Synthesis, characterization and in vitro anticancer evaluations of two novel derivatives of deferasirox iron chelator

Article abstract of DOI:10.1016/j.ejphar.2016.04.026

Iron (Fe) chelation therapy was initially designed to alleviate the toxic effects of excess Fe evident in Fe-overload diseases. However, the novel toxicological properties of some Fe chelator-metal complexes have shifted significant attention to their application in cancer chemotherapy. The present study investigates the new role of deferasirox as an anticancer agent due to its ability to chelate with iron. Because of aminoacids antioxidant effect, deferasirox and its two novel amino acid derivatives have been synthesized through the treatment of deferasirox with DCC as well as glycine or phenylalanine methyl ester. All new compounds have been characterized by elemental analysis, FT-IR NMR and mass spectrometry. Therefore, the cytotoxicity of these compounds was screened for antitumor activity against some cell lines using cisplatin as a comparative standard by MTT assay and Flow cytometry. The impact of iron in the intracellular generation of reactive oxygen species was assessed on HT29 and MDA-MB-231 cells. The potential of the synthesized iron chelators for their efficacy to protect cells against model oxidative injury induced was compared. The reactive oxygen species intracellular fluorescence intensity were measured and the result showed that the reactive oxygen species intensity after iron incubation increased while after chelators incubation the reactive oxygen species intensity were decreased significantly. Besides, the effect of the synthesized compounds on mouse fibroblast cell line (L929) was simultaneously evaluated as control. The pharmacological results showed that deferasirox and its two novel aminoacid derivatives were potent anticancer agents.

Full text of DOI:10.1016/j.ejphar.2016.04.026

Author’s Accepted Manuscript
Synthesis, characterization and in vitro anticancer
evaluations of two novel derivatives of deferasirox
iron chelator
Samie Salehi, Amir. Sh. Saljooghi, Ali Shiri
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PII:
S0014-2999(16)30241-2
http://dx.doi.org/10.1016/j.ejphar.2016.04.026
EJP70594
DOI:
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To appear in: European Journal of Pharmacology
Received date: 27 January 2016
Revised date: 11 April 2016
Accepted date: 13 April 2016
Cite this article as: Samie Salehi, Amir. Sh. Saljooghi and Ali Shiri, Synthesis,
characterization and in vitro anticancer evaluations of two novel derivatives o
deferasirox
iron
chelator, European Journal of Pharmacology
http://dx.doi.org/10.1016/j.ejphar.2016.04.026
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Synthesis, characterization and in vitro anticancer evaluations of two novel derivatives of
deferasirox iron chelator

Samie Salehi, Amir. Sh. Saljooghi , Ali Shiri
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, 91775-1436
Mashhad, Iran.
Corresponding author. Amir. Shokooh Saljooghi, Ferdowsi University of Mashhad, Azadi
Square, Mashhad, Iran. Postcode: 91735.654. saljooghi@um.ac.ir
Abstract
Iron (Fe) chelation therapy was initially designed to alleviate the toxic effects of excess Fe
evident in Fe-overload diseases. However, the novel toxicological properties of some Fe
chelator-metal complexes have shifted significant attention to their application in cancer
chemotherapy. The present study investigates the new role of deferasirox as an anticancer agent
due to its ability to chelate with iron. Because of aminoacids antioxidant effect, deferasirox and
its two novel amino acid derivatives have been synthesized through the treatment of deferasirox
with DCC as well as glycine or phenylalanine methyl ester. All new compounds have been
characterized by elemental analysis, FT-IR, NMR and mass spectrometry. Therefore, the
cytotoxicity of these compounds was screened for antitumor activity against some cell lines
using cisplatin as a comparative standard by MTT assay and Flow cytometry.
impact of
MDA-MB-

2
31
Besides, the effect of the synthesized compounds
on mouse fibroblast cell line (L929) was simultaneously evaluated as control. The
pharmacological results showed that deferasirox and its two novel aminoacid derivatives were
potent anticancer agents.
Keywords: Iron chelating therapy, Deferasirox,
Apoptosis, Confocal laser scanning microscope.
ROS), MTT assay,
1
. Introduction
Iron chelation therapy (ICT) has attracted many attentions due to their important roles as
chelators (Kontoghiorghes et al., 2005; Nathan and Ann, 2005). Although ICT was first designed
to reduce the toxic effects of extra Fe evident in Fe-overload diseases, the novel toxicological
properties of some Fe-chelator complexes have shifted their intended application to cancer
chemotherapy (Liu and Hider, 2002)(Fig. 1).
β
Lovejoy and Richardson, 2000)
The other major effect of overloaded iron is the increase of cancer risk due to the generation
of reactive oxygen species (Frey and Reed, 2012; Kell, 2009). Iron can catalyze the production
of reactive oxygen species in terms of redox cycling when it is available in a labile, redox-active

form (Dixon and Stockwel, 2014; Britton et al., 2002; Liochev, 1999; Burkitt, 2003; Dunford,
002). Iron and are increasingly recognized as important initiators and
2
mediators of cell death in a variety of organisms and pathological situations (Griendling and
FitzGerald, 2003; Terman and Brunk, 2006; Kalinowski and Richardson, 2005; Olivieri and
Brittenham, 1997).
Fig. 1
Some Fe chelators are able to bind and inhibit the redox activity of Fe, making them ideal
candidates for the treatment of Fe-overload conditions (Chaston and Richardson, 2003;
Richardson et al., 2006). Such ligands are able to prevent excess Fe from participating in Fenton
chemistry (Chaston et al., 2003) and inhibit the formation of reactive oxygen species, such as the
hydroxyl radical, which initiates oxidative damage. Hence, Fe chelators that remove excess Fe
and form redox-inactive complexes provide a useful method of treatment in preventing the toxic
effects of Fe overload (Galey 2001; Halliwell and Gutteridge, 2007).
Torti and Torti, 2013)
the fact that cancer
cells typically require more iron than normal cells to mediate their generally rapid DNA
synthesis and growth. Therefore, iron is a target for cancer therapy (Lui et al., 2013).
Two broad strategies in the use of iron chelators in cancer treatment have been explored. The
first has been to use iron chelators to reduce cancer cells of iron. A second, more recent strategy
has been to use chelators that help the redox cycling of iron to generate cytotoxic
within tumors. Both methods are currently being pursued (Coombs et al., 2012).
In this regard, the choice of donor atom play an important role in the ability of a Fe chelator
complex to redox cycle and consequently determines its toxicological profile. Those chelators
which contains “hard” donor atoms such as oxygen form a stable complex with high-spin Fe (III)

and stabilize the ferric state, are not able to form redox-active cycle in the biological conditions
Liu and Hider, 2002) so these class of chelators enter tumor cells and bind intracellular iron,
(
depriving these cells of this important metal and cause to tumor death (Kalinowski and
Richardson, 2007) whereas chelators that are able to bind both Fe (II) and Fe (III) have the
potential to redox cycle. Iron complexes of chelators applying “soft” donor atoms, such as the
sulfur donor atoms, can be enzymatically reduced under physiological conditions. Furthermore,
these agents can also form redox-active iron complexes, the resulting Fe (II) can participate in
Fenton chemistry, generating reactive oxygen species and oxidative damage. (Chaston and
Richardson, 2003). The metal ions key role in both reactive oxygen species production and
clearance, have been proved (Jomova and Valko, 2011). Metal ions such as iron readily bind
reactive oxygen species ligands, and produce hydroxyl radical in solvent-exposed cellular
environments, notably via variations of the simplest form of the Fenton reaction. The actual
mechanisms can differ substantially (Kepp, 2012) but commonly aggravate oxidative stress by
converting H O to much more potent hydroxyl. A simplified type of Fenton reaction, is shown
2
2
in eqn. 1. (Yu et al., 2012; Liu and Hider, 2002)
Some examples of these chelators include the commercially available ligands: deferiprone
®
®
(
Ferriprox ), deferasirox (Exjade ) and desferrioxamine (Desferal). These agents which easily
coordinate with iron are bidentate, tridentate and hexadentate ligands, respectively (Bendova et
al., 2010).
3
+
Deferasirox is a chiral, tridentate ligand for Fe which has been recently reported as oral iron
chelator with efficacy similar to deferoxamine (Steinhauser et al., 2004; Heinz et al., 1999).
Early studies examined the anti-cancer efficacy of DFO in neuroblastoma cell lines where

exposure to the ligand (60 µM) for 72 h resulted in approximately 90% cell death (Estrov et al.,
987; Nick et al., 2002; Hegetschweiler, 1999). Considering its oral-activity and low toxicity, a
number of recent studies have examined the potential of deferasirox as an anticancer agent
1
(Donfrancesco et al., 1990; Chantrel-Groussard et al., 2006; Lescoat et al., 2007).
Further studies found us that deferasirox has been shown high anti-proliferative activity
against human hepatoma cells (Vazana-Barad et al., 2013; Nick et al., 2003). In fact, deferasirox
was able to reduce cell viability and inhibit the cell cycle at the G /G and S-phase in human
0
1
hepatoma cell line, HUH7 (Epsztejn et al., 1999). Moreover, the anti-tumor effects of deferasirox
were more pronounced in hepatoma cells relative to primary cultures of human hepatocytes
(Song et al., 2011).
Amino acids like glycine or their corresponding methyl esters alone or accompanying with
some bioactive molecules have been proved to show antimycobacterial activity (Stavrakov et al.,
016) and antioxidant effects (Mauriz et al., 2001) It has also been shown that deferasirox
2
combined with glycine can reduce blood lead content in animal models. (Najarnezhad and Asri-
Rezaei, 2015). These finding prompted us to synthesize combined deferasirox–glycine or
phenylalanine methyl esters and evaluate the possible enhancement effects of them as iron
chelating in chemotherapy. In fact, the present study is conducted to compare the efficacy of the
deferasirox as a chelator and its glycine or phenylalanine methyl ester derivatives to investigate
their anticancer activity against human breast cancer cells (MDA-MB-231), human cervix
epithelial carcinoma (HeLa), human colon cancer cell line (HT-29), human leukemia cell line
(K-562), bladder cancer cell line (T-24), non-small cell lung carcinoma (A-549), mouse
neuroblastoma cell line (Neuro-2a) and mouse fibroblast L-929 cell lines. The results were
compared with cisplatin as standard through MTT assay. Also

measurement was performed using H DCFDA to evaluate the impact of iron on the
2
concentration and the role of synthesized chelator to
quenching in the MDA-MB-231 and HT-29 cells.
2
2
. Materials and methods
.1. Chemicals and analysis
All material and compounds were purchased commercially from Sigma-Aldrich. Reactions were
analyzed on precoated silica gel 60 F254 plates (Merck) and the compounds were visualized with
a UV lamp (254 nm). Melting points were determined on a Barnstead Electrothermal 9300
apparatus. IR measurements were performed on an FT/IR AVATAR 370 Thermo Nicolet
spectrometer using KBr discs. Elemental analyses were carried out with a Thermo Finnigan
1
13
Flash EA-1112 micro analyzer and Perkin-Elmer 2004(II) apparatus. H and C NMR spectra
were recorded on a Bruker 400 or 300 MHz spectrometer. Chemical shifts (δ) are reported in
parts per million (ppm) and the signals are quoted as s (singlet), br (broad), d (doublet) and m
(multiplet). Mass spectra were scanned on a Varian Mat CH-7 at 70 eV.
2
2
2
.2. Synthesis
.2.1. 4-[3,5-Bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoicAcid (Deferasirox) (DFX)
-(2-Hydroxyphenyl)-4H-3,1-benzoxazin-4-one was prepared according to the previous reported
procedure with a little modifications (Lattmann and Acklin, 1997; Ryabukhin et al., 1983). 4-
Hydrazino-benzoic acid (11.5 mmol, 1.75 g) and Et N (11.5 mmol, 1.16 g) were dissolved in
3
boiling EtOH (80 ml). Then, 2-(2-Hydroxyphenyl)-4H-1,3-benzoxazin-4-one (10.45 mmol, 2.50
g) was added to the clear solution, and the reaction mixture was refluxed for an additional 2 h.

After the completion of the reaction, the solution was cooled to room temperature, and water was
added until the first sign of precipitation was observed. The mixture was concentrated to a total
volume of 50% under reduced pressure and aqueous 6M HCl (40 ml) was added. The resulting
solid was filtered, washed with water and dried for 24 h in vacuo (3.11 g, Yield = 80%).
−
1
1
IR (KBr, cm ) ν 33317, 2540, 1680 (ν ), 1607, 1517, 1495, 1431, 1351, 1221, 988, 752. H
C=O
NMR (CD COCD , ppm):δ= 7.00 (s, 1 H), 7.01-7.04 (m, 3 H), 7.39 (m, 2 H), 7.48 (d, 1 H), 7.53
3
3
1
3
(
d, 2 H), 8.15 (d, 2 H), 8.19 (d, 1 H) 10.00 (s, OH), 10.78 (s, OH ) ppm. C NMR (CD COCD ,
3 3
ppm) δ= 113.7, 113.9, 116.6 (CH), 117.0 (CH), 119.5 (CH), 119.8 (CH), 124.0 (2 CH), 126.9
CH), 130.4 (2 CH), 130.5, 130.7 (CH), 131.4 (CH), 132.6, 141.9 (CH), 152.1, 155.6, 156.4,
(
1
6
60.4, 165.7 (C=O ) ppm. MS (m/z) for C H N O = (374) Anal. Calc. for C H N O : C
21 15 3 4 21 15 3 4
7.56, H 4.05, N 11.25; found C 67.16, H 4.09, N 10.86.
Gly
Phala
2
.2.2. General coupling procedure for preparation of compounds DFX , DFX
A solution of carboxylic acid (1.1 equiv. per equiv. of NH group), DCC (1.1 equiv. per equiv. of
2
NH group), and DMAP (equiv. per equiv. of NH group) in CH Cl (10 ml per mmol of
2
2
2
2
aminoacid) was stirred at RT for 15 min. The methyl ester of aminoacid was added and the
reaction was stirred at RT overnight (Rouge et al., 2012). The reaction was diluted with CH Cl
2
2
(100 ml per mmol of aminoacid) and this organic phase was washed successively with
1
Maqueous HCl, saturated aqueous NaHCO , water, andbrine. The organic phase was dried over
3
anhydrous Na SO and then evaporated by rotary evaporation under reduced pressure. The crude
2
4
product was purified by column chromatography using CHCl / MeOH (19:1) as eluent and then
3
recrystallized in EtOH.

2
.2.2.1. Methyl2-(4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)benzamido)-acetate
Gly
(
DFX )
This compound was obtained from glycine methyl ester hydrochloride (0.391 mmol, 0.20 g). A
−
1
pale red solid, (172 mg, Yield = 51 %). M.p. 190–195;. IR (KBr, cm ) ν 3248, 2929, 2847, 1745
(νC=O glaycinat), 1670 (νC=O deferasirox), 1608, 1589, 1516, 1409, 1352, 1252, 1158, 1068, 989,
1
8
6
57, 757, 690. H NMR (DMSO-d , ppm) δ=3.33 (s, 3H,CH ), 3.66 (s, 2H, CH ), 6.85 (d, 1 H),
6
3
2
.96-7.04 (m, 3 H), 7.38 (m, 2 H), 7.52 (d, 1 H), 7.55 (d, 2 H), 8.06 (d, 2 H), 8.19 (d, 1 H), 9.09
1
3
(
br s, 1H, -NH), 10.41 (br s, 1H, -OH), 10.76 (br s, 1H, -OH). C NMR (DMSO-d , ppm): δ=
6
4
2.3 (CH3), 52.4(CH2), 113.6, 114.0, 116.8 (CH), 117.0 (CH), 119.4 (CH), 119.8 (CH), 123.8
2CH), 127.1 (CH), 130.8 (2CH), 130.9, 131.0 (CH), 131.8 (CH), 132.0, 141.9 (CH), 152.1,
55.6, 156.4, 159.4 (C=O ), 179.3 (C=O glaycinat); MS (m/z) 444 for C H N O (444): Anal.
(
1
24
20
4
5
Calc. for C H N O : C 64.86, H 4.54, N 12.61; found C 64.68, H 4.38, N 12.52
2
4
20
4
5
2
.2.2.2. Methyl 2-(4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)benzamido)
Phala
phenylpropanoate (DFX
)
This compound was obtained from phenylalanine methyl ester hydrochloride (0.391 mmol, 0.20
−
1
g). Yellow solid, (172 mg, Yield = 37 %), m.p. = 180–185; IR (KBr, cm ) ν 3248, 2929, 2847,
1
1
4
745 (νC=O phenylalanine), 1670 (νC=O Deferasirox), 1608, 1589, 1516, 1409, 1352, 1252, 1158,
1
068, 989, 857, 757, 690. H NMR (DMSO-d , ppm) δ=3.33 (s, 3H, CH ), 3.63 (d, 2H,CH ),
6
3
2
.31 (t, 1H), 6.90-7.17(m, 3H), 7.38 (m, 2H), 7.28(d, 3H), 8.02 (m, 5H), 9.02 (s, NH), 10.49 (s,
OH), 10.80 (s, OH ) ppm. MS (m/z) Calc. for C H N O (534); Anal. Calc. for C H N O : C
24
20
4
5
31 26
4
5
6
9.65, H 4.90, N 10.48; found C 69.73, H 4.89, N10.52.

The structureof these synthesis compounds shown in Fig. 2.
Fig. 2
2
.3. Cell culture methods
human breast cancer cells MDA-MD-231 (ATCC HTB-26), human cervix epithelial carcinoma
HeLa (ATCC CCL-2), human colon cancer cell line HT-29 (ATCC HTB-38), human leukemia
cell line K-562 (ATCC CCL-243), bladder cancer cell line T-24 (ATCC TCP-1020), non-small
cell lung carcinoma A-549 (ATCC CCL-185), mouse neuroblastoma cell line Neuro-2a (ATCC
CCL-131), mouse fibroblast L-929 cell line (ATCC CCL-1) were obtained from the American
Type Culture Collection (ATCC; Manassas, VA, USA) and cultured at 37 °C in a humidified
atmosphere of 5% CO in air HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium
2
(DMEM) with 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.0 mM sodium pyruvate
and 5 % fetal bovine serum, at 37 °C in an atmosphere of 5 % CO . Cells were plated in 96-well
2
4
sterile plates at a density of 1×10 cells/well in 100 μL of medium and incubated for 24 h. Also
MDA-MD-231, HT-29, Neuro-2a and T-24 cells were cultured in DMEM containing 10 % fetal
bovine serum, 100 units/ ml of penicillin and 100 μg/ ml of streptomycin. L-929 and K-562 cells
were cultured in RPMI-1640 medium containing 10 % fetal bovine serum, 100 units/ ml of
penicillin and 100 μg/ ml of streptomycin. A-549 cells were grown in F-12K Ham's medium
supplemented with 1 % glutamine, 1 % antibiotic/antimycotic, 2 % NaHCO and 10 % fetal calf
3
serum.
3
3
. Results
.1. MTT assay in human cancer cell lines

Synthesis compounds were screened for antitumor activity against human breast cancer cells
MDA-MD-231) human cervix epithelial carcinoma (HeLa), human colon cancer cell line (HT-
9), human leukemia cell line (K-562), bladder cancer cell line (T-24), non-small cell lung
(
2
carcinoma (A-549), mouse neuroblastoma cell line (Neuro-2a), mouse fibroblast L-929 cell line
and using cisplatin as a comparative standard. Cell viability was evaluated by using a
colorimetric method based on the tetrazolium salt MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]), which is reduced by living cells to yield purple formazan
4
crystals. Cells were seeded in 96-well plates at a density of 2-5×10 cells of MDA-MD-231,
HeLa, HT-29, T-24, K-562, A-549, Neuro-2a and L-929 per well in 200 μL of culture medium
and left to incubate overnight for optimal adherence. After careful removal of the medium, 200
μL of a dilution series of the compounds in fresh medium were added and incubation was
Gly
Phala
performed at 37 °C/5% CO for 72 h. DFX, DFX , DFX
were first solubilized in DMSO,
2
diluted in medium and added to the cells in final concentrations between 20 nM and 200 μM.
The percentage of DMSO in cell culture medium did not exceed 0.3%. Cisplatin was first
solubilized in saline and then added at the same concentrations used for the other compounds. At
the end of the incubation period, the compounds were removed and the cells were incubated with
2
00 μL of MTT solution (500 μg/ ml). After 3–4 h at 37 °C/5% CO , the medium was removed
2
and the purple formazan crystals were dissolved in 200 μL of DMSO by shaking. The cell
viability was evaluated by measurement of the absorbance at 570 nm by using a STAT FAX-
2
100 microplate reader (Awareness Technology, Palm City, FL, USA). The cell viability was
calculated dividing the absorbance of each well by that of the control wells (cells treated with
medium containing 1% DMSO). Each experiment was repeated at least three times and each
point was determined in at least four replicates.

3
.2. Intracellular reactive oxygen species Generation Measurement Using H DCFDA.
2
To assess reactive oxygen species production, HT29 and MDA-MB-231 cells were treated with
the oxidation-sensitive dye, 2’,7’-dichlorodihydrofluorescein diacetate (H DCF-DA, Invitrogen,
2
Cat. D399). The oxidation product of H DCF-DA generates the fluorescent 2’,7’-
2
dichlorofluorescein (DCF) which has excitation/emission maxima of ~488/523 nm. Following
the addition of the Fe donor, ferric ammonium citrate (30 µM) the changes in intracellular
°
fluorescence intensity (λ =488 nm; λ =530 nm) were followed every 15 min for 1 h/37 C with
ex
em
H O as positive control (Claudia et al., 2012).
2
2
Gly
Phala
3
.3. Apoptosis assay for compounds DFX, DFX and DFX
by flow cytometry
In order to study in which way deferasirox and its aminoacid derivatives produced the cellular
death (necrosis or apoptosis) studies of flow cytometry were performed on synthesized
compounds, and cisplatin as a reference. These compounds were incubated for 24 h at a
concentration close to the IC and the results were shown in Fig. 4. Four areas in the diagrams
5
0
stand for necrotic cells (Q1, low Annexin V-FITC and high PI signal, left square on the top), late
apoptosis or necrosis cells (Q2, high Annexin V-FITC and high PI signal, right square on the
top), live cells (Q3, low Annexin V-FITC and low PI signal, left square at the bottom), apoptosis
cells (Q4, high Annexin V-FITC and low PI signal, right square at the bottom), respectively. As
shown in Fig. 4 and tables 2-4, deferasirox and its two novel aminoacid derivatives could induce
apoptosis against MDA-MB-231 and HT-29 cancer cell lines. But the proapoptotic property
needs further investigation to better understand the precise mechanism of action of the
complexes.
Also apoptosis was detected using an in situ cell death detection kit (Boehringer Mannheim
Corp., Indianapolis, IN) as described by Narla et al. and Zhu et al. Cells were incubated with

compounds 1-3 in 0.3% DMSO or 1:16-diluted plasma samples from DFX-treated mice for 48 h
at 37 °C, and were fixed, permeabilized, incubated with the reaction mixture containing TdT-
and FITC-conjugated dUTP, and counterstained with propidium iodide. Cells were transferred to
slides and viewed with a confocal laser scanning microscope (Bio-Rad MRC 1024) mounted on
a Nikon Eclipse E800 series upright microscope as reported previously (Narla et al., 2000; Zhu
et al., 1998).
3
.4. Statistical analysis
IC values expressed as mean ± standard deviation (S.D.) from at least three independent
5
0
experiments. Statistical tests including One way ANOVA, Tukey multiple comparison or
unpaired Student’s t tests were performed using SPSS, ver.17 software. A p value of less than
0
4
.05 was considered as significant.
. Discussion
Iron is crucially involved in the formation and destruction of
, a blanket
term for a collection of partially reduced oxygen containing molecules, including superoxide
•
–
•
•
(
O ), peroxides (H O and ROOH) and free radicals (HO and RO ). Although certain amounts
2 2 2
of iron and reactive oxygen species are crucial for normal cell and organismal function (Lim et
al., 2008; Lambeth et al., 2014), the unusual accumulation of iron, or
both is linked to a staggering number of acute traumas and chronic degenerative conditions.
Iron chelators such as deferasirox, have been implemented or proposed as treatments for
diverse pathologies linked to iron or
accumulation (Szakács et al., 2004).
A potential problem associated with iron chelation is the generation of oxidative stress.
have a considerable potential to damage a variety of cellular components. Early
studies confirmed that in view of a possible role in redox catalysis, reduction of

III
3-
[
Fe (Defereasirox) ] is not possible in a physiological medium, and the 1:2 complex is thus
2
redox inactive (Steinhauser et al., 2004) So this class of iron chelator could do their
antiproliferative activities by entering tumor cells and bind intracellular iron, depriving these
cells of this important metal and stop the tumor growth without producing
(Ohyashiki et al., 2009).
Synthesis of deferasirox and its aminoacid derivatives prepared by an easy protocole.
deferasirox and two novel of its aminoacid derivatives synthesized and the structures of
Gly
Phala
synthesized title compounds (DFX, DFX , DFX
) were confirmed by FT-IR, NMR, CHN-S
and Mass techniques.
MDA-MB-231
Yuan et al., 2004
′
′
Ming-Gao et al.,
production, the
(30 µM)
Richardson and Baker, 1992), either alone or in combinations with synthesized chelators at their
binding ratio (2:1).
2
009 To evaluate the Fe role in increasing the
MDA-MB-231 cells were incubated with the Fe donor,
(
µM)

in
MDA-MB-231.
Fig. 3
In this study, the potential of interesting iron chelators, deferasirox and its two aminoacid
derivatives as effective anticancer compounds have been investigated in vitro. In order to gain
this aim, the in vitro cytotoxiciy of these three compounds, against MDA-MB-231, HeLa, HT-
2
9, K-562, T-24, A-549, Neuro-2a, and L-929 cell lines were determined by MTT-based assays
Gly
Phala
(
Table 1). For compounds DFX, DFX , DFX
and cisplatin as a comparative standard, we
have measured their IC values in all cell lines. Such measurements were done after 72 h of
5
0
incubation and using concentrations of the several compounds in the range 20 nM and 200 μM.
The values determined for these compounds spanned between 4.4 and 183.2 μM for DFX, 3.7
Gly
Phala
and 172.5 μM for DFX , 3.4 and 239.4 μM for DFX , while those found for the
comparative standard ranged between 0.5 an up to 200 μM (Table 1).
Deferasirox exhibits the highest selectivity against MDA-MB-231 and HT-29. It is notable
Gly
Phala
that DFX and DFX
exhibits the highest selectivity against HT-29. Deferasirox in the same
range is nearly two times more cytotoxic than cisplatin against MDA-MB-231 while it is nearly
Gly
Phala
the same as cisplatin against HT-29. Compounds DFX and DFX
are nearly five times
more cytotoxic than cisplatin against HT-29. Also, the compounds are inactive against all cancer
cell lines (except MDA-MB-231 and HT-29) and L-929. The effect of synthesized compound on
mouse fibroblast cell line (L-929) was evaluated as control, simultaneously. Our results
confirmed that the synthesized compounds have not any cytotoxicity effects on L-929.

Based on the results of in vitro cytotoxicity studies of deferasirox and its two aminoacid
derivatives, the highly active compounds were selected for apoptosis assay by flow cytometry.
The results were shown in Tables 2-4 and Fig. 4. Four areas in the diagrams stand for necrotic
cells (Q1, left square on the top), late apoptosis or necrosis cells (Q2, right square on the top),
live cells (Q3, left square at the bottom), apoptosis cells (Q4, right square at the bottom),
respectively. As it could be seen in tables 2 and Fig. 4, DFX showed a high population of
apoptotic cell (69.3%) and nearly 1.2-fold higher than cisplatin (58.1%) at the same
Gly
Phala
concentration. Also according to tables 3 and 4, DFX and DFX
apoptotic cell (58.0%) and nearly 1.6-fold higher than cisplatin (36.8%) and (73.1%) and nearly
-fold higher than cisplatin (36.8%) for cat the same concentration, respectively.
showed high population of
2
Fig. 4
The results demonstrated that the newly synthesized compounds could induce apoptosis
against MDA-MB-231 and HT-29 cancer cell lines. But the pro apoptotic property needs further
investigation to better understand the precise mechanism of action of these compounds and basic
pre-clinical research is needed before they could be recommended for human administration.
Also synthesis compounds induced cell death was confirmed to be apoptotic using the TUNEL
of exposed 3'-OH termini of DNA with dUTP-FITC. As shown in the confocal laser scanning
microscopy images in Fig. 5, DFX-treated MDA-MB-231 breast cancer cells, examined for
dUTP-FITC incorporation (green fluorescence) and propidium iodide counterstaining (red
fluorescence), exhibited many apoptotic yellow nuclei (superimposed green and red
Gly
Phala
fluorescence) at 24 h after treatment. The same results obtained for DFX and DFX
.
Fig. 5

5
. Conclusion
The following study has reported synthesis, spectroscopic characterization and anticancer
activity of deferasirox iron chelators and its two new aminoacid derivatives. The anticancer
effect of two new derivations was compared with parent compound and cis platin as standard.
The results can be summarized as follows:
1
. Besides the main use of deferasirox in reducing chronic iron overload in patients receiving
long-term blood transfusions (i.e., Beta-thalassemia and chronic anemia) this well-known iron
chelator also exhibits a number of other therapeutic effects including anti-cancer activity. Here
our results have confirmed the potent cytotoxic activity of these derivatives against MDA-MB-
2
31 and HT-29 compared to other cell lines. Also the results demonstrated that the newly
synthesized compounds could induce apoptosis against MDA-MB-231 and HT-29 cancer cell
lines.
2
. The result showed that synthesized compounds have not any cytotoxicity effects on L-929,
which indicate deferasirox and its derivatives could be considered as high potential Iron
chelators to treat in cancer therapy. These results also verify the previous study which considered
new role for iron chelators as anticancer agents.
3
. The result confirmed the potential of deferasirox and its derivatives ability to reduce the
concentration induced by iron incubation in the treated cells. The
florescent intensity measurement showed that the
concentration
concentration and
increased meaningfully after iron incubation while the
the florescent intensity after chelator incubation decreased in compare to control and free Fe.
. Furthermore, synthesis compounds have been successfully used for bio imaging by using a
confocal laser scanning microscope, which may be favorable for biological applications.
4

Figure legends:
Fig. 1. The role of iron in
metabolism and iron chelators antiproliferative
2
+
activity mechanism. The reaction of peroxides with Fe to yield soluble hydroxyl (HO•) radicals
is referred to as the Fenton reaction. Key enzymes that contribute to
formation or detoxifiation are shown. Those whose function is iron dependent are colored blue,
and the relevant iron species is in parentheses. Clinical iron chelator such as deferasirox are used
to enter tumor cells and bind intracellular iron, depriving these cells of this important metal and
stop the tumor growth.
Fig. 2. Three synthesis compounds as anticancer agents.
Fig. 3. Intracellular
induced by
production in HT-29 and MDA-MB-231 cells
(30μM), and various synthesized Fe chelators at their iron-
binding ratio concentrations (60μM). Following the intracellular acetate group cleavage, an
increase in fluorescence intensity (λ =488; λ =520 nm) is proportional to probe oxidation by
ex
em
to green fluorescent DCF. effects of synthesized iron chelators on
quenching induced by incubation with 30 µM
in HT29 (A) and MDA-MB-231 (B).
Fig. 4. Cell death mechanism induced by safingol treatment in human breast (MDA-MB-231)
Gly
Phala
and human colon (HT-29) cells to the active compounds DFX, DFX , DFX
and cisplatin.
Four areas in the diagrams represent four different cell states: necrotic cells (Q1), late apoptotic
or necrotic cells (Q2), living cells (Q3) and apoptotic cells (Q4). (A) Flow cytometric analysis of
Gly
MDA-MB-231 by DFX. (B) Flow cytometric analysis of HT29 by DFX . (C) Flow cytometric
Phala
analysis of HT29 by DFX . (Controls were obtained in absence of DMSO)

Fig. 5. Deferasirox and its two amino acid derivatives, induces apoptosis in human breast (MDA-
MD-231) and human colon (HT-29) cancer cell lines. MDA-MB-231 breast cancer (A: control,
A': After treatment with DFX ) and HT-29 human colon (B: control, B': After treatment with
Gly
Phala
DFX ) and (C: control, C': After treatment with DFX ) cells were incubated with 10 mM of
Gly
Phala
DFX, DFX , DFX
for 24 h, fixed, permeabilized and visualized for DNA degradation in a
TUNEL assay using dUTP-labeling. Red fluorescence, nuclei stained with propidium iodide.
Green or yellow (i.e., superimposed red and green) fluorescence, apoptotic nuclei containing
fragmented DNA. When compared with controls, treated with 0.3% DMSO (A, B and C),
Gly
Phala
several of the cells incubated with DFX, DFX and DFX
(A', B' and C') exhibited apoptotic
nuclei.
Acknowledgments
This work was supported by a grant number 3.28507, from Ferdowsi University of Mashhad,
Mashhad, Iran. We are grateful for help provided by Buali Research Institute, Faculty of
Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
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Gly
Phala
Table 1 Cytotoxic activities DFX, DFX , DFX
tested against MDA-MB-231, HeLa, HT-
2
9, K-562, T-24, A-549, Neuro-2a and L-929 cell lines after 72 h continuous Exposure.
a
IC ± S.D. (ìM)
5
0
Compound MDA-MB-231 HeLa
T-24
35.5 ± 3.9 55.8 ± 6.3 183.2 ± 11.7 18.3 ± 4.0 28. 3 ± 2.9 105.1 ± 9.8
37.5 ± 4.1 67.0 ± 7.8 106.7 ± 9.5 3.7 ± 0.5
42.1 ± 4.9 81.5 ± 7.3 165.4 ± 16.3 3.4 ± 0.8
0.8 ± 0.1 15.3 ± 4.2 22.3 ± 3.8 17.6 ± 2.4 1.5 ± 0.4
K-562
HT-29
A-549
Neuro-2a
L-929
DFX
DFX
DFX
4.4 ± 1.2
15.3 ± 4.5
14.9 ± 4.2
9.2 ± 1.1
100.7 ± 9.4
Gly
25.7 ± 3.2 172.5 ± 14.5 133.9 ± 11.8
34.5 ± 2.8 166.5 ± 17 239.4 ± 16.4
243.4 ± 17.2 0.5 ± 0.1
Phala
Cisplatin
a
The concentration of the complex required to inhibit cell growth by 50%. The experiments
were done in triplicate. Data were expressed as the mean of the triplicate.

Table 2 Percentages of the cell death pathways observed by the flow cytometry assay for DFX.
Treatment % Vital cells % Apoptotic cells % Late apoptotic/necrotic cells % Necrotic cells
Control
77.5
16.8
30.8
37.6
4.6
1.1
7.8
6.5
Cisplatin
34.1
27.3
31.7
Deferasirox 24.2
Table 3 Percentages of the cell death pathways observed by the flow cytometry assay for
Gly
DFX .
Treatment
Control
% Vital cells % Apoptotic cells % Late apoptotic/necrotic cells % Necrotic cells
98.4
58.3
41.15
0.37
0.74
27.9
35.4
0.49
Cisplatin
8.9
1.6
Gly
DFX
22.6
0.85
Table 4 Percentages of the cell death pathways observed by the flow cytometry assay for
Phala
DFX
.
Treatment
Control
% Vital cells % Apoptotic cells % Late apoptotic/necrotic cells % Necrotic cells
98.4 0.37 0.74 0.49

Cisplatin
58.3
26.8
8.9
27.9
37.9
1.6
0.1
Phala
DFX
35.2
Fig. 1.
Fig. 2.

Fig. 3.

Fig. 4.