Synthesis, cytotoxicity and apoptosis of cyclotriphosphazene compounds as anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.03.018

In the present study, a number of new dispirobino and dispiroansa spermine derivatives of cyclotriphosphazene (8-10, 13) were synthesized and characterized by elemental analysis, mass spectrometry, ¹H and ³¹P NMR spectroscopy. At fir

Full text of DOI:10.1016/j.ejmech.2012.03.018

European Journal of Medicinal Chemistry 52 (2012) 213e220
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, cytotoxicity and apoptosis of cyclotriphosphazene compounds as
anti-cancer agents
,
b
c
c
c
Tuba Yıldırım a , Kemal Bilgin , Gönül Yenilmez Çiftçi , Esra Tanrıverdi Eçik , Elif S¸ enkuytu ,
*
e
c
ꢀ d
Yıldız Uludag , Leman Tomak , Adem Kılıç
a Department of Biology, Faculty of Art and Science, Amasya University, 05100 Amasya, Turkey
b Department of Medical Microbiology, Faculty of Medicine, Ondokuz Mayıs University, 55139 Samsun, Turkey
c Department of Chemistry, Gebze Institute of Technology, 41400 Gebze/Kocaeli, Turkey
d UEKAE e The Scientific and Technological Research Council of Turkey (TUBITAK), 41470 Gebze/Kocaeli, Turkey
e Department of Biostatistic, Faculty of Medicine, Ondokuz Mayıs University, 55139 Samsun, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, a number of new dispirobino and dispiroansa spermine derivatives of cyclo-
triphosphazene (8e10, 13) were synthesized and characterized by elemental analysis, mass spectrom-
etry, 1H and 31P NMR spectroscopy. At first, in vitro cytotoxic activity of cyclotriphosphazene compounds
(1e14) against HT-29 (human colon adenocarcinoma), Hep2 (Human epidermoid larynx carcinoma), and
Vero (African green monkey kidney) cell lines was investigated. Our study showed that most of these
compounds stimulate apoptosis and they have cytotoxic effects for HT-29 and Hep2 cells. Additionally,
these compounds (1e14) were investigated for their antibacterial activity against gram-positive
(Staphylococcus aureus), gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria and for their
antifungal activity against Candida albicans, and were shown to be inactive.
Received 23 September 2011
Received in revised form
7 March 2012
Accepted 9 March 2012
Available online 19 March 2012
Keywords:
Cyclotriphosphazene
Spermine derivatives
Biological activity
Cytotoxic activity
Apoptosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
cyclotriphosphazenes as anti-cancer agents, previously mentioned
by Labarre et al. [2], has been enhanced by the finding of aziridino-
Cancer is a leading cause of death worldwide and accounted for
7.6 million deaths (around 13% of all deaths) in 2008 [1]. Treatment
options for a cancer patient vary depending on the type of cancer,
its location and the stage at which it is diagnosed. The adminis-
tration of anti-cancer drugs is one of the effective cancer treatment
options, and search for new anti-cancer agents is a great task as not
only many cancer types exist but also the response of each indi-
vidual to different anti-cancer drugs may vary considerably.
Another challenge is to design chemicals that have minimum
adverse effects on normal cells.
Many compounds with different structures have been tested by
researchers in the search for anti-cancer drugs. Although a number
of compounds exhibit therapeutical properties and are currently
used for treatment, the search for new drugs with improved
efficiency and minimum side effects still continues. Cyclo-
triphosphazene derivatives have attracted the attention of
researchers to be used as potential anti-cancer agents. Interest in
and polyamine-linked cyclotriphosphazenes which are active on
a large series of tumour cells [3,4]. A number of studies have
been devoted to describe the antitumour activity of cyclo-
triphosphazenes [5e8]. Cyclotriphosphazenes, having a variety of
applications in science and technology, are an important class of
inorganic ring system [9]. Chemical and physical properties of
cyclotriphosphazenes change with the substituted side groups. So,
it is possible to design materials with special properties such as
anti-cancer agents [5e7], liquid crystals [10,11], electrical conduc-
tivity [12], hydraulic fluids and lubricants [13,14], flame retardant
properties [15], elastomers [16], rechargeable batteries [17] and
biomedical materials [18,19]. In addition, biological activities of
cyclotriphosphazene derivatives against various bacterial and
fungal species are known [20,21].
The native polyamines are essential growth factors for cells and
polyamine derivatives have been shown to have cytotoxic activity
against numerous human cancer cell lines (e.g., breast, prostate,
etc) [22e24]. Polyamine-based small molecules have been devel-
oped for use as biochemical probes and as potential therapies for
a variety of diseases [25]. The polyamines inhibit cell growth and
* Corresponding author. Tel.: þ90 358 2421613; fax: þ90 358 2421616.
E-mail address: yildirimt55@gmail.com (T. Yıldırım).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.018

214
T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
kill cancer cells both in tissue culture and experimental animal
models [26e29]. One important property in the treatment of any
cancer is to kill the tumour cells rather than just inhibiting their
growth, although stable growth inhibition might be an acceptable
alternative. In mammals, polyamines directly affect apoptosis in
several cell types [25]. Polyamines are used in the protection of cells
from external toxic conditions, such as oxidative stress, radiation,
acidic pH, and other toxic agents [30]. In addition, antiparasitic and
antibacterial effects of polyamine-linked analogues have been
reported [31,32]. Spermine, found in all mammalian cells, can act as
a regulator of DNA synthesis (replication, transcription, translation,
and enzyme activities) and in the inhibition of cellular proliferation.
Several studies have concluded that spermine-linked analogues
affect gene expression at the transcriptional level and this effect is
most probably due to the direct interaction of spermine with DNA
and interacting protein factors [33e36].
2. Results and discussion
2.1. Chemistry: synthesis and characterization of the compounds
Reaction of N3P3Cl6 (1) with spermine aprotic solvents such as
dichloromethane results in a series of dispirobino spermine deriva-
tives of cyclotriphosphazene compounds, whereas, in protic solvents
such as chloroform proceeds toyield thedispiroansa derivatives [37].
In this work, firstly disubstituted derivatives of cyclotriphosphazene
(2e6) were synthesized from the reactions of 1 with the aniline,
thiophenol, 2,2,3,3-tetrafluorobutane-1,4-diol, 2,2-dimethyl-1,3-
propanediol, respectively [38e41]. And then, the reactions of gem-,
spiro-, ansa-substituted cyclotriphosphazene derivatives N3P3Cl4R2
[R ¼ NHPh, SPh, (OCH2(CF2)2CH2O)0.5, (OCH2C(CH3)2CH2O)0.5], (2e6)
with spermine in dichloromethane results in dispirobino spermine
derivatives of cyclotriphosphazene (7e11), namely spermine
bridged compounds. But, if chloroform is used, the reaction results in
dispiroansa derivatives (12e14), namely tetracyclic cyclo-
triphosphazene compounds (Scheme 1). The synthesis and charac-
terization of compounds (7, 11, 12, 14) were reported in our previous
study [37], here we describe the biological activities of these
compounds (7, 11, 12, 14) and novel compounds (8e10, 13).
In this study, hexachlorocyclotriphosphazene, N3P3Cl6 (1),
disubstituted derivatives of cyclotriphosphazene (2e6) and sper-
mine derivatives of cyclotriphosphazene compounds (7e14) were
synthesized and their cytotoxic properties against cell lines were
investigated to assess their anti-cancer capabilities. Additionally,
the antimicrobial effects of the compounds were assessed.
X
X
Cl
Cl
P
N
P
N
N
N
N
P
N
P
Cl
Cl
Cl
Cl
Cl
Cl
X
P
P
X
X
X
+
(2) NHPh
(5) [OCH2(CF2)2CH2O]0.5
(3) SPh
H
N
H2N
(4) [OCH2(CF2)2CH2O]0.5
(6) [OCH2C(CH3)2CH2O]0.5
N
H
NH2
SPM=Spermine
Et3N
CH2Cl2
SPM
CHCl3
Et3N
CH2Cl2
X
X
P
N
H
N
HN
N
H
N
NH
N
HN
N
N
N
NH
N
N
N
P
N
P
P
P
P
N
P
N
N
N
N
N
N
N
Cl
Cl
X
N
Cl
Cl
X
X
N
Cl
Cl
Cl
Cl
X
P
P
P
P
P
P
P
P
N
X
X
X
X
X
X
X
(10) [OCH2(CF2)2CH2O]0.5
(7) NHPh
(8) SPh
(9) [OCH2(CF2)2CH2O]0.5
(11) [OCH2C(CH3)2CH2O]0.5
(12) NHPh
(13) [OCH2(CF2)2CH2O]0.5
(14) [OCH2C(CH3)2CH2O]0.5
Scheme 1. Dispirobino and dispiroansa spermine derivatives of cyclotriphosphazenes.

T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
215
Table 2
The exact synthesis of these compounds (8e10, 13) is described
in the Experimental section and graphical presentation is given in
Scheme 1. Each of the new compounds (8e10, 13) has been char-
acterized by MS, elemental analysis, 1H and 31P NMR spectroscopy.
The 1H NMR results are provided as part of the analytical data in the
synthesis section. Spermine bridged analogues with gem-
substituted cyclotriphosphazene rings (e.g., compounds 8, 9)
exhibit stereoisomerism [37] because the three phosphorus atoms
of each cyclotriphosphazene ring have different substitution
patterns and those that are part of the bridge, >P(N-spiro), are
stereogenic, i.e., there are R and S forms. As seen from the Table 1,
the proton decoupled 31P NMR spectrum of compound 8 is
observed as AMX and compound 9 is observed as ABX spin system
due to the different environments for the three different phos-
phorus nuclei of the cyclotriphosphazene ring. The 31P NMR spectra
of compounds 10, 13 are analysed as A2X and AX2 spin systems,
respectively.
In vitro cytotoxic activity of the cell lines were examined by MTT assay after treating
HT-29, Hep2 and Vero cells with varying concentrations of cyclophosphazenes
compounds (0.2e50 mg/mL) for 24 h. The obtained data were evaluated using Probit
analysis and described as ID50 values. (‘Negative’ in the table shows the compounds
that have no toxic effect for the stated cell line.).
Compound
Cell Line ID50
HT-29
[m
M] ꢂ SE
Hep2
Vero
ID50 ꢂ SE
ID50 ꢂ SE
Negative
Negative
5.43 ꢂ 0.07
3.30 ꢂ 0.05
2.70 ꢂ 0.05
2.30 ꢂ 0.03
Negative
ID50 ꢂ SE
1
2
3
4
5
6
7
8
7.14 ꢂ 0.05
3.16 ꢂ 0.03
Negative
Negative
3.78 ꢂ 0.03
4.31 ꢂ 0.03
5.59 ꢂ 0.05
4.09 ꢂ 0.03
Negative
1.96 ꢂ 0.01
2.33 ꢂ 0.02
Negative
3.02 ꢂ 0.03
3.55 ꢂ 0.05
5.84 ꢂ 0.05
2.96 ꢂ 0.03
Negative
3.35 ꢂ 0.03
1.31 ꢂ 0.01
1.61 ꢂ 0.01
2.65 ꢂ 0.02
6.55 ꢂ 0.06
5.41 ꢂ 0.04
5.88 ꢂ 0.06
Negative
Negative
9
10
11
12
13
14
2.63 ꢂ 0.03
2.75 ꢂ 0.02
4.58 ꢂ 0.04
Negative
Negative
Negative
2.2. Biology
3.39 ꢂ 0.04
Negative
3.23 ꢂ 0.04
2.2.1. The cytotoxic effects of cyclotriphosphazene compounds
In this study, cyclotriphosphazene compounds were tested for
their cytotoxic activity in vitro against human colon adenocarci-
noma (HT-29), human epidermoid larynx (Hep2) cancer cell lines
and as control African green monkey kidney (Vero) cell line. In
order to test the cytotoxic effects, the cells were incubated with
cyclotriphosphazene compounds at different concentrations
(Vero) was also examined. The anti-proliferative activity of some
compounds (1e7, 9e11) was much higher against the cancer cells
than the normal cells. Although the compounds 8, 12e14 had
cytotoxic effects on HT-29 and Hep2 cells, they did not have any
effect on the Vero cells.
(0.2e50 m
g/mL) for 24 h at 37 ꢀC. The cytotoxic effects of these
compounds were determined by MTT assay (Table 2).
2.2.2. Induction of apoptotic cell death in cells by the compounds
Flow cytometry was used to investigate analogue-induced
apoptosis and cell death. The compounds (1e14) with cytotoxic
effects were investigated for the effects on the apoptosis of HT-29
and Hep2 cells. To clarify the mechanism of cyclotriphosphazene-
induced cell death in cells, we determined both early and late
apoptosis using annexin V-FITC and PI (Propidium iodide) labelling
of live cells. Annexin V binds to phosphatidylserine which is
exposed on the cell membrane and it is one of the earliest indica-
tors of cellular apoptosis. PI is used as a DNA stain for both flow
cytometry to evaluate cell viability or DNA content in cell cycle
analysis and microscopy to visualise the nucleus and other DNA
Probit analysis was used to evaluate the data and the calculated
ID50 values (the dose of compound that inhibits cell proliferation by
50%) of the compounds against cell lines can be found from Table 2.
Cytotoxicity of 1, 3e6, 8, 9, 11, 12 compounds against HT-29 cells
was detected after incubation for 24 h. Under the same conditions,
cytotoxicity was detected on Hep2 cells after incubation with the
compounds 3e6, 10, 13, 14. However, compounds 2, 7, 10, 13, 14 did
not exhibit any cytotoxic effect on the HT-29 cells and the
compounds 1, 2, 7e9, 11, 12 did not show any cytotoxic effect on the
Hep2 cells. These results showed that cyclotriphosphazene
compounds have selective and significant effect on the cell lines.
For comparison, activity of compounds (1e14) against normal cells
Table 1
31P NMR parameters of compounds 2e14.a
Chemical Shifts/ppm
R
2J(PP)/Hz
Ref.
Compound
P(N) Spiro
PCl(R)
PR2
PCl2
2J1,2
2J1,3
2J2,3
2b
e
e
ꢁ1.1
48.8
9.3
23.0
20.3
25.9
25.6
24.5
24.1
22.6
26.7
e
NHPh
SPh
[OCH2C(CF2)2CH2O]0.5
[OCH2C(CF2)2CH2O]0.5
[OCH2C(CH3)2CH2O]0.5
NHPh
e
49.2
5.4
ee
e
[38]
[42]
[40]
[40]
[41]
[37]
This work
This work
This work
[37]
3c
e
e
e
4b
e
e
e
74.3
48.0
68.7
57.2
9.2
63.1
51.6
69.9
e
e
5d
e
24.1
e
e
e
e
6b
e
3.2
2.9
46.5
15.7
e
e
e
7e,h
8e,h
9f,h
10b
11e,h
12b
13c
14g
14.9
13.2
15.4
15.7
15.9
17.0
17.8
18.0
e
38.1
6.2
57.1
e
44.5
36.3
e
e
SPh
e
[OCH2C(CF2)2CH2O]0.5
[OCH2C(CF2)2CH2O]0.5
[OCH2C(CH3)2CH2O]0.5
NHPh
[OCH2C(CF2)2CH2O]0.5
[OCH2C(CH3)2CH2O]0.5
28.1
e
e
9.4
9.4
24.4
17.7
25.1
e
37.0
e
64.4
50.8
71.6
54.7
e
[37]
This work
[37]
e
e
e
e
e
e
e
e
a
202.38 MHz 31P NMR measurements in CDCl3 solutions at 298 K. Chemical shifts referenced to external H3PO4.
31P NMR spectrum analysed as an A2X spin system.
31P NMR spectrum analysed as an AX2 spin system.
31P NMR spectrum analysed as an AB2 spin system.
31P NMR spectrum analysed as an AMX spin system.
31P NMR spectrum analysed as an ABX spin system.
31P NMR spectrum analysed as an A2B spin system.
Diastereoisomer.
b
c
d
e
f
g
h

216
T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
containing organelles. It can be used to differentiate necrotic,
apoptotic and normal cells.
The apoptotic cell rates were determined in the Hep2 cells that
were stimulated for 24 h by the compounds (4e6, 13, 14) with
cytotoxic effects at different concentrations and the results shown
in Fig. 2. From these compounds 4, 6 and 14 were determined to
stimulate apoptosis compared to positive (cisplatin) and negative
controls. The results obtained from compound 6 attract extra
attention as it stimulates apoptosis for 92% of the Hep2 cells.
The majority of drugs used for the treatment of cancer today are
cytotoxic drugs that induce apoptosis in susceptible cells. Over the
last fifty years thousands of chemical compounds have been tested
for anti-cancer activity, but only a few of these are being used for
cancer treatment and are in wide use today. Therefore, studies in
this area continue to grow in importance. Some of the compounds
synthesized in the study attract particular attention as anti-cancer
agents (1, 4, 5, 9, 12 for HT-29 cells and 4, 6, 14 for Hep2 cells) and
further studies are required to explore their potential in this area.
In the present study, the apoptotic cell rates were determined
for the HT-29 cells that were stimulated for 24 h by the compounds
(1, 4e6, 9, 11, 12) with cytotoxic effects at different concentrations.
The graphical values of the results were given in Fig. 1. From the
results, it was found that these compounds stimulate apoptosis for
the HT-29 cells compared to positive (etoposide) and negative (un-
stimulated cells by compounds) controls, for example compounds
1, 9 and 12 induced apoptosis in 80% of the cells. As seen with the
control compound (etoposide), most of the cytotoxic anti-cancer
drugs in current use induce apoptosis in susceptible cells [47];
Compounds 4 and 5, however, are of special interest as these
compounds seem to drive HT-29 cells directly from early apoptosis
into necrosis. The fact that the cell populations are so cleanly
sequestered into the upper left (necrosis) and lower right quadrants
(early apoptotic cells) is interesting. Either these compounds give
a divergent result where some cells are killed directly (necrosis)
and some are induced to undergo apoptosis, or they speed up the
apoptosis process so that we start observing necrosis before the test
ends and perhaps if time allowed others would have followed soon.
2.2.3. Antimicrobial activities
Infectious diseases caused by Pseudomonas aeruginosa, Escher-
ichia coli, Staphylococcus aureus and Candida albicans microorgan-
isms represent a serious threat to the health of hospitalized
Fig. 1. Effect of cyclotriphosphazene compounds on apoptosis of HT-29 cells. Apoptotic cells were analyzed by flow cytometry, after being stained with Annexin V-FITC together
with PI. The percentage of cells positive for PI and/or Annexin V-FITC are reported inside the quadrants. Lower left quadrant, viable cells (annexin Vꢁ/PIꢁ); lower right quadrant,
early apoptotic cells (annexin Vþ/PIꢁ); upper right quadrant, late apoptotic cells (annexin Vþ/PIþ); upper left quadrant, necrotic cells (annexin Vꢁ/PIþ). The percentage of cells
positive for PI and/or Annexin V-FITC was reported inside the quadrants.

T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
217
Fig. 2. Effect of cyclotriphosphazene compounds on apoptosis of Hep 2 cells. Apoptotic cells were analyzed by flow cytometry, after being stained with Annexin V-FITC together
with PI. The percentage of cells positive for PI and/or Annexin V-FITC are reported inside the quadrants.
patients. The discovery of new antimicrobial agents against these
microorganisms has great importance, therefore, in the study the
antimicrobial effects of the newly synthesized cyclotriphosphazene
compounds were investigated as no previous reference has been
found related with the biologic activity of these compounds.
All these compounds (1e14) were investigated for their in vitro
antibacterial activities against P. aeruginosa (ATCC 27853), E. coli
(ATCC 35218) and S. aureus (ATCC 25923), and for antifungal
activities against C. albicans (ATCC 10231). The minimum inhibitory
concentration (MIC) values of these compounds were determined
importance. In the present study, as potential anti-cancer agents,
disubstituted derivatives (2e6) and spermine derivatives of cyclo-
triphosphazene compounds (7e14) were synthesized and charac-
terized by elemental analysis, mass spectrometry, 1H and 31P NMR
spectroscopy. Later the cytotoxic and apoptotic effects of the
cyclotriphosphazene compounds on cancer cell lines were deter-
mined. It was detected that the Compounds 1, 4, 5, 9, 12 and 4, 6, 14
have selective and significant cytotoxic effects on HT-29 and Hep2
cells, respectively. Therefore, these compounds may be considered
as the agents with highest potential anti-cancer activity and appear
to be good candidates for more advanced screening. As the findings
of this work revealed the anti-cancer properties of specific cyclo-
triphosphazene derivatives, investigation of these materials will
continue in our laboratories.
by the broth microdilution method (range, 2e1024
compounds investigated gave an ID50 above 250
m
g/mL). All the
m
g/mL, the high
MIC values of these compounds indicated that the synthesized
cyclotriphosphazene compounds do not have any significant
antimicrobial activity against standard strains of gram-positive
S. aureus, gram-negative E. coli, P. aeruginosa and yeast like fungi
C. albicans.
4. Experimental
4.1. Chemistry
3. Conclusions
Hexachlorocyclotriphosphazene (Otsuka Chemical Co., Ltd) was
purified by fractional crystallization from hexane. Tetrahydrofuran
(ꢃ99.0%), n-hexane (ꢃ96.0%), methanol (ꢃ99.0%), sodium hydride
Cancer is a leading cause of death worldwide and hence studies
to find anti-cancer agents for its treatment continue to grow in

218
T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
(60% dispersion in mineral oil), 2,2-dimethyl-1,3-propanediol
(ꢃ98.0%), dichloromethane (ꢃ99.0%), aniline (ꢃ99.0%), chloro-
form (ꢃ99.0e99.4%), triethylamine (ꢃ99.0%), thiophenol (ꢃ99.0%)
were obtained from Merck. Spermine (ꢃ99.0%) was obtained from
Fluka. 2,2,3,3-tetrafluoro-1,4-butanediol (ꢃ98.0%) was obtained
from Aldrich. All solvents were purified by conventional methods.
THF was distilled over a sodiumepotassium alloy under an atmo-
sphere of dry argon. CDCl3 for NMR spectroscopy was obtained
from Merck. Elemental analyses were obtained using a Thermo
Finnigan Flash 1112 Instrument. Mass spectra were recorded on
a Bruker MicrOTOF LC-MS spectrometer using the electro spray
ionization (ESI) method; 35Cl values were used for calculated
masses. Analytical Thin Layer Chromatography (TLC) was per-
formed on Merck silica gel plates (Merck, Kieselgel 60, 0.25 mm
thickness) with F254 indicator. Column chromatography was per-
formed on silica gel (Merck, Kieselgel 60, 230e400 mesh; for 3 g
crude mixture, 100 g silica gel was used in a column of 3 cm in
diameter and 60 cm in length). 1H and 31P NMR spectra were
recorded in CDCl3 solutions on a Varian INOVA 500 MHz spec-
trometer using TMS as an internal reference for 1H NMR and 85%
H3PO4 as an external reference for 31P NMR.
three-necked round-bottomed flask. The reaction mixture was
cooled in an ice-bath and spermine (0.2 g, 0.9 mmol) in 10 mL of
dichloromethane was added to the stirred solution under argon
atmosphere. The reaction was stirred for 23 h at room temperature
and followed by TLC on silica gel plates using n-hexane-THF (2:1) as
the mobile phase. The reaction mixture was filtered to remove the
triethylamine hydrochloride formed, the dichloromethane was
removed and the resulting colourless oil product subjected to
column chromatography using n-hexane-THF (2:1) as mobile
phase. Compound 10 was isolated as white powder (0.3 g, 35%, mp
214e216 ꢀC). Anal Calc. C18H30Cl4F8N10O4P6 requires: C, 23.24; H,
3.25; N, 15.06%, M, 930, Found: C, 23.21; H, 3.22; N, 15.00%, [M þ H]
þ, 931. 1H NMR, CDCl3, 298 K: 4.1e4.0 m, 8H, OCH2, 3.8e3.5 4H,
NHCH2; 3.9e2.7 m, 8H, NCH2; 2.8 broad, 2H, NH; 2.3e1.6 m, 8H,
CCH2 (spiro and bridge).
4.1.4. Synthesis of compound 13
Compound 4 [40] (1.0 g, 2.3 mmol) was dissolved in 50 mL of
CHCl3 in a 250 mL three-necked round-bottomed flask. The reac-
tion mixture was cooled in an ice-bath and spermine (0.9 g,
4.6 mmol) in 20 mL of chloroform was added to the stirred solution
under an argon atmosphere. The reaction was stirred for 23 h at
room temperature and followed by TLC on silica gel plates using
THF-dichloromethane (2:1) as the mobile phase. The reaction
mixture was filtered to remove the spermine tetrahydrochloride
formed, the chloroform was removed under reduced pressure and
the resulting colourless oil was subjected to column chromatog-
raphy using THF-dichloromethane (2:1) as mobile phase.
Compound 13 was isolated as white powder (0.6 g, 53%, mp
250e251 ꢀC). Anal Calc. C14H26F4N7O2P3 requires: C, 34.09; H, 5.31;
N, 19.87%, M, 493, Found: C, 34.05; H, 5.28; N, 19.81%, [M þ H]þ,
494. 1H NMR, CDCl3, 298 K: 4.5e4.3 m, 4H, OCH2, 3.9 4H, NHCH2;
2.9e2.8 m, 8H, NCH2; 2.81 broad, 2H, NH; 1.8e1.5 m, 8H, CCH2
(spiro and ansa).
Compounds (2e7, 11, 12, 14) were prepared as described in the
literature [38e41].
4.1.1. Synthesis of compound 8
Compound 3 [39] (2.0 g, 4.0 mmol) and triethylamine (2.3 mL,
17.2 mmol) were dissolved in 100 mL of dichloromethane in
a 250 mL three-necked round-bottomed flask. The reaction mixture
was cooled in an ice-bath and spermine (0.8 g, 4.0 mmol) in 10 mL
of dichloromethane was added to the stirred solution under an
argon atmosphere. The reaction was stirred for four days at room
temperature and followed by TLC on silica gel plates with n-
hexane-THF (3:1) as mobile phase. The reaction mixture was
filtered to remove the triethylamine hydrochloride, the dichloro-
methane was removed and the resulting colourless oil subjected to
column chromatography, using n-hexane-THF (3:1) as mobile
phase. Compound 8 was isolated as oil (1.5 g, 72%). Anal Calc.
C34H42Cl4N10P6S4 requires: C, 39.02; H, 4.04; N, 13.38%, M, 1046,
Found: C, 39.00; H, 4.01; N, 13.31%, [MeH þ Na]þ,1069. 1H NMR,
CDCl3, 298 K: 7.9e7.3 m, 20H, AreCH; 4.2m, 4H, NHCH2; 3.7e2.8 m,
8H, NCH2; 2.7 broad, 2H, NH; 1.7e2.1 m, 8H, CCH2 (spiro and
bridge).
4.2. Biological assays
4.2.1. Cytotoxicity of the compounds
Cell Lines. The following in vitro human cancer cell lines were
used: HT-29 (human colon adenocarcinoma), Hep2 (Human
epidermoid larynx carcinoma) and Vero (African green monkey
kidney). The cell lines (HT-29/An1-9704220, Hep2/An1-92041501,
Vero/An10-97121501) were purchased from the Institute of Foot
and Mouth Diseases, Virology Laboratory, Ankara, Turkey.
4.1.2. Synthesis of compound 9
Compound 4 [40] (2.0 g, 4.6 mmol) and triethylamine (2.6 mL,
18.4 mmol) were dissolved in 100 mL of dichloromethane in
a 250 mL three-necked round-bottomed flask. The reaction mixture
was cooled in an ice-bath and spermine (0.9 g, 4.6 mmol) in 10 mL
of dichloromethane was added to the stirred solution under argon
atmosphere. The reaction was stirred for 23 h at room temperature
and followed by TLC on silica gel plates n-hexane-THF (3:2) as
mobile phase. The reaction mixture was filtered to remove the
triethylamine hydrochloride formed, the dichloromethane was
removed and the resulting colourless oil product subjected to
column chromatography using n-hexane-THF (3:2) as mobile
phase. Compound 9 was isolated as a white powder (1.9 g, 45%, mp
>250 ꢀC). Anal Calc. C18H30Cl4F8N10O4P6 requires: C, 23.24; H, 3.25;
N,15.06%, M, 930, Found: C, 23.20; H, 3.21; N,15.00%, [M þ H]þ, 931.
1H NMR, CDCl3, 298 K: 4.3e4.2 m, 8H, OCH2, 4.2e4.1 4H, NHCH2;
3.9e2.8 m, 8H, NCH2; 2.80 broad, 2H, NH; 2.0e1.6 m, 8H, CCH2
(spiro and bridge).
Cell Culture. HT-29 and Vero cells were cultured in Dulbecco
Modified Eagle Medium (DMEM) (Sigma, Steinheim, Germany)
containing 2 mM Na2CO3 supplemented with 10% (v/v) foetal
bovine serum (FBS) (Sigma, Steinheim, Germany). Hep2 cells were
cultured in EMEM (Sigma, Steinheim, Germany) and 10% (v/v) of
foetal bovine serum. The cell culture media was supplemented with
penicillin/streptomycin at 100 Units/mL (Sigma, Steinheim,
Germany) as adherent monolayers. Cell lines were incubated at
37 ꢀC under 5% CO2 and 95% air in a humidified atmosphere. Stock
solutions were prepared in dimethyl sulfoxide (DMSO) (Sigma,
Steinheim, Germany) and further dilutions were made with fresh
culture medium. The concentration of DMSO in the final culture
medium was 1%, which had no effect on the cell viability.
MTT assay. The cytotoxic response to the cell lines and the
growth-inhibitory effects of compounds were determined by MTT
assay using a colorimetric substrate tetrazolium salt, 3-[4,5-
dimethyl-thiazollyl-2-yl]-2,5-diphenyltetrazolium bromide (Appli-
Chem, Darmstadt, Germany) [43]. Hep2 (2 ꢄ 104 cells/mL), Vero
(2 ꢄ 104Darmstadtcells/mL) and HT-29 (3 ꢄ 104Darmstadtcells/mL)
4.1.3. Synthesis of compound 10
Compound 5 [40] (0.4 g, 0.9 mmol) and triethylamine (0.5 mL,
3.7 mmol) were dissolved in 40 mL of dichloromethane in a 100 mL
cells were incubated with the compounds (0.2e50 mg/mL) in 96-
well plates (TPP, Switzerland) for 24 h. After incubation, culture

T. Yıldırım et al. / European Journal of Medicinal Chemistry 52 (2012) 213e220
219
mediumwas removed and 100
m
L of MTTreagent (1 mg/mL in serum
C. albicans (ATCC 10231) standard strain was tested. Compounds
were dissolved in dimethyl sulfoxide and sterilized by membrane
filter. Compounds were tested at 2, 4, 8, 16, 32, 64, 128, 256, 512 and
free medium) was added to each well. The plates were incubated for
2 h at 37 ꢀC. MTT containing medium was removed and each well
was washed gently with 100
was dissolved by addition of 100
m
L of PBS. The blue formazan product
L of 100% DMSO per well. The
1024 mg/mL concentrations. Standard strains were incubated on
m
Sabouraud dextrose agar (Merck, Darmstadt, Germany) plates at
37 ꢀC for 48 h. After incubation, the yeast suspension was adjusted
to a turbidity of 0.5 McFarland. Yeast suspension was diluted seri-
ally by 1:50 and then 1:20 in RPMI 1640 media (Sigma, Steinheim,
plates were swirled gently for 10 min to dissolve the precipitate, and
quantified by measuring the optical density (OD) of the plates at
a wavelength of 540 nm on a micro plate reader (Bio-Tek ELX808 IU).
The mean OD values for the negative control (DMSO treated cells)
were standardized as 100% OD (i.e. no growth inhibition) and results
were displayed as OD (% of control) concentrations of the
compounds. ID50 value is the concentration of compounds required
to inhibit growth by 50%. Each concentration was repeated in three
wells and control cell viability was considered as 100%. The same
experimental conditions were provided for all compounds and MTT
analysis was repeated three times for each cell line.
Germany). 50 mL of inoculum were inoculated into each well. The
plates were incubated at 35 ꢀC for 48 h. All tests were performed
twice. MIC’s were defined as the lowest concentrations of
compounds at which 50% of the fungi were inhibited compared to
the positive control.
4.3. Statistics
Determination of apoptosis. Apoptosis or necrosis was discrimi-
nated with the annexin V-FITC/propidium iodide test [44]. Cells
were seeded at 2 ꢁ 3 ꢄ 106/well in 10% FBSeDMEM into 75 cm2
tissue culture flasks (Greiner, Bio-one) and treated with
compounds for 24 h. Phosphatidylserine externalisation was
determined by annexin V staining. Culture flasks were washed with
PBS 3 times. Cells were pelleted and re-suspended in 1 mL annexin
binding buffer (10 mM HEPES [pH: 7.4], 140 mM NaCl, 2.5 mM
CaCl2) (Sigma, Steinheim, Germany). The cells were stained with
100 ng/mL annexin V-FITC-conjugated (fluorescein isothiocyanate)
Statistical analyses were performed with SPSS 18.0 for
Windows. Probit analysis was used to detect the value of ID50. The
results were shown as ID50 ꢂ standard error of mean (SE) for three
cell lines.
Acknowledgments
The authors thank the Scientific and Technical Research Council
of Turkey for financial support TUBITAK (Grant 108T355). We are
grateful to Dilek USTA for technical assistance for use of the flow
cytometer at the Department of Hematology, Ondokuz Mayis
University Hospital, Samsun.
(BD, Pharmingen) and 0.1 mg/mL of propidium iodide (PI) (Calbio-
chem, USA). Cells were incubated for 15 min at room temperature
in the dark and cells were analyzed with a flow cytometer (FACS-
Calibur, BD Biosciences). Etoposide (12.5e25e50
mg/mL) (Calbio-
chem, Darmstadt, Germany) and cisplatin (200
mM) (Sigma,
Steinheim, Germany) were used as internal positive controls. The
spectral overlap was electronically compensated for with single-
colour control cells stained with annexin V-FITC or PI. Analysis was
performed with the system software (CellQuest; BD Biosciences).
Lower left quadrant, viable cells (annexin Vꢁ/PIꢁ); lower right
quadrant, early apoptotic cells (annexin Vþ/PIꢁ); upper right
quadrant, late apoptotic cells (annexin Vþ/PIþ); upper left quad-
rant, necrotic cells (annexin Vꢁ/PIþ). The percentage of cells
positive for PI and/or Annexin V-FITC was reported inside the
quadrants. The experiment was repeated three times in duplicate,
with similar results.
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