Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds having perchlorate, tetrafluoridoborate and bis(chlorido)argentate anions

Article abstract of DOI:10.1016/j.jinorgbio.2015.08.030

Three new compounds containing the bis(triphenylphosphane)iminium cation (PPN⁺) with ClO₄^-, BF₄^- and [AgCl₂]^- as counter anions have been synthesized and structurally characteriz

Full text of DOI:10.1016/j.jinorgbio.2015.08.030

JIB-09801; No of Pages 9
Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium
compounds having perchlorate, tetrafluoridoborate and bis(chlorido)argentate anions
a,1
b,1
c
c
c
Alessandra Folda , Valeria Scalcon , Mohamed Ghazzali , Mohammed H. Jaafar , Rais Ahmad Khan ,
d
a
b
a,
c
Angela Casini , Anna Citta , Alberto Bindoli , Maria Pia Rigobello ⁎, Khalid Al-Farhan ,
Ali Alsalme , Jan Reedijk ⁎⁎
Department of Biomedical Sciences, University of Padova, via Ugo Bassi 58/b, 35131 Padova, Italy
Institute of Neuroscience (CNR), viale G. Colombo 3, 35131 Padova, Italy
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Kingdom of Saudi Arabia
Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands
c
c,e,
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Three new compounds containing the bis(triphenylphosphane)iminium cation (PPN+) with ClO 4− , BF 4− and
Article history:
Received 26 July 2015
Accepted 28 August 2015
Available online xxxx
−
[
AgCl
2
]
as counter anions have been synthesized and structurally characterized. The two derivatives with
−
−
4 4
ClO and BF were found to be isostructural by single crystal X-ray diffraction. Interestingly, the three com-
pounds show extremely potent antiproliferative effects against the human cancer cell line SKOV3. To gain in-
sights into the possible mechanisms of biological action, several intracellular targets have been considered.
Thus, DNA binding has been evaluated, as well as the effects of the compounds on the mitochondrial function.
Furthermore, the compounds have been tested as possible inhibitors of the seleno-enzyme thioredoxin
reductase.
Keywords:
Isomorphism
Anti-cancer compounds
Crystal structure
DNA binding
© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Mitochondria
Thioredoxin reductase
1
. Introduction
The bis(triphenylphosphane)iminium cation (often abbreviated as
unsurprising that 1453 crystal structures incorporating PPN+ are count-
ed in the Cambridge Structural Database [5], of which a majority of
ca. 90% is incorporating anionic metal complexes. The rest of reported
+
+
PPN ) is a classical cation, known to be useful in the isolation of reactive
anions. The distinguished ability of PPN in stabilizing air-sensitive,
less-stable metallic anions is the reason for its popularity in the synthe-
sis of many unique organometallic and coordination compounds. For
example, it is known that many important and unique chemo-physical
phenomena can arise when trapping an interesting anion in a chemical-
PPN compounds with simple metallic-free anions is also of remark-
+
able importance. For example, the bis(triphenylphosphane)iminium
nitrite is reported as a versatile nitrosylating reagent [6], and the
bis(triphenylphosphane)iminium nitrate is a known nitrate-sensitive
electrode [7].
Unfortunately, structurally-reported compounds of PPN+ contain-
ing small anions are scarce, namely those with thiocyanate [8], mono-
thionitrate [9], perthionitrite [10], iodate [11], tri-iodide [12] and
ly inert solid phase. Thus, the first known crystal structures of fullerene
C
+
60 were only achievable using the PPN as the counter ion [1,2], while
+
+
the compound PPN -(bipyridyl)tetracyanidoruthenate is a technologi-
cally efficient humidity sensor [3].
chloride [13] anions. Also, the crystal structure of PPN with tetra-
fluoridoborate mono(dichloromethane) is known [14]. None of these
compounds has ever been characterized for its biological properties
and its effects on cancer cell lines. We hypothesized that PPN+ may
behave as a so-called membrane permeable Delocalized Lipophilic
Cation (DLC) which can target mitochondria and exert cytotoxic effects
[15].
Another reason of the growing uses of PPN+ arose after its degree of
bent vs. linear geometrical flexibility had been disclosed [4]. In fact, it is
⁎
Correspondence to: M. P. Rigobello, Dipartimento di Scienze Biomediche, University of
Padova, via Ugo Bassi 58/b, 35131 Padova, Italy.
Correspondence to: J. Reedijk, Leiden Institute of Chemistry, Leiden University, P.O.
Mitochondria are known to play a major role in cell energy metabo-
lism as they generate ATP (adenosine triphosphate) following oxidation
of respiratory substrates. Furthermore, they are also involved in the
modulation of Ca2 homeostasis, in ROS (Reactive Oxygen Species)
production and in stimulation of the intrinsic pathway to cell apoptosis.
⁎
⁎
Box 9502, 2300 RA Leiden, The Netherlands.
E-mail addresses: mariapia.rigobello@unipd.it (M.P. Rigobello),
reedijk@chem.leidenuniv.nl (J. Reedijk).
+
1
These two authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030
0162-0134/© 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

2
A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Mitochondria, once energy generated by the oxidation of substrates is
stored as electrochemical potential, develop a membrane potential of
about 180 mV, that is used for ATP formation and ion transport. As
this membrane potential is negative inside, the molecules endowed
with a positive charge distributed over a broad surface area such as
DLC, can be accumulated in the mitochondrion. These molecules are
first driven into the cell by the plasma membrane potential and then,
from the cytosol, they can concentrate in the mitochondrial matrix [16].
Several cancer cell lines present a higher plasma and mitochondrial
membrane potential in comparison to normal cells; this condition has
been exploited for targeting lipophilic cations with potential anticancer
action to mitochondria [17–20]. In the mitochondrial matrix, the
thioredoxin system represents a major pathway involved in the remov-
al of dihydrogen peroxide and in thiol-dependent redox regulation.
The thioredoxin system is composed of NADPH, thioredoxin reductase
and thioredoxin. NADPH, through thioredoxin reductase, maintains
thioredoxin reduced, in turn able to act as a substrate of peroxiredoxin
that very efficiently removes dihydrogen peroxide. Thioredoxin reduc-
tase is reported to be inhibited by a large number of molecules most
of which are anticancer agents [21].
Synthesis of [N(PPh
1 mmol) was added to a solution of KCN (0.065 g, 1 mmol) in methanol
(40 mL). After a clear solution had been obtained, [N(PPh ) ] Cl
3 2
(0.574 g, 1 mmol) was added to the reaction mixture. Stirring was con-
tinued for 15 min. The solution was evaporated to dryness and the
white residue was treated with acetone (30 mL). The remaining solid,
KCl (0.065 g), was filtered off. Addition of hexane to the filtrate resulted
3 2 2 n
) ][Ag(CN) ]. Silver cyanide [Ag(CN)] (0.134 g,
+
−
in white crystals of [N(PPh
Elemental analysis: found: C, 65.99; H, 4.55; N, 5.18. Calc. for:
30AgN , C, 65.33; H, 4.30; and N, 6.01.
Compound 1: silver perchlorate AgClO
added to a clear solution of [N(PPh ][Ag(CN)
3 2 2
) ][Ag(CN) ] (0.663 g, 95%), m.p. 90 °C.
C
38
H
3 2
P
4
(0.208 g, 1 mmol) was
] (0.698 g, 1 mmol) in
)
3 2
2
acetone (20 mL). Immediately, a white precipitate was formed. Stirring
was continued for 90 min at room temperature, the precipitate was
filtered off, washed with acetone (10 mL), and collected as Ag(CN)
(0.260 g, 97%), m.p. decomposition at 300 °C. Addition of hexane
(30 mL) to the filtrate resulted in white crystals of [N(PPh
(0.5 g, 94%), m.p. 265 °C. Elemental analysis: found: C, 67.70; H, 4.29;
N, 1.96. Calc. for C36 30NClO : C, 67.87; H, 4.71; and N, 2.19%. The IR
3 2 4
) ]ClO
H
4 2
P
spectrum was found to be typical for the PPN cation and a strong peak
assigned to perchlorate (near 1090 cm ).
Compound 2: silver tetrafluoridoborate AgBF
was added to a clear solution of [N(PPh ][Au(CN)
1 mmol) in acetone (25 mL). Immediately a white precipitate was
formed. Stirring was continued for 90 min at room temperature, the
solid was filtered off, washed with acetone (10 mL), and identified as
1
Within this frame, we are reporting here the synthesis, characteriza-
tion, crystal structures, anti-proliferative and DNA-binding studies, the
effects on mitochondria functions and mitochondrial thioredoxin reduc-
tase activity of bis(triphenylphosphane)iminium perchlorate, onwards
denoted as 1, bis(triphenylphosphane)iminium tetrafluoridoborate,
onwards denoted as 2 and of bis(triphenylphosphane)iminium
4
(0.195 g, 1 mmol)
] (0.787 g,
3
)
2
2
[
dichloridoargentate(I)] monoethanol, onwards denoted as 3, see Fig. 1
AuAg(CN)
hexane (30 mL) to the filtrate gave white crystals of [N(PPh
0.590 g, 94.4%), m.p. 240 °C. Elemental analysis: found: C, 69,70; H,
4.75; N, 2.19. Calc. for C36 30NBF : C, 69.12; H, 4.8; and N, 2.22%.
2
(0.325 g, 91%), m.p. decomposition at 335 °C. Addition of
for the schematic structures.
3
)
2
]BF
4
(
2
. Experimental
H
4 2
P
The IR spectrum was found to be typical for the PPN cation and a strong
1
2
.1. Materials and methods
peak assigned to tetrafluoridoborate (near 1050 cm ).
Compound 3: silver(I) chloride AgCl, (0.143 g, 1 mmol) was
'
Melting points were determined on an electrothermal s IA9000
added to a clear solution of bis(triphenylphosphane)iminium
+
−
series digital capillary melting point apparatus and are uncorrected.
FT-IR spectra were measured using a KBr pressed disk on a Shimadzu
3 2
chloride, [N(PPh ) ] Cl (0.574 g, 1 mmol), in methanol (35 mL).
Stirring continuously under nitrogen gas and heating at 60 °C for
30 min, resulted in a clear solution. Addition of diethyl ether
(20 mL) to this clear solution resulted in a crystalline solid, which
was filtered off and from which light pale yellow crystals were
−
1
IR-Affinity spectrophotometer (4000–400 cm ). X-ray powder diffrac-
tion was carried out for the sake of confirming phase purity, using
Rigaku Ultima-IV equipment with copper radiation and Bragg–Brentano
geometry. The elemental analyses were performed by using a Perkin
Elmer Series II-2400 analyzer.
+
−
collected as [N(PPh
at 170 °C and melting at 272 °C. Elemental analysis: found: C,
60.00; H, 4.65; N, 2.19. Calc. for. C36 36AgNCl OP : C, 59.76; H,
4.72; N, 1.83%. The IR spectrum was found to be typical for the PPN
3 2 3 2
) ] [ClAgCl] ·CH CH OH, (0.712 g, 95%), dec.
The starting chemicals, [N(PPh
3
)
2
]+Cl, KCN, Ag(CN) and Au(CN)
H
2
2
and solvents were used as commercially available (BDH-Analar
grade). Hexane and acetone were kept over molecular sieves. The com-
−
1
cation. Bands due to [ClAgCl] were not found above 400 cm .
3 2 2 3 2 2
pounds [N(PPh ) ][Ag(CN) ] and [N(PPh ) ][Au(CN) ] were prepared
as described below.
2.3. X-ray crystallography
2
.2. Synthesis
Suitable colorless plate crystals of the compounds 1, 2 and 3 were se-
Synthesis of [N(PPh
mmol) was added to a solution of KCN (0.065 g, 1 mmol) in methanol
20 mL). After a clear solution had been obtained, [N(PPh
0.574 g, 1 mmol) was added to the reaction mixture. Stirring was con-
3
)
2
][Au(CN)
2
]: gold cyanide [Au(CN)]
n
(0.223 g,
lected under an optical microscope, glued and mounted onto a thin glass
capillary. Diffraction data were collected for 1 and 2 at 294 K, and for 3 at
173 K using Rigaku R-axis diffractometer equipped with an imaging
1
(
(
+
−
3 2
) ] Cl
plate detector, utilizing graphite-monochromatised Cu-K
α
in 1–2 and
tinued for 15 min. The solution was evaporated to dryness and the
white residue was treated with acetone (25 mL). The remaining solid,
KCl (0.050 g), was filtered off. Addition of hexane to the filtrate resulted
Mo-K radiations in 3. Full spheres of reciprocal spaces were using the
α
ω-scan. Preliminary orientation matrices, unit cell determination and
data reduction and corrections were performed using the CrystalClear
package [22]. The structures were solved by direct methods and refined
in white crystals of [N(PPh
Elemental analysis: found: C, 57.64; H, 4.19; N, 4.12. Calc. for:
30AuN : C, 57.94; H, 3.81; and N, 5.3.
3 2 2
) ][Au(CN) ] (0.750 g, 95%), m.p. 209 °C.
2
by full-matrix least squares on all |F | data, using the SHELX package
38
C H
P
3 2
[23]. Hydrogen atoms were isotropically refined and constrained to
ideal geometry, using their appropriate riding model and all non-
hydrogen atoms were anisotropically refined. The perchlorates and
tetrafluoridoborates in 1 and 2 were constrained to ideal geometry,
using appropriate geometrical restraints. The relatively high R value
(
9.8%) for compound 1 is due to poor crystal quality; the isomorphism
Fig. 1. Schematic representation of [PPN]+X− compounds; in reality the P-N-P angle is
often bent by some 140°.
with compound 2 gives confidence for its accuracy. Fig. 1 was created
using the DIAMOND package [24]. Crystallographic parameters of
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
3
compounds 1–3 are summarized in Table 1, while selected geometrical
parameters are discussed and presented below.
by dissolving an appropriate amount of the compound and DNA stock
solutions, while maintaining the total volume constant (3 mL). The
ethidium bromide displacement assay was carried out by fixing the
excitation wavelength. The DNA was pretreated with ethidium bro-
mide, [DNA/EthBr] = 10 for 30 min at 25 °C. Then the compounds
were added to this mixture and their effect on the emission intensity
was measured.
2
.4. Antiproliferative assays
The human ovarian cancer SKOV3 cells, (obtained from the
European Centre of Cell Cultures ECACC, Salisbury, UK) were cultured
in DMEM containing GlutaMAX-I supplemented with 10% FBS and
1
% penicillin/streptomycin (all from Invitrogen), at 37 °C in a humidi-
fied atmosphere of 95% of air and 5% CO (Heraeus, Germany). For eval-
uation of growth inhibition, cells were seeded in 96-well plates
Costar, Integra Biosciences, Cambridge, MA) at a concentration of
0,000 cells/well and grown for 24 h in complete medium. Solutions
of the compounds were prepared by H O and appropriate dilutions in
2.6. Mitochondrial functions
2
2.6.1. Preparation of rat liver mitochondria
Mitochondria were prepared from rat liver by differential centrifu-
gations following the procedure of Myers and Slater [31] in a medium
(
1
2
containing 220 mM mannitol, 70 mM sucrose, 0.1 mM H
4
edta and
DMEM were added to the wells (200 μL) to obtain a final concentration
ranging from 0.01 to 50 μM. Following 72 h drug exposure, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
5 mM Hepes/Tris buffer (pH 7.0). H edta was omitted in the final wash-
ing and in the mitochondrial suspension. Mitochondrial proteins were
estimated with the biuret procedure [32].
4
−
1
added to the cells at a final concentration of 0.50 mg·mL and incubat-
ed for 3–4 h, time after which the culture medium was removed and the
violet formazan crystals dissolved in DMSO. The optical density of each
well (96-well plates) was quantified in quadruplicate at 540 nm, using a
multi-well plate reader, and the percentage of surviving cells was calcu-
lated from the ratio of absorbance between treated and untreated cells.
The IC50 value was calculated as the concentration reducing the prolifer-
ation of the cells by 50% and is presented as an average (± SD) of at least
three independent experiments.
2.6.2. Estimation of mitochondrial swelling
Mitochondrial swelling was followed spectrophotometrically as a
decrease of the optical density at 540 nm. Briefly, rat liver mitochondria
(0.25 mg/mL) were incubated at 25 °C in 213 mM mannitol, 71 mM su-
crose, 5 mM Hepes/Tris buffer (pH 7.4), 5 mM succinate, 5 μM rotenone
and 2.5 μM oligomycin in the presence of compounds 1–3.
2.6.3. Mitochondrial dioxygen consumption
Dioxygen uptake was measured polarographically, utilizing a Clark-
type oxygen electrode inserted in a water jacketed chamber (25 °C)
with constant stirring. Data were acquired and analyzed as described
in [33]. Briefly, rat liver mitochondria (1 mg/mL) were incubated at 25
2
.5. DNA binding
The interaction of the compounds (1–3) with CT-DNA was carried
out in buffer containing 5 mM tris(hydroxymethyl)aminomethane buff-
er adjusted to pH 7.2 with 1 M hydrochloric acid. The CT-DNA dissolved
in this Tris–HCl buffer was dialyzed against the same buffer overnight.
Solutions of CT-DNA gave ratios of UV absorbance at 260 and 280 nm
above 1:1.9, indicating suitable DNA (free of protein). Absorption spec-
troscopy was used to determine the DNA concentration, using the molar
absorption coefficient 6600 dm ·mol cm per nucleotide at 260 nm.
The stock solution was kept at 4 °C. The Shimadzu UV-2450 UV–Vis
spectrophotometer was used to record absorption and the ethidium
bromide displacement assay using a JASCO F 6500 spectrofluoro-
photometer. The DNA-binding experiments include absorption spectral
studies and fluorescence conform to standard methods [25–27] and
have been reported earlier [28–30]. Absorption spectral titration exper-
iments were performed by upholding a constant concentration of the
complexes and varying the calf thymus DNA (CT DNA) concentration
°C in 0.1 M sucrose, 50 mM KCl, 1 mM MgCl
20 mM Hepes/Tris buffer (pH 7.4) and 1 mM H
2
, 1 mM NaH
2 4
PO and
4
egta. Compounds 1–3
were added and, after 1 min, respiration was started by 7.5 mM
succinate. The consumption of dioxygen was calculated in nmoles of
−
1
−1
2
O sec ·mg of protein.
3
−1
−1
2.6.4. Determination of membrane potential in isolated mitochondria and
in cancer cell
The mitochondrial membrane potential was estimated by
means of the fluorescent dye rhodamine 123. Mitochondrial pro-
teins (0.6 mg/mL) were incubated at 25 °C in 100 mM sucrose,
50 mM KCl, 20 mM Hepes/Tris buffer (pH 7.4), 1 mM NaH
2 4
PO , 1 mM
MgCl , 12.5 μM rotenone, and 0.5 μM oligomycin. Mitochondria were
2
treated with 5 mM succinate as an oxidizable substrate. After 4 min,
complexes 1–3 were added. Fluorescence was estimated in a microplate
Table 1
Crystal data and refinement details for compounds 1–3.
Crystallographic parameters
1
C
2
C
3
C
Formula
Formula weight
Crystal system
Space group
a [Å]
36
H30NP
2
.ClO
4
36
H30NP
2
.BF
4
36 2 2 6
H30NP , AgCl2,C H O
638.00
Monoclinic
P2/n
15.3547(3)
14.0504(3)
16.2237(3)
112.340(1)
3237.40(11)
4
625.36
Monoclinic
P2/n
15.3592(3)
13.9495(3)
16.2115(3)
112.454(1)
3210.04(11)
4
763.39
Monoclinic
P2 /c
9.1448(8)
23.7695(18)
17.0796(14)
101.480(2)
3638.3(5)
4
1
b [Å]
c [Å]
beta [°]
3
V [Å ]
Z
3
D(calc) [g/cm ]
1.309
1.294
1.394
Radiation [Å]
Theta min-max [°]
Dataset
Tot., uniq. data, R(int)
Observed data [I N 2δ(I)]
Nref, Npar, restraints
1.54187
6.6–72.3
−18:18;−17:17;−18:18
33220, 5994, 0.042
3406
1.54187
6.6–72.3
−18:18;−16:17;−17:18
41145, 5915, 0.031
2599
0.71075
3.1, 27.5
−11:11;−30:30;−22:22
53543, 8317, 0.134
4773
5994, 400, 16
5915, 400, 16
8317, 409, 0
R, wR
Min., max. resd. dens.
2
, S
0.098, 0.2483, 1.23
−1.22, 0.85
0.075, 0.2233, 1.16
−0.70, 0.66
0.0746, 0.2280, 1.04
−1.04, 0.97
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

4
A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Table 2
The protein content of isolated enzymes was estimated according to
Lowry et al. [36]. Thioredoxin reductase activity was measured at 25 °C
in 0.2 M Tris/HCl buffer (pH 8.1), and 0.25 mM NADPH. The reaction was
started by the addition of 1 mM DTNB (DNTB = 5,5′-dithiobis-2-
nitrobenzoic acid; Ellman's reagent) and followed spectrophotometri-
cally at 412 nm.
Selected bond distances (Å) and angles (°) for compounds 1–3.
Geometrical parameters
1
2
3
P1–N1
P1–C1
P1–C7
P1–C13
P2–N2
1.577(3)
1.800(6)
1.756(8)
1.785(6)
1.573(3)
……
1.808(7)
1.772(6)
1.774(7)
1.343(11)
1.320(8)
……
1.575(3)
1.792(6)
1.805(6)
1.778(7)
1.578(3)
……
1.778(6)
1.803(6)
1.797(6)
……
1.589(5)
1.774(7)
1.788(8)
1.807(7)
……
1.590(5)
1.779(8)
1.783(7)
1.787(7)
……
3. Results and discussion
P2–N1
P2–C19
P2–C25
P2–C31
Cl1–O1
Cl1–O2
F1–B1
3.1. Synthesis and 3D structure of the compounds
The synthesis of the three compounds went rather straightforward,
despite the unusual recipe starting from bis(cyanido)metallates(I)
for compounds 1 and 2 and yielded pure, crystalline homogeneous
materials, from which crystals for X-ray diffractions were easily obtain-
ed. Using alternative synthetic methods, by using (PPN)Cl and silver
salts, did not yield pure compounds. Compounds 1 and 2 were found
to be isostructural with no interactions between the cation and
the anion. The crystal data and refinement details for all 3 com-
pounds were mentioned in Table 1 (above). Relevant bond lengths
and angles are in Table 2. The molecular structures and packing are
given in Fig. 2.
In addition to the intrinsic intramolecular C–H…N hydrogen
bonding in the PPN molecular structures, the molecular packing in 1
and 2 are supported with weak C–H…O and C-H…F intermolecular hy-
drogen bonding. The molecular arrangement in 3 is dominated by weak
C–H…Cl hydrogen bonding interactions.
……
……
……
……
1.258(14)
1.235(10)
……
F2–B1
……
……
……
Ag1–Cl1
Ag1–Cl2
N1–P1–C1
N1–P1–C7
N1–P1–C13
C1–P1–C7
C1–P1–C13
2.563(8)
2.570(7)
107.6(3)
112.9(3)
113.4(3)
108.9(4)
105.6(3)
……
136.2(4)
……
……
……
……
……
……
……
……
……
……
……
……
……
113.0(3)
109.9(2)
107.3(3)
109.6(3)
107.1(3)
137.7(5)
……
111.5(7)
100.7(7)
110.2(6)
110.2(6)
112.2(5)
111.5(7)
……
……
……
……
……
……
……
112.5(3)
114.4(3)
108.3(3)
106.2(3)
107.8(3)
141.4(4)
……
……
……
……
……
i
P1–N1–P1
P1–N1–P2
O1–Cl1–O2
O1–Cl1–O1
O1–Cl1–O2
O2–Cl1–O1
i
i
i
O2–Cl1–O2
……
……
i
i
O1 –Cl1–O2
F1–B1–F2
F1–B1–F1
F1–B1–F2
F2–B1–F1
112.2(6)
97.4(11)
108.8(6)
108.8(6)
115.9(11)
112.2(6)
……
iii
The intramolecular distances for the cation and the anion, are all
iii
uneventful. Compound 3 contains an ethanol solvent molecule filling a
iii
−
gap in the crystal lattice; in fact this compound having the [AgCl
2
]
iii
F2–B1–F2
iii
iii
anion is a new compound, compared to reported similar examples
37,38].
F1 –B1–F2
Cl1–Ag1–Cl2
[
178.8(2)
Symmetry codes: (1) i: 1/2-x,y,3/2-z and (2) i: 3/2-x,y,1/2-z, and iii: 5/2-x,y,3/2-z.
3.2. Antiproliferative effects
reader (Tecan Infinite® M200 PRO), in a final volume of 0.250 mL, at
The antiproliferative effects of compounds 1–3 were tested against
4
85 nm (excitation wavelength) and 527 nm (emission wavelength).
Membrane potentials of human cisplatin-sensitive ovarian cancer
the human breast cancer cell line SKOV3. The compounds present ex-
tremely potent effects on cell viability, with IC50 values in the nM
range. Specifically, compound 3 is the most potent of the series with
an IC50 = 0.20 ± 0.05 μM, while 1 and 2 have slightly higher values
(ca. 0.4 μM for both compounds).
cells A2780 were analyzed using flow cytometry. Cells were grown
at 37 °C in a 5% CO using RPMI 1640 medium, supplemented with
5
2
−
1
−1
0 units·mL
penicillin and 50 μg·mL
streptomycin, 2 mM L-
glutamine, containing 10% fetal calf serum. The changes of the
membrane potential induced by the compounds were estimated
with a FACSCanto™ II (Becton Dickinson) cytofluorometer using
tetramethylrhodamine (TMRM) as a fluorescent dye, with an argon
laser at 585 nm.
3.3. DNA binding studies
The primary intracellular target of antitumor drugs is DNA; so, drug–
DNA interaction is of paramount importance and can lead to the prima-
ry mechanism of tumor inhibition for the treatment of cancer. There-
fore, to obtain an insight into the binding propensity and binding
mode, interaction of compounds 1–3 with calf thymus DNA (CT-DNA)
was carried out by using absorption spectroscopy. Interaction of
compounds with CT-DNA may result in an increase in absorbance
(hyperchromism), or a decrease in absorbance (hypochromism). The
2
.6.5. Thioredoxin reductase inhibition studies
Cytosolic thioredoxin reductase (TrxR1) was prepared from rat
liver according to Luthman and Holmgren [34], and mitochondrial
thioredoxin reductase (TrxR2) was purified according to Rigobello and
Bindoli [35].
Fig. 2. Molecular depiction of the compounds 1–3. From left to right thermal ellipsoidal of 1, packing and hydrogen bonding of 2, thermal ellipsoidal of 3 and packing with hydrogen
bonding of 3.
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
5
Fig. 3. Absorption spectra of compounds 1 (left), 2 (middle), and 3 (right) in Tris–HCl buffer (pH = 7.2) and in the presence of increasing amounts of CT-DNA. Arrows indicate the increase
in the intensity upon increasing DNA concentration.
former suggests the rupture of the secondary structure of DNA, while
the latter is the characteristic of intercalation [39] attributed to the in-
teraction between the electronic states of the chromophore in the com-
pound and those of the DNA bases [40].The binding interaction between
the cationic compounds and CT-DNA leads to diffusion-limited ion-pair
formation at higher concentration of the compound, such that the mol-
ecule is fitted to the contour of DNA double helix in an induced-fit fash-
ion. Thus, the compounds preferably bind to the DNA helix via groove
binding interactions. This groove binding results in structural reorgani-
zation of CT-DNA, which entails partial unwinding or damage of the
double helix at the exterior phosphate backbone leading to the forma-
tion of a cavity to accommodate the molecule. Consequently, uptake
occurs with partial melting of the double helix and generation of an ap-
propriate binding pocket. The absorption spectra of compound 1–3 in
the presence of CT-DNA are depicted in Fig. 3.
3.4. Ethidium bromide displacement assay
The competitive fluorescence displacement assay involves the addi-
tion of molecule to pretreated CT-DNA with ethidium bromide (EthBr),
and the intensity of EthBr emission was measured. The addition of a sec-
ond DNA-binding molecule can quench the emission intensity of DNA–
EthBr adduct by either replacing EthBr and/or by accepting the excited-
state electron of the EthBr through a photoelectron transfer mechanism
[41].
The emission spectra of EthBr–DNA in the absence and presence of
compounds 1–3 are depicted in Fig. 4. The addition of increasing
amounts of compounds 1–3 to CT-DNA pretreated with ethidium bro-
mide ([DNA] is 100 μM, [EthBr] = 10 μM) resulting in the quenching
of emission intensity, that indicates displacement of the bound
ethidium bromide from the CT-DNA by the complexes. This observation
may suggest binding of complexes 1–3 to DNA via a groove binding
mode and liberating few EthBr molecules from the EthBr–DNA system.
The quenching of the fluorescence of ethidium bromide-bound DNA
reveals the magnitude of DNA binding of compounds. Thus, we have
evaluated quantitatively the quenching extents of complexes 1–3,
using the classical Stern–Volmer Eq. (2),
−
4
Upon the addition of increasing amounts (0.067–0.33 × 10 M) of
−
4
CT-DNA to 1–3 of fixed concentration (0.067 × 10 M), concomitant
increase in the absorption intensity was observed “hyperchromaticity”.
The observed hyperchromic effect suggests that the binding of the com-
pounds to CT-DNA may be through an external contact (electrostatic
and/or groove binding).
Furthermore, to evaluate quantitatively the binding strength of
compounds 1–3 with CT-DNA, the intrinsic binding constant, K
determined using Eq. (1) by monitoring the changes in absorbance of
the π–π* bands with increasing concentration of CT-DNA,
b
, was
Io=I ¼ 1 þ KSV
Where, I
r
ð2Þ
o
and I give the fluorescence intensities in the absence
and the presence of complex, respectively. The concentration ratio of
the complex to DNA is the value for “r”. KSV values are used to evalu-
½
DNAꢀ=jεa−ε j ¼ ½DNAꢀ=jε −ε j þ 1=K =jε −ε j
ð1Þ
b
b
f
b
b
f
ate the quenching efficiency and are obtained as the slope of I
o
/I vs. r.
The KSV values for compounds 1–3 were found to be 1.68, 1.22, and
−
1
0
.95 M , respectively. The DNA binding affinities of the compounds,
where [DNA] represents the concentration of DNA, ε
a
, ε
f
and ε
b
are the
therefore, follows the order 1 N 2 N 3. Also, the apparent DNA binding
constants (Kapp) of the complexes 1–3 to CT DNA were calculated
using the equation.
apparent extinction coefficients Aobs/[complex], the extinction coeffi-
cient for the free compound and the extinction coefficient for the com-
pound in the fully bound form, respectively. The intrinsic binding
4
−1
4
−1
constants were found to be: 1 (2.96 × 10 M ), 2 (1.78 × 10 M
)
4
−1
and 3 (0.9 × 10 M ).
KEthBr½EthBrꢀ ¼ Kapp½complexꢀ
Fig. 4. Effect of addition of complexes 1 (left), 2 (middle) and 3 (right), on the emission intensity of DNA-EthBr in 5 mM Tris–HCl buffer at pH 7.1.
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

6
A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
where KEthBr (4.94 × 10 M−1) is the DNA binding constant of EthBr,
EthBr] is the concentration of EthBr (10 μM), and [complex] is the
concentration of the complex which reduces the EthBr florescence
5
m
cytoplasm, owing to the large Δψ generated by the respiratory chain
[
[42]. Thus, the effects on isolated mitochondria of compounds 1–3
were evaluated by estimation of the principal parameters of mitochon-
drial functions. As shown in Fig. 5, mitochondrial swelling (A) and
membrane potential (B) have been examined.
4
4
intensity to 50%.The Kapp values for 1–3 are 2.49 × 10 , 2.21 × 10 , and
.53 × 10 M , giving an order 1 N 2 N 3, in conformity from absorption
4
−1
1
spectral studies.
Compounds 1 and 2 are able to induce swelling at a relatively high
concentration (40 μM), while compound 3 causes a marked swelling
at lower concentrations (5–10 μM). With the same compound, swelling
occurs also at very low concentrations (1, 2.5 μM) and is preceded by a
lag of about 4 to 8 min. All three compounds, at 10 μM concentration,
determine a large and rapid decrease of the membrane potential that
is particularly extensive for compound 3. This decrease might be re-
ferred to the nature of delocalized lipophilic cations.
Finally, mitochondrial respiration in the presence of compounds 1–3
was also measured. As reported in Fig. 6, addition of compound 1 and 2,
at 10 μM concentration, induces an increase of succinate-dependent res-
piration that is not further stimulated by the subsequent addition of the
classical uncoupling agent CCCP (Carbonyl cyanide m-chlorophenyl
3
.5. Analysis of mitochondrial functions
Since the PPN+ moiety could behave as a Delocalized Lypophilic
Cation (DLC) type of compound, we decided to investigate the effects
of 1–3 on the mitochondria. It is worth mentioning that DLCs have
been explored as an approach to cancer chemotherapy that exploits
their selective accumulation in mitochondria of cancer cells, as a conse-
quence of the elevated transmembrane mitochondrial potential
Δψ [20] In fact, DLCs can pass easily through the lipid bilayer and
m .
their positive charge then directs them to the mitochondria where
they accumulate at significantly higher concentrations than in the
Fig. 5. Induction of mitochondrial swelling by increasing concentrations of 1–3 (left) and the effect of compounds 1–3 on the mitochondrial membrane potential (right). Rat liver mito-
chondria (0.25 mg/mL) were incubated at 25 °C in 213 mM mannitol, 71 mM sucrose, 5 mM Hepes/Tris buffer (pH 7.4), 5 mM succinate, and 3 mg/mL rotenone. Mitochondrial swelling
was followed spectrophotometrically at 540 nm. Compounds 1–3 were used at the indicated concentrations. The mitochondrial membrane potential was determined with rhodamine 123,
as described in the experimental section. Compounds 1–3 were added where arrows are indicated.
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
7
hydrazone), but instead an inhibitory effect takes place and increases
with time.
potential exerted by the PPN+ compounds on isolated mitochondria
and cultured cells (see Figs. 5 and 7). Among the potential targets sub-
jected to the action of PPN we have examined mitochondrial
+
Compound 3 markedly inhibits respiration, and is also completely
insensitive to the uncoupling agent CCCP. The inset of Fig. 6 shows a
control experiment reporting the stimulation of respiration by ADP
and the subsequent oxygen uptake by the uncoupler CCCP. It should
be noted that the increase of respiration occurring in the presence of
compounds 1 and 2, at least at the beginning, is independent of the
swelling of the mitochondria, as the latter is a slower event with respect
to respiration. However, by comparing the rates of respiration occurring
after CCCP addition in the control experiment with those obtained in the
presence of compounds 1 and 2, it is clearly evident that, after an initial
stimulation, compounds 1 and 2 progressively inhibit mitochondrial
respiration.
thioredoxin reductase. This enzyme is endowed with a C-terminal
motif characterized by the sequence -Cys-SeCys-Gly, which is also the
site of inhibition of several agents, especially gold and platinum com-
plexes and electrophilic compounds many of which are also anticancer
agents [21].
However, also cations such as Ca2+ and La3+ were shown to act
as inhibitors of TrxR although possibly at a site different from the
C-terminal. Their effect was attributed to an interaction with anionic
groups of the enzyme as also apparent from the preventive effect
4
exerted by the ion chelator H edta [45,46]. In the present study we
+
show that also PPN compounds can act as inhibitors of thioredoxin re-
ductase showing values for IC50 in the range of 5–10 μM. Although these
figures are higher in comparison to those obtained with other inhibi-
tors, such as gold compounds that act at nanomolar levels [47], it should
be noted that DLC concentrate hundreds of times in the mitochondrial
The mitochondrial membrane potential was also analyzed in cancer
5
cells. A2780 cells (6 × 10 ) were incubated with 5 μM of compound 1–3
for 18 h and then subjected to flow cytometry analysis using 25 nM
TMRM (tetramethylrhodamine). As reported in Fig. 7 all three com-
pounds lead to a strong decrease in the number of fluorescent cells
and, in particular, compound 3 causes a dramatic loss of mitochon-
drial membrane potential. Table 3 summarizes the percentage of cells
with high and low fluorescence, reported as an average ± SD of five
experiments.
+
matrix [16]. Therefore, inside the mitochondrion the PPN compounds
under study should easily reach concentrations able to completely pre-
2
+
3+
vent the activity of thioredoxin reductase. The effect of Ca and La is
+
electrostatic in nature and PPN compounds might act in a similar way
by binding to anionic sites located inside tumor cell mitochondria.
Considering the differential inhibitory effect on thioredoxin reduc-
+
3
.6. Mitochondrial thioredoxin reductase inhibition
tase exhibited by the PPN compounds containing different anions
(
Fig. 8), it is apparent that these play a relevant role in eliciting the
In mitochondria, thioredoxin reductase (TrxR2) is present in the ma-
inhibitory properties on thioredoxin reductase. It is interesting to note
that the anions ClO and BF alone are completely ineffective
4 4
on thioredoxin reductase at concentrations until 100 μM (data not
shown).
−
−
trix fraction. As apparent in Fig. 8 compounds 1 and 2 are able to inhibit
TrxR2 at micromolar level (as results from their IC50) and this inhibition
is possibly related to their cationic structure. Compound 3 determines a
strong inhibition on TrxR2 at nanomolar level (IC50 = 2.75 nM). The lat-
Furthermore, we observed that PPNCl still inhibits thioredoxin
reductase, although at concentrations higher (IC50 N 40 μM) than
those observed with the other compounds (data not shown). The effects
of compounds 1–3 were also tested on cytosolic thioredoxin reductase
(TrxR1) and on glutathione reductase (GR) (see Table S1 of the Elec-
tronic Supporting Information). As apparent, compounds 1–3 inhibit
TrxR1 at concentrations comparable to those observed for TrxR2. In
−
ter effect might partly depend on the presence of the [AgCl
2
] moiety, as
silver compounds are known as effective inhibitors of thioredoxin
+
reductase [43,44]. PPN compounds after entering mitochondria by
exploiting the membrane potential can act on different intramito-
chondrial functional targets. A marked energetic impairment takes
place, as shown by the very effective decrease of the membrane
Fig. 6. Effects of compounds 1–3 on mitochondrial dioxygen uptake. Rat liver mitochondria were incubated with 10 μM compounds 1–3 in the presence, where indicated, of 7.5 mM suc-
−
1
−1
cinate and 0.1 μM CCCP. Numbers by each line indicate dioxygen consumption rate (nmoles O
2
sec mg protein). Inset: control experiment showing the stimulation of respiration by
0.2 mM ADP.
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

8
A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
Fig. 8. Mitochondrial thioredoxin reductase inhibition by compounds 1, 2 and 3. Aliquots
of highly purified TrxR2 were incubated in the presence of increasing concentrations of
compounds 1–3, and the reaction was followed at 412 nm, as indicated under experimen-
tal methods.
contrast, compounds 1 and 2 are ineffective on GR, while compound 3
markedly inhibits this enzyme.
TrxR is considered a major target of the inhibitory effect of a large
number of anticancer agents and its inhibition leads to cell death [21].
+
However, it should also be considered that PPN compounds, once
inside the mitochondrion, can exert their inhibitory action on other
targets in addition to TrxR. It is noted that other DLCs, such as
tetraphenylphosphanium or rhodamine-123 tested in the same experi-
mental conditions are ineffective on TrxR activity. However, brilliant
green, which is a cationic triphenylmethane, was shown to exert a disrup-
tive effect on the mitochondrial thioredoxin system [48].
4
. Concluding remarks
Fig. 7. Decrease of membrane potential in A2780 cells in the presence of compounds 1–3.
A2780 cells, treated for 18 h with 5 µM of compounds 1-3, were incubated with 25 nM
TMRM for 20 min and then analyzed by flow cytometry. A, control; B, compound 1, C, com-
pound 2; D, compound 3.
The results described and discussed above strongly suggest that salts
of the bis(triphenylphosphane)iminium cation do show significant
anticancer activity, DNA binding and also inhibition of the enzyme
thioredoxin reductase, as well as causing mitochondrial damage. The
role of the used counter anions, i.e. perchlorate (1), tetrafluoridoborate
Table 3
Percentage of cells with high and low mitochondrial membrane potential (MMP) after
treatment with compounds 1–3.
(2) and dichloridoargentate(I) (3), was relevant for the specific proper-
Sample
High MMP
Low MMP
ties of each compound, as they differentiate their reactivity in a biolog-
ical context. Compound 1 appeared to be less effective than compound
2, however, the strongest effects were found for compound 3. These
differences observed for thioredoxin reductase inhibition and mito-
chondrial functions alteration, were found also reflected in other
Control
91.2 ± 2.6
40.3 ± 4.2
36.6 ± 3.4
13.7 ± 1.3
08.1 ± 1.2
59.2 ± 5.4
61.4 ± 3.2
84.1 ± 2.1
1
2
3
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030

A. Folda et al. / Journal of Inorganic Biochemistry xxx (2015) xxx–xxx
9
[
[
[
[
11] M.A. Beckett, P.N. Horton, M.B. Hursthouse, J.L. Timmis, Acta Crystallogr. Sect. E:
Struct. Rep. Online 66 (2010) O319–U2063.
12] K.F. Tebbe, N. Krauss, Acta Crystallogr. Sect. C: Cryst. Struct. Commun. 46 (1990)
measurements, like DNA binding. The effect of compound 3 toward
thioredoxin reductases and glutathione reductase is significantly larger
than that of compounds 1 and 2, and is most likely due to the role played
878–880.
+
13] C. Knapp, R. Uzun, Acta Crystallogr. Sect. E: Struct. Rep. Online 66 (2010)
O3185–U3783.
14] R.Y. Liau, H. Ehlich, A. Schier, H. Schmidbaur, Z. Naturforsh. B 57 (2002) 1085–1089.
by the Ag ions known to inhibit TrxR [43,44]. In fact the selenocysteine
residue present in the C-terminal motif of TrxR is sensitive to several in-
hibitors, including some metal ions [21].
[15] J.T. Madak, N. Neamati, Curr. Top. Med. Chem. 15 (2015) 745–766.
[16] M.P. Murphy, Biochim. Biophys. Acta Bioenerg. 1777 (2008) 1028–1031.
+
The present results have also made clear that the PPN cation
[
[
17] S.J. Berners-Price, A. Filipovska, Aust. J. Chem. 61 (2008) 661–668.
18] S. Rodriguez-Enriquez, A. Marin-Hernandez, J.C. Gallardo-Perez, L. Carreno-Fuentes,
R. Moreno-Sanchez, Mol. Nutr. Food Res. 53 (2009) 29–48.
can determine an energetic impairment, resulting in a strong decrease
of the mitochondrial membrane potential, which, associated with
thioredoxin reductase inhibition, may play a relevant role in the possi-
ble toxic effects of these compounds in cancer cells, together with
other mechanisms (e.g. DNA damage). Further studies are necessary
to demonstrate the selectivity of such biological activity in cancer cells
[19] M. Kurtoglu, T.J. Lampidis, Mol. Nutr. Food Res. 53 (2009) 68–75.
[
[
20] J.S. Modica-Napolitano, J.R. Aprille, Adv. Drug Deliv. Rev. 49 (2001) 63–70.
21] A. Bindoli, M.P. Rigobello, G. Scutari, C. Gabbiani, A. Casini, L. Messori, Coord. Chem.
Rev. 253 (2009) 1692–1707.
[22] Rigaku, Crystal Clear. Crystal Structure Analysis Package, Rigaku, 9009 New Trails
+
Dr., The Woodlands TX 77381, 2007.
with respect to non-cancerous ones, and to develop PPN compounds
[
[
23] G.M. Sheldrick, Acta Crystallogr. A 64 (2008) 112–122.
24] K. Brandenburg, Diamond, 3.1–6, DIAMOND, ver 3.1d, Crystal Impact GbR, Bonn,
Germany, 2006.
25] J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161–4170.
26] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
especially targeted to mitochondria of diseased tissues therefore ad-
dressing cancer cell to death.
[
[
Acknowledgments
3047–3053.
[
[
27] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392–6396.
28] R.A. Khan, F. Arjmand, S. Tabassum, M. Monari, F. Marchetti, C. Pettinari, J.
Organomet. Chem. 771 (2014) 47–58.
The authors are indebted to several sources of funding for this pro-
ject. The authors acknowledge PRIN20107Z8XBW granted by Ministero
dell'Istruzione, Università e Ricerca (MIUR) Italy, CPDA130272 granted
by University of Padova (Italy) and COST ActionsCM1105. Also thanks
are due to the Distinguished Scientific Fellows Programme of King
Saud University, for financial support.
[
29] R.A. Khan, A. Asim, R. Kakkar, D. Gupta, V. Bagchi, F. Arjmand, S. Tabassum, Organo-
metallics 32 (2013) 2546–2551.
[30] R.A. Khan, S. Yadav, Z. Hussain, F. Arjmand, S. Tabassum, Dalton Trans. 43 (2014)
534–2548.
[
[
[
2
31] D.K. Myers, E.C. Slater, Biochem. J. 67 (1957) 558–572.
32] A.G. Gornall, C.J. Bardawill, M.M. David, J. Biol. Chem. 177 (1949) 751–766.
33] F. Cazzaro, M.P. Rigobello, A. Bindoli, Comput. Methods Prog. Biomed. 51 (1996)
1
41–151.
Appendix A. Supplementary data
[
[
34] M. Luthman, A. Holmgren, Biochemistry 21 (1982) 6628–6633.
35] M.P. Rigobello, A. Bindoli, in: E. Cadenas, L. Packer (Eds.), Methods in Enzymology,
Thiol Redox Transitions in Cell Signaling, Pt B: Cellular Localization and Signaling,
Vol 474, Elsevier Academic Press Inc., San Diego 2010, pp. 109–122.
36] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275.
37] S. Aboulkacem, W. Tyrra, I. Pantenburg, J. Chem. Crystallogr. 36 (2006) 141–145.
[38] G.A. Bowmaker, M.I. Bruce, B.W. Skelton, N. Somers, A.H. White, Z. Anorg. Allg.
Chem. 633 (2007) 1024–1030.
39] T. Afrati, A.A. Pantazaki, C. Dendrinou-Samara, C. Raptopoulou, A. Terzis, D.P.
Electronic supplementary information (ESI) available: Crystallo-
graphic, structural data and figures of compound 1, 2 and 3. See CCDC
reference numbers 1,403,185 (1), 1,403,186 (2) and 1,403,187 (3).
These data can be obtained free of charge via http://www.ccdc.cam.ac.
uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223–
[
[
[
Kessissoglou, Dalton Trans. 39 (2010) 765–775.
[40] B.D. Wang, Z.Y. Yang, P. Crewdson, D.Q. Wang, J. Inorg. Biochem. 101 (2007)
3
36–033; or e-mail: deposit@ccdc.cam.ac.uk. Supplementary data to
this article can be found online at http://dx.doi.org/10.1016/j.jinorgbio.
015.08.030.
1
492–1504.
[
[
[
41] B. Selvakumar, V. Rajendiran, P.U. Maheswari, H. Stoeckli-Evans, M. Palaniandavar, J.
2
Inorg. Biochem. 100 (2006) 316–330.
42] M.F. Ross, G.F. Kelso, F.H. Blaikie, A.M. James, H.M. Cocheme, A. Filipovska, T. Da Ros,
T.R. Hurd, R.A.J. Smith, M.P. Murphy, Biochem. Mosc. 70 (2005) 222–230.
43] A. Citta, E. Schuh, F. Mohr, A. Folda, M.L. Massimino, A. Bindoli, A. Casini, M.P.
Rigobello, Metallomics 5 (2013) 1006–1015.
[44] M. Srivastava, S. Singh, W.T. Self, Environ. Health Perspect. 120 (2012) 56–61.
[45] A. Citta, A. Folda, G. Scutari, L. Cesaro, A. Bindoli, M.P. Rigobello, J. Inorg. Biochem.
117 (2012) 18–24.
[46] M.P. Rigobello, F. Vianello, A. Folda, C. Roman, G. Scutari, A. Bindoli, Biochem.
Biophys. Res. Commun. 343 (2006) 873–87
8.
[47] S. Gromer, L.D. Arscott, C.H. Williams, R.H. Schirmer, K. Becker, J. Biol. Chem. 273
References
[
[
1] P. Paul, Z.W. Xie, R. Bau, P.D.W. Boyd, C.A. Reed, J. Am. Chem. Soc. 116 (1994)
145–4146.
2] H. Moriyama, H. Kobayashi, A. Kobayashi, T. Watanabe, J. Am. Chem. Soc. 115 (1993)
185–1187.
4
1
[
[
[
[
[
[
[
3] J.K. Evju, K.R. Mann, Chem. Mater. 11 (1999) 1425–1433.
4] R.D. Wilson, R. Bau, J. Am. Chem. Soc. 96 (1974) 7601–7602.
5] CSD, CSD, Cambridge Crystallographic Data Center, 2015.
6] R.E. Stevens, T.J. Yanta, W.L. Gladfelter, J. Am. Chem. Soc. 103 (1981) 4981–4982.
7] G. Werner, I. Kolowos, J. Senkyr, Talanta 36 (1989) 966–968.
8] C. Glidewell, H.D. Holden, J. Organomet. Chem. 226 (1982) 171–181.
9] F. Seel, R. Kuhn, G. Simon, Z. Naturforsh. B 39 (1984) 1622–1623.
(1998) 20096–20101.
[48] X. Zhang, Y.J. Zheng, L.E. Fried, Y.T. Du, S.J. Montano, A. Sohn, B. Lefkove, L. Holmgren,
J.L. Arbiser, A. Holmgren, J. Lu, Free Radic. Biol. Med. 50 (2011) 811–820.
[10] F. Seel, R. Kuhn, G. Simon, M. Wagner, B. Krebs, M. Dartmann, Z. Naturforsh. B 40
(1985) 1607–1617.
Please cite this article as: A. Folda, et al., Insights into the strong in-vitro anticancer effects for bis(triphenylphosphane)iminium compounds
having perchlorate, tetrafluorid..., J. Inorg. Biochem. (2015), http://dx.doi.org/10.1016/j.jinorgbio.2015.08.030