Synthesis and spectroscopic characterizations of hexakis[(1-(4′-oxyphenyl)-3-(substituted-phenyl)prop-2-en-1-one)]cyclotriphosphazenes: their in vitro cytotoxic activity, theoretical analysis and molecular docking studies

Article abstract of DOI:10.1080/07391102.2020.1846621

The hexachlorocyclotriphosphaze compound (N₃P₃Cl₆, HCCP) was reacted with excess (E)-(1-(4′-oxyphenyl)-3-(substituted-phenyl)prop-2-en-1-ones (2-11) to produce hexakis[(1-(4-oxyphenyl)-3-(substituted-phenyl)prop-2-en-1-one

Full text of DOI:10.1080/07391102.2020.1846621

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis and spectroscopic characterizations
of hexakis[(1-(4′-oxyphenyl)-3-(substituted-
phenyl)prop-2-en-1-one)]cyclotriphosphazenes:
their in vitro cytotoxic activity, theoretical analysis
and molecular docking studies
Hacer Doğan , Mehmet Refik Bahar , Eray Çalışkan , Suat Tekin , Harun Uslu ,
Feride Akman , Kenan Koran , Süleyman Sandal & Ahmet Orhan Görgülü
To cite this article: Hacer Doğan , Mehmet Refik Bahar , Eray Çalışkan , Suat Tekin , Harun
Uslu , Feride Akman , Kenan Koran , Süleyman Sandal & Ahmet Orhan Görgülü (2020):
Synthesis and spectroscopic characterizations of hexakis[(1-(4′-oxyphenyl)-3-(substituted-
phenyl)prop-2-en-1-one)]cyclotriphosphazenes: their inꢀvitro cytotoxic activity, theoretical
analysis and molecular docking studies, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1846621
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1846621
Synthesis and spectroscopic characterizations of hexakis[(1-(40-oxyphenyl)-3-
(substituted-phenyl)prop-2-en-1-one)]cyclotriphosphazenes: their in vitro
cytotoxic activity, theoretical analysis and molecular docking studies
a
b
c
b
d
, Feride Akmane, Kenan Koranf,
ꢀ
Hacer Dogan , Mehmet Refik Bahar , Eray C¸alıs¸kan , Suat Tekin , Harun Uslu
b
€
€
€ €a
and Ahmet Orhan Gorgulu
Suleyman Sandal
a
b
ꢀ
Chemistry Department, Science Faculty, Firat University, Elazıg, Turkey; Physiology Department, Faculty of Medicine, Inonu University,
c
d
Malatya, Turkey; Department of Chemistry, Faculty of Science, Bingol University, Bingol, Turkey; Department of Anesthesiology, Vocational
e
ꢀ
School of Health Services, Firat University, Elazıg, Turkey; Vocational School of Technical Sciences, Bingol University, Bingol, Turkey;
f
ꢀ
Department of Food Processing, Karakoc¸an Voc. School, Firat University, Elazıg, Turkey
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
The hexachlorocyclotriphosphaze compound (N3P3Cl6, HCCP) was reacted with excess (E)-(1-(40-oxy-
phenyl)-3-(substituted-phenyl)prop-2-en-1-ones (2-11) to produce hexakis[(1-(4-oxyphenyl)-3-(substi-
tuted-phenyl)prop-2-en-1-one)]cyclotriphosphazenes (CP 2-11). The structures of products (CP 2-11)
were confirmed using elemental analysis, FT-IR, MS spectral analysis as well as 31P, 1H and 13C-APT
NMR techniques and their thermal properties determined by TGA and DSC techniques. The HOMO-
LUMO energy gap and chemical reactivity identifiers were calculated and HOMO and LUMO images
were viewed. According to the calculations, all the chemical potential values of CP 2-11 are negative
and it shown that the molecules are stable. The in vitro cytotoxic of CP 2-11 investigated and their
activity potentials were evaluated by molecular docking studies with Autodock Vina softwares. CP 2-
11 compounds were found to demonstrate cytotoxic activity against human cancer cell lines (A2780,
LNCaP and PC-3). The CP 2-11 compounds reduced the cell viability against all cancer cell lines in the
range 36%–90% especially. The results showed that these compounds are powerful candidate mole-
cules for pharmaceutical applications.
Received 31 May 2020
Accepted 1 November 2020
KEYWORDS
MTT assay; spiro-
phosphazene; cytotoxicity;
molecular docking;
structural characterizations
Synthesis, spectroscopic and thermal properties, in vitro cytotoxic properties, theoretical analysis and
molecular docking studies of hexa substituted cyclotriphosphazenes CP 2-11 were investigated.
1. Introduction
Phosphazene compounds containing phosphorus–nitrogen
bonds in their structure are bounded by two organic or inor-
ganic side groups (R) in each phosphorus atom in repeated
Phosphazenes, which is an important class in inorganic com-
pounds, are synthesized by the reaction of phosphorus com-
pounds and nucleophiles containing nitrogen atoms. –P ¼ N– units. Their general structure can be straight chain,
ꢀ
Department of Food Processing, Karakoc¸an Voc. School, Firat University, Elazıg 23600, Turkey
CONTACT Kenan Koran
kenan.koran@gmail.com
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
H. DOĞAN ET AL.
cyclic or polymeric (Allcock, 1972; Gleria & De Jaeger, 2004). synthesized according to Claisen-Schmidt condensation by
the method in the literature (Bist et al., 2017; Brian et al.,
1989; Chaudhary et al., 2020, 2020; Chimenti et al., 2004;
Ingle & Upadhyay, 2005; Jeyasheela & Subramanian, 2019;
Mirzaei et al., 2020; Satyanarayana et al., 2004; Shenvi et al.,
2013; Xie et al., 2011). In the second stage, hexa substituted
cyclophosphazene compounds CP 2-11 were obtained by
reaction of the compounds 2-11 obtained in the previous
step with the compound of hexachlorocyclotriphosphazene
(HCCP). The structures of these pure compounds were char-
Reactive –Cl atoms in their structure provide many important
reactions such as alcoholism, phenolysis, hydrolysis, aminoly-
sis and Friedel-Crafts (Akbas¸ et al., 2013; C¸os¸ut et al., 2009;
Kuan & Lin, 2004; McBee et al., 1965; Siwy et al., 2006). Due
to the chlorine atoms in its structure, substitution reactions
with -di, -tri, tetra, penta or hexa function can be performed
with aliphatic or aromatic nucleophiles (Chen et al., 2019;
_
ꢀ
Ibis¸oglu et al., 2020; Lee et al., 2020). The cyclic phosphos-
phazene was chosen as the main skeleton because of its
characteristics such as the easy functioning of the cyclic
phosphazene compounds, the stability of the substituted
phosphazenes, the possibility of different biological or phys-
ical properties depending on the characteristics of the substi-
tuted side groups, and the possibility of multiple functional
1
acterized using 31P, H-NMR, 13C-APT NMR, FT-IR and MALTI-
TOF MS spectroscopic methods. In addition, thermal stability
of the compounds was determined by taking DSC and TGA
thermograms. The HOMO-LUMO energy gap and chemical
reactivity identifiers were calculated using the Gaussian 09W
package in DFT (B3LYP) methods set on the basis of 6-31G
(d, p), and HOMO and LUMO images were viewed using
GaussView 5.0. In the last step, hexa substituted cyclotriphos-
phazenes CP 2-11 at different concentrations (1, 5, 25 and
50 mM) are employed against human ovarian (A2780) and
human prostate (PC-3 and LNCaP) cancer cell lines to investi-
gate % cell viability and in vitro cytotoxicity activities using
the MTT assay method. LogIC50/IC50 values of the com-
pounds were calculated. CP 2-11 compounds were found to
demonstrate cytotoxic activity against human cancer cell
lines (A2780, LNCaP and PC-3). The results showed that these
compounds are powerful candidate molecules for pharma-
ceutical applications. Disruption of the microtubules can lead
to apoptosis induction as it has vital importance to the con-
tinuation of the cell cycle (Carlson, 2008). Therefore, the lit-
erature emphasizes that tubulin inhibitors can be used as
effective anticancer drugs (Prota et al., 2014; Sengupta &
Thomas, 2006). Binding sites for colchicine is well determined
in the tubule (Fleming, 2010). Potential tubulin inhibition
effects with synthesized molecules were determined by
Tubulin–Colchicine complex. The interactions of compounds
with the Tubulin–Colchicine complex were put forth via
molecular docking studies.
groups to coexist (Andrianov, 2008; Gleria
Jaeger, 2004).
&
De
Substitution reactions, one of the most important reac-
tions in phosphazene chemistry, take place with nucleophiles
such as alcohol, phenol and diol. The most important reason
why these reactions take place in numerous studies is that
the by-product formation is minimal and the products
formed are stable, which allows the products to be purified
and characterized easily. It is another factor in the study that
the aryloxy substituted phosphazene compounds have very
high thermal and hydrolytic stability (Allcock, 1972; Koran &
€
€ €
Gorgulu, 2018; Shaw, 1986). Such properties have made
these structures important compounds that can be used
both in obtaining high temperature resistant materials and
in synthesizing new polymers. Halocyclotriphosphazene com-
pounds can be easily obtained in a suitable organic solvent
medium using sodium or potassium salts of alcohol or phe-
nols or a base such as triethylamine, potassium or sodium
€
€ €
carbonate (Carriedo et al., 1996; Gorgulu et al., 2015; Koran
et al., 2016). Depending on the properties of the substituted
groups on the cyclotriphosphazene compound, these com-
pounds can display many different properties, including non-
linear optics, solar cell and photodiode, electrical properties,
chemical sensor, tribological behavior and OFET application,
ꢀ
antimicrobial and cytotoxic activities (Alidagı et al., 2013;
2. Experimental section
€
Asmafiliz et al., 2019; Bolink et al., 2008; Destegul et al., 2019;
Elgazzar et al., 2017; Fei et al., 2008, 2011; Hadji & Rahmouni,
2016; Koran, 2019; Ozcan et al., 2019; Tanrıverdi Ec¸ik
2.1. Materials and measurements
€
All the chemicals were bought from Sigma-Aldrich and
Merck. 1H and 13C-APT NMR spectra were recorded via
Bruker DPX-400 MHz spectrometer and Perkin Elmer FT-IR
spectrometer was used for Infrared analysis. Microanalysis
was acquired by a LECO 932 CHNS-O apparatus. Mass spectra
were obtained by a Bruker microflex LT MALDI-TOF MS spec-
trometer. The thermal behavior of hexa substituted cyclotri-
phosphazenes was investigated by differential scanning
calorimetry (DSC, 20 ꢀC/min) and thermogravimetric analysis
(TGA, 10 ꢀC/min) using a SHIMADZU. Human prostate (PC-3
and LNCaP) and ovarian cancer cell lines required for cell cul-
ture studies were purchased from ATCC (American Type
Culture Collection). Penicillin, trypsin, streptomycin, newborn
calf serum and Dulbecco’s modified Eagle’s medium (DMEM)
et al., 2019).
The articles on the synthesis of cyclic phosphazenes and
the investigation of their various biological and physical
properties have been reported in the literature (Bobrov et al.,
2020; Dagdag et al., 2019; Januszewski et al., 2018; Jiang
€
et al., 2015; Nikovskii et al., 2018; Tumer et al., 2018; Wang
et al., 2018; Yang et al., 2019). There are, however, no studies
about synthesis, in vitro cytotoxic activity, theoretical analysis
and molecular docking studies of hexa substituted cyclotri-
phosphazene compounds CP 2-11.
The aim of this study is to prepare hexa substituents of
cyclotriphosphazenes, which constitute an important class of
thermally stable inorganic heterocyclic compounds, and to
investigate in vitro cytotoxicity properties, theoretical analysis were purchased from Hyclone (Waltham, MA, USA).
and molecular docking studies. Firstly, compounds 2-11 were Panasonic was used as a CO2 incubator, Nuve MN-120 as a

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
biological safety cabinet, BioTEK spectrophotometer as a
microplate reader, Reverse Microscope SOIF-XDS for cell
maintenance and control, and Nuve was preferred for
sterilization.
Microanalysis: Found: C 66.79, H 4.96%. requires C 66.77, H
4.94%. FT-IR (KBr) ꢀmax (cmꢁ1): 3008 and 3058 ꢀAr–CH, 2837 and
2941 ꢀAliphatic-CH, 1655 ꢀC¼O, 1500, 1572 and 1596 ꢀC¼C, 1158
and 1180 ꢀP¼N, 946 ꢀP–O–Ph
.
31P-NMR (DMSO-d6, ppm) d ¼ 8.25
(3P, s, PA, A3). H NMR (DMSO-d6, ppm) d ¼ 3.85 (18H, s, H17
(–OCH3)), 3.91 (18H, s, H16 (–OCH3)), 6.54–6.58 (12H, m, Ar–H12,
Ar–H14), 7.12–7.14 (12H, d, J ¼ 7.6 Hz, Ar–H3,5), 7.57–7.61 (6H, d,
J ¼ 15.6 Hz, H8 (–CH¼)), 7.80–7.82 (6H, d, J ¼ 8.4 Hz, Ar–H15),
7.92–7.96 (6H, d, J ¼ 15.6 Hz, H9 (¼CH–)), 7.98–8.00 (12H, d,
1
2.2. Synthesis and characterization
Starting compounds 2-11 were synthesized according to the
procedure reported in the literature. A similar method was
used in the synthesis of CP 2-11. The detailed procedure of
the reaction is given only in the synthesis of the compound
CP-2.
J ¼ 7.6,
Ar–H2,6).
13C-APT
NMR
(DMSO-d6,
ppm)
d ¼ 135.99 Ar–C1, 130.78 Ar–C2,6, 118.76 Ar–C3,5, 153.09 Ar–C4,
188.10 C7 (–C ¼ O), 121.15 C8 (–CH¼), 139.59 C9 (¼CH–),
116.31 Ar–C10, 160.43 Ar–C11, 098.55 Ar–C12, 163.60 Ar–C13,
106.71 Ar–C14,
130.47 Ar–C15,
56.16 C16
(–OCH3),
2.2.1. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(3-methoxy-
phenyl)prop-2-en-1-one)]cyclotriphosphazene (CP-2)
55.93 C17 (–OCH3).
After the argon and airless medium reaction apparatus was
prepared, 0.58 mmol (0.2 g) HCCP was added to the three
necked reaction flask containing 40 mL of dry acetone at
0 ꢀC. 4.03 mmol (0.56 g) of anhydrous K2CO3 was added to
this mixture and the reaction was stirred for 30 min. Finally,
the solution of 2 (3.49 mmol, 0.89 g) in acetone was added
dropwise to the mixture. The reaction was then stirred for
about 1 h. Then it was mixed with magnetic stirrer by heat-
ing at the boiling point of acetone. The reaction was
stopped by following thin layer chromatography. Reaction
time is about 12 h. After the reaction was stopped, the solid
in the reaction medium was filtered off. After some of the
acetone was evaporated, it was precipitated by adding 3%
to 250 mL NaOH solution. The precipitated solid was filtered
off and washed with plenty of water until pH 7. Finally, it
was washed with ethyl alcohol and dried. The dried solid
was again dissolved in chloroform and precipitated in n-hex-
ane. It was obtained as pure in 85% yield. White solid; m.p.
125–126 ꢀC. MALDI-MS: N3P3O18C96H78 m/z calc. 1654.60;
found: 1655.10 [M þ H]þ. Microanalysis: Found: C 70.02, H
4.79%. requires C 69.69, H 4.75%. FT-IR (KBr) ꢀmax (cmꢁ1):
2.2.3. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(3,4-dime-
thoxyphenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-4)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol), 4 (1 g, 3.49 mmol) and K2CO3 (0.56 g, 4.03 mmol).
Yield: 76%; white solid. m.p. 189–190 ꢀC. MALDI-MS:
N3P3O24C102H90 m/z calc. 1834.76; found: 1834.499 and
1857.468 [M þ Na]. Microanalysis: Found: C 66.80, H 4.98%.
requires C 66.77, H 4.94%. FT-IR (KBr) ꢀmax (cmꢁ1): 3001 and
3072 ꢀAr–CH, 2837 and 2935 ꢀAliphatic-CH, 1653 ꢀC¼O, 1512, 1575,
1594 and 1625 ꢀC¼C, 1159 and 1184 ꢀP¼N, 948 ꢀP–O–Ph.
31P-
1
NMR (DMSO-d6, ppm) d ¼ 8.21 (3P, s, PA, A3). H NMR (DMSO-
d6, ppm) d ¼ 3.84 (18H, s, H16 (–OCH3)), 3.81 (18H, s, H17
(–OCH3)), 6.95–6.97 (6H, d, J ¼ 8.4 Hz, Ar–H15), 7.14–7.16 (12H,
d, J ¼ 7.6 Hz, Ar–H3,5), 7.24–7.26 (6H, d, J ¼ 8.4 Hz, Ar–H14), 7.42
(6H, s, Ar–H11), 7.62–7.65 (12H, m, H8, H9), 8.04–8.06 (12H, d,
J ¼ 8.4 Hz, Ar–H2,6). 13C-APT NMR (DMSO-d6, ppm)
d ¼ 135.74 Ar–C1, 130.99 Ar–C2,6, 121.16 Ar–C3,5, 153.22 Ar–C4,
188.00 C7 (–C ¼ O), 119.31 C8 (–CH¼), 145.39 C9 (¼CH–),
137.74 Ar–C10, 111.27 Ar–C11, 149.34 Ar–C12, 151.79 Ar–C13,
111.85 Ar–C14,
124.34 Ar–C15,
56.02 C16
(–OCH3),
56.06 C17 (–OCH3).
3001 and 3065 ꢀAr–CH, 2837 and 2961 ꢀAliphatic-CH, 1658 ꢀC¼O
1501, 1578 and 1597 ꢀC¼C, 1158 and 1178 ꢀP¼N, 949 ꢀP–O–Ph
,
.
31P-NMR (DMSO-d6, ppm) d ¼ 8.10 (3P, s, PA, A3). 1H NMR
(DMSO-d6, ppm) d ¼ 3.81 (18H, s, H16 (–OCH3)), 7.0–7.03 (6H,
d, Ar–H13), 7.16–7.18 (12H, d, J ¼ 8.4 Hz, Ar–H3,5), 7.33–7.38
(18H, m, Ar–H11,14,15), 7.60–7.64 6, d, J ¼ 15.6 Hz, H8 (–CH¼)),
7.79–7.83 (6H, d, J ¼ 15.6 Hz, H9 (¼CH–)) and 8.06–8.08 (12H,
d, J ¼ 8.4 Hz, Ar–H2,6).13C-APT NMR (DMSO-d6, ppm)
d ¼ 135.41 Ar–C1, 131.15 Ar–C2,6, 121.19 Ar–C3,5, 153.40 Ar–C4,
188.18 C7 (–C ¼ O), 122.16 C8 (–CH¼), 144.79 C9 (¼CH–),
136.35 Ar–C10, 121.19 Ar–C11, 160.04 Ar–C12, 113.95 Ar–C13,
130.31 Ar–C14, 122.08 Ar–C15 and 55.71 C16 (–OCH3).
2.2.4. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(2,3,4-trime-
thoxyphenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-5)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol), 5 (1.1 g, 3.49 mmol) and K2CO3 (0.56 g, 4.03 mmol).
Yield: 81%; white solid. m.p. 142–143 ꢀC. MALDI-MS:
N3P3O30C108H102 m/z calc. 2014.92; found: 2014.306.
Microanalysis: found: C 66.41, H 5.12%. requires C 64.38, H
5.10%. FT-IR (KBr) ꢀmax (cmꢁ1): 3000 and 3065 ꢀAr–CH, 2837 and
2938 ꢀAliphatic–CH, 1654 ꢀC¼O, 1512, 1572, 1586 and 1618 ꢀC¼C
,
1155 and 1180 ꢀP¼N, 942 ꢀP–O–Ph
.
31P-NMR (DMSO-d6) d ¼ 8.20
2.2.2. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(2,4-dime-
thoxyphenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-3)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol), 3 (1 g, 3.49 mmol) and K2CO3 (0.56 g, 4.03 mmol).
Yield: 77%; white solid. m.p. 135–136 ꢀC. MALDI-MS:
N3P3O24C102H90 m/z calc. 1834.76; found: 1834.286.
1
(3P, s, PA, A3). H NMR (DMSO-d6, ppm) d ¼ 3.75 (18H, s, H18
(–OCH3)), 3.79 (18H, s, H17 (–OCH3)), 3.85 (18H, s, H16 (–OCH3)),
6.84–6.86 (6H, d, J ¼ 8.8 Hz, Ar–H14), 7.14–7.16 (12H, d,
J ¼ 8.4 Hz, Ar–H3,5), 7.33–7.38 (18H, m, Ar–H11, Ar–H14, Ar–H15),
7.66–7.70 (6H, d, J ¼ 16 Hz, H8 (–CH¼)), 7.70–7.73 (6H, d,
J ¼ 8.8 Hz, Ar–H15), 7.85–7.89 (6H, d, J ¼ 15.6 Hz, H9 (¼CH–)),
8.01–8.03 (12H, d, J ¼ 8.4 Hz, Ar–H2,6). 13C-APT NMR (DMSO-d6,

4
H. DOĞAN ET AL.
ppm)
d ¼ 135.71 Ar–C1,
130.91 Ar–C2,6
,
121.20 Ar–C3,5
,
4.03 mmol). Yield: 62%; white solid. m.p. 121–122 ꢀC. MALDI-
MS: N3P3O12C90H54F12 m/z calc. 1690.33; found: 1690.226.
Microanalysis: Found: C 63.99, H 3.19%. requires C 63.95, H
3.22%. FT-IR (KBr) ꢀmax (cmꢁ1): 3005 and 3075 ꢀAr–CH, 2854
156.26 Ar–C4, 188.01 C7 (–C ¼ O), 120.09 C8 (–CH¼), 139.17 C9
(¼CH–),
121.32 Ar–C10,
142.06 Ar–C11,
153.17 Ar–C12,
153.56 Ar–C13, 108.74 Ar–C14, 123.85 Ar–C15, 61.70 C16 (–OCH3),
60.86 C17 (–OCH3), 56.40 C18 (–OCH3).
and 2945 ꢀAliphatic-CH, 1667 ꢀC¼O, 1502, 1595 and 1618 ꢀC¼C
,
1160 and 1183 ꢀP¼N, 946 ꢀP–O–Ph
.
31P-NMR (DMSO-d6)
d ¼ 7.93 (3P, s, PA, A3). 1H-NMR (DMSO-d6, ppm)
d ¼ 7.18–7.20 (12H, d, J ¼ 8.8 Hz, Ar–H3,5), 7.25–7.27 (6H,
Ar–H13), 7.56–7.61 (18H, m, H8 (–CH¼), Ar–H11, Ar–H15),
7.90–7.94 (6H, d, J ¼ 15.6 Hz, H9 (¼CH–)), 8.07–8.09 (12H, d,
J ¼ 8.8 Hz, Ar–H2,6). 13C-APT NMR (DMSO-d6, ppm)
d ¼ 135.01 Ar–C1, 131.31 Ar–C2,6, 121.12 Ar–C3,5, 153.50 Ar–C4,
187.78 C7 (–C ¼ O), 124.49 C8 (–CH¼), 141.94 C9 (¼CH–),
138.72 Ar–C10, 112.26 Ar–C11, 164.30 Ar–C12, 105.97 Ar–C13,
161.86 Ar–C14, 112.26 Ar–C15.
2.2.5. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(2,4,5-trime-
thoxyphenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-6)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol),
6
(1.1 g, 3.49 mmol) and K2CO3 (0.56 g,
4.03 mmol). Yield: 69%; white solid. m.p. 113–114 ꢀC. MALDI-
MS: N3P3O30C108H102 m/z calc. 2014.92; found: 2014.224.
Microanalysis: Found: C 66.42, H 5.08%. requires C 64.38, H
5.10%. FT-IR (KBr) ꢀmax (cmꢁ1): 3001 and 3065 ꢀAr–CH, 2834
and 2938 ꢀAliphatic-CH, 1654 ꢀC¼O, 1505, 1564 and 1597 ꢀC¼C
,
31P-NMR (DMSO-d6)
2.2.8. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(4-chloro-2-
fluorophenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-9)
1158 and 1180 ꢀP¼N, 945 ꢀP–O–Ph
.
d ¼ 8.25 (3P, s, PA, A3). 1H NMR (DMSO-d6, ppm) d ¼ 3.81
(18H, s, H18 (–OCH3)), 3.82 (18H, s, H17 (–OCH3)), 3.87 (18H, s,
H16 (–OCH3)), 6.64 (6H, d, Ar–H12), 7.09–7.11 (12H, d, J ¼ 8 Hz,
Ar–H3,5), 7.40 (6H, s, Ar–H15), 7.58–7.62 (6H, d, J ¼ 15.6 Hz, H8
(–CH¼)), 7.98–8.01 (6H, d, J ¼ 16 Hz, H9 (¼CH–)), 8.01–8.03
(12H, d, J ¼ 7.2 Hz, Ar–H2,6). 13C-APT NMR (DMSO-d6, ppm)
d ¼ 136.06 Ar–C1, 130.86 Ar–C2,6, 121.12 Ar–C3,5, 154.78 Ar–C4,
188.05 C7 (–C ¼ O), 118.29 C8 (–CH¼), 139.54 C9 (¼CH–),
114.62 Ar–C10, 143.40 Ar–C11, 97.64 Ar–C12, 153.38 Ar–C13,
153.07 Ar–C14, 111.28 Ar–C15, 56.65 C16 (–OCH3), 56.27 C17
(–OCH3), 56.22 C18 (–OCH3).
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol),
9
(0.97 g, 3.49 mmol) and K2CO3 (0.56 g,
4.03 mmol). Yield: 71%; white solid. m.p. 113–114 ꢀC. MALDI-
MS: N3P3O12C90H54F6Cl6 m/z calc. 1789.04; found: 1789.449.
Microanalysis: Found: C 60.42, H 3.04%. requires C 60.46, H
3.10%. FT-IR (KBr) ꢀmax (cmꢁ1): 3005 and 3075 ꢀAr-CH, 2854
and 2945 ꢀAliphatic-CH, 1663 ꢀC¼O, 1504, 1595 and 1600 ꢀC¼C
,
1158 and 1181 ꢀP¼N, 946 ꢀP–O–Ph
.
31P-NMR (DMSO-d6)
d ¼ 8.06 (3P, s, PA, A3). 1H-NMR (DMSO-d6, ppm)
d ¼ 7.16–7.18 (12H, d, Ar–H3,5), 7.28–7.31 (6H, d, Ar–H14),
7.42–7.45 (6H, d, Ar–H15), 7.61–7.65 (6H, d, J ¼ 16 Hz, H8
(–CH¼)), 7.76–7.88 (12H, m, H9 (¼CH–), Ar–H12), 7.99–8.02
(12H, d, Ar–H2,6). 13C-APT NMR (DMSO-d6, ppm)
d ¼ 134.79 Ar–C1, 131.0 Ar–C2,6, 121.51 Ar–C3,5, 154.06 Ar–C4,
187.95 C7 (–C ¼ O), 124.04 C8 (–CH¼), 135.76 C9 (¼CH–),
122.69 Ar–C10, 162.65 Ar–C11, 116.64 Ar–C12, 160.15 Ar–C13,
125.38 Ar–C14, 129.57 Ar–C15.
2.2.6. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(4-phenyl-
phenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-7)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol),
7
(1.05 g, 3.49 mmol) and K2CO3 (0.56 g,
4.03 mmol). Yield: 74%; white solid. m.p. 256–257 ꢀC. MALDI-
MS: N3P3O12C126H90 m/z calc. 1931.04; found: 1931.561.
Microanalysis: Found: C 78.39, H 4.73%. requires C 78.37, H
4.70%. FT-IR (KBr) ꢀmax (cmꢁ1): 3001 and 3065 ꢀAr–CH, 2834 and
2938 ꢀAliphatic-CH, 1661 ꢀC¼O, 1504, 1558 and 1597 ꢀC¼C, 1161
2.2.9. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(3-(trifluoro-
methyl)phenyl)prop-2-en-1-one)]cyclotriphospha-
zene (CP-10)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol), 10 (1 g, 3.49 mmol) and K2CO3 (0.56 g,
4.03 mmol). Yield: 79%; white solid. m.p. 115–116 ꢀC. MALDI-
MS: N3P3O12C96H60F18 m/z calc. 1882.44; found: 1882.555.
Microanalysis: Found: C 61.29, H 3.25%. requires C 61.25, H
3.21%. FT-IR (KBr) ꢀmax (cmꢁ1): 3005 and 3075 ꢀAr–CH, 2854
and 1177 ꢀP¼N, 969 ꢀP–O–Ph
.
31P-NMR (DMSO-d6) d ¼ 8.21 (3P,
s, PA, A3). 1H NMR (DMSO-d6, ppm) d ¼ 7.35–7.7 (12H, d,
J ¼ 8.4 Hz, Ar–H3,5), 7.41–7.42 (6H, d, Ar–H19), 7.46–7.49 (12H, t,
Ar–H18,20), 7.70–7.72 (18H, m, H8 (–CH¼) ve Ar–H11,15),
7.73–7.78 (12H, d, Ar–H12,14), 7.91–7.93 (12H, d, J ¼ 8.4 Hz,
Ar–H17,21), 7.93–7.96 (6H, d, J ¼ 15.2 Hz, H9 (¼CH–)), 8.22–8.24
(12H, d, J ¼ 8.8 Hz, Ar–H2,6). 13C-APT NMR (DMSO-d6, ppm)
d ¼ 135.58 Ar–C1, 131.28 Ar–C2,6, 121.38 Ar–C3,5, 153.58 Ar–C4,
188.19 C7 (–C ¼ O), 122.03 C8 (–CH¼), 144.29 C9 (¼CH–),
134.17 Ar–C10, 128.47 Ar–C11,15, 130.08 Ar–C12,14, 142.57 Ar–C13,
139.63 Ar–C16, 127.19 Ar–C17,21, 129.48 Ar–C18,20, 127.47 Ar–C19.
and 2945 ꢀAliphatic-CH, 1663 ꢀC¼O, 1501, 1561 and 1598 ꢀC¼C
,
1157 and 1181 ꢀP¼N, 957 ꢀP–O–Ph
.
31P-NMR (DMSO-d6)
d ¼ 8.03 (3P, s, PA, A3). 1H-NMR (DMSO-d6, ppm)
d ¼ 7.19–7.21 (12H, d, J ¼ 8.8 Hz, Ar–H3,5), 7.59–7.63 (6H, t,
Ar–H14), 7.66–7.70 (6H, d, J ¼ 15.6 Hz, H8 (–CH¼)), 7.72–7.74
(6H, d, J ¼ 8 Hz, Ar–H13), 7.94–7.98 (6H, d, J ¼ 15.6 Hz, H9
(¼CH–)), 8.02–8.04 (6H, d, J ¼ 8 Hz, Ar–H15), 8.09–8.11 (12H, d,
J ¼ 8.8 Hz, Ar–H2,6), 8.15 (6H, s, Ar–H11). 13C-APT NMR (DMSO-
2.2.7. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(3,5-difluor-
ophenyl)prop-2-en-1-one)]cyclotriphosphazene
(CP-8)
The procedure is similar to that of CP-2, using HCCP (0.2 g, d6, ppm) d ¼ 135.11 Ar–C1, 131.31 Ar–C2,6
, ,
121.14 Ar–C3,5
0.58 mmol),
8
(0.91 g, 3.49 mmol) and K2CO3 (0.56 g, 153.50 Ar–C4, 187.89 C7 (–C ¼ O), 125.57 C8 (–CH¼), 142.82 C9

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
(¼CH–),
136.01 Ar–C10,
123.65 Ar–C11,
130.00 Ar–C12,
2.4. Determination of in vitro cytotoxic activity with
MTT assay
127.12 Ar–C13, 130.23 Ar–C14, 133.10 Ar–C15, 125.80 Ar–C16.
In this study, in vitro cytotoxic activities of compounds CP 2-
11 were tested by colorimetric MTT assay against human
ovarian (A2780), prostate (LNCaP and PC-3) cancer cell lines.
All the cell lines were put into 25 cm2 culture flasks contain-
ing RPMI-1640 medium (prepared using 10% FCS, 100 U/mL
penicillin and 0.1 mg/mL streptomycin) and then were kept
in a CO2 incubator conditioned with 5% CO2 at 37 ꢀC for
24 h. The medium of cells maintained in a humid environ-
ment was changed twice a week. When cells are confluent,
the cell culture medium was removed from flasks using tryp-
sin-EDTA solution. And then the cells were transferred into
to 96-well plates and used in MTT analysis. Paclitaxel and
Docetaxel were used as the positive control at similar con-
centrations in only medium. The plates containing RPMI-1640
medium with 10% FCS, 100 U/mL penicillin and 0.1 mg/mL
streptomycin were incubated at 37 ꢀC in an incubator for
24 h. Following 24 h incubation under the same conditions,
0.5 mg/mL MTT reagent (prepared in sterile PBS) was added
into 96-well plates. After waiting for 3 h in the incubator, the
optical density of cells in plates were immediately read in an
Elisa Microplate Reader (Synergy HT, USA) at 550 nm wave-
length and the cell viability percentage of each group was
calculated based on the definition of the control cell viability
as 100%. Each value represents an average of 10 measure-
2.2.10. Synthesis of hexakis[(1-(40-oxyphenyl)-3-(3-(trifluor-
omethyl)phenyl)prop-2-en-1-one)]cyclotriphospha-
zene (CP-11)
The procedure is similar to that of CP-2, using HCCP (0.2 g,
0.58 mmol), 11 (1.14 g, 3.49 mmol) and K2CO3 (0.56 g,
4.03 mmol). Yield: 81%; white solid. m.p. 159–160 ꢀC. MALDI-
MS: N3P3O12C96H54Cl6F18 m/z calc. 2089.09; found: 2089.152.
Microanalysis: Found: C 55.21, H 2.58%. requires C 55.19, H
2.61%. FT-IR (KBr) ꢀmax (cmꢁ1): 3045 and 3072 ꢀAr-CH, 2898
and 2975 ꢀAliphatic-CH, 1665 ꢀC¼O, 1502 and 1599 ꢀC¼C, 1160
and 1178 ꢀP¼N, 950 ꢀP–O–Ph
.
31P-NMR (DMSO-d6) d ¼ 8.01 (3P,
s, PA, A3). 1H-NMR (DMSO-d6, ppm) d ¼ 7.18–7.20 (12H, d,
J ¼ 8.4 Hz, Ar–H3,5), 7.63–7.70 (12H, m, H8 (–CH¼), Ar–H14),
7.90–7.94 (12H, m, H9 (¼CH–), Ar–H15), 8.06–8.08 (12H, d,
J ¼ 8.8 Hz, Ar–H2,6), 8.18 (6H, s, Ar–H11). 13C-APT NMR (DMSO-
d6, ppm) d ¼ 132.63 Ar–C1, 131.29 Ar–C2,6
, ,
121.10 Ar–C3,5
153.51 Ar–C4, 187.73 C7 (–C ¼ O), 128.37 C8 (–CH¼), 141.66 C9
(¼CH–),
134.64 Ar–C10,
134.05 Ar–C11,
127.43 Ar–C12,
128.37 Ar–C13, 124.15 Ar–C14, 132.43 Ar–C15, 124.44 Ar–C16.
2.3. The frontier molecular orbitals (FMO’s)
The highest occupied molecular orbital (HOMO) and the low-
est unoccupied molecular orbital (LUMO) are the most
important orbitals in a molecule and are called frontier mole-
culer orbitals (FMO’s). Frontier Molecular orbitals (FMO’s)
generally play an essential role in chemical reactions, UV-vis
spectra, optical and electrical properties (Politzer et al., 1985).
Also recently, the energy gap (Eg) of HOMO-LUMO has been
used to prove the chemical activity and the bioactivity from
intra molecular charge transfer (Frisch et al., 2010; Politzer
et al., 1985). The HOMO-LUMO energy gap and chemical
reactivity identifiers were calculated using the Gaussian 09W
package (Dennington & Millam, 2010) in DFT (B3LYP) meth-
ods set on the basis of 6–31G (d, p), and HOMO and LUMO
images were viewed using GaussView 5.0 (Koopmans, 1934).
The ability of electron accepting and giving is defined for
LUMO and HOMO, respectively. Besides, the HOMO-LUMO
energies and their energy gap (Eg) are calculated with the
B3LYP/6-31G(d,p) method. The global chemical reactivity and
density functional descriptors of molecules such as electron
affinity (A), electronegativity (v), chemical potential (l), ion-
ization potential (I), global hardness (g), electrophilicity index
(x) and global softness (f) are calculated from the energies
of the HOMO and the LUMO at B3LYP/6-31G (d,p) basis set.
These parameters can be calculated as follows (Mosmann
et al., 1986; Mulliken, 1934; Parr et al., 1999; Parr & Pearson,
1983; Pearson, 1985; Yang & Parr, 1985):
€
€ €
ments (Gorgulu et al., 2015; Morris et al., 2009; Singh &
Singh, 2002). The compounds dissolved in in dimethyl sulfox-
ide (DMSO). The same amounts of solvent (DMSO) with 1, 5,
25, 50 and 100 mM concentrations of the compounds were
added to the wells containing the cells and incubated at
37 ꢀC for 24 h in the CO2 incubator (Panasonic/Japan). After
incubations, viability of the cells was determined using 0.4%
tryphan blue in a hemocytometer.
IBM SPSS Statistics 22.0 (Windows) package program was
used in Statistical Analysis. The normality distribution of the
data was determined by the Shapiro Wilk test. Comparison
of variables between groups was analysed with
Kruskal–Wallis H test. Multiple Bonferroni corrected was used
with Mann–Whitney U test. Data are expressed as mean-
standard deviation. p < 0.05 value was considered statistic-
ally significant. LogIC50 values were calculated using
Graphpad prism
6 program in computer environment
according to the obtained MTT results.
2.5. Molecular docking studies
Ligands were energy-minimized using ChemOffice on
Windows 10 operating system. Grid box points as size of
96 ꢂ 96 ꢂ 96 Å3 and a regular space of 0.375 Å were deter-
mined by centring on Colchicine. “Tubulin–Colchicine com-
plex” pdb file (PDB ID: 4O2B) was get (https://www.rcsb.org/)
Energy gap, DE ¼ ELUMO – EHOMO; electron affinity, A ¼
–ELUMO; ionization potential, I ¼ –EHOMO; chemical hardness,
g ¼ (I ꢁ A)/2; chemical potential, lo ¼ ꢁ(I þ A)/2; electro-
negativity, v ¼ (I þ A)/2; chemical softness, f ¼ 1/g; electro-
philicity index, x ¼ l2/2g.
€
and was modified using the Maestro (Maestro, Schrodinger,
LLC, New York, NY, 2020.). Since our compounds are large-
sized molecules and the program allows up to 32 rotatable
bonds, the number of rotatable bonds has been reduced to
32 for all ligands when preparing ligands with

6
H. DOĞAN ET AL.
Scheme 1 Synthetic pathway of 2-11 and CP 2-11 and their numbering for 31P, 1H and 13C-APT NMR characterizations
AutoDockTools4 (Trott & Olson, 2010). Docking scores were of A3. When the 31P-NMR spectra were examined, the structure
formed was symmetrical and the peak of equivalent phosphor-
ous observed at 21.12 ppm belonging to the trimer (HCCP)
was not observed by obtaining the target compounds CP 2-
11. As a result of obtaining the target compounds, phosphorus
peaks were observed as singlets in the range 7.93–8.25 ppm
for each compound. In addition, the phosphorus peak has
shifted to the high field due to the substitution effect attached
to the phosphazene ring. This means that the electron density
on the phosphorus atom increases. This can be explained by
the fact that the substituent attached to the phosphazene ring
attracts electrons, causing the electrons in the P ¼ N bond to
intensify on the phosphorous.
obtained using AutoDock Vina software (Table 6) (Elmas
et al., 2017).
3. Results and discussion
3.1. Synthesis and characterization
The synthetic pathway of 2-11 and CP 2-11 and their num-
bering for 31P, 1H and 13C-APT NMR characterizations is
shown in Scheme 1. The characteristic peaks of the com-
pounds in the FT-IR spectrum are given in detail in the
experimental section. It appears that the –OH stretching
vibrations present in the starting compounds 2-11 are not
observed in the FT-IR spectrum by obtaining target com-
pounds CP 2-11. In addition, the peaks of the C ¼ O stretch-
ing vibration, which is observed between 1639–1657 shift to
1653–1667 cmꢁ1 in target compounds. The P ¼ N stretching
vibration peaks are seen in 1155 and 1184 cmꢁ1. The peaks
of the P–O–Ph bond formed by binding the starting com-
pounds to the cyclotriphosphazene ring were observed in
the range 942–969 cmꢁ1. As a result of binding of the sub-
stituents to the trimer (HCCP) ring, the P ¼ N bond is weak-
ened and the P ¼ N stretching vibrations of the ring have
shifted to low energy.
A
proton –OH peaks observed in the range
1
10.29–10.52 ppm in the H-NMR spectra of the starting com-
pounds 2-11 were not observed in after formation of target
products CP 2-11. This is the first proof that substitution has
occurred. The olefin proton peaks of the starting compounds
at 7.63–7.80 (1H, d, J ¼ 15.6 Hz) and 7.79–8.14 (1H, d,
J ¼ 15.6 Hz) ppm were shifted to 7.57–7.70 (6H, d, J ¼ 15.6,
H8) and 7.70–8.01 (6H, d, J ¼ 15.6, H9) ppm ranges after the
formation of target compounds. In addition, the obtained
integral heights are compatible with the structure and the
aromatic (for CP 2-11) and aliphatic (for CP 2-6) protons in
the structure of the starting compounds are observed with
low shifts and changes in the protons next to the phos-
phorus atom are the proof for formation of the tar-
The single peak seen in the 31P-NMR spectra shows that the
phosphorous in the structure have the same chemical environ-
ment, symmetrical structure and the spin system is in the form get structure.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 1. (A) 31P, (B) 1H and (C) 13C-APT NMR spectrum in DMSO-d6 and (D) MALDI-TOF MS spectrum of compound CP-6.
it is seen that there are significant shifts in carbon peaks due
to interactions in other carbon atoms.
Table 1. TGA data for the decomposition and DSC values of compounds CP
2-11.
The experimental values of the compounds in the MALDI-
TOF MS spectrum appear to be almost exactly the same as
the theoretically calculated values. This is one of the most
important evidence that the compounds are formed
(Figure 1).
The thermal characteristics of the target compounds were
determined by thermogravimetric analysis (TGA), and the
results showed that the compounds are highly stable to high
temperatures. Detailed results are given in Table 1.
TGA results
DSC results
Compounds
CP 2-11
Residue
Melting
point
Ti (ꢀC)a
T50% (ꢀC)b
(%, 800 ꢀC)
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
306.15
329.41
311.14
331.46
318.50
321.91
261.21
290.54
293.27
290.54
664.28
654.73
613.81
643.14
629.50
648.59
614.49
613.13
564.02
570.84
26.44
20.66
19.72
18.44
19.17
21.82
17.52
22.15
28.42
18.07
125–126
135–136
189–190
142–143
113–114
256–257
121–122
113–114
115–116
159–160
aInitial decomposition temperature.
3.2. The determination of frontier molecular
bDecomposition temperature at 50% mass loss.
orbitals (FMO’s)
The HOMO-LUMO energy gap and chemical reactivity identi-
fiers for CP 2-11 were calculated using the Gaussian 09W
package in DFT (B3LYP) methods set on the basis of 6-31G
(d, p), and HOMO and LUMO images were viewed using
GaussView 5.0. The 3D plots of the HOMO and LUMO orbitals
of molecules were shown in Figure 2. As can be seen in
Figure 2, there is a charge transfer within the molecules. The
results of ELUMO, EHOMO, Eg, electron affinity, ionization poten-
tial, chemical softness, chemical potential, chemical hardness,
electrophilicity index and electronegativity parameters for CP
2-11 are given in Table 2 according to the obtained
calculations.
In the 13C-APT NMR spectrum of the compounds, C ¼ O
carbon was seen in the range 187.16–187.69 ppm (C8) for
the starting compounds 2-11, while this peak shifted to the
range 187.73–188.19 ppm (C7) in the target compounds CP
2-11. This situation shows the change of the structure after
it is bounded to the phosphazene ring. While the ipso car-
bon (C4) of the starting compounds bound to the –OH
group was observed in the range 162.32–163.28 ppm, this
carbon peak shifted to the range 153.09–156.26 ppm (C4)
after binding to the cyclotriphosphazene ring. This is an indi-
cation that formation of substitution is based on the result
When the chemical hardness is considered, a molecule
from the 13C-APT NMR spectrum. Apart from these examples, having a small HOMO–LUMO energy gap means a soft

8
H. DOĞAN ET AL.
Figure 2. Molecular orbital representation of the molecules (CP 2-11).
Table 2. Some electronic structure parameters for the molecules (CP 2-11) calculated at B3LYP/6-31G (d,p) basis set.
Values (eV)
Parameters
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
ELUMO
EHOMO
Eg
A
I
–2.3845
–5.7290
3.3445
2.3845
5.7290
0.5980
–4.0567
1.6722
4.9205
4.0567
–1.9575
–5.3783
3.4207
1.9575
5.3783
0.5846
–3.6679
1.7103
3.9329
3.6679
–2.2218
–5.2591
3.0373
2.2218
5.2591
0.6585
–3.7404
1.5186
4.6062
3.7404
–2.0511
–5.6330
3.5818
2.0511
5.6330
0.5583
–3.8420
1.7909
4.1211
3.8420
–1.9080
–5.1173
3.2093
1.9080
5.1173
0.6232
–3.5126
1.6046
3.8445
3.5126
–2.2035
–5.9040
3.7005
2.2035
5.9040
0.5404
–4.0537
1.8502
4.4406
4.0537
–2.8041
–6.4218
3.6177
2.8041
6.4218
0.5528
–4.6129
1.8088
5.8818
4.6129
–2.4174
–6.3998
3.9824
2.4174
6.3998
0.5022
–4.4086
1.9912
4.8804
4.4086
–2.7195
–6.6107
3.8912
2.7195
6.6107
0.5139
–4.6651
1.9456
5.5929
4.6651
–2.7535
–6.7217
3.9682
2.7535
6.7217
0.5040
–4.7376
1.9841
5.6561
4.7376
Z
M
H
X
X
Eg ¼ ELUMO – EHOMO; A, electron affinity; I, ionization potential; f, chemical softness; l, chemical potential; g, chemical hardness; x, electrophilicity index; v,
electronegativity.
molecule and large energy gap means a hard molecule. It CP 2-11 molecules are 3.34, 3.42, 3.03, 3.58, 3.20, 3.70, 3.61,
may also relate to the stability of the molecule against hard- 3.98, 3.89 and 3.96 eV, respectively. Taking into account the
ness. That is, the molecule with least energy gap is less sta- values of the energy between HOMO and LUMO, it is shown
ble and more reactive. The calculated energy gap values of that the energy gap of CP-4 molecule smaller than the other

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 3. The cell viability results of PC-3 cells after a 24-h treatment with CP-5, CP-6, CP-8 and CP-10. The changes on the cell viability (%) caused by hexa substi-
ꢃ
tuted cyclotriphosphazene derivatives are compared with the control data. Each data point is an average of 10 viabilities ( p < 0.05).
molecules. That is, this molecule is less stable and more cytotoxic effect on A2780 cell lines at different concentra-
ꢃ
reactive than others. Besides, as shown in Table 2, all the tions ( p < 0.05). Compound CP 3, 4 and 11 were exhibited
chemical potential values are negative and it means that the significant cytotoxic effect on the cell viability at only 100 mM
ꢃ
molecules are stable.
concentration ( p < 0.05). The cell viability of compounds CP
5, 6, 8 and 10 against A2780 cells decreased in a dose-
ꢃ
dependent manner (Figure 3 and Table 3, p < 0.05). All con-
centrations of these compounds showed significant cytotoxic
effect ( p < 0.05). At 25, 50 and 100 mM concentrations for
compound CP-2, at 50 and 100 mM concentrations for com-
pound CP-7 and at 5, 25, 50 and 100 mM concentrations for
compound CP-9 were exhibited significant cytotoxic effect
3.3. In vitro cytotoxic activity
ꢃ
3.3.1. Cytotoxicity of the compounds on cell lines
In vitro cytotoxic activities of CP 2-11 were analysed via MTT
assay against human ovarian (A2780), prostate (LNCaP and
PC-3) cancer cell lines. The percentages of viable cells were
determined at different concentrations (1, 5, 25, 50
and 100 mM).
The articles on the cytotoxic activity of substituted phos-
phazene compounds against different cancer cells are avail-
able in the literature (Akbas¸ et al., 2013; Binici et al., 2019;
ꢃ
(Table 3, p < 0.05).
The results of CPN 2-11 on human prostate cancer cell
lines are given in Table 4. When the cytotoxic activities of
target compounds against human prostate cancer cell lines
are examined, compounds CPN-2, 3, 6 and 7 have been sig-
nificantly reduced cell viability in a dose-dependent manner
against the PC-3 cell lines at all concentrations ( p < 0.05).
The graphics of CPN-2, 3, 6 and 7 showing dose-dependent
effects are given in Figure 4.
At only 100 mM concentration for CP-2 and CP-6, at 25,
50 and 100 mM concentrations for CP-3, at 50 and 100 mM
concentrations for CP-7 were exhibited significant cytotoxic
effect against LNCaP cancer cell lines ( p < 0.05).
Compounds CPN-8, has been significantly reduced cell via-
€
€ €
€
Gorgulu et al., 2015; Tumer et al., 2018). In these studies, it is
stated that the substituted phosphazene compounds show
significant cytotoxic effects. In addition, toxic effects of com-
pounds against normal cells were investigated and it has
been found that substituted phosphazene compounds do
not show toxic effects (Koran et al., 2017). It is stated in the
literature that degradation products of phosphazene com-
pounds are non-toxic (Andrianov, 2008). In conclusion, these
compounds can be expressed as strong candidate molecules
for pharmaceutical applications.
ꢃ
ꢃ
bility in a dose-dependent manner against the LNCaP cell
ꢃ
In terms of MTT results, compounds CP 2-11 appear to lines at all concentrations ( p < 0.05). The logIC50/IC50 val-
be highly effective at high doses ( p < 0.05). Applied com- ues of CP 2-11 against all cancer cell lines are given in
ꢃ
pounds CP 2-11, except CP 3, 4 and 11, showed significant Table 5.

10
H. DOĞAN ET AL.
ꢃ
Table 3. % Cell viability of compounds CP 2-11 against human ovarian cancer cell lines ( p < 0.05).
A2780 human ovarian cancer cell lines
Control
Solvent
1 mM
5 mM
25 mM
50 mM
100 mM
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
100 9.67
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
91.25 8.16
86.23 12.63
90.14 12.69
92.26 10.33
88.02 7.99
92.68 15.76
100.84 15.6
69.22 6.91
88.16 19.8
93.64 10.9
60.83 7.75
54.40 6.23
90.26 9.12
52.86 8.69
41.21 7.26
55.48 7.39
79.83 7.06
60.23 9.12
91.71 10.16
98.75 8.29
30.14 5.81
26.43 7.96
7.23 1.09
54.16 7.19
35.32 7.41
32.16 2.36
44.03 9.17
22.09 5.74
53.35 9.41
52.31 8.81
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
62.93 8.16
55.15 7.26
93.26 10.26
60.21 9.66
60.99 5.23
52.45 8.72
89.26 11.06
60.13 6.83
56.19 5.86
ꢃ
ꢃ
ꢃ
ꢃ
66.31 6.19
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
64.19 10.22
45.06 8.81
ꢃ
ꢃ
82.42 11.26
62.19 8.12
58.00 8.81
30.81 6.03
50.96 8.24
ꢃ
ꢃ
ꢃ
60.81 9.16
79.50 9.23
81.34 10.41
80.20 10.92
mM, micromolar.
*It is statistically significant.
ꢃ
Table 4. % Cell viability of compounds CP 2-11 against human prostate cancer cell lines ( p < 0.05).
LNCaP human prostate cancer cell lines
Control
Solvent
1 mM
5 mM
25 mM
50 mM
100 mM
ꢃ
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
100 8.99
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
93.17 10.12
96.58 11.8
92.26 9.23
97.41 8.26
87.06 8.29
95.13 8.21
100.02 14.
68.29 8.69
76.12 10.2
94.33 6.69
88.10 9.26
84.05 10.29
73.08 10.12
84.93 10.13
77.74 11.16
83.43 7.46
80.94 9.33
62.75 6.15
80.08 9.64
72.25 10.2
72.56 9.21
93.31 12.3
63.14 9.12
39.48 9.26
77.04 8.19
75.92 10.8
88.06 9.67
61.03 5.23
70.39 5.12
63.56 8.85
72.04 12.36
44.11 8.77
67.27 7.45
39.69 10.13
44.30 10.21
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
57.91 8.81
62.21 7.26
63.87 6.69
55.73 8.17
11.38 2.41
63.84 8.88
31.12 7.94
ꢃ
ꢃ
ꢃ
108.85 16.2
69.96 6.78
51.39 8.99
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
91.44 8.22
77.69 7.26
63.38 7.25
68.49 6.90
50.83 10.16
ꢃ
ꢃ
62.54 8.19
PC-3 human prostate cancer cell lines
Control
Solvent
1 mM
5 mM
25 mM
50 mM
100 mM
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
100 9.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
92.36 8.13
72.97 6.89
60.42 7.29
94.49 10.2
96.21 12.6
54.60 5.23
40.73 6.72
76.68 9.32
90.06 8.74
95.64 5.36
108.47 5.3
65.87 6.13
56.73 5.69
60.78 5.94
65.16 6.44
48.59 8.91
37.39 6.29
52.26 7.19
47.65 8.17
47.96 9.41
52.74 7.93
69.39 7.32
97.49 16.6
101.68 14.4
35.13 4.11
18.49 3.51
11.64 4.52
10.91 2.76
20.54 2.29
45.26 5.36
28.58 4.38
31.34 3.41
39.06 5.29
64.17 8.91
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
50.72 10.14
102.56 18.4
85.16 10.81
52.61 5.26
47.49 8.90
71.06 6.49
91.22 12.51
100.03 13.2
50.98 6.21
40.44 7.94
73.63 7.22
82.95 9.34
105.06 8.91
106.81 12.0
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
74.10 10.41
105.09 12.7
109.11 18.8
mM, micromolar.
*It is statistically significant.
€
€ €
When the cytotoxic activity results of the target com- (Gorgulu et al., 2015; Koran, Tekin, Biryan, et al., 2017; Koran
pounds CP 2-11 on human prostate cancer cell lines were Tekin, C¸alıs¸kan, et al., 2017). While the dose-dependent effect
examined, CP 2-11 were more effective against PC-3 cancer was not observed in the compounds used in previous stud-
ies, most of the compounds in hexa-substituted structures
decreased dose-dependent cell viability. Also, in previous
studies, the compounds generally showed a high dose
(100 mM) cytotoxic effect. While the results obtained in previ-
ous studies caused a decrease in cell viability by 50%–60%
at this dose, the results in our current study showed that the
cell lines. According to the information given above, some
substances have a cytotoxic effect against PC-3 cell lines at
almost all concentrations, whereas the same effect has not
been observed against LNCaP cell lines (e.g. CP-2 com-
pound). Against this cell line, CP 2-11 compounds generally
showed high doses of cytotoxic effects.
In this study, it was observed that there were significant decrease in cell viability was 70%–90% (e.g. CP 2-4, CP-7, 8).
differences in cell viability compared to the cytotoxicity activ-
ity results of the disubstituted and tetra-substituted phos-
phazene compounds of the hexa-substituted phosphazene
3.4. Molecular docking studies
compounds with the cytotoxic activities of our study team.
In our previous study, the cytotoxicity activity results of
disubstituted and tetra substituted phosphazene containing
According to the X-ray crystallographic structure of
Tubulin–Colchicine complex (PDB ID:4O2B), main binding site
has been determined around Colchicine (Ligand ID: LOC) and
different organic groups were compared with the results of Guanosine-50-Triphosphate (Ligand ID: GTP) in receptor
hexa-substituted phosphazene in this study and it was deter- (https://www.rcsb.org/). It has been declared that colchicine
mined that there were significant differences in cell viability interacts with active site in tubulin as binding site. It has

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 4. The cell viability results of PC-3 cells after a 24-h treatment with CP-2, CP-3, CP-6 and CP-7. The changes on the cell viability (%) caused by hexa substi-
ꢃ
tuted cyclotriphosphazene derivatives are compared with the control data. Each data point is an average of 10 viabilities ( p < 0.05).
Table 5. LogIC50/IC50 (50% inhibition-causing concentration) values (mM) of
CP 2-11 against human ovarian (A2780) and prostate (LNCaP, and PC-3) can-
cer cell lines.
produced showed by Maestro software (Maestro,
€
Schrodinger, LLC, New York, NY, 2020). Binding modes of
these compounds were revealed to be similar to colchicine,
but it was found that these large chemical structured ligands
did not settle inside the receptor and were docked externally
with the corresponding residues (Figure 6).
A2780
LNCaP
PC-3
Comp.
LogIC50/IC50 (mM)
LogIC50/IC50 (mM)
LogIC50/IC50 (mM)
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
Paclitaxel
Docetaxel
1.749/56.1
2.021/104.9
1.971/93.62
1.794/62.25
1.524/33.41
1.911/81.44
1.526/33.57
1.182/15.22
1.652/44.86
2.089/122.9
0.7516/5.645
–
2.09/123.2
1.886/76.98
2.118/131.1
2.023/105.3
2.029/106.9
1.721/52.56
1.978/95.15
1.174/14.92
1.961/91.45
2.07/117.6
–
1.709/51.13
1.326/21.2
1.698/49.87
1.707/50.98
1.297/19.82
1.285/19.28
1.665/46.23
1.835/68.34
2.237/172.5
2.571/372.4
–
4. Conclusion
In conclusion, a new series hexa substituted cyclicphospha-
zenes CP 2-11 obtained and their investigated in vitro cyto-
toxicity properties, theoretical analysis and molecular
docking studies. The structures of CP 2-11 were character-
ized using 31P, 1H-NMR, 13C-APT NMR, FT-IR and MALTI-TOF
MS spectroscopic methods. In addition, thermal stability of
the compounds was determined by taking DSC and TGA
thermograms. The HOMO-LUMO energy gap and chemical
reactivity identifiers were calculated and HOMO and LUMO
images were viewed. According to the calculations, all the
chemical potential values of CP 2-11 are negative and it
shown that the molecules are stable. Molecular docking stud-
ies exhibited the interaction mode of all compounds with
tubulin including some hydrophobic, H-bond, Pi–Pi stacking,
Pi-cation, Halogen bond interactions. In the last step, hexa
substituted cyclotrophosphazenes CP 2-11 at different con-
centrations (1, 5, 25 and 50 mM) are employed against human
ovarian (A2780) and human prostate (PC-3 and LNCaP) can-
0.7792/6.014
1.229/16.95
mM, micromolar.
been previously established that colchicine interacts with
SER178A, THR179A, ALA180A, VAL181A, CYS241B, LEU242B,
LEU248B, ALA250B, ASP251B, LYS254B, LEU255B, ASN258B,
MET259B, THR314B, VAL315B, ALA316B, ILE318B, ASN350B,
LYS352B, ILE378B residues (https://www.ebi.ac.uk/pdbe/).
Hydrophobic, H-bond, Pi–Pi stacking, Pi-cation, Halogen
bond interactions of all ligands were given in detail with
interacting residues (Table 6).
Docking studies were performed for the all compounds.
Interaction modes for compounds CP 2-11 with enzyme
active sites were determined. 2D Interaction diagram for
compound CP-5 and CP-6 at the Tubulin-binding cavity are cer cell lines to investigate % cell viability and in vitro cyto-
given in Figure 5. The binding types and residues were toxicity activities using the MTT assay method. CP 2-11

12
H. DOĞAN ET AL.
Table 6. Molecular docking-binding scores of CP 2-11, within the Tubulin-Colchicine complex (PDB ID: 4O2B) active site.
Residues participating
Comp.
H-bond
Pi–Pi stacking
Pi-cation
Halogen bond
Best docking score
CP-2
CP-3
CP-4
CP-5
CP-6
CP-7
CP-8
CP-9
CP-10
CP-11
ASN18A SER178A TYR224A THR225A
THR82A ARG229A GLY246B
TYR224A
ARG229A
–
LYS352B
–
ARG158B ARG164B ARG253B
ARG229A
ARG164B
ARG229A
ARG121A
–
–
–
–
–
–
–9.2
–11.1
–10.6
–11.3
–10.8
–8.9
–10.3
–9.6
–8.3
–11.6
–
ARG214A GLN336B
ASN18A
ARG253B
ARG229A
–
ASN228A
LEU132B
–
HIS393A
TYR224A
–
–
TYR108A TRP407A
–
THR82A ARG84A
–
–
–
–
LYS304A LYS394A
GLU429A PHE343B VAL344B
nM, nanomolar; mM, micromolar.
Residues participating H-bond, Pi–Pi stacking, Pi-cation, halogen-bond interactions with the compounds are shown.
Figure 5. 2D interaction diagram for compounds CP-5 and CP-6 at the Tubulin-binding cavity, respectively.
Figure 6. The positioning of CP-6 (red), Colchicine (black) and Guanosine-5’-Triphosphate (blue) compounds with in Tubulin-Colchicine complex (green)
was presented.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
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Acknowledgements
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Disclosure statement
ꢁ
ꢁ
ꢁ
Carriedo, G. A., Fernandez-Catuxo, L., Garcıa Alonso, F. J., Gomez-Elipe,
ꢁ
P., & Gonzalez, P. A. (1996). Preparation of a new type of phospha-
No potential conflict of interest was reported by the authors.
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Chaudhary, M., Kumar, N., Baldi, A., Chandra, R., Arockia Babu, M., &
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condensates augmented anticancer activity against human cervical
cancer cells: Design, synthesis, in silico docking and in vitro cytotox-
icity analysis. Journal of Biomolecular Structure & Dynamics, 38(1),
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Funding
The Scientific and Technological Research Council of TurkeyThis work
was supported by
Research Council of Turkey (Grant No: 116Z758).
a grant from The Scientific and Technological
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ORCID
Harun Uslu
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€
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