 31P NMR (400 MHz, DMSO-d6) δ = 6.78 (d, 2P,
PB), 22.15 (t, 1P, PA). 1H NMR (400 MHz, DMSO-d6) δ =
7.23–7.39 (16H, m, H6, H14, H16, Ar–H), 7.35–7.37 (4H, m,
H3, Ar–H), 7.43–7.52 (10H, m, H4, H17, Ar–H and H10,
–CH=), 7.76–7.88 (8H, m, H2, Ar–H and H11, =CH–),
7.98–8.0 (8H, d, J = 8.8 Hz, H7, Ar–H), 8.04–8.06 (4H, t,
H15, Ar–H). 13C NMR (400 MHz, DMSO-d6) δ = 133.15
C1 (P–C(Ar)), 128.82–128.95 C2 (Ar–CH), 130.27–130.38
C3 (Ar–CH), 132.25 C4 (Ar–CH), 154.06 C5 (Ar–C),
121.51 C6 (Ar–CH), 131.0 C7 (Ar–CH), 133.24 C8 (Ar–C),
187.95 C9 (C=O), 124.04 C10 (–CH=), 135.76 C11
(=CH–), 122.58 C12 (Ar–C), 160.15–162.65 C13 (Ar–C),
116.64 C14 (Ar–CH), 131.0 C15 (Ar–CH), 125.38 C16
(Ar–CH), 129.57 C17 (Ar–CH).
1
d6) δ = 6.93 (d, 2P, PB), 22.10 (t, 1P, PA). H NMR (400
MHz, DMSO-d6) δ = 7.24–7.26 (8H, d, J = 8.8 Hz, H6,
Ar–H), 7.34–7.37 (4H, H3, Ar–H), 7.44–7.47 (14H, m, H4,
H15–17, Ar–H), 7.56 (4H, d, J = 15.6 Hz, H10, –CH=),
7.86–7.90 (4H, d, J = 15.6 Hz, H11, =CH–), 7.98–8.04
(12H, m, H2, H7, Ar–H), 8.17–8.19 (4H, d, H14, Ar–H).
13C NMR (400 MHz, DMSO-d6) δ = 132.62 C1 (P–C(Ar)),
128.83–128.97 C2 (Ar-CH), 130.26–130.37 C3 (Ar–CH),
132.48 C4 (Ar–CH), 154.14 C5 (Ar–C), 121.49 C6
(Ar–CH), 132.48 C7 (Ar–CH), 134.89 C8 (Ar–C), 187.96
C9 (C=O), 124.67 C10 (–CH=), 139.14 C11 (=CH–),
132.62 C12 (Ar–C), 134.76 C13 (Ar–C), 131.10 C14
(Ar–CH), 129.01 C15 (Ar–CH), 130.46 C16 (Ar–CH),
128.08 C17 (Ar–CH).
2,2,4,4-Tetra(4′-oxy-3-chlorochalcone)-6,6-diphenylcyclotr
iphosphazene (6)
2,2,4,4-Tetra(4′-oxy-3-fluorochalcone)-6,6-diphenylcyclotr
iphosphazene (8)
For the synthesis of compound 6 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1d (3.6 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 24 h. Yield: 1.98 g (65%). M.p.: 145–146 °C. Anal.
Calcd. For C72H50Cl4N3O8P3: C, 65.52; H, 3.82; N, 3.18.
Found: C, 66.01; H, 3.89; N, 3.27. MALDI-MS: m/z calc.
1319.92. Found: 1319.98. FT-IR (KBr, cm−1) ν 3003 and
3059 (C–H aromatic), 2836 and 2967 (C–H Aliphatic),
1662 (C=O), 1503, 1580, and 1597 (C=C), 1179 and 1203
(P=N), 932 (P-O-Ph). 31P NMR (400 MHz, DMSO-d6) δ
= 6.68 (d, 2P, PB), 22.12 (t, 1P, PA). 1H NMR (400 MHz,
DMSO-d6) δ = 7.23–7.25 (8H, d, J = 8.4 Hz, H6, Ar–H),
For the synthesis of compound 8 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol), 1f
(3.37 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol) for
30 h. Yield: 1.93 g (66%). M.p.: 169–170 °C. Anal. Calcd.
For C72H50Cl4N3O8P3: C, 68.96; H, 4.02; N, 3.35. Found: C,
69.03; H, 4.11; N, 3.29. MALDI-MS: m/z calc. 1254.1.
Found [M+H]+: 1255.6. FT-IR (KBr, cm−1) ν 3001 and
3060 (C–H aromatic), 2838 and 2969 (C–H Aliphatic), 1662
(C=O), 1504, 1581, and 1597 (C=C), 1179 and 1203
(P=N), 930 (P-O-Ph). 31P NMR (400 MHz, DMSO-d6) δ =
1
7.34–7.37 (4H, H3, Ar–H), 7.43–7.49 (14H, m, H14–17
,
6.91 (d, 2P, PB), 22.15 (t, 1P, PA). H NMR (400 MHz,
Ar–H), 7.64–7.68 (4H, d, J = 15.6 Hz, H10, –CH=),
7.76–7.78 (4H, d, H2, Ar–H), 7.91–7.95 (4H, d, J = 15.6
Hz, H11, =CH–), 8.02–8.06 (12H, m, H7, H13, Ar–H). 13C
NMR (400 MHz, DMSO-d6) δ = 134.25 C1 (P–C(Ar)),
128.84–128.97 C2 (Ar–CH), 130.24–130.35 C3 (Ar–CH),
130.66 C4 (Ar–CH), 154.12 C5 (Ar–C), 121.42 C6
DMSO-d6) δ = 7.24–7.26 (8H, d, J = 8.8 Hz, H6, Ar–H),
7.28–7.37 (8H, m, H3, H13, Ar–H), 7.43–7.52 (10H, m, H4,
H15, H16, Ar–H), 7.64–7.71 (8H, m, H17, Ar–H and H10,
-CH=), 7.79–7.81 (4H, d, H2, Ar–H), 7.89–7.93 (4H, d, J =
15.6 Hz, H11, =CH–), 8.04–8.06 (8H, d, J = 8.8 Hz, H7,
Ar–H). 13C NMR (400 MHz, DMSO-d6) δ = 134.88 C1

Med Chem Res
(P–C(Ar)), 128.83–128.96 C2 (Ar–CH), 130.23–130.34 C3
(Ar–CH), 131.25–131.34 C4 (Ar–CH), 154.11 C5 (Ar–C),
121.45 C6 (Ar–CH), 130.10 C7 (Ar–CH), 137.54 C8 (Ar–C),
188.05 C9 (C=O), 123.45 C10 (–CH=), 143.13 C11 (=CH–),
137.62 C12 (Ar–C), 115–02–115.24 C13 (Ar–CH),
161.72–164.14 C14 (Ar–C), 117.71–117.92 C15 (Ar–CH),
130.10 C16 (Ar–CH), 126.10 C17 (Ar–CH).
J = 15.6 Hz, H10, –CH=), 7.97–8.01 (4H, d, J = 15.6 Hz,
H11, =CH–), 8.04–8.06 (8H, d, J = 7.6 Hz, H7, Ar–H),
8.30–8.32 (4H, dd, H14, Ar–H), 8.62 (4H, s, H13, Ar–H).
13C NMR (400 MHz, DMSO-d6) δ = 133.76–135.58 C1
(P–C(Ar)), 128.83–128.96 C2 (Ar–CH), 130.25–130.36 C3
(Ar–CH), 132.25 C4 (Ar–CH), 153.14 C5 (Ar–C), 121.47
C6 (Ar–CH), 131.10 C7 (Ar–CH), 134.82 C8 (Ar–C),
187.95 C9 (C=O), 123.92 C10 (–CH=), 141.15 C11
(=CH–), 130.86 C12 (Ar–C), 151.51 C13 (Ar–CH), 150.81
C14 (Ar–C), 124.36 C15 (Ar–CH), 135.58 C16 (Ar–CH).
2,2,4,4-Tetra(1-(4′-oxyphenyl)-3-(2-pyridine)-2-propene-1-
one)-6,6-diphenylcyclotriphosphazene (9)
For the synthesis of compound 9 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1g (3.14 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 24 h. Yield: 1.76 g (64%). M.p.: 156–157 °C. Anal.
Calcd. For C68H50N7O8P3: C, 68.86; H, 4.25; N, 8.27.
Found: C, 68.92; H, 4.30; N, 8.31. MALDI-MS: m/z calc.
1186.08. Found [M+H]+: 1187.10. FT-IR (KBr, cm−1) ν
3006 and 3065 (C–H aromatic), 2838 and 2930 (C–H
Aliphatic), 1661 (C=O), 1503, 1579, and 1597 (C=C),
1177 and 1203 (P=N), 928 (P-O-Ph). 31P NMR (400 MHz,
DMSO-d6) δ = 7.03 (d, 2P, PB), 22.14 (t, 1P, PA). 1H
NMR (400 MHz, DMSO-d6) δ = 7.26–7.28 (8H, d, J = 8.8
Hz, H6, Ar–H), 7.34–7.35 (4H, H3, Ar–H), 7.41–7.46 (10H,
m, H2, H 4, H14, Ar–H), 7.65–7.69 (4H, d, J = 15.6 Hz, H10,
–CH=), 7.87–7.89 (8H, m, H15, H16, Ar–H), 7.95–7.97
(8H, d, J = 8.8 Hz, H7, Ar–H), 8.03–8.07 (4H, d, J = 15.6
Hz, H11, =CH–). 13C NMR (400 MHz, DMSO-d6) δ =
133.71–135.07 C1 (P–C(Ar)), 128.81–128.94 C2 (Ar–CH),
130.27–130.38 C3 (Ar–CH), 132.24 C4 (Ar–CH), 153.13
C5 (Ar–C), 121.64 C6 (Ar–CH), 130.95 C7 (Ar–CH),
134.86 C8 (Ar–C), 188.54 C9 (C=O), 125.26 C10 (–CH=),
143.61 C11 (=CH–), 154.07 C12 (Ar–C), 150.51 C13
(Ar–CH), 130.95 C14 (Ar–C), 137.63 C15 (Ar–CH), 130.95
C16 (Ar–CH).
Thermal behaviors of compounds 3–10
The melting points of the synthesized cyclotriphosphazene
compounds were determined by heating them up at the
heating rate of 20 °C min−1 until it reached 250 °C and by
recording the DSC curves from DSC thermograms. The
thermal degradation of compounds 3–10 were determined
by heating them up at the heating rate of 10 °C min−1 until
it reached 900 °C and then by recording the TGA curves.
TGA measurements were carried out on approximately 5
mg samples at a heating rate of 10 °C min−1 under condi-
tions of nitrogen flow (10 cm3 min−1). DSC and TGA
curves of 3–10 were given in Figs. S1 (A) and (B),
respectively. In general, the starting degradation tempera-
tures of compounds is higher than 250 °C and 50% weight
loss are generally higher than 500 °C. According to the
results of TGA measurements, the temperatures at which
degradation started on as well as the temperature ratings at
which degradation proved to be 50%, and the residue per-
centages at 900 °C are given in Table 1.
Dielectric properties of compounds 3–10
2,2,4,4-Tetra(1-(4′-oxyphenyl)-3-(3-pyridine)-2-propene-1-
one)-6,6-diphenylcyclotriphosphazene (10)
Dielectric constant, loss factors, and electrical conductivity
To examine the dielectric behaviors of the synthesized
cyclotriphosphazenes, compounds 3–10 were turned into
tablets with the help of a disc under the pressure of 4 tons,
and the disc thickness was measured, in addition to which
the Cp,DF, and Gp values parameters were measured with
the help of golden conductors. The measurements were
performed at the range of 100 Hz–20 kHz. The dielectric
constant, dielectric factors, and ac conductivity of 3–10
were calculated with the help of the following Eqs. (1), (2),
and (3), respectively (Singh and Gupta 1998; Biryan and
Demirelli 2016). As for the obtained compounds, the results
of the dielectric constant (ε′), dielectric loss (ε″), and con-
ductivity (logσ) that were measured against the frequency
are given in Fig. 1. In Table 1, the dielectric results at the
For the synthesis of compound 10 was used the synthesis
procedure in 3 and was prepared using 2 (1 g, 2.32 mmol),
1h (3.16 g, 13.92 mmol) and K2CO3 (1.92 g, 13.92 mmol)
for 30 h. Yield: 2.06 g (75%). M.p.: 188–189 °C. Anal.
Calcd. For C68H50N7O8P3: C, 68.86; H, 4.25; N, 8.27.
Found: C, 68.93; H, 4.28; N, 8.33. MALDI-MS: m/z calc.
1186.08. Found [M+H]+: 1187.11. FT-IR (KBr, cm−1) ν
3036 and 3058 (C–H aromatic), 2930 and 2963 (C–H
Aliphatic), 1663 (C=O), 1504, 1569, and 1604 (C=C),
1174 and 1200 (P=N), 934 (P-O-Ph). 31P NMR (400 MHz,
DMSO-d6) δ = 6.92 (d, 2P, PB), 22.16 (t, 1P, PA). 1H
NMR (400 MHz, DMSO-d6) δ = 7.24–7.26 (8H, d, J = 7.6
Hz, H6, Ar–H), 7.34–7.36 (6H, m, H3, H6, Ar–H),
7.47–7.50 (12H, m, H2, H 15, H16, Ar–H), 7.70–7.74 (4H, d,

Med Chem Res
fixed frequency (1000 Hz) are shown.
the cell types. All the cells in 25 cm2 culture flasks were fed
within RPMI-1640 medium (that which was prepared by
adding 10% FCS, 100 U/mL penicillin and 0.1 mg/mL
streptomycin into it). The media of the cells kept in a humid
atmosphere at 37 °C and in a carbon dioxide-incubator (5%
CO2) were changed twice a week. When the cells became
confluent, they were extracted from the flasks by using
trypsin-EDTA solution, after which they were transferred
into the plaques with 96 wells and were used in the analyses
of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bro-
mide (MTT). The solutions of the substances in DMSO
solvent were used in cell culture. Thus, in the comparison of
the results obtained, the effects of the substances on DMSO
were determined by performing a statistical analysis. The
toxic effect of DMSO on the cell was determined, and it was
seen that this was statistically insignificant in spite of its
toxic effect. 1, 5, 25, 50, and 100 μM-concentrations per-
taining to the tested compounds (3–10) as well as the sol-
vent with the same amount (DMSO) were added into the
wells where cells were put, and then it was left for incu-
bation in CO2 incubator (Panasonic/Japan) at 37 °C for
24 h. In the wake of the incubations, the viability rate of the
cells was determined on a hemocytometer by using 0.4%
tryphan blue (Singh and Singh 2002).
The compliance of the groups with the normal distribu-
tion was evaluated through Kolmogorov Smirnov test. In
the comparison of the groups, on the other hand, one-way
analysis of variance was used. The homogeneity of the
variances was analyzed through Levene’s test. It was
observed after one-way analysis of variance that the var-
iances were not homogeneous. For multiple comparisons,
TAMHANE T2 test was used. The value p < 0.05 was
accepted as statistically significant. The data were expressed
in the form of mean standard error. The LogIC50 values
were calculated through a Graphpad prism 6 programs on a
computer.
d
ε0 ¼ Cp
Aε0
ð1Þ
ð2Þ
ð3Þ
ε00 ¼ ε0DF
d
σ ¼ Gp
A
where ɛ′ is dielectric constant, σ is ac conductivity, ɛ0 is the
dielectric constant of vacuum (8.854 × 10−12), d is the
thickness (m) and A is effective area (m2) of the sample, ɛ″
is dielectric loss factors and C is the capacitance (F) of test
device.
In vitro assay for cytotoxic activities
The % changes in cell viability rates that were caused by 1,
5, 25, 50, and 100 μM concentrations specified for the
synthesized compounds were determined against human
ovarian cancer (A2780) and human prostate cancer (PC-3
and LNCaP) cell lines. The cytotoxic activities of the
substituted-cyclotriphosphazene compounds was analyzed
by MTT assay which is based on reduction of MTT salt by
mitochondria of alive cells. This method is based on the
principle that the MTT dye is capable of breaking down the
tetrazolium ring. In this method, MTT is actively absorbed
into the live cells, and the reaction is catalyzed by mito-
chondrial succinate dehydrogenase and is, then, reduced to
blue-purple colored, water-insoluble formazan. The forma-
tion of formazan is only observed in the live cells in which
active mitochondria are found, which is also accepted as the
indicator of cell viability, and the value specified spectro-
photometrically is associated with the number of living
cells. 0.5 mg/mL of MTT study solution was prepared from
out of the stock MTT solution prepared within sterile PBS,
and it was added into the plaques with 96 wells. After
keeping it waiting for 3 h in the incubator, the optical
densities of the cells in the plaques were made to be read at
550 nm- wave lengths on the ELISA device (Synergy HT
ABD). By having the control wells read, the average of the
obtained absorbance values was taken, and this value was
accepted as 100% live cell. The absorbance values obtained
from the solvent (the group into which only dimethylsulf-
oxide (DMSO) was added) as well as agent-applied wells
were proportioned to the control absorbance values, in
addition to which the percentage (%) viability values were
calculated (Mosamann et al. 1986; Singh and Singh 2002;
Özen et al. 2016; Kucukbay et al. 2016). These experiments
were carried out on different days by being repeated for at
least 10 times, independent from each other.
Acknowledgments This work was supported financially by The
Scientific & Technological Research Council of Turkey (TUBITAK)
(Project Number: 115Z101).
Compliance with ethical standards
Conflict of interest The authors declare that they have no com-
peting interest.
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Med Chem Res
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