Enhancing the Cytotoxic Activity of Anticancer PtIV Complexes by Introduction of Lonidamine as an Axial Ligand

Article abstract of DOI:10.1002/ejic.201600857

The synthesis and in vitro cytotoxicity of a series of Pt^IV complexes with lonidamine as a ligand coordinated in axial position are described. Lonidamine was found to affect strongly the in vitro cytotoxic activity of these new complexes, lowering the IC₅₀ values down to the nanomolar range. Lipophilicity assessed in terms of log P showed no direct correlation with cytotoxicity.

Full text of DOI:10.1002/ejic.201600857

A Journal of
Accepted Article
Title: Enhancing the cytotoxic activity of anticancer Pt(IV) complexes by introduction
of lonidamine as an axial ligand
Authors: Yulia N. Nosova; Lidia S. Foteeva; Ilia V. Zenin; Timur I. Fetisov; Kirill I.
Kirsanov; Marianna G. Yakubovskaya; Taisya A. Antonenko; Viktor A. Tafeen-
ko; Leonid A. Aslanov; Anna A. Lobas; Mikhail V. Gorshkov; Markus Galanski;
Bernhard K. Keppler; Andrei R. Timerbaev; Elena R. Milaeva; Alexey A. Na-
zarov
This manuscript has been accepted after peer review and the authors have elected
to post their Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
blished to ensure accuracy of information. The authors are responsible for the content
of this Accepted Article.
To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201600857
Link to VoR: http://dx.doi.org/10.1002/ejic.201600857

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
Enhancing the cytotoxic activity of anticancer Pt(IV) complexes
by introduction of lonidamine as an axial ligand.
[
a]
[b]
[a]
[c]
[c]
Yulia N. Nosova , Lidia S. Foteeva , Ilia V. Zenin , Timur I. Fetisov , Kirill I. Kirsanov , Marianna G.
[
c]
[a]
[a]
[a]
[d]
Yakubovskaya , Taisya A. Antonenko , Viktor A. Tafeenko , Leonid A. Aslanov , Anna A. Lobas ,
[
d]
[e]
[e]
[b]
Mikhail V. Gorshkov , Markus Galanski , Bernhard K. Keppler , Andrei R. Timerbaev , Elena R.
[
a]
[a]*
Milaeva and Alexey A. Nazarov
Abstract: The synthesis and in vitro cytotoxicity of a series of Pt(IV)
complexes with lonidamine as a ligand coordinated in axial position
are described. The lonidamine was found to affect strongly the in
vitro cytotoxic activity of these novel complexes, lowering the IC50
values down to the nanomolar range. Lipophilicity assessed in terms
of log P showed no direct correlation with cytotoxicity.
that such ligands exhibit their own activity and act as carriers in
drug targeting (e.g., folic acid, estradiol, aspirin, and short
[9]
[10]
[11]
[
12]
[13]
peptides ), inhibit a key enzyme (ethacrynic acid
inhibitor of glutathione-S-transferase, dichloroacetic acid
as an
[
14]
for
– histone
[
15]
pyruvate dehydrogenase kinase, or valproic acid
deacetylase inhibitor), or inhibit immune checkpoint.
[
16]
As a selective aerobic glycolysis inhibitor, lonidamine was
evaluated in clinical trials, being administrated as a single agent
or in combination with other drugs against lung, prostate, breast,
Introduction
[17]
and in non-small cell lung cancer. Phase II/III clinical trials for
several chemotherapeutic programs that include lonidamine are
still in progress. Selectivity of lonidamine due to inhibiting
aerobic glycolysis in cancer cells while simultaneously
enhancing aerobic glycolysis in normal cells might be attributed
Nowadays platinum(II)-based chemotherapeutic agents are in
[
1]
the first-line for treatment of different types of cancers. Much
effort was devoted to understanding the mechanism of action of
[
2]
platinum drugs and overcoming side effects and resistant
to the inhibition of mitochondria bound hexokinase, which is
[
3]
phenomena. Pt(IV) complexes have received considerable
interest due to their kinetical inertness and specific reactivity in
the reducing milieu of the cancer cell (activation by reduction),
[18]
highly specific to malignant cells.
To our expectations,
integration of a platinum moiety and a releasable organic
inhibitor in one compound can provide a stronger tumor-
inhibiting effect than that observed when anticancer platinum
[
4]
lower general toxicity, and possibility of oral administration.
[
19]
complexes were co-administrated with lonidamine.
Here, we
report the synthesis, chemical characterization and evaluation of
drug-like properties, such as lipophilicity and in-vitro
antiproliferative activity, of the first anticancer complexes with
the aerobic glycolysis inhibitor, lonidamine, covalently linked to
the platinum(IV) center.
Figure 1. Examples of Pt(IV) complexes evaluated in clinical trials.
Results and Discussion
Several Pt(IV) compounds (see e.g. Figure 1) were tested in
clinical trials, however none of them so far reached the approval
stage. Tetraplatin was rejected due to a high toxicity that
We combined the known pharmacophore lonidamine with Pt(IV)
moiety by introduction of organic part as an axial ligand.
Complexes 5–7 (Scheme 1) were prepared by reaction of
[
5]
probably stemmed from its fast reduction in the blood, whereas
iproplatin demonstrated a low activity associated with slow
dihydroxidoplatinum(IV)
diamminedichloridoplatinum(II) (cisplatin), dichlorido(ethane-1,2-
diamine)platinum(II), and [(1R,2R)-cyclohexane-1,2-
diamine](oxalato)platinum(II) (oxaliplatin) with an excess of 1-
(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid chloride
derivatives
based
on
[
6]
reduction.
For satraplatin, evaluated in the treatment of
hormone-refractory prostate cancer, lack of convincing benefit
for overall survival did not let the drug to enter the clinic, though
clinical trials in combination with various organic drugs are still
obtained in situ from lonidamine and oxalyl chloride. Complex 8
was made up in a similar way, starting from acetato(1R,2R-
[
7]
undergoing.
One of the synthetic advantages of Pt(IV) complexes is the
possibility to covalently link biologically active ligands at the
cyclohexanediamine)hydroxido(oxalato)platinum(IV).
In
all
reactions, pyridine was used as an acceptor of HCl. Complex 6
precipitated during the reaction because of its low solubility in
acetone, while 5, 7, and 8 precipitated after evaporating the
reaction mixture. Finally, pyridine hydrochloride was removed by
washing the precipitates with water, hence yielding pure
complexes.
[
4c]
respective axial positions.
In cancer chemotherapy, drug
combinations are widely applied, and therefore incorporation of
a bioactive ligand can, by the rule of thumb, increase the activity
or selectivity profile of new complexes and tune the controlled
[
8]
release of an active organic moiety. There is a proven record
[
a]
Lomonosov Moscow State University, Department of Medicinal Chemistry and Fine Organic Synthesis, Leninskie Gory 1/3, 119991 Moscow, Russian
Federation E-mail: alexey.nazarov@me.com
[
[
[
[
b]
c]
d]
e]
Vernadsky Institute of Geochemistry and Analytical Chemistry, Kosygin St. 19, 119991 Moscow, Russian Federation
Blokhin Cancer Research Center RAMS, Kashirskoye Shosse 24, 115478 Moscow, Russian Federation
Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, Leninsky Pr. 38, Bld.2, Moscow, 119334, Russian Federation
Institute of Inorganic Chem
T
is
h
tr
i
y
s
, U
a
n
r
iv
t
e
i
rs
c
it
l
y
e
of
i
V
s
ie
p
nn
r
a
o
, Wteae
c
h
t
r
e
ing
d
er
b
S
y
tr.
c
42
o
,
p
A-
y
10
r
1
i
9
g
, V
h
ie
t
n
.
n
A
a,
l
A
l rig
iahts reserved
u
s
tr

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
Figure 2. Molecular structure of complex 7 with the atom numbering scheme.
Thermal displacement ellipsoids are given at the 50% probability level.
Complex 7 was found to be a monomer in solid state, with
distorted octahedron geometry around the Pt center (Figure 2).
The bond distances Pt‒O and Pt‒N vary between 2.007(3) and
2
8
.055(4) Å. The bond angles around Pt(IV) vary between
3.29(16) and 98.35(15) and 171.75(14) and 177.96(17)°. The
crystal structure of 7 contains two acetone molecules without
any interactions with the complex; this finding explains low
stability of the crystals at room temperature.
Table 1. Selected bond lengths and angles for complex 7.
Bond lengths (Å)
2.007(3)
Angles (°)
86.06(15)
Pt1‒O1
Pt1‒O3
Pt1‒O4
Pt1‒O1A
Pt1‒N4
Pt1‒N3
O1‒Pt1‒O3
O1‒Pt1‒O4
O3‒Pt1‒O4
O1‒Pt1‒O1A
O3‒Pt1‒O1A
O4‒Pt1‒O1A
O1‒Pt1‒N4
O3‒Pt1‒N4
O4‒Pt1‒N4
O1A‒Pt1‒N4
O1‒Pt1‒N3
O3‒Pt1‒N3
O4‒Pt1‒N3
O1A‒Pt1‒N3
N4‒Pt1‒N3
2.012(3)
2.013(3)
2.024(3)
2.037(4)
2.055(4)
92.33(14)
84.40(14)
171.75(14)
86.36(15)
83.69(14)
85.64(15)
95.68(15)
177.96(17)
98.35(15)
96.44(16)
177.21(16)
96.72(16)
91.21(15)
83.29(16)
Scheme 1. Synthesis of Pt(IV) complexes 5-8.
1
13
1
15
195
Complexes 5–8 were characterized by H, C{ H}, N, Pt 1D
and 2D NMR spectroscopy, ESI mass spectrometry and
13
1
elemental analysis. In the C{ H} spectra, we observed a shift of
the carboxylic group that confirms the attachment of lonidamine
to the platinum center. In addition, in the proton and carbon
spectra for complexes 5–7, only one set of signals was recorded
confirming formation of the bis-substituted complexes. In ESI
mass spectra in methanol, the most abundant peaks were
assigned to the [M+H ] ion in the positive ion mode or to [M-H ]
ion in the negative ion mode, respectively, containing the
characteristic isotope pattern for such type of platinum
complexes. For all mass spectra, experimental isotopic patterns
are in good agreement with calculated ones.
Cytotoxicity of complexes 5–8, as well as of lonidamine and
cisplatin, was determined in human MCF7 (breast cancer) cell
line and its doxorubicin/cisplatin resistant analogue, MCF7D, in
colon carcinoma SW480 cells, in the non-small cell lung
carcinoma cell line A549, and in the immortalized human
keratinocyte cell line HaCat (nonmalignant) by means of
standard MTT colorimetric assay. For comparison, cytotoxicity
data taken from literature for the precursor complexes. Cytotoxic
activity of the compounds was evaluated as the IC50 value after
72 h of incubation (Table 2).
In MCF7, MCF7D, SW480, and A549 cell lines, complexes 5–8
showed considerably higher toxicity than lonidamine or cisplatin.
In immortalized keratinocytes HaCat, compound 5 exhibited the
same cytotoxic activity as lonidamine showing low degree of
specificity towards the cancer cells. A similar effect was
observed for complex 7, while complexes 6 and 8 displayed
+
+
+ -
Monitoring of the coordination sphere around the platinum(IV)
1
95
center using
Pt NMR spectroscopy is well described in the
literature. For complexes 5 and 6, the respective resonance at
842 and 2633 ppm indicates the formation of Pt(IV)Cl
configured complexes. Likewise, for complexes and
resonances at 3235 and 3240 ppm indicate a Pt(IV)N O
4
[
20]
2
2 2 2
N O
7
8
2
coordination sphere. As shown earlier, the nature of
carboxylates in the axial position has no influence on the
1
95
[21]
resonance in Pt spectra.
The molecular structure of 7 was determined by X-ray diffraction
analysis at 200 K (Figure 2). Re-crystallization of the complex
from acetone and diethyl ether gave crystals suitable for
crystallographic analysis. Selected bond distances and angles
for complex 7 are reported in Table 1. Crystal data and the
structure refinement details are given in Table 3.
stronger
cytotoxicity
(0.04±0.015
and
0.21±0.06 µM,
respectively). The highest activity of all the complexes was
found against breast cancer cells, with IC50 values in the MCF7
cell line falling into the nanomolar range. In spite of the fact that
IC50 values for cisplatin and lonidamine were 3–4 times higher
against doxorubicin/cisplatin-resistant MCF7D in comparison
with MCF7 measurements, no difference between cytotoxicity of
the tested compounds against both MCF7 and MCF7D was
observed.
In summary, compound 6 showed the highest cytotoxic activity
against MCF7, MCF7D, HaCat, and SW480 cell lines, with
nanomolar IC50 values. Consequently, this compound can be
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
recommended for a more detailed investigation directed toward
the assessment of its cytotoxic profile, interactions with
important biomolecules, and in vivo evaluation.
platinum(IV) prodrugs, as well as to the positive control cisplatin.
Of the tested compounds, complex 6 displays the most attractive
biological performance, thus being worth of further investigations
the mechanism of action and in vivo study.
Table 2. Cytotoxicity of complexes 5–8, lonidamine and precursor complexes
in different cell lines and log P values
a
compounds
IC50 (µM)
MCF7
log P
Experimental Section
SW480
A549
MCF7D
HaCat Shake- MEKC
flask
Materials
5
6
7
8
1±0.5
2.1±0.7
0.45±0.3 0.6±0.15 2.5±1.1
0.03±0.008 0.07±0.024 0.04±0.015 1.31
0.5±0.25 0.33±0.01 1.2±0.5 1.79
0.21±0.06 1.84
1.76
1.69
1.60
1.77
1.84
1.30
-
[28]
All solvents were purified and degassed prior to use. Methanol (HPLC
0.13±0.06 1.5±0.9
0.7±0.2 1.2±0.4
2.1±1.0 3.9±2.4
grade, Fisher) and Millipore water (18.2 MΩ; Millipore, Molsheim, France)
were used for ESI-MS and MEKC studies.(OC-6-33)-Dichlorido(ethane-
1,2-diamine)bis[(4-propylamino)-4-oxobutanato]platinum(IV) (9, log P –
0.43 ) and (OC-6-33)-dichlorido(ethane-1,2-diamine)bis[(4-propyloxy)-
-oxobutanato]platinum(IV) (10, log P 0.70 ) used as reference points
in MEKC measurements were synthesized in the University of Vienna.
SDS (Sigma-Aldrich, St. Louis, USA), Sudan III and oxalyl chloride (Fluka,
St. Louis, USA) were used as purchased.
0.5±0.2
8.5±2.1
0.5±0.1
27±14
64±7
[
22]
lonidamine˃30
cisplatin 10±6
˃40
2.5±0.9
56±15
1.30
-
[
29]
21.0±1.7 16±5
[
29]
4
[23]
[23]
[25]
Oxaliplatin 0.9±0.3 11.5±3.9 43.8
[24]
Pt(en)Cl
2
20.6±4.1
a
Errors represent the standard error of the mean.
Physical Measurements
Liphophilicity, expressed in the terms of log P, is one of
important parameters in drug discovery. This characteristic is
included in the Lipinsky rule of 5 and can be used as an
indication for bioavailability of drug-like molecules and the
possibility of oral administration.
Two different methods (shake-flask and micellar electrokinetic
chromatography, MEKC), applied according to previously
NMR spectra were recorded on a Bruker FT-NMR Avance III 500 MHz
1
13
15
195
instrument at 500.32 ( H), 125.81 ( C), 50.70 ( N) and 107.57 ( Pt)
MHz. 2D NMR measurements were carried out using standard pulse
programs. Chemical shifts were referenced relative to the solvent signal
[26]
1
13
15
195
for H and C spectra. For N and Pt spectra, the external standards,
NH Cl an K [PtCl ], were used, respectively. Elemental analysis was
4
2
4
developed experimental protocols, were used for log
determination of new complexes. The log P values of complexes
–8 and lonidamine are collected in Table 2. (Note that because
of a fairly high hydrophobicity of newly developed complexes, an
adjustment of MEKC conditions toward lower surfactant
concentration was necessary. As can be seen from Table 2, the
data of both methods used to determine log P are in good
agreement. However, for such a limited number of compounds,
it was not possible to observe any strict relationship between log
P and the structure of equatorial ligands (see Scheme 1).
P
performed by the Microanalytical Laboratory of the University of Vienna
using a 2400 CHN elemental analyzer (Perkin–Elmer) and at Moscow
State University with MicroCube Elementar analyzer. ESI mass spectra
were recorded using a LC/MSn ion trap mass spectrometer amaZon SL
5
(
Bruker, Bremen, Germany) with MeOH as solvent. ICP-MS
measurements were performed on a X-7 quadrupole mass-spectrometer
Thermo Elemental, USA) equipped with a standard low-volume glass
spray chamber (Peltier, cooled at 3 °C) and a concentric glass nebulizer
(
–1
at a sample up-take rate of 0.8 mL min . Before analysis, the instrument
was tuned to achieve maximum Pt count rate and In served as internal
standard. All MEKC separations were carried out on a CAPEL 105M
115
(Lumex Instruments, St. Petersburg, Russia) equipped with an on-
It should be noted that for metal-based drugs with low to
moderate lipophilicity, the order of log P is equivalent to the
order of increased drug accumulation inside the cell and hence
cytotoxicity. However, when the cytotoxicity of all compounds
column UV-vis detector and controlled by an Elphoran software. Fused-
silica capillaries of 60 cm total length (50 cm effective length), 75 μm
inner diameter, were provided by Polymicro Technologies (Phoenix,
USA). Further details concerning MEKC system operational parameters
was compared to log P, no correlation was evident. Furthermore, and protocols for capillary initialization and pre- and between-run
[
29]
the least lipophilic complex 6 showed the highest activity. This
most plausible explanation behind this observation is that neither
lipophilicity nor accumulation corresponds to the activation of
bioreductive Pt(IV) prodrugs in a reducing environment of cancer
conditioning can be found elsewhere.
log P
[
27]
cells, which is likely dependent on the rate of reduction.
Using the shake-flask method log P values of complexes 5–8 were
obtained similarly to other sparingly soluble metal complexes, as
described elsewhere. Briefly, the saturated solution of each compound
in water (pre-saturated with n-octanol) was prepared by agitating in the
ultrasonic bath. The supernatant was filtrated through
[
30]
Conclusions
a 0.45 μm
membrane filter and shaken with an equal volume of water-saturated n-
octanol for 2 h using a mechanical shaker. Platinum concentrations in the
aqueous phase after phase separation (Caq) and in the initial saturated
In this study, a unique approach to increasing the anticancer
activity of platinum(IV) complexes was proposed, taking
advantage of their axial ligation by lonidamine. The in vitro
0
solution (C ) were determined by ICP-MS. Partition coefficients were
calculated from the equation:
results for
a
series of complexes with
a
cisplatin,
dichlorido(ethane-1,2-diamine)platinum(II), or oxaliplatin core
indicate that lonidamine exhibits remarkable influences on their
activity, which is superior to that of the majority of known
log P = log [(C
0
– Caq) / Caq
]
(1)
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
-
4
In addition, log P values were determined with a MEKC method. A 6×10
M solution of each compound in acetone, as well as of two previously
studied Pt(IV) complexes (9 and 10), was introduced hydrodynamically
into the capillary filled with the micellar background electrolyte (30 mM
SDS in 20 mM sodium tetraborate solution) and run separately by
applying a high voltage. Acetone and Sudan III were used as markers for
the electroosmotic flow (EOF) and micelles, respectively. Migration times
of each analyte and marker were used to calculate the retention factor, k,
according to the following equation
Table 3. Selected bond lengths and angles for complex 7.
Empirical formula
Fw
Space group
Syngony
a (Å)
38 4 6 8 3 6
C H32 Cl N O Pt, 2(C H O)
1153.74
Pꢉ̅
Triclinic
12.7080(10)
13.5260(10)
14.2440(10)
101.883(6)
93.235(6)
106.228(7)
2283.5(3)
1.67
b (Å)
c (Å)
α (°)
β (°)
ꢀ
ꢁ
k = ꢁ
(2)
ꢅ
ꢂꢃꢄꢀ
ꢈ
ꢆꢇ
(
t
0
, tmc and t
R
are migration times of EOF marker, micelles, and the
γ (°)
3
complex, respectively). The corresponding log P value was assessed
V (Å )
calc., g/cm
3
similarly to the previously established approach to ascertain the
[
29]
Z
2
relationship between log P and log k .
3
max
/
min (e/Å )
2.109/-1.561
CuKα
8.445
0.0397/0.0875
0.999
−1
Cell lines and culture conditions
μ (mm
/wR (I ≥ 2σ(I)
GOOF
)
R
1
2
MCF7, MCF7D (a kind gift of N.I. Moiseeva), HaCat, A549, and SW480
cell lines were cultured in DMEM (PanEco, Russia) with 10% FBS
Synthesis
2
(HyClone, USA) and antibiotics (PanEco, Russia) in 5% CO at 37ºC.
The compounds were pre-dissolved at 20 mM in DMSO and then added
to the cell culture medium at the required concentration with a maximum
DMSO content of 0.5 v/v%. At these concentrations, DMSO has no effect
on cell viability. Cells were cultured in 96-well plates (7×103 cells/well)
and treated with various concentrations of complexes 5–8 (from 0.01µM
to 50µM) as well as with lonidamine and cisplatin at 37ºC for 72 h. Cell
viability was determined using the MTT assay, which quantifies the
mitochondrial activity Then, the cells were incubated at 37ºC for 4 h with
(
OC-6-33)-(diammine)dichloridobis(1-(2,4-dichlorobenzyl)-1H-
20 µl of 5 mg/ml solution of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (Sigma-Aldrich, St. Louis, USA). The
supernatant was discarded and cells were dissolved in 150 µl of dimethyl
sulfoxide. The optical density of the solution was measured at 550 nm
using a multiwell plate reader (Multiskan FC, Thermo Fisher Scientifics,
USA) and the percentage of surviving cells was calculated from the
absorbance of untreated cells. Each experiment was repeated at least
three times and each concentration was tested in at least three replicates.
indazole-3-carboxylate)platinum(IV) 5
Crystallographic data collection and structure determination
Oxalyl chloride (1.2 mL, 14 mmol), one-two drops of DMF was added to a
stirred suspension of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic
The data of 7 were collected by using STOE diffractometer Pilatus100K
detector, focusing mirror collimation CuKα (1.54086Å) radiation, rotation
method mode at 200 K. STOE X-AREA software was used for cells
refinement and data reduction. Data collection and image processing
was performed with X-Area 1.67 (STOE & Cie GmbH, Darmstadt,
Germany, 2013). Intensity data were scaled with LANA (part of X-Area)
in order to minimize differences of intensities of symmetry-equivalent
reflections (multi-scan method). The structures were solved with direct
[33]
acid
2 2
(321 mg, 1 mmol) in CH Cl (25 mL). The reaction mixture was
refluxed for 1 h until a clear solution was formed. Solvent and unreacted
oxalyl chloride were removed under reduced pressure to yield the acid
chloride as a pale yellow solid that was used without purification. A
solution of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbonyl chloride in
acetone (10 mL) was added to a stirred suspension of (OC-6-33)-
[34]
(diammine)dichloridodihydroxidoplatinum(IV)
(65 mg, 0.2 mmol) and
pyridine (120 μl, 1.5 mmol) in acetone (10 mL). The reaction mixture was
stirred for 12h at room temperature, concentrated to ~3 mL and a formed
white precipitate was filtered off, washed with diethyl ether (2 x 5 mL),
water (3 x 3mL) and dried under reduced pressure. Yield: 78 mg, (42%),
m.p. . 210–212 °C (decomp), elem. anal. calc (%) for (С30H24Cl N O Pt):
6 6 4
C 38.32, H 2.57, N 8.94, found: C 38.21, H 2.91, N 9.03. 1H NMR ([d6]-
2
methods and refined by full-matrix least-squares procedures on F with
[
31]
SHELXL97.
The non-hydrogen atoms were refined by using the
anisotropic full matrix least-square procedure; hydrogen atoms were
located at calculated positions and refined via the “riding model”.
Refinement was made against 8251 reflections. 604 parameters were
refined using 20 restraints. CCDC 1492004 contain the supplementary
crystallographic data for this paper. These data can be obtained free of
DMSO) δ: 8.38 (d, 2H, J = 8.2 Hz, H4), 7.75 (d, 2H, J = 8.6 Hz, H7), 7.71
(
d, 2H, J = 2.1 Hz, H12), 7.47 (t, 2H, J = 7.3 Hz, H6), 7.40 (dd, 2H,
J = 2.1, 8.4 Hz, H14), 7.31 (t, 2H, J = 7.6 Hz, H5), 6.90 (brs, 6H, NH ),
.86 (d, 2H, J = 8.4 Hz, H15), 5.83 (s, 4H, H9) ppm. 13C{1H} ([d6]-
DMSO) δ: 169.5 (С1), 141.2 (C8), 137.3 (C2), 134.1 (C10),
33.7 (C11/13), 133.6 (C11/13), 131.1 (C15), 129.6 (C12), 128.2 (C14),
27.0 (C6), 124.1 (C4), 123.6 (C3), 122.8 (C5), 110.5 (C7), 50.0 (C9)
charge
from
The
Cambridge
Crystallographic
Data
via
3
www.ccdc.cam.ac.uk/data_request/cif. The structures of complexes were
6
[
32]
drawn using the MERCURY CSD 3.1 program.
1
1
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
1
5
ppm. 195Pt NMR ([d6]-DMSO): δ = 2842 ppm. N NMR ([d6]-DMSO):
0.56 mmol) and pyridine (50 μl, 0,62 mmol). The reaction mixture was
stirred for 12h at room temperature, concentrated to ~3 mL and a formed
white precipitate was filtered off, washed with diethyl ether (2 x 5 mL),
water (3 x 3 mL) and dried under reduced pressure. Yield: 410 mg, (71%),
+
+
2
δ = -40.6 (NH ) ppm. ESI-MS: m/z: 963 [M + H ] .
(
OC-6-33)-dichloridobis(1-(2,4-dichlorobenzyl)-1H-indazole-3-
m.p. 170–172 °C (decomp), elem. anal. calc (%) for (С38
H
32Cl
4 6 8
N O Pt): C
1
43.99, H 3.11, N 8.10, found: C 43.86, H 3.28, N 7.92. H NMR ([d6]-
2 2
DMSO) δ: 8.74 (d, 2H, J = 7.5 Hz, NH ), 8.60 (t, 2H, J = 10.0, NH ),
8.07 (d, 2H, 8.2, H4), 7.82 (d, 2H, J = 8.6 Hz, H7), 7.71 (d, 2H, J = 2.1 Hz,
H12), 7.51-7.47 (m, 2H, H6), 7.41 (dd, 2H, J = 2.2, 8.4 Hz, H14), 7.33-
7
2
4
.28 (m, 2H, H5), 7.02 (d, 2H, J = 8.4 Hz, H15), 5.82 (s, 4H, H9),
.89 (brs, 2H, H16, H21), 2.18 (d, 2H, J = 10.5 Hz, H17, H20), 1.53 (d,
H, J = 6.2 Hz, H17, H18, H19, H20), 1.16 (t, 2H, J = 11.7 Hz, H18,
13
1
H19) ppm.
C{ H} ([d6]-DMSO) δ: 169.6 (С1), 163.9 (C22, C23),
141.2 (C8), 136.6 (C2), 133.9 (C10), 133.9 (C11/13), 133.8 (C11/13),
131.7 (C15), 129.6 (C12), 128.2 (C14), 127.4 (C6), 123.2 (C3),
1
23.2 (C5), 122.8 (C4), 110.9 (C7), 61.8 (C16, C21), 50.1 (C9),
195
31.4 (C17, C20), 24.1 (C18, C19) ppm.
Pt NMR ([d6]-DMSO):
15
δ = 3235 ppm.
ESIMS: m/z: 1037 [M + H ] .
N NMR ([d6]-DMSO): δ = -5.9 (NH ), -6.1 (NH ) ppm.
2
2
+
+
(
OC-6-44)-Acetato(trans-1R,2R-diaminocyclohexane)(1-(2,4-
carboxylato)(ethane-1,2-diamine)platinum(IV) 6
dichlorobenzyl)-1H-indazole-3-carboxylato)(oxalato)platinum(IV) 8
Complex 6 was prepared similar to complex 5 starting from 1-(2,4-
dichlorobenzyl)-1H-indazole-3-carboxylic acid (635 mg, 2 mmol), oxalyl
chloride
(2.5 mL,
29 mmol),
(OC-6-33)-dichlorido(ethane-1,2-
(144 mg, 0.4 mmol) and pyridine
[
35]
diamine)dihydroxidoplatinum(IV)
(
254 μl, 3.2 mmol). The reaction mixture was stirred overnight and
formed white solid was filtered off, washed with cold acetone (2 x 5 mL),
diethyl ether (2 x 5 mL) and dried under reduced pressure. Yield: 147 mg,
(38%), m.p.
.
215–217 °C (decomp), elem. anal. calc (%) for
(
C
32
H
26Cl
6
1
N
6
O
4
Pt): C 39.77, H 2.71, N 8.70, found: C 39.80, H 3.03,
N 8.31.
2
H NMR ([d7]-DMF) δ: 9.17 (brs, 1H, NH ), 8.55 (d, 2H,
J = 8.1 Hz, H4), 7.85 (d, 2H, J = 8.6 Hz, H7), 7.71 (d, 2H, J = 2.1 Hz,
H12), 7.52 (t, 2H, J = 7.6 Hz, H6), 7.45 (dd, 2H, J = 2.1, 8.5 Hz, H14),
7
.36 (t, 2H, J = 7.54 Hz, H5), 7. 09 (d, 2H, J = 8.3 Hz, H15), 5.90 (s, 4H,
13
1
H9), 3.23 (s, 4H, H16) ppm. C{ H} ([d7]-DMF) δ: 170.7 (С1), 141.3 (C8),
1
37.3 (C2), 133.9 (C10), 133.8 (C11/13), 133.7 (C11/13), 131.2 (C15),
Complex 8 was prepared similar to complex 5 starting from of 1-(2,4-
dichlorobenzyl)-1H-indazole-3-carboxylic acid (220 mg, 0.69 mmol),
129.3 (C12), 127.9 (C14), 126.9 (C6), 124.0 (C4), 123.7 (C3), 122.5 (C5),
1
95
110.1 (C7), 50.0 (C16), 49.9 (C9) ppm.
Pt NMR ([d7]-DMF): δ = 2633
ppm. ESI-MS: m/z: 967
oxalyl chloride (1.0 mL, 11.7 mmol), (OC-6-44)-acetato(trans-1R, 2R--
1
5
ppm.
N NMR ([d7]-DMF): δ = -3.7 (NH
2
)
[37]
diaminocyclohexane)hydroxido(oxalato)platinum(IV)
(156 mg, 0.33
+
+
[M + H ] .
mmol) and with pyridine (57 μl, 3.2 mmol). The reaction mixture was
stirred for 12h at room temperature, concentrated to ~3 mL and a formed
white precipitate was filtered off, washed with diethyl ether (2 x 5 mL),
water (3 x 3 mL) and dried under reduced pressure. Yield: 120 mg, (47%),
(
OC-6-33)-(trans-1R,2R-diaminocyclohexane)bis(1-(2,4-
dichlorobenzyl)-1H-indazole-3-carboxylate)oxalatoplatinum(IV) 7
m.p.
C 38.67, H 3.38, N 7.22, found: C 38.32, H 3.71, N 7.15. H NMR ([d6]-
DMSO) δ: 8.53 (brs, 3H, NH ), 8.35 (brs, 1H, NH ), 8.02 (d, 1H,
. 2 4 8
195-200 °C (decomp), elem. anal. calc (%) for (C25H26Cl N O Pt):
1
2
2
J = 8.2 Hz, H4), 7.78 (d, 1H, J = 8.6 Hz, H7), 7.68 (d, 1H, J = 2.0 Hz,
H12), 7.47 (t, 1H, J = 7.8 Hz, H6), 7.39(dd, 1H, J = 2.0, 8.3 Hz, H14),
7.28 (t, 1H, J = 7.6 Hz, H5), 6. 99 (d, 1H, J = 8.4 Hz, H15), 5.80 (s, 2H,
H9), 2.83-2.74 (m, 1H, H16/21), 2.69-2.61 (m, 1H, H16/21), 2.19-2.11 (m,
2
H, H17, H20), 2.00 (s, 1H, H25), 1.54-1.46 (m, 4H, H17, H18, H19,
13
1
H20), 1.25-1.15 (m, 2H, H18, H19) ppm.
C{ H} ([d6]-DMSO) δ:
1
1
1
1
3
78.6 (C24), 169.9 (С1), 163.9 (C22/23), 163.9 (C22/23), 141.2 (C8),
36.8 (C2), 133.9 (C10), 133.8 (C11/13), 133.7 (C11/13), 131.65 (C15),
29.53 (C12), 128.20 (C14), 127.35 (C6), 123.3 (C3), 123.09 (C5),
22.8 (C4), 110.9 (C7), 61.9 (C16/21), 61.5 (C16/21), 50.1 (C9),
1.4 (C17/20),
31.3 (C17/20),
Pt NMR ([d6]-DMSO): δ = 3240 ppm.
2
.4 (NH ), 7.6 (NH ) ppm. ESI-MS: m/z: 775 [M - H ] .
24.0 (C18, C19),
23.4 (C25) ppm.
1
95
15
N NMR ([d6]-DMSO): δ = -
+
-
7
2
Complex 7 was prepared similar to complex 5 starting from 1-(2,4-
dichlorobenzyl)-1H-indazole-3-carboxylic acid (789 mg, 2.46 mmol),
oxalyl
chloride
(3.5 mL,
40.8 mmol),
(OC-6-33)-(trans-1R,2R-
[
36]
diaminocyclohexane)dihydroxido(oxalato)platinum(IV)
(240 mg,
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
Martino, W. Malorni, M. L. Marcante, B. Silvestrini, A. Floridi, Exp. Mol.
Pathol. 1985, 42, 194-205.
Acknowledgements
[
[
19] S. Di Cosimo, G. Ferretti, P. Papaldo, P. Carlini, A. Fabi, F. Cognetti,
Drugs Today 2003, 39, 157-174.
We thank the Russian Foundation for Basic Research (grant No.
16-33-00373, L.F.), (grant No. 16-03-00743 Y.N.N., I.V.Z.,
20] a) J. Banfić, A. A. Legin, M. A. Jakupec, M. Galanski, B. K. Keppler, Eur.
J. Inorg. Chem. 2014, 2014, 484-492; b) M. R. Reithofer, S. M. Valiahdi,
M. A. Jakupec, V. B. Arion, A. Egger, M. Galanski, B. K. Keppler, J.
Med. Chem. 2007, 50, 6692-6699; c) A. C. Tsipis, I. N. Karapetsas,
Dalton Trans. 2014, 43, 5409-5426.
A.A.N.), Russian science foundation (grant No. 14-03-00483
Y.N.N, K.I.K, A.A.N, E.R.M (biological study)) for financial
support. The authors also acknowledge support from Moscow
State University Program of Development (NMR measurements).
[
21] a) D. Höfer, H. P. Varbanov, A. Legin, M. A. Jakupec, A. Roller, M.
Galanski, B. K. Keppler, J. Inorg. Biochem. 2015, 153, 259-271; b) B. R.
Hoffmeister, M. Hejl, M. S. Adib-Razavi, M. A. Jakupec, M. Galanski, B.
K. Keppler, Chem. Biodiversity 2015, 12, 559-574.
Keywords: Platinum complexes • lonidamine • log P •
cytotoxicity
[
[
22] G. Giorgioni, S. Ruggieri, A. Di Stefano, P. Sozio, B. Cinque, L. Di
Marzio, G. Santoni, F. Claudi, Bioorg. Med. Chem. Lett. 2008, 18,
[
[
1]
2]
a) S. Dilruba, G. V. Kalayda, Cancer Chemother. Pharmacol. 2016, 77,
103-1124; b) T. C. Johnstone, G. Y. Park, S. J. Lippard, Anticancer
2445-2450.
1
23] U. Jungwirth, D. N. Xanthos, J. Gojo, A. K. Bytzek, W. Körner, P.
Heffeter, S. A. Abramkin, M. A. Jakupec, C. G. Hartinger, U.
Windberger, M. Galanski, B. K. Keppler, W. Berger, Mol. Pharmacol.
Res. 2014, 34, 471-476; c) A. V. Klein, T. W. Hambley, John Wiley &
Sons Ltd., 2014, pp. 9-45; d) E. Shaili, Sci. Prog. (St. Albans, U. K.)
2014, 97, 20-40; e) N. R. Farrell, Drugs Future 2012, 37, 795-806; f) J.
2012, 81, 719-728.
Zhang, Y. Ke, J. Shen, Shanghai Yiyao 2013, 34, 52-59.
[
[
[
[
24] L. Qiu, G. Lv, Y. Cao, L. Chen, H. Yang, S. Luo, M. Zou, J. Lin, JBIC
Journal of Biological Inorganic Chemistry 2015, 20, 1263-1275.
25] H. Xiao, D. Zhou, S. Liu, R. Qi, Y. Zheng, Y. Huang, X. Jing,
Macromolecular Bioscience 2012, 12, 367-373.
a) M. W. Kellinger, G. Y. Park, J. Chong, S. J. Lippard, D. Wang, J. Am.
Chem. Soc. 2013, 135, 13054-13061; b) R. C. Todd, S. J. Lippard,
Chem. Biol. (Cambridge, MA, U. S.) 2010, 17, 1334-1343; c) B. Wu, G.
E. Davey, A. A. Nazarov, P. J. Dyson, C. A. Davey, Nucleic Acids Res.
26] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Adv. Drug
Deliv. Rev. 2001, 46, 3-26.
2011, 39, 8200-8212.
[
[
3]
4]
M. A. Fuertes, C. Alonso, J. M. Pérez, Chem. Rev. 2003, 103, 645-662.
a) T. C. Johnstone, K. Suntharalingam, S. J. Lippard, Chem. Rev.
27] E. Schreiber-Brynzak, V. Pichler, P. Heffeter, B. Hanson, S. Theiner, I.
Lichtscheidl-Schultz, C. Kornauth, L. Bamonti, V. Dhery, D. Groza, D.
Berry, W. Berger, M. Galanski, M. A. Jakupec, B. K. Keppler,
Metallomics 2016, 8, 422-433.
(Washington, DC, U. S.) 2016, 116, 3436-3486; b) J. X. Ong, S. Q. Yap,
D. Y. Q. Wong, C. F. Chin, W. H. Ang, Chimia 2015, 69, 100-103; c) J.
J. Wilson, S. J. Lippard, Chem. Rev. (Washington, DC, U. S.) 2014,
[
[
[
28] W. L. F. Armarego, C. Chai, Purification of Laboratory Chemicals, 5th
ed., Butterworth-Heinemann, Oxford, 2003.
1
14, 4470-4495; d) N. Graf, S. J. Lippard, Adv. Drug Deliv. Rev. 2012,
4, 993-1004.
6
29] M. Matczuk, L. S. Foteeva, M. Jarosz, M. Galanski, B. K. Keppler, T.
Hirokawa, A. R. Timerbaev, J. Chromatogr. A 2014, 1345, 212-218.
30] A. V. Rudnev, L. S. Foteeva, C. Kowol, R. Berger, M. A. Jakupec, V. B.
Arion, A. R. Timerbaev, B. K. Keppler, J. Inorg. Biochem. 2006, 100,
[
[
5]
6]
H. Anderson, J. Wagstaff, D. Crowther, R. Swindell, M. J. Lind, J.
McGregor, M. S. Timms, D. Brown, P. Palmer, Eur. J. Cancer Clin.
Oncol. 1988, 24, 1471-1479.
N. J. Wheate, S. Walker, G. E. Craig, R. Oun, Dalton Trans. 2010, 39,
1
819-1826.
31] G. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64,
12-122.
8113-8127.
[
[
[
[
7]
8]
H. Choy, C. Park, M. Yao, Clin. Cancer Res. 2008, 14, 1633-1638.
W. H. Ang, L. J. Parker, A. De Luca, L. Juillerat-Jeanneret, C. J. Morton,
M. Lo Bello, M. W. Parker, P. J. Dyson, Angew. Chem., Int. Ed. 2009,
1
32] C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields,
R. Taylor, M. Towler, J. van de Streek, J. Appl. Crystallogr. 2006, 39,
4
8, 3854-3857.
S. Dhar, Z. Liu, J. Thomale, H. Dai, S. J. Lippard, J. Am. Chem. Soc.
008, 130, 11467-11476.
453-457.
[
9]
[
[
33] J.-X. Duan, Threshold Pharmaceuticals, Inc., USA . 2005, p. 35 pp.
34] Y. Shi, S.-A. Liu, D. J. Kerwood, J. Goodisman, J. C. Dabrowiak, J.
Inorg. Biochem. 2012, 107, 6-14.
2
[
[
10] K. R. Barnes, A. Kutikov, S. J. Lippard, Chem. Biol. 2004, 11, 557-564.
11] R. K. Pathak, S. Marrache, J. H. Choi, T. B. Berding, S. Dhar, Angew.
Chem., Int. Ed. Engl. 2014, 53, 1963-1967.
[
[
35] M. Galanski, B. K. Keppler, Met.-Based Drugs 1995, 2, 57-63.
36] G. M. Cruise, C. G. Harris, M. J. Constant, MicroVention, Inc., USA .
[
[
[
[
12] S. Mukhopadhyay, C. M. Barnés, A. Haskel, S. M. Short, K. R. Barnes,
S. J. Lippard, Bioconjug. Chem. 2008, 19, 39-49.
2015, p. 23pp.
[
37] B. Moreau, M. T. Bilodeau, K. Whalen, K. Meetze, S. Singh, R. Wooster,
C.-A. Lemelin, Blend Therapeutics, Inc., USA . 2015, p. 139pp.
13] W. H. Ang, I. Khalaila, C. S. Allardyce, L. Juillerat-Jeanneret, P. J.
Dyson, J. Am. Chem. Soc. 2005, 127, 1382-1383.
14] S. Dhar, S. J. Lippard, Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 22199-
22204.
15] V. Novohradsky, L. Zerzankova, J. Stepankova, O. Vrana, R.
Raveendran, D. Gibson, J. Kasparkova, V. Brabec, Biochem.
Pharmacol. 2015, 95, 133-144.
[
[
16] S. G. Awuah, Y.-R. Zheng, P. M. Bruno, M. T. Hemann, S. J. Lippard, J.
Am. Chem. Soc. 2015, 137, 14854-14857.
17] a) U. Gatzemeier, F. Cavalli, K. Haussinger, E. Kaukel, G. Koschel, G.
Martinelli, R. Neuhauss, J. von Pawel, Semin Oncol 1991, 18, 42-48; b)
C. Gridelli, F. De Marinis, A. Rossi, E. Tucci, M. D'Aprile, R. Cioffi, E.
Cortesi, R. Migliorino, A. Pisano, F. Scognamiglio, R. Di Giacomo, A. R.
Bianco, Anticancer Res. 1997, 17, 1277-1279.
[
18] a) A. Floridi, M. G. Paggi, M. L. Marcante, B. Silvestrini, A. Caputo, C.
de Martino, J. Natl. Cancer Inst. 1981, 66, 497-499; b) A. Nista, C. De
This article is protected by copyright. All rights reserved

European Journal of Inorganic Chemistry
10.1002/ejic.201600857
FULL PAPER
Layout 2:
FULL PAPER
Key Topic: metals in medicine
Yulia N. Nosova, Lidia S. Foteeva, Ilia V. Zenin, Timur I.
Fetisov, Kirill I. Kirsanov, Marianna G. Yakubovskaya,
Taisya A. Antonenko, Viktor A. Tafeenko, Leonid A.
Aslanov, Anna A. Lobas, Mikhail V. Gorshkov, Markus
Galanski, Bernhard K. Keppler, Andrei R. Timerbaev,
*
Elena R. Milaeva and Alexey A. Nazarov
A series of Pt(IV) complexes with lonidamine as a ligand
coordinated in axial position is reported. The lonidamine was
found to affect strongly the in vitro cytotoxic activity lowering the
IC50 values down to the nanomolar range.
Page No. – Page No.
Enhancing the cytotoxic activity of anticancer
Pt(IV) complexes by introduction of lonidamine as
an axial ligand.
This article is protected by copyright. All rights reserved