Novel methylene modified cyclohexyl ethylenediamine-N,N′-diacetate ligands and their platinum(IV) complexes. Influence on biological activity

Article abstract of DOI:10.1016/j.jinorgbio.2012.01.012

This paper focuses on the synthesis, characterization and biological activity of new N,N′-methylene modified cyclohexyl ethylenediamine-N, N′-diacetate (edda)-type ligands and their Pt(IV) complexes. Both the ligands and complexes were characterized by infrared, UV-vis, ESI-MS, 1D ( ¹H, ¹³C, ¹⁹⁵Pt) and 2D (COSY, HSQC, HMBC) NMR spectroscopy and elemental analysis. The possible correlation between the reduction potentials and the cytotoxicity of the complexes was examined. The potential antitumoral activity of all compounds was tested in vitro on human melanoma A375, human glioblastoma U251, human prostate cancer PC3, human colon cancer HCT116, mouse melanoma B16 and mouse colon cancer CT26CL25 cells, as well as primary fibroblasts and keratinocytes. The results obtained revealed strong antitumor potential of the newly synthesized drugs with preserved efficacy against cisplatin resistant lines and less toxicity towards nonmalignant counterparts. The mechanism found to be responsible for the observed tumoricidal action of each synthesized compound was induction of apoptosis generally accompanied with caspase activation. Taken together, the effective response to the treatment of a wide range of different cell lines, including cisplatin resistant subclones, as well as induction of apoptosis, as the mechanism suggested to be the most desirable way of eliminating malignant cells, represents a great advantage of this novel group of drugs in comparison to other members in this metallo-drug family.

Full text of DOI:10.1016/j.jinorgbio.2012.01.012

Journal of Inorganic Biochemistry 109 (2012) 40–48
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel methylene modified cyclohexyl ethylenediamine-N,N′-diacetate ligands and
their platinum(IV) complexes. Influence on biological activity
Ljiljana E. Mihajlović a, Aleksandar Savić a, Jelena Poljarević a, Ivan Vučković a, Marija Mojić b,
Mirna Bulatović b, Danijela Maksimović-Ivanić b, Sanja Mijatović b, Goran N. Kaluđerović c,
a,
Stanislava Stošić-Grujičić b, Đorđe Miljković b, Sanja Grgurić-Šipka a, , Tibor J. Sabo
⁎
⁎
a
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
b
Department of Immunology, Instititute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia
Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straβe 2, 06120 Halle, Germany
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 June 2011
Received in revised form 29 December 2011
Accepted 20 January 2012
Available online 31 January 2012
This paper focuses on the synthesis, characterization and biological activity of new N,N′-methylene modified
cyclohexyl ethylenediamine-N,N′-diacetate (edda)-type ligands and their Pt(IV) complexes. Both the ligands
and complexes were characterized by infrared, UV–vis, ESI-MS, 1D (1H, 13C, 195Pt) and 2D (COSY, HSQC,
HMBC) NMR spectroscopy and elemental analysis. The possible correlation between the reduction potentials
and the cytotoxicity of the complexes was examined. The potential antitumoral activity of all compounds was
tested in vitro on human melanoma A375, human glioblastoma U251, human prostate cancer PC3, human
colon cancer HCT116, mouse melanoma B16 and mouse colon cancer CT26CL25 cells, as well as primary
fibroblasts and keratinocytes. The results obtained revealed strong antitumor potential of the newly synthe-
sized drugs with preserved efficacy against cisplatin resistant lines and less toxicity towards nonmalignant
counterparts. The mechanism found to be responsible for the observed tumoricidal action of each synthe-
sized compound was induction of apoptosis generally accompanied with caspase activation. Taken together,
the effective response to the treatment of a wide range of different cell lines, including cisplatin resistant
subclones, as well as induction of apoptosis, as the mechanism suggested to be the most desirable way of
eliminating malignant cells, represents a great advantage of this novel group of drugs in comparison to
other members in this metallo-drug family.
Keywords:
Platinum(IV)
Amine ligands
Anticancer
Apoptosis
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
leading to biological effects [11]. Platinum occurs in two oxidation
states. The Pt(II) square planar complexes show several limitations
Much interest in developing metal based drugs appeared in the
mid-1960s [1]. As frequently used anticancer agents, platinum con-
taining compounds have a unique place in metal coordination chem-
istry [2,3]. Almost five decades after Rosenberg's discovery of cisplatin
[4,5], significant progress has been made in alleviating the cytotoxic
properties of these compounds. In order to overcome severe side
effects, such as nephrotoxicity, neurotoxicity, ototoxicity, nausea,
etc. [6], numerous platinum based complexes have been synthesized
[7]. Although the search for ideal anticancer agents continues, only
two cisplatin analogues (carboplatin, oxaliplatin) [8] have led to pro-
gress in cancer treatment [9]. Rigorous clinical trials eliminated other
potential drugs, such as ipro- and tetraplatin, so intensive research in
this field continues. So far, satraplatin has shown the most beneficial
results, although it is still undergoing active clinical trials [10]. The in-
fluence of complex geometry plays a great role in specific interactions
compared with Pt(IV) octahedral complexes, such as less kinetic in-
ertness, cis geometry and low coordination number [12]. Indisputable
structural differences between Pt(II) and Pt(IV) complexes enable
more lipophilic compounds to be obtained [13], which can be con-
firmed by greater cellular uptake and increased cytotoxicity [14].
The use of orally administrable anticancer drugs is desirable for
numerous reasons (noninvasive therapies, reduced hospital costs),
which are the main indications for synthesizing novel complexes.
Considerable progress has been made using axial ligands with sev-
eral advantages [15], involving specific prodrug targeting and combi-
nation with bioactive compounds [16,17]. Selecting amine ligands
[18–20] that are biologically active themselves, made a visible break-
through in this field [21,22]. Ethylenediamine-N,N′-diacetate (edda)-
type of ligands [23], together with their derivatives exhibit structure–
activity relationships based on numerous in vitro tests [24]. While
Pt(II) and Pt(IV) complexes with edda type ligands show significant
cytotoxicity but are less efficient than cisplatin [25], Pt(IV) complexes
linked to esterified edda type ligands have greater anticancer activity
than cisplatin [16]. Moreover, intensive research has indicated two
⁎
Corresponding authors. Fax: +381 1 2184330.
E-mail addresses: sanjag@chem.bg.ac.rs (S. Grgurić-Šipka), tsabo@chem.bg.ac.rs
(T.J. Sabo).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2012.01.012

L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
41
different mechanisms of cell death. The above mentioned Pt(IV)
complexes with esterified edda type ligands induce rapid death
by necrosis [26], while cisplatin causes slower apoptotic cell death
[27].
and propidium iodide (PI) were obtained from Sigma (St. Louis, MO).
Annexin V-FITC (AnnV) was from Biotium (Hayward, CA). Tested
compounds were dissolved in DMSO and kept at −20 °C. The U251
glioblastoma cell line was
a kind gift from Dr. Pedro Tranque
Structure–activity relationships describe various structural
changes in the amine ligands. Earlier investigations by Keppler and
co-workers evaluated the way that modifying ligands influences the
cytotoxic properties [18,28]. Hence, the main concept of our study
was to examine whether methylation via both nitrogen donor
atoms of (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic
acid affects the biological activity of the corresponding ligands and
complexes. In this work, the synthesis, characterization and biological
activity of Pt(IV) complexes with methylated cyclohexyl functiona-
lized edda-type ligands are reported. In addition, their biological
activity was evaluated through assessment of cell viability and subse-
quent basic analysis of the mechanism of drug action. The results
presented here reveal the strong antitumor potential of these com-
pounds evident even in cell lines resistant to conventional cisplatin.
Finally, in comparison to non-methylated forms of esterified edda
type Pt(IV) complexes, the novel drugs act through induction of apo-
ptosis followed by caspase activity.
(Universidad de Castilla-La Mancha, Albacete, Spain). The B16 murine
melanoma was a kind gift from Dr. Siniša Radulović (Institute for
Oncology and Radiology of Serbia, Belgrade, Serbia). Human melanoma
A375, colon cancer HCT116 and SW620, prostate PC3 and mouse colon
CT26CL25 cell lines were a kind gift from Prof. Ferdinando Nicoletti
(Department of Biomedical Sciences, University of Catania, Italy). Cells
were routinely maintained in HEPES-buffered RPMI-1640 medium
supplemented with 10% FCS, 2 mM L-glutamine, 0.01% sodium
pyruvate and antibiotics (culture medium) at 37 °C in a humidified
atmosphere with 5% CO2. For the experiments, cells were detached
by standard trypsinization, resuspended in culture medium and seed-
ed at 1×104/well in 96-well plates for viability determination and
2.5×105/well in 6-well plates for flow cytometry.
2.2.2. Preparation of primary cell cultures of keratinocytes and adult lung
fibroblasts
Primary keratinocyte cell culture was prepared from mouse ear
skin. Briefly, excised ears were rinsed in 70% ethanol, washed in PBS
and split, the skin was spread dermis side down in a sterile tissue cul-
ture dish and left to float on 0.5% trypsin-PBS solution for 1 h at 37 °C.
The epidermis was removed, cut into small pieces and transferred to a
conus tube. Cells were vigorously resuspended to obtain single cell
suspensions and filtered through nylon mesh. Cells were washed
twice to remove trypsin, and finally resuspended in 15% FCS-RPMI
and immediately used for experiments. The primary fibroblast cell
culture was prepared from adult mouse lungs. In brief, lungs were
aseptically removed, rinsed in PBS, cut into small pieces and incubat-
ed in collagenase IV from Clostridium histolyticum (0.7 mg/ml) in ad-
dition to DNase (30 μg/ml) with slow stirring for 30 to 90 min at
37 °C. The solution containing tissue fragments was centrifuged, the
pellet was washed three times and resuspended in 15% FCS-RPMI.
After incubation for 24 h in the tissue culture dish, nonadherent
cells and tissue fragments were removed and the medium was
replaced. At 80–90% confluence, the cells were split and used for
experiments.
2. Experimental
2.1. Chemistry
2.1.1. Starting agents
The precursor substances, methyl, ethyl, n-propyl and n-butyl esters
of (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid were
synthesized starting from (S)-2-amino-3-cyclohexylpropanoic acid hy-
drochloride purchased from Senn Chemicals (Dielsdorf, Switzerland).
The entire method was previously described and published [26]. K2
[PtCl6] was synthesized by the standard procedure [29].
2.1.2. Measurements
Elemental analyses were performed on an Elemental Vario EL III
microanalyzer. A Nicolet 6700 FT-IR spectrometer and ATR technique
were used for recording infrared spectra. 1H and 13C spectra were
obtained using a Varian Gemini 200 spectrometer, while Burker
Avance III 500 spectrometer was employed for 1D (1H, 13C), 2D
COSY (Correlation Spectroscopy) and 2D 1H–13C heteronuclear corre-
lation spectra using CDCl3 and TMS as the reference. The 195Pt NMR
spectra were recorded on a Varian Unity 400 NMR spectrometer
and the shifts were determined using hexachloroplatinic acid as
external standard (δ 0 ppm). Recording ESI spectra was noted on a
6210 Time-of-Flight LC–MS instrument (G1969A, Agilent Technolo-
gies) in methanol. A GBC UV/Vis Cintra 6 spectrometer was used for
electronic spectra of complexes dissolved in methanol at 1×10−4 M.
Reagents and solvents were of commercial reagent grade quality and
used without further purification. An electrothermal melting point ap-
paratus was used for determining the melting points of each complex.
Electrochemical studies were performed by differential pulse voltam-
metry using CHI-760B instrument with a scan rate of 20 mV/s at
room temperature. The working electrode was a glassy carbon elec-
trode (Model 6.1204.300), the reference electrode was non-aqueous
Ag/AgCl electrode (Model CHI 112) and the counter electrode was a
platinum wire (Model CHI 115). The complexes, C1–C4 were dissolved
in DMSO in concentrations of 1.0 mM containing LiClO4 as supporting
electrolyte.
2.2.3. Cell viability evaluation by crystal violet and MTT assay
Cell viability was estimated colorimetrically by crystal violet (CV)
assay for adherent cells, or by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay for nonadherent cells.
Tumor cell lines (1×104/well) were treated for 24 h with a broad
range of drug concentrations, whereas lung fibroblasts (3×104/
well) and keratinocytes (8×104/well) were treated for 24 h with
IC50 doses of each drug. For the CV assay, at the end of treatment
cells were rinsed with PBS, fixed for 10 min with 4% paraformalde-
hyde and stained with 2% crystal violet-PBS for 15 min at room tem-
perature. The absorbance of dye dissolved in 33% acetic acid, which
correlates with the number of viable cells, was measured in a micro-
plate reader at 540 nm, while the background was measured at
640 nm. For nonadherent cells, MTT was added to cell suspensions
in a final concentration of 0.5 mg/ml. After 2 h incubation at 37 °C
cells were collected, centrifuged and pellets were dissolved in
DMSO. The absorbance, which correlates with the level of
mitochondrial-dependent MTT reduction to formazan, was measured
in a microplate reader at 540 nm with the reference wavelength at
640 nm. The results are presented as a percentage of the value for
untreated cells (control) which was arbitrarily set at 100%.
2.2. Biology
2.2.1. Reagents and cells
2.2.4. Cell cycle analysis
Fetal calf serum (FCS), RPMI-1640, phosphate-buffered saline
(PBS), DMSO 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT), collagenase IV from Clostridium hystoliticum, DNase,
After treatment with the IC50 dose of each compound for 24 h,
cells were fixed for 45 min with 70% ethanol at 4 °C, washed twice
with cold PBS and incubated with 20 μg/ml PI and 0.1 mg/ml

42
L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
RNase for 30 min at 37 °C in the dark. Quantification of cellular
DNA stained with red fluorescent PI was analyzed on a FACS Calibur
flow cytometer (BD, Heidelberg, Germany) using a peak fluores-
cence gate to discriminate aggregates, while cell distribution in
cell cycle phases was determined with Cell Quest Pro software
(BD) [30].
2.3.2. Synthesis of the diethyl ester of N,N′-methylene-(S,S)-
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid, L2
A suspension of the precursor, ethyl ester of (S,S)-ethylenediamine-
N,N′-di-2-(3-cyclohexyl)propanoic acid (0.2 g, 0.40 mmol) in methanol
(10 ml) was mixed and heated up to 40 °C for 20 min on a steam bath
until the mixture was well homogenized. The methylation mixture
was made by dissolving sodium triacetoxyborohydride (0.25 g,
1.2 mmol) in 10 ml methanol followed by adding 36% aqueous formal-
dehyde (0.12 ml, 4.0 mmol). The obtained solution for methylation was
poured dropwise into the previously made suspension. In order to ad-
just the pH value to 4–5, glacial acetic acid (0.25 ml) was slowly
added to the reaction mixture. The next portion of the same volume
was added after 2 h and stirring was continued for the following
30 min. The whole reaction solution was washed out with diethylether
(40 ml) followed by rinsing the ether extract with three equal portions
(10 ml) of 1 M KOH and a 10 ml portion of brine. The combined ether
solutions were dried overnight using anhydrous K2CO3 and evaporated
in vacuo the next day to obtain a colorless, oily liquid. Total yield:
103 mg, 58.68%. Anal. Calc. for C25H44N2O4∙0.75CH3OH (Mr=460.66):
C, 67.14; H, 10.28; N, 6.08%; Found: C, 66.88; H, 10.77; N, 6.32%.
2.2.5. AnnexinV-FITC/PI staining
Apoptotic cells, which are characterized by phosphatidylserine ex-
posed to the extracellular environment while cell membrane integrity
remains intact, were detected using an annexinV-FITC/PI staining kit
(Biotium, Hayward, CA). Cells were treated for 14 h with IC50 doses
of each compound, trypsinized and stained according to the manufac-
tureur's instructions. Cells were analyzed on a FACS Calibur flow cyt-
ometer using Cell Quest Pro software.
2.2.6. Caspase detection
For detection and quantification of caspase activity in apoptotic
cells ApoStat (R&D Systems, Minneapolis, MN USA) was used. Briefly,
cells were treated with IC50 doses of selected compounds for 24 h,
collected and stained with ApoStat in phenol red free RPMI supple-
mented with 5% FCS for 15 min at 37 °C. After washing, cells were
analyzed on a FACS Calibur flow cytometer using Cell Quest Pro
software.
ν
max(ATR)/cm−1: 2920, 2848, 1727, 1681, 1446, 1155, 1098; ESI-MS
(methanol), positive: m/z 425.35 [M-CH2+3H]+ 1H NMR
.
(500.26 MHz, CDCl3) δH 0.82 (m, C5a′, C5b′, 4H), 1.01–1.28 (m, C7′,
C6′, CH3CH2OOC\, C4, 14H), 1.45 (m, \CH2′\Cy, 2H), 1.52–1.64 (m,
\CH2\Cy, C7, C5a, C6, 10H), 1.70 (br d, C5b, 2H), 2.79 (m, \N\CH2-
CH2\N\, 2H), 2.93 (m, \N\CH2\CH2\N\, 2H), 3.30 (dd,
\OOC\CH\N\, 2H), 3.57 (s, \N\CH2\N\, 2H), 4.10 (m, CH3CH2-
OOC\, 4H); 13C NMR (125.80 MHz, CDCl3) δC 14.31 (CH3CH2OOC\),
26.10 (C6), 26.43 (C7), 33.07 (C5b), 33.52 (C5a), 34.33 (C4), 38.63 (\CH2-
Cy), 48.05 (\N\CH2\CH2\N\), 60.15 (CH3CH2OOC\), 61.90
(\OOC\CH\N\), 69.38 (\N\CH2\N\), 172.98 (CH3CH2OOC\).
2.2.7. Statistical analysis
The results are presented as mean SD of triplicate observations
from one representative of at least three experiments with similar re-
sults, except if indicated otherwise. The significance of the differences
between various treatments was assessed by ANOVA followed by the
Student Newman–Keuls test. Pb0.05 was considered to be significant.
2.3.3. Synthesis of the dipropyl ester of N,N′-methylene-(S,S)-
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid, L3
2.3. Synthesis of the ligands
A
suspension of the precursor, n-propyl ester of (S,S)-
2.3.1. Synthesis of the dimethyl ester of N,N′-methylene-(S,S)-
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid, L1
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid (0.2 g,
0.38 mmol) in methanol (10 ml) was mixed and heated up to
40 °C for 20 min on a steam bath until the mixture was well
homogenized. The methylation mixture was made by dissolving
sodium triacetoxyborohydride (0.24 g, 1.14 mmol) in 10 ml meth-
anol followed by adding 36% aqueous formaldehyde (0.11 ml,
3.8 mmol). The obtained solution for methylation was poured
dropwise into the previously made suspension. In order to adjust
the pH value to 4–5, glacial acetic acid (0.25 ml) was slowly
added to the reaction mixture. The next portion of the same
volume was added after 2 h and stirring was continued for the
following 30 min. The whole reaction solution was washed out
with diethylether (40 ml) followed by rinsing the ether extract
with three equal portions (10 ml) of 1 M KOH and a 10 ml portion
of brine. The combined ether solutions were dried overnight
using anhydrous K2CO3 and evaporated in vacuo the next day to
obtain a colorless, oily liquid. Total yield: 98 mg, 55.42%. Anal.
A suspension of the precursor, methyl ester of (S,S)-ethylenediamine-
N,N′-di-2-(3-cyclohexyl)propanoic acid (0.2 g, 0.43 mmol) in methanol
(10 ml) was mixed and heated up to 40 °C for 20 min on a steam bath
until the mixture was well homogenized. The methylation mixture was
made by dissolving sodium triacetoxyborohydride (0.27 g, 1.29 mmol)
in 10 ml methanol followed by adding 36% aqueous formaldehyde
(0.13 ml, 4.3 mmol). The obtained solution for methylation was poured
dropwise into the previously made suspension. In order to adjust the
pH value to 4–5, glacial acetic acid (0.25 ml) was slowly added to the
reaction mixture. The next portion of the same volume was added after
2 h and stirring was continued for the following 30 min. The whole
amount of the reaction solution was washed out with diethylether
(40 ml) followed by rinsing the ether extract with three equal portions
(10 ml) of 1 M KOH and a 10 ml portion of brine. The combined ether so-
lutions were dried overnight using anhydrous K2CO3 and evaporated in
vacuo next day to obtain a colorless, oily liquid. Total yield: 98 mg,
56.31%. Anal. Calc. for C23H40N2O4∙0.25CH3OH (Mr=416.58): C, 67.03;
Calc. for
C27H48N2O4·0.5CH3OH (Mr=480.70): C, 68.71; H,
10.48; N, 5.83%; Found: C, 69.09; H, 10.97; N, 6.06%. νmax(ATR)/
H, 9.92; N, 6.72%; Found: C, 66.77; H, 9.98; N, 6.82%. νmax(ATR)/cm−1
2923, 2850, 1734, 1683, 1448, 1193, 1161; ESI-MS (methanol), positive:
m/z 397.34 [M-CH2+3H]+ 1H NMR (500.26 MHz, CDCl3) δH 0.89 (m,
:
cm−1: 2924, 2851, 1733, 1682, 1450, 1258, 1165; ESI-MS (methanol),
positive: m/z 453.37 [M-CH2+3H]+ 1H NMR (199.97 MHz, CDCl3) δH
.
.
0.74–1.02 (m, C5a′, C5b′, CH3CH2CH2OOC\, 10H), 1.03–1.40 (m, C7′,
C6′, C4, 8H), 1.42–1.85 (m, \CH2′\Cy,\CH2\Cy, CH3CH2CH2OOC\,
C7, C5a, C6, C5b, 18H), 2.80–3.08 (m, \N\CH2\CH2\N\, 4H),
3.39 (dd, \OOC\CH\N\, 2H), 3.65 (s, \N\CH2\N\, 2H), 4.05
(t, CH3CH2CH2OOC\, 4H); 13C NMR (50.28 MHz, CDCl3) δC 10.20
(CH3CH2CH2OOC\), 21.74 (CH3CH2CH2OOC\), 25.86 (C6), 26.18
(C7), 32.81 (C5b), 33.30 (C5a), 34.09 (C4), 38.46 (\CH2\Cy), 47.81
(\N\CH2\CH2\N\), 61.62 (CH3CH2CH2OOC\), 65.58 (\OOC\
CH\N\), 69.12 (\N\CH2\N\), 172.86 (CH3CH2CH2OOC\).
C5a′, C5b′, 4H), 1.08–1.34 (m, C7′, C6′, C4, 8H), 1.52 (m, \CH2′ \Cy,
2H), 1.59–1.71 (m, \CH2\Cy, C7, C5a, C6, 10H), 1.76 (br d, C5b, 2H),
2.85 (m, \N\CH2\CH2\N\, 2H), 2.98 (m, \N\CH2\CH2\N\,
2H), 3.39 (dd, \OOC\CH\N\, 2H), 3.62 (s, \N\CH2\N\, 2H), 3.70
(s, CH3OOC\, 6H); 13C NMR (125.80 MHz, CDCl3); δC 26.10 (C6), 26.44
(C7), 33.12 (C5b), 33.47 (C5a), 34.32 (C4), 38.59 (\CH2\Cy), 48.07
(\N\CH2\CH2\N\), 51.31 (CH3OOC\), 61.82 (\OOC\CH\N\),
69.42 (\N\CH2\N\), 173.49 (CH3OOC\).

L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
43
2.3.4. Synthesis of the dibutyl ester of N,N′-methylene-(S,S)-
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid, L4
acid, (0.10 g, 0.23 mmol), dissolved in 10 ml of dichloromethane was
stirred under reflux at 40 °C for 3 days. During that period, the whole
amount of dichloromethane evaporated and the formed solid product
was filtered off, rinsed with diethyl ether and air dried. The complex
was obtained as an orange powder. Total yield: 89 mg, 50.24%. mp:
90 °C. Anal. Calcd for C25H44N2O4PtCl4∙0.5H2O(Mr=782.52): C, 38.37;
H, 5.80; N, 3.58%; Found: C, 38.52; H, 6.29; N, 4.06%. νmax(ATR)/
A suspension of the precursor, n-butyl ester of (S,S)-ethylenediamine-
N,N′-di-2-(3-cyclohexyl)propanoic acid (0.2 g, 0.36 mmol) in methanol
(10 ml) was mixed and heated up to 40 °C for 20 min on a steam bath
until the mixture was well homogenized. The methylation mixture
was made by dissolving sodium triacetoxyborohydride (0.23 g,
1.08 mmol) in 10 ml methanol followed by adding 36% aqueous formal-
dehyde (0.11 ml, 3.6 mmol). The obtained solution for methylation was
poured dropwise into the previously made suspension. In order to ad-
just the pH value to 4–5, glacial acetic acid (0.25 ml) was slowly
added to the reaction mixture. The next portion of the same volume
was added after 2 h and stirring was continued for the following
30 min. The whole reaction solution was washed out with diethylether
(40 ml) followed by rinsing the ether extract with three equal portions
(10 ml) of 1 M KOH and a 10 ml portion of brine. The combined ether
solutions were dried overnight using anhydrous K2CO3 and evaporated
in vacuo the next day to obtain a colorless, oily liquid. Total yield:
106 mg, 59.55%. Anal. Calc. for C29H52N2O4·0.5CH3OH (Mr=508.75):
C, 69.64; H, 10.70; N, 5.51%; Found: C, 69.48; H, 10.90; N, 5.68%. ν-
max(ATR)/cm−1: 2925, 2851, 1730, 1680, 1450, 1252, 1163; ESI-MS
cm−1
(CH3OH): λmax (ε, 2802 M−1 cm−1): 213 nm; ESI-MS (methanol),
positive: m/z 436.07 [M-PtCl4+H]+ 1H NMR (199.97 MHz, CDCl3) δH
: 2922, 2849 1735, 1635, 1447, 1260, 1195, 851; UV/Vis
.
0.74–1.02 (m, C5a′, C5b′, CH3CH2OOC\, 10H), 1.04–1.40 (m, C7′, C6′,
C4, 8H), 1.42–1.86 (m, \CH2′\Cy,\CH2\Cy, C7, C5a, C6, C5b, 14H),
2.78–3.08 (m, \N\CH2\CH2\N\, 4H), 3.38 (dd, \OOC\CH\N\,
2H), 3.64 (s, \N\CH2\N\, 2H), 4.07 (m, CH3CH2OOC\, 4H); 13C NMR
(50.28 MHz, CDCl3) δC 10.40 (CH3CH2OOC\), 26.16 (C6), 26.38 (C7),
32.99 (C5b), 33.52 (C5a), 34.30 (C4), 38.69 (\CH2\Cy), 48.05
(\N\CH2\CH2\N\), 61.91 (CH3CH2OOC\), 65.84 (\OOC\CH\N\),
69.41 (\N\CH2\N\), 173.17 (CH3CH2OOC\). 195Pt NMR (85.98 MHz,
CDCl3): 150.9.
2.4.3. Synthesis of N,N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-
(3-cyclohexyl)-dipropylpropanoato-tetrachlorido-platinum(IV), C3
A mixture of K2[PtCl6] (0.11 g, 0.22 mmol) dissolved in 10 ml of
warm water and an equimolar amount of ligand, dipropyl ester of N,N
′-methylene-(S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic
acid, (0.10 g, 0.22 mmol), dissolved in 10 ml of dichloromethane were
stirred under reflux at 40 °C for 3 days. During that period, the whole
amount of dichloromethane evaporated and the formed solid product
was filtered off, rinsed with diethyl ether and air dried. The complex
was obtained as an orange powder. Total yield: 87 mg, 50.43%. mp:
93 °C. Anal. Calcd for C27H48N2O4PtCl4 (Mr=801.569): C, 40.46; H,
(methanol), positive: m/z 481.40 [M-CH2+3H]+
.
1H NMR
(199.97 MHz, CDCl3) δH 0.78–1.01 (m, C5a′, C5b′, CH3CH2CH2CH2OOC\,
10H), 1.05–1.47 (m, C7′, C6′, CH3CH2CH2CH2OOC\, C4,\CH2′\Cy,
14H), 1.49–1.86 (m, \CH2\Cy, CH3CH2CH2CH2OOC\, C7, C5a, C6, C5b,
16H), 2.79–3.06 (m, \N\CH2\CH2\N\, 4H), 3.37 (dd,
\OOC\CH\N\, 2H), 3.63 (s, \N\CH2\N\, 2H), 4.08 (t, CH3CH2-
CH2CH2OOC\, 4H); 13C NMR (50.28 MHz, CDCl3) δC 13.64 (CH3CH2-
CH2CH2OOC\), 19.14 (CH3CH2CH2CH2OOC\), 26.11 (C6), 26.42
(C7), 30.64 (CH3CH2CH2CH2OOC\), 33.03 (C5b), 33.58 (C5a), 34.34
(C4), 38.71 (\CH2\Cy), 48.11 (\N\CH2\CH2\N\), 61.98 (CH3-
CH2CH2CH2OOC\), 64.09 (\OOC\CH\N\), 69.44 (\N\CH2-
N\), 173.19 (CH3CH2CH2CH2OOC\).
6.04; N, 3.49%; Found: C, 40.27; H, 6.35; N, 3.78%. νmax(ATR)/cm−1
:
2925, 2850, 1738, 1640, 1448, 1242, 1198, 887; UV/Vis (CH3OH): λmax
(ε, 1818 M−1 cm−1): 208 nm; ESI-MS (methanol), positive: m/z 464.89
2.4. Synthesis of the complexes
[M-PtCl4+H]+ 1H NMR (199.97 MHz, CDCl3) δH 0.78–1.01 (m, C5a′,
.
C5b′, CH3CH2CH2OOC\, 10H), 1.02–1.40 (m, C7′, C6′, C4, 8H), 1.42–1.85
(m, \CH2′\Cy, \CH2\Cy, CH3CH2CH2OOC\, C7, C5a, C6, C5b, 18H),
2.78–3.06 (m, \N\CH2\CH2\N\, 4H), 3.39 (dd, \OOC\CH\N\,
2H), 3.65 (s, \N\CH2\N\, 2H), 4.07 (t, CH3CH2CH2OOC\, 4H); 13C
NMR (50.28 MHz, CDCl3) δC 10.20 (CH3CH2CH2OOC\), 21.74 (CH3CH2-
CH2OOC\), 25.85 (C6), 26.18 (C7), 32.80 (C5b), 33.30 (C5a), 34.09
(C4), 38.45 (\CH2\Cy), 47.82 (\N\CH2\CH2\N\), 61.62 (CH3CH2-
CH2OOC\), 65.59 (\OOC\CH\N\), 69.12 (\N\CH2\N\), 172.85
(CH3CH2CH2OOC\).
2.4.1. Synthesis of N,N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-
(3-cyclohexyl)-dimethylpropanoato-tetrachlorido-platinum(IV), C1
A mixture of K2[PtCl6] (0.12 g, 0.24 mmol) dissolved in 10 ml of
warm water and an equimolar amount of the ligand, dimethyl ester
of N,N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)
propanoic acid, (0.10 g, 0.24 mmol), dissolved in 10 ml of dichloro-
methane was stirred under reflux at 40 °C for 3 days. During that pe-
riod, the whole amount of dichloromethane evaporated and the
formed solid product was filtered off, rinsed with diethyl ether and
air dried. The complex was obtained as an orange powder. Total
yield: 86 mg, 47.13%. mp: 88 °C. Anal. Calcd for C23H40N2O4PtCl4∙0.5-
H2O (Mr=754.47): C, 36.61; H, 5.48; N, 3.71%; Found: C, 37.05; H,
5.97; N, 3.50%. νmax(ATR)/cm−1: 2924, 2851, 1742, 1634, 1444,
1271, 1218, 886; UV/Vis (CH3OH): λmax (ε, 1872 M−1 cm−1):
2.4.4. Synthesis of N,N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-
(3-cyclohexyl)-dibutylpropanoato-tetrachlorido-platinum(IV), C4
A mixture of K2[PtCl6] (0.10 g, 0.20 mmol) dissolved in 10 ml of
warm water and an equimolar amount of ligand, dibutyl ester of N,
N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic
acid, (0.10 g, 0.20 mmol), dissolved in 10 ml of dichloromethane was
stirred under reflux at 40 °C for 3 days. During that period, the whole
amount of dichloromethane evaporated and the formed solid product
was filtered off, rinsed with diethyl ether and air dried. The complex
was obtained as an orange powder. Total yield: 93 mg, 55.23%. mp:
96 °C. Anal. Calcd for C29H52N2O4PtCl4∙0.25H2O (Mr=834.13): C, 41.76;
207 nm; ESI-MS (methanol), positive: m/z 408.29 [M-PtCl4+H]+ 1H
.
NMR (199.97 MHz, CDCl3) δH 0.90 (m, C5a′, C5b′, 4H), 1.06–1.40 (m,
C7′, C6′, C4, 8H), 1.42–2.00 (m, \CH2′\Cy,\CH2\Cy, C7, C5a,
C6, C5b, 14H), 2.78–3.06 (m,\N\CH2\CH2\N\, 4H), 3.38 (dd,
\OOC\CH\N\, 2H), 3.62 (s, \N\CH2\N\, 2H), 3.70 (s, CH3OOC\,
6H); 13C NMR (50.28 MHz, CDCl3); δC 22.59 (C6), 22.91 (C7),
29.54 (C5b), 30.02 (C5a), 30.82 (C4), 35.15 (\CH2\Cy), 44.56
(\N\CH2\CH2\N\), 56.69 (CH3OOC\), 58.42 (\OOC\CH\N\),
65.92 (\N\CH2\N\), 169.56 (CH3OOC\). 195Pt NMR (85.98 MHz,
CDCl3): 145.6.
H, 6.34; N, 3.36%; Found: C, 41.47; H, 6.25; N, 3.85%. νmax(ATR)/cm−1
:
2924, 2851, 1737, 1635, 1451, 1251, 1195, 887; UV/Vis (CH3OH): λmax
(ε, 2160 M−1 cm−1): 211 nm; ESI-MS (methanol), positive: m/z 492.38
[M-PtCl4+H]+ 1H NMR (199.97 MHz, CDCl3) δH 0.78–1.00 (m, C5a′,
.
C5b′, CH3CH2CH2CH2OOC\, 8H), 1.04–1.46 (m, C7′, C6′, CH3CH2CH2CH2-
OOC\, C4,\CH2′\Cy, 14H), 1.49–1.88- (m, \CH2\Cy, CH3CH2CH2CH2-
OOC\, C7, C5a, C6, C5b, 16H), 2.78–3.07 (m, \N\CH2\CH2\N\, 4H),
3.37 (dd, \OOC\CH\N\, 2H), 3.62 (s, \N\CH2\N\, 2H), 4.10 (t, CH3-
CH2CH2CH2OOC\, 4H); 13C NMR (50.28 MHz, CDCl3) δC 13.63 (CH3CH2-
CH2CH2OOC\), 19.13 (CH3CH2CH2CH2OOC\), 26.11 (C6), 26.41 (C7),
2.4.2. Synthesis of N,N′-methylene-(S,S)-ethylenediamine-N,N′-di-2-
(3-cyclohexyl)-diethylpropanoato-tetrachlorido-platinum(IV), C2
A mixture of K2[PtCl6] (0.11 g, 0.23 mmol) dissolved in 10 ml of
warm water and an equimolar amount of ligand, diethyl ester of N,N′-
methylene-(S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic

44
L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
30.63 (CH3CH2CH2CH2OOC\), 33.02 (C5b), 33.57 (C5a), 34.34 (C4),
38.71 (\CH2\Cy), 48.10 (\N\CH2\CH2\N\), 61.97 (CH3CH2CH2CH2-
OOC\), 64.09 (\OOC\CH\N\), 69.44 (\N\CH2\N\), 173.18
(CH3CH2CH2CH2OOC\).
group. As these bands could also be found in the IR spectra of the
corresponding complexes around 1740 cm−1, coordination of the
carboxylic oxygen to the metal center is excluded. Asymmetric CH2
stretching vibrations were found around 2920 and 2850 cm−1 in
the form of two strong bands for both ligands and complexes. Charac-
teristic bands for tertiary amines identified in the area between 1210
and 1150 cm−1 were confirmed in the IR spectra of all ligands and
complexes. Due to the ligand coordination, C\N vibrations of com-
plexes were shifted toward higher wavenumbers.
3. Results and discussion
3.1. Synthesis of ligands
The reductive methylation of methyl, ethyl, n-propyl and n-butyl
esters of (S,S)-ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic
acid was typical [31] with slight modifications. Precursor esters
were well homogenized in methanol. The mixture for methylation
containing 36% aqueous formaldehyde and sodium triacetoxyborohy-
dride dissolved in methanol, was slowly added to the ligand solution
which was stirred for the following 2 h. The pH value was adjusted
using glacial acetic acid. The obtained solution was treated with ap-
propriate amounts of diethylether, 1 M KOH and brine. After drying
ether solutions overnight, ligands were obtained by evaporation in
vacuo. All ligands were soluble in methanol, ethanol, dimethyl sulfox-
ide, diethyl ether, acetone, dichloromethane and chloroform.
Mass spectra of each ligand gave the strongest peak originating
from the [M-CH2 +3H]+ fragment and matched the calculated mo-
lecular mass. The observed fragmentation indicated that the formed
\N\CH2\N\ bond was the weakest one in ligands, L1–L4. On the
other hand, mass spectra in positive mode for all complexes detected
a [M-PtCl4 +H]+ fragment as the most intensive peak [34,35].
1H NMR spectra gave well-dissolved signals characteristic of a
cyclohexyl moiety in the range 0.8–1.6 ppm for both ligands and
complexes (Supplementary material Fig. S1–S4). Two sets of signals
were observed for the –CH2–(C5) from the cyclohexyl group, due to
the diastereotopic nature of these protons induced by the chiral C
atom. The shifts assignable to \N\CH2\CH2\N\ protons were
found around 2.90 ppm in the form of two separated multiplets but
were slightly moved to a lowered chemical shift in the spectra of
complexes. The obvious doublet of doublets located around
3.40 ppm was related to (ROOC)CH–proton. All protons originating
from alkyl groups of the complexes showed similar shifts to those of
the free ligands. The central confirmation of the \CH2\ group bond-
ed to both nitrogen atoms was the singlet around 3.6 ppm in the 1H
NMR spectra of all ligands and complexes. The corresponding signal
around 69 ppm in the 13C NMR spectra and proper C\H correlation
in the 2D NMR spectra are additional confirmation of the presence
of the methylene bridge in ligand and complex structures. In the
195Pt NMR spectra complexes C1 and C2 exhibit chemical shifts at
145 and 150 ppm, respectively. Thus, indicating on the octahedrally
configured Pt(IV) complex with [N2, Cl4] donor set [36].
3.2. Synthesis of complexes
The prepared K2[PtCl6] [29] dissolved in warm water was mixed
with an equimolar amount of ligand solution in dichloromethane
and stirred under reflux at 40 °C for 3 days. The corresponding com-
plexes were filtered off, rinsed with diethyl ether and air dried. All
products were obtained as an orange powder. The complexes were
soluble in methanol, ethanol, dimethyl sulfoxide, acetone, dichloro-
methane and chloroform (Fig. 1).
3.3. Spectroscopic studies
IR spectra of the free ligands, L1–L4, showed characteristic strong
bands around 1730 cm−1 [32,33], which correspond to the C_O
Electronic spectra of all complexes showed a peak around 29
903 cm−1 (210 nm), which indicated 1A1g–1T1g d–d transitions (38
300–28 000 cm−1) [37] and octahedral geometry of Pt(IV) complexes.
Based on the spectroscopic data, we confirmed octahedral geome-
try around the Pt(IV) center with N,N′ coordinated ligands and four
chloride ions for all complexes.
3.4. Electrochemistry
The reduction potentials of the complexes, C1–C4, are in the range
from −816 to −924 mV and the data are given in Table 1. The similar
reduction potentials (−705 and −846 mV) were found for analog
Pt(IV) complexes having R2eddp (R=Et, n-Pr; eddp=ethylenedia-
mine-N,N′-di-3-propionate) ligands [38]. The Pt(IV) to Pt(II) reduction
is irreversible process followed by the loss of the chloride ligands
[39,40]. As the length of the alkyl chain increase, small differences in
Ep values are observed (Supplementary material Fig. S5). Overall, the
strong correlation between reduction potentials and biological activity
was not detected.
3.5. Ligands, L1–L4 and complexes, C1–C4 strongly down-regulated
tumor cell growth
To evaluate the potential antitumor effect of newly synthesized
ligands and the corresponding Pt(IV) complexes, cells were treated
with a broad range of doses for 24 h and viability was assessed by
CV test. As shown in Fig 2, both ligands and complexes down-
regulated the growth of different cell lines, such as human melanoma
A375, human glioblastoma U251, human prostate cancer PC3, human
colon cancer HCT116, mouse melanoma B16 and mouse colon cancer
Fig. 1. A) Synthesized ligands L1–L4 and B) Pt(IV) complexes C1–C4.

L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
45
Table 1
complexes were similarly cytotoxic except for C1, which displayed at
least two-fold less potential in decreasing tumor cell viability. In com-
parison to the anticancer activity of non-methylated forms of esteri-
fied edda type of Pt(IV) complexes, IC50 values of the novel
compounds were similar or slightly higher [26]. Moreover, drug activ-
ity was not limited to the type and origin of the tumor cell line and
even occurred in cell lines resistant or poorly responsive to treatment
with cisplatin, such as HCT116 and CT26CL25, respectively (Table 2).
Therefore, these two cell lines were selected for further analysis.
Reduction potential values (vs. Ag/AgCl) of the complexes C1–C4.
Ep/mV
C1
C2
C3
C4
−816
−900
−884
−924
CT26CL25. As expected, complexes C1–C4 were more efficient at re-
ducing cell viability in comparison with the corresponding ligands.
Thus, complexes reached their IC50 values at doses in which ligands
were completely ineffective (Table 2). These values were even
lower than those for the conventional cytostatic drug, cisplatin, in
all cell lines tested. The activity of the ligands was influenced by the
length of the alkyl chain and should range from L4, which was ineffec-
tive, to L1–2 whose activity was similar. However, the corresponding
3.6. Ligands, L1–L4 and complexes, C1–C4 were less toxic to primary cells
In order to determine the toxicity of drugs towards primary cells,
mouse fibroblasts and keratinocytes were treated with the dose se-
lected for each compound. This was the highest IC50 dose of each
compound chosen from the list of IC50 doses determined on different
tumor cells. Analysis of cell viability by MTT and CV tests after 24 h
Fig. 2. The effect of ligands L1–L4 and complexes C1–C4 on viability of tumor cell lines. Cells (1×104/well) were treated with different doses of the tested compounds for 24 h, and
cell viability was determined by CV assay. The data are presented as mean SD from a representative of three independent experiments.

46
L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
Table 2
cisplatin treatment. L4 was excluded from further analysis because
its IC50 dose was higher than 200 μM. Cell cycle distribution was an-
alyzed first. As shown in Fig. 4A, the IC50 doses of both ligands and
complexes elevated the percentage of cells in the sub G compartment
after 24 h of treatment, which indicated accumulation of cells with
fragmented DNA. In comparison to control cells where less than 5%
of apoptotic cells were detected, there were 30% hypodiploid cells
upon treatment with the experimental drugs. These data suggested
that their mode of action is preferentially induction of apoptosis rath-
er than cell cycle arrest and subsequent inhibition of proliferation. To
delineate the apoptotic process, the cells were stained with Ann/PI
14 h after drug addiction. The results revealed significant accumula-
tion of Ann+ PI− cells with inverted phosphatidylserine as a marker
of early apoptosis (Fig. 4B) in all treatments applied. Subsequently,
the amount of double Ann+PI+ positive cells was elevated. Cells
accumulated in this compartment are marked as necrotic cells due
to disturbed permeability or membrane rupture [44]. However, LDH
release, as a marker of necrotic cell death, was not detected before
24 h of cultivation in the presence of the drugs, indicating that this
path to death is not the primary target of drug action (not shown).
To assess the mechanism of the triggered apoptotic process, total
caspase activity was determined in the treated cultures. In concor-
dance with the observed apoptosis, caspase activation was observed
in almost all treated cultures (Fig. 4C). The intensity of caspase activa-
tion varied significantly from treatment to treatment. Interestingly,
the highest caspase activation was observed in cultures exposed to
L2, L3, C3 and C4, which were found to be more toxic for primary
cells. This indicated that the intensity of caspase activity could not
be extrapolated with the rate of apoptosis. Thus, different tumor
cells showed diverse sensitivities to apoptotic cell death not neces-
sarily correlated with the intensity of caspase activation [45,46].
Moreover, apoptosis induction by L1 was not followed by caspase ac-
tivation, suggesting that this type of cell death may be independent of
caspase activation (Fig. 4C). To clarify this, HCT116 cells were ex-
posed to L1 in the presence of 20 μM of ZVAD-fmk inhibitor for 24 h
and viability was measured by CV test. The presence of this caspase
inhibitor did not abolish the toxicity of L1 (viability 50 7.6% in L1
treated cells vs 56 3.1% in co-treatment) confirming the irrelevance
of caspase activation.
In vitro cytotoxicity of examined compounds determined by CV test. IC50 (μM) values
are from representative of three independent experiments.
Cell line/
compound
Cisplatin L1
L2
L3
L4
C1
C2
C3
C4
PC3
U251
A375
B16
HCT116
CT26CL25
12.5
20
88.3
44.1
89.2 >200
51
>200
8.1
6.2
2.9 12.5 11.8
7.2 5.7 6.2
>200 21.3 13.8 9.1 10.2
5.4
5.1
98.3 >200 17.5
45
94.3
127.3 134.8 >200
108.3 135.5 >200
>200
9.1
>120
120
74.1
114
45.9
65.9
127.1 >200
179.9 >200 11.3
8.8
4.1
5.2
6.3
6.3
5.9
6.3
revealed that the applied doses were significantly less toxic for pri-
mary keratinocytes and fibroblasts than for tumor cells (Fig 3). Except
in the case of treatment with C2 and L3, the decrease of viability did
not exceed 30% of control and was similar in both types of primary
cells. In accordance with data in the literature, the IC50 dose of cis-
platin obtained on keratinocytes and fibroblasts under the same con-
ditions was approximately 30 μM. This is significantly lower than
doses found to be efficient in tumor cell lines, underlining the great
toxicity of cisplatin towards primary tissues [41,42]. Taken together,
apart from strong tumoricidal potential the newly synthesized
drugs affected the viability of normal cells to a lower extent.
3.7. Ligands, L1–L4 and complexes, C1–C4 induced caspase dependent
apoptosis
Previous data showed that the synthesized ligands and their
Pt(IV) complexes significantly diminished cell viability [43]. To ex-
plore further the cause of viability reduction upon drug treatment,
the presence of different modes of cell death was examined in
HCT116 and CT26CL25 cells, which are resistant to conventional
Octahedral Pt(IV) complexes with the esterified edda type of
ligands expressed antitumor potential similar to cisplatin but with
different mechanisms involved. In contrast to cisplatin, these drugs
triggered rapid necrotic death [24]. Necrotic cell death was described
as accidental cell death, which promoted local inflammation and fur-
ther tissue damage, as well as recruitment of immune inflammatory
cells which support tumor progression through angiogenesis inside
neoplastic tissue, cancer cell proliferation and invasiveness [47]. On
the other hand, apoptosis finalized by consumption of apoptotic bodies
by neighboring cells prevented an inflamed tumor microenvironment
[24]. The more important presence of apoptotic debris stimulated
dendritic cell maturation with subsequent priming of a desirable specif-
ic antitumor immune response [48]. In this light, the feature that meth-
ylation via both nitrogen donor atoms enables alkyl esters of (S,S)-
ethylenediamine-N,N′-di-2-(3-cyclohexyl)propanoic acid to trigger
apoptosis instead of necrosis is a great advantage of this family of
drugs in comparison to earlier ones.
4. Conclusion
Platinum(IV) complexes derived from cyclohexyl edda derivatives
are attracting attention as they allow a variety of substitutions favor-
able for potential anticancer drugs. In this context, we examined the
influence of a \CH2\ group introduced ethylenediamine chain of
the cyclohexyl-edda precursor on spectroscopic and biological fea-
tures of ligands and their corresponding complexes. The applied
chemical intervention generated a novel class of molecules found to
Fig. 3. The effect of ligands L1–L4 and complexes C1–C4 on viability of normal cells.
(A) Fibroblasts (3×104/well) and (B) keratinocytes (8×104 cells/well) were treated for
24 h with 200 μM of L1–L4, 20 μM of C1 or 12.5 μM of C2–C4, after which cell viability
was determined by CV and MTT assay, respectively. The data are presented as mean SD
from a representative of three independent experiments. *Pb0.05, refers to untreated
cultures.

L.E. Mihajlović et al. / Journal of Inorganic Biochemistry 109 (2012) 40–48
47
Fig. 4. Apoptotic cell death induced by ligands L1–L4 and complexes C1–C4. HCT116 (left panel) and CT26CL26 cells (right panel) (2.5×105/well) were exposed to the IC50 dose of
each compound. Cell cycle distribution was assessed after 24 h (A), Ann/PI double staining after 14 h (B) and caspase activity after 24 h (C). Results from one of three representative
flow cytometric analyses are presented.
be efficient against numerous cell lines of different specificity
regarding their origin or the relevant signaling pathways involved
in cancerogenesis (prostate, glioblastoma, melanoma, colon cancer).
Moreover, their efficacy was not compromised by initial resistance
to the original drug, cisplatin, as found for HCT116 and CT26CL25
colon cancer cells. On the other hand, primary keratinocytes and
fibroblasts were less affected by treatment with the modified com-
pounds indicating their selectivity toward cancer cells. Similarly to
cisplatin, the novel compounds induced apoptosis of cancer cells but
at a lower range of doses. Except in the case of L1, the induced apo-
ptotic process correlated with caspase activation in all treatments.
Apoptosis as the proposed mechanism of action, together with an in-
tensified anticancer potential accompanied with less toxicity towards
primary cells qualify the novel methylated forms of esterified edda
type of Pt(IV) complexes and ligands as suitable candidates for
further evaluation in this field.
ANOVA analysis of variance
CV crystal violet
Acknowledgments
This research was supported by the Ministry of Science of the
Republic of Serbia, grant numbers 172035 and 173013. We are also
greatful to Professor Dr. Dragan D. Manojlović and his associate,
Msc. Dalibor Stanković who contributed the electrochemistry part of
the manuscript.
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