New in vitro highly cytotoxic platinum and palladium cyanoximates with minimal side effects in vivo

Article abstract of DOI:10.1016/j.jinorgbio.2020.111082

Several biologically active bivalent Pd and Pt complexes with two structurally similar cyanoxime ligands abbreviated as H(DECO): 2-oximino-2-cyano-N,N′-diethylacetamide, and H(PyrCO): 2-oximino-2-cyan-N-pyrrolidine acetamide were synthesized and characterized using spectroscopic methods, thermal analysis and X-ray crystallography. Structures revealed planar cis-geometry of studied complexes. Freshly obtained Pt(DECO)₂, Pd(DECO)₂, Pt(PyrCO)₂ and Pd(PyrCO)₂ complexes were used in for in vitro cytotoxicity assays using two different etiology human cancer cell lines HeLa and WiDr cells. Investigated compounds showed cytotoxicity levels at or above cisplatin. Pt(DECO)₂ was also tested in vivo in healthy C57BL/6 mice. The complex was administered at three different dosage (0, 7.5, 15 mg/kg, i.p. once/week), over a total period of 8 weeks. No changes were observed in the animal weight in the treated mice compared to the control dextrose-treated group. The levels of erythrocytes, leukocytes, and hemoglobin were within the normal level suggesting low myelotoxicity. Negligible cardiotoxicity was observed from the histological evaluation of the hearts from the treated animals. Results from the tail nerve conduction velocity (NCV) and nerve histomorphometry suggested no impact of Pt(DECO)₂ on peripheral nerves. The complex, however, induced certain hepatotoxicity and lead to the elevation of IL-6, a pro-inflammatory cytokine. Overall, Pt(DECO)2 showed minimal in vivo toxicity, thus presenting a promising candidate for future testing in animal models of cancer.

Full text of DOI:10.1016/j.jinorgbio.2020.111082

JournalofInorganicBiochemistry208(2020)111082
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
New in vitro highly cytotoxic platinum and palladium cyanoximates with
minimal side effects in vivo
Stephanie D. Dannena, Lauren Cornelisonb, Paul Durhamb, John E. Morleyc, Kiana Shahverdid,
Junwei Due, Haiying Zhoud, Leland C. Sudlowd, Daniel Hunterf, Matthew D. Woodf,
⁎
⁎
Mikhail Y. Berezind,e, , Nikolay Gerasimchuka,
a Department of Chemistry, Temple Hall 431, Missouri State University, 901 S. National, Springfield, MO 65897, USA
b Department of Biology, Missouri State University, MC/Center for Biomedical & Life Sciences, Springfield, MO 65897, USA
c Division of Geriatric Medicine, Saint Louis University, St. Louis, MO 63110, USA
d Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
e Institute of Materials Science and Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA
f Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Several biologically active bivalent Pd and Pt complexes with two structurally similar cyanoxime ligands ab-
breviated as H(DECO): 2-oximino-2-cyano-N,N′-diethylacetamide, and H(PyrCO): 2-oximino-2-cyan-N-pyrroli-
dine acetamide were synthesized and characterized using spectroscopic methods, thermal analysis and X-ray
crystallography. Structures revealed planar cis-geometry of studied complexes. Freshly obtained Pt(DECO)2, Pd
(DECO)2, Pt(PyrCO)2 and Pd(PyrCO)2 complexes were used in for in vitro cytotoxicity assays using two different
etiology human cancer cell lines HeLa and WiDr cells. Investigated compounds showed cytotoxicity levels at or
above cisplatin. Pt(DECO)2 was also tested in vivo in healthy C57BL/6 mice. The complex was administered at
three different dosage (0, 7.5, 15 mg/kg, i.p. once/week), over a total period of 8 weeks. No changes were
observed in the animal weight in the treated mice compared to the control dextrose-treated group. The levels of
erythrocytes, leukocytes, and hemoglobin were within the normal level suggesting low myelotoxicity. Negligible
cardiotoxicity was observed from the histological evaluation of the hearts from the treated animals. Results from
the tail nerve conduction velocity (NCV) and nerve histomorphometry suggested no impact of Pt(DECO)2 on
peripheral nerves. The complex, however, induced certain hepatotoxicity and lead to the elevation of IL-6, a pro-
inflammatory cytokine. Overall, Pt(DECO)2 showed minimal in vivo toxicity, thus presenting a promising can-
didate for future testing in animal models of cancer.
Pd/Pt-cyanoximates
Crystal structures
Human cancer cells
in vitro studies
Animal model
in vivo studies
1. Introduction
platinum-based therapies with high anticancer efficiency and low side ef-
fects is an ongoing clinical and research goal.
Platinum-based compounds, such as cisplatin, carboplatin, and ox-
aliplatin are among the most commonly prescribed chemotherapeutic
agents [1]. One of the common problems with many chemotherapy drugs,
including platinum complexes, is their strong side effects that weaken the
immune system [2], trigger anemia [3], damage cardiovasculature, induce
muscle loss [4], and cause neurological deficits and pain [5]. The responses
are usually drug-dependent. For example, cisplatin has been specifically
connected to neurotoxicity, adverse gastrointestinal effects, and renal
function impairment [6]. Carboplatin-containing chemotherapy has been
more associated with myelosuppression, and high incidence of severe
neutropenia and anemia [6]. Finding suitable alternatives to the existing
Organometallic complexes with cyanoxime ligands might present a
viable option. Cyanoximes are low molecular weight ampolydentate
organic ligands of general formula NC-(C=N-OH)–R which have re-
ceived attention in recent years due to their pronounced biological
activity and interesting properties [7]. Previous publications have
shown that pairing these ligands with platinum(II) and palladium(II)
has resulted in the formation of complexes with substantial in vitro
cytotoxic activity against HeLa (cervical adenocarcinoma) and WiDr
(colon adenocarcinoma) cell lines [8,9]. Of the 42 cyanoximes known
today [10], there are still a large number of complexes that have not yet
been evaluated in biological settings.
⁎ Corresponding authors.
E-mail addresses: berezinm@wustl.edu (M.Y. Berezin), NNGerasimchuk@MissouriState.edu (N. Gerasimchuk).
https://doi.org/10.1016/j.jinorgbio.2020.111082
Received 16 February 2020; Received in revised form 28 March 2020; Accepted 28 March 2020
Availableonline06May2020
0162-0134/©2020ElsevierInc.Allrightsreserved.

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
CN
cytokine level, and nerve conduction velocity were recorded in live
animals. The study was completed by histological evaluation of the
sciatic nerve and the heart to estimate the level of peripheral neuro-
pathy and cardiotoxicity.
O
CN
O
N
N
OH
N
N
OH
2. Experimental section
H(PyrCO)
H(DECO)
2.1. Synthesis and characterization of complexes
2.1.1. General considerations
The common chemicals and solvents were all reagent grade
(Aldrich, WI) and used without further purification. Sources of Pd and
Pt were available as tetrachloro-salts of K2[MCl4] (M = Pd, Pt) com-
position from Pressure Chemical Company (Pittsburgh, PA). The TLC
for ligands' identification were carried out on silica-coated glass plates
(Merck) with a 256 nm fluorescent indicator. Elemental analyses on C,
H, N content were performed at Atlantic Microlab (Norcross, GA).
Melting points for organic compounds were measured on a Digimelt™
apparatus without correction.
CN
CN
R2N
R2N
N
O
O
O
M
O
N
O
O
M = Pd, Pt
M
O
O
N
N
NR2
trans-
R2N
NC
CN
cis-
2.1.2. Synthesis of H(DECO) and H(PyrCO) ligands
Both these cyanoximes were prepared at room temperature under N2
atmosphere as a result of reaction within several minutes between neat
cyanoacetic ester and HN(C2H5)2 (for H(DECO)) and pyrrolidine (for H
(PyrCO)) (Scheme 1). Resulting white waxy solids were filtered off, washed
with cold methanol, then with anhydrous benzene and then dried in va-
cuum desiccator over H2SO4(c). The next step was the nitrosation reaction
that was carried out by slow bubbling of gaseous methylnitrite CH3-ONO
within ~15 min through n-propanol solutions of appropriate substituted
acetonitrile in the presence of freshly prepared in situ sodium propoxide
[11]. The reaction mixture was concentrated on a rotary evaporator to give
yellow sludge that was solidified under an oil pump vacuum. To the yellow
solid ~50 mL of distilled water were added at once with the formation of a
bright-yellow solution. Diluted HCl(aq) solution (1:5) was then added
dropwise to bring pH of the mixture from 12 to ~3. Excess of solid NaCl
was added to that solution to form brine, and extraction of desired cya-
noxime was performed using three portions of 20, 30 and 50 mL of ether.
Slightly yellow colored organic layers were combined and dried over an-
hydrous Na2SO4 overnight at +4 °C in a refrigerator. Ether was removed on
a rotary evaporator to give off-white solids of H(DECO) and H(PyrCO)
oximes. The solids were then re-dissolved in dry ether, mixed with a spatula
of Norite© and then filtered through Celite© to give transparent solutions.
Solvent removal provided colorless crystalline compounds.
Fig. 1. Structures of cyanoximes used in this study. Each ligand molecule can
form metallocomplexes of cis- and trans- geometries. DECO: 2-oximino-2-
cyano-N,N′-diethylacetamide, PyrCO: 2-oximino-2-cyan-N-pyrrolidine acet-
amide. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Given the potential of the cyanoximate complexes in cancer treat-
ment, testing the in vitro activity and in vivo toxicity of these molecules
is instrumental in discovering a new class of novel chemotherapy drugs
with potentially low side effects.
Herein, we investigated several existing and new members of the Pt
and Pd cyanoxime complexes (Fig. 1) in vitro using common HeLa and
WiDr cancer cell lines. To assess the toxic side effects in live animals, we
administered multiple doses of Pt(II) 2-oximino-2-cyano-N,N′-diethy-
lacetamide abbreviated as Pt(DECO)2 to healthy C57BL/6 mice at two
different levels (7.5 mg/kg and 15 mg/kg) once per week over an
8 week period. The highest dose (15 mg/kg) corresponded to a typical
maximal tolerable single dose of cisplatin that can be administered to
normal mice. Control animals were injected with the equivalent volume
of a dextrose solution with no platinum complex added. The established
markers of side effects such as weight change, complete blood count,
O
CH3
CH3
O
+ CH3ONO
R.T.
CH3
CH3
N
N
NC
HO
NC
N
)
2
H 5
C 2
H
(
O
H
N
H 5
C 2
+
1, H(DECO)
-
;
T
-
O
CH3
O
HN
NC
2, H(PyrCO)
2
O
N
5
O
+ CH3ONO
R.T.
N
NC
NC
N
HO
Scheme 1. Synthetic routes to H(DECO) and H(PyrCO) amide-based cyanoximes.
2

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
white powders
CN
O
R = C2H5, ligand: H(DECO)
N OH
R2N
R =
,
ligand: H(PyRCO)
N
in H2O, 35o C
+KOH
CN
O
yellow solutions
(DECO)-, (PyrCO)-
O
N
R2N
+ K2[PdCl4]
H2O, R.T.
+ K2[PtCl4]
H2O, R.T.
+ stirring
24 hrs
5 min
O
N
O
O
O
CN
NC
CN
NC
N
N
N
Pt
Pd
O
O
NR2
R2N
O
O
NR2
R2N
fine dark green solids
Pt(DECO)2, Pt(PyrCO)2
bright yellow crystalls
Pt(DECO)2, Pt(PyrCO)2
Scheme 2. Synthesis of Pt and Pd -based cyanoximines.
2.1.3. Synthesis of metal complexes
(PyrCO)2 complex was observed after an extended period of time. After
~24 h, a very fine dark-green precipitate was filtered (see ESI 2),
washed three times with water, and then dried in a vacuum desiccator
under concentrated H2SO4. Elemental analysis: found (calcd) for green-
form PtC14H16N6O4: N, 15.75 (15.78); C, 31.52 (31.46); H, 4.16 (4.03).
IR (KBr pellet, cm−1): 2985 [νas(CH)], 2943 [νs(CH)], 2213 [ν(C^N)],
1709 [ν(CO, carbonyl)], 1622; 1442 [ν(CNO), oxime], 1585 [ν(CO,
amide II)], 1227 [ν(CNO)]. The dark-green form is not soluble in water
but has some solubility in alcohols, slowly dissolves in DMSO, forming
at first a dark-red solution of a dimer [Pt(PyrCO)2]2. There are three
differently colored polymorphs (ESI 2).
The general procedure for other complexes presented in Scheme 2.
2.1.3.1. Pd(DECO)2. H(DECO) is not soluble in water, but after
deprotonation with base it becomes soluble which enables reactions
with transition-metal salts. Thus, the stoichiometric amount of a 1.01 M
KOH solution (1.93 mL) was added to the H(DECO) (1) sludge in water
that immediately dissolved with the formation of a bright yellow
solution. Another solution of 0.318 g (0.98 mM) of K2[PdCl4] in 5 mL
of water was added dropwise into the yellow DECO− solution under
intense stirring. A thick yellow precipitate formed instantaneously, and
after ~5 min of stirring, it was filtered, washed three times with water,
and then dried in a desiccator charged with concentrated H2SO4.
Elemental analysis: found (calcd) for PdC14H20N6O4: N, 18.24
(18.24); C, 37.05 (37.05); H, 4.50 (4.50). IR (KBr pellet, cm−1): 2983
[νas(CH)], 2940 [νs(CH)], 2211 [ν(C^N)], 1631 [ν(CO, amide I)], 1440
[ν(CNO), oxime], 1579 [ν(CO, amide II)], 1200 [ν(CNO)]. The complex
is soluble in CH3CN, DMF, DMSO, pyridine, and its homologues, but not
well soluble in acetone and alcohols.
2.1.3.4. Pd(PyrCO)2. Pd(PyrCO)2 was obtained in a similar fashion.
Elemental analysis: found (calcd) for PdC14H16N6O4: N, 19.24 (19.16);
C, 38.05 (38.33); H, 3.95 (3.68).
2.1.4. IR-spectroscopy
The IR-spectra of all compounds reported herein were recorded in
KBr disks at room temperature using the Bruker R70 FT-IR-Raman
complex. All spectra were recorded with 64 repetitions at the resolution
of 4 cm−1. Data tabulated in the ESI 3.
2.1.3.2. Pt(DECO)2. The Pt(DECO)2 was obtained in a similar fashion
that was reported early [12]. Elemental analysis: Found (calcd) for
PtC14H20N6O4: N, 18.24 (18.24); C, 37.05 (37.05); H, 4.50 (4.50).
There were three polymorphs obtained from Pt(II) – DECO system:
yellow, red and dark-green (Electronic Supporting Information section,
ESI: 1).
2.1.5. IR-spectral data
Vibrational spectra of synthesized Pd, Pt complexes contain bands of
the ν(C=O) amide and ν(NCO) nitroso groups shifted accordingly to the
anions' binding modes. Thus, the amide group frequency is lower than
that for uncomplexed cyanoximes, while the band with participation of
the C-N-O fragment is shifted to higher wavenumbers as compared to
free ligands since it gained nitroso character [13].
2.1.3.3. Pt(PyrCO)2. For the synthesis of Pt(PyrCO)2, 0.217
g
(1.28 mM) of H(PyrCO) (2) in 8 mL of water was treated with
1.28 mL of 1.01 M KOH, forming a yellow solution of deprotonated
cyanoxime PyrCO−, to which 0.268 g (0.65 mM) of K2PtCl4 in 5 mL was
added. When all components were mixed, the formation of the Pt
2.1.6. UV–visible spectra
The electronic spectra of the compounds were recorded at room
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S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
temperature using the HP 8354 diode array UV–visible spectro-
photometer operating in the range of 200–1100 nm. The absorbance
spectra from samples were recorded in solutions and in fine suspensions
in mineral oil squeezed between 1 × 4 cm quartz plates. Data of
spectroscopic studies are presented in ESI 4–6.
all structures, H-atoms were placed in calculated positions in ac-
cordance with the hybridization state of a hosting carbon atom and
were refined isotropically. No apparent problems or complications were
encountered during the structures' solutions and refinement as evident
from a very positive PLATON checkCIF reports. The crystal data for
monomeric Pd(DECO)2, Pt(PyrCO)2 and [Pt(PyrCO)2]2 are presented in
ESI 9; Table 3, with selected bond lengths and valence angles presented
in ESI 10,11; Tables 4,5. Details of the crystal packing for these three
complexes are presented in ESI 12–17. A representative drawing of the
crystal structures and packing diagrams was done using the ORTEP and
Mercury software packages. Reported structures have been deposited at
CCDC at the following numbers: Pt(PyrCO)2 1,984,335 and [Pt(Pyr-
CO)2]2(DMSO)2 1,984,334, Pd(DECO)2 1,984,333 with PLATON
checkCIF reports presented in at the end of Supporting Information
Section in ESI 22.
2.1.7. X-ray crystallography
Suitable single crystals of Pt(DECO)2, Pt(PyrCO)2, and [Pt
(PyrCO)2]2 were grown from the DMSO solutions. Large red crystals of
the latter dimer appeared overnight. The crystals were collected and
placed for handling and selection in thick paratone oil. The yellow
crystals of the monomeric complex appeared upon monomerization of
the dimeric crystals at ambient conditions from red ones. This fact, in
essence, represents a rather unusual crystal-to-crystal conversion of
polymorphs spontaneously breaking down from more complex struc-
ture to more simple monomeric units. All attempts to grow suitable
Structures of all crystallographically characterized here in co-
ordination compounds contain cyanoxime anions in cis-orientation to
central atoms of Pd or Pt (Figs. 2, 4). This finding is in line with pre-
viously determined structures of other cyanoximates of these transition
metals [14].
crystals of the dark-green polymeric [PtL2]n complexes (L = DECO−
,
PyrCO−) were unsuccessful (ESI 7). The crystal structures for the DECO
compounds were reported earlier [12]. Structures for the yellow
monomeric Pt(PyrCO)2 and its red [Pt(PyrCO)2]2 dimer are shown
below. All crystals selected and suitable for studies were placed on
plastic MiTeGen holders attached to the copper-pin on the goniometer
head of the Bruker APEX-2 diffractometer, equipped with a SMART
CCD area detector. The intensity data were collected at low tempera-
ture. Data collection was done in ω scan mode using the Mo tube (Kα
radiation; λ = 0.71073 Å) with a highly oriented graphite mono-
chromator. Intensities were integrated from 4 series of 364 exposures,
each covering 0.5o steps in ω at 20–60 s of exposition time, with the
total data set being a sphere. The space group determination was done
with the aid of the XPREP software. The absorption correction was
performed by a crystal face indexing procedure with a help of a video
microscope (ESI 8) followed by numerical input into the SADABS pro-
gram that was included in the Bruker AXS software package. All
structures were solved by direct methods and refined by least squares
on weighted F2 values for all reflections using the SHELXTL program. In
Anions of DECO− and PyrCO− are in the nitroso-form, and central
atoms adopt distorted square-planar cis-MN2O2 structures of coordina-
tion polyhedrons. The nitroso-character of anions is evident from sig-
nificantly shorter NeO bonds as compared to CeN bonds in the C-N-O
fragment (ESI 10, Table 4). In the structure of
a red [Pt
(PyrCO)2]2·(DMSO)2 complex monomeric units are arranged via short
platinophilic interactions into “head-to-tail” dimer (Fig. 3). Red crystals
of the dimer left for 20–30 h change to yellow monomer that surpris-
ingly keep the same shape, but lose the solvent molecules. Single crystal
specimen suitable for the X-ray analysis was selected from a series of
yellow crystals in paratone oil that were red just a day before. The
structure of the yellow monomer show no solvent molecules present,
DMSO apparently went into the oil.
Indeed, close analysis of the structure of the red dimer evidences
importance of the role of DMSO that stabilizes the dimer via short
A
B
Fig. 2. Crystal structure of yellow monomeric Pt(PyrCO)2: A – top, and B – side views showing ligands' cis-arrangement and planar metal core environment. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
A
B
Fig. 3. Crystal structure of red dimeric [Pt(PyrCO)2]2(DMSO)2: A – top view showing dimer's head-to-tail arrangement, and B – side view demonstrating short
metallophilic interaction at 3.221 Å. Solvent molecules are omitted for clarity. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
electrostatic C-H—O contacts (ESI 17). This is a similar situation re-
cently observed for the other red dimeric [Pt(MCO)2]2·(DMSO)2 com-
plex [14].
antibiotic, and 10% fetal bovine serum (Sigma Aldrich). Both cell lines
were cultured in 24 well plates at a seeding density of 8·104 cells per
cm2. The cells on the plates were grown for 24 h in normal media before
any compounds were introduced.
Synthesized Pt and Pd cyanoximates have limited water-solubility.
In order to bring them into solution for subsequent mixing with buf-
fered cell culture media DMSO was used in amount not exciding 1% by
volume in the final mixture. Platinum and palladium complexes were
prepared in 0.1 M concentrations in DMSO, and then further diluted by
growth media to 1 mM. Cisplatin was used as a positive control and
prepared at the same concentrations as the Pt/Pd complexes. Normal
growth media was removed from the wells of each culture plate and
replaced with the solutions of interest. Cells were exposed to 1 mM
complex in their growth media in two duplicate wells.
2.1.8. Thermal Stability Studies
Thermal stability of the obtained metal complexes was assessed
using Q-600 TG/DSC analyzer (TA Instruments) under N2 flow of
100 mL/min at 10o/min heat rate. Heating of samples was carried out
to 1000 °C at which the samples' complete decomposition was attained.
Results of thermal analyses studies are presented in detail in ESI 18–21.
2.2. In vitro and in vivo evaluation
2.2.1. In vitro cytotoxicity studies
Platinum and palladium cyanoximates Pt(DECO)2, Pd(DECO)2, Pt
(PyrCO)2, and Pd(PyrCO)2 were tested in vitro for cytotoxicity on cul-
tured HeLa and WiDr cells. The HeLa and WiDr cancer cells (American
Type Culture Collection, CCL-2 and CCL-218, respectively) were cul-
tured according to the vendor's recommended procedures. They were
maintained at 37 °C with no CO2 in the incubator in L-15 growth media
(Sigma Aldrich, Inc.) containing HEPES buffer, penicillin/streptomycin
2.2.2. Viability assay
The plates of each cell line were incubated for 24 h in the new
media. The cells were then assayed using Trypan Blue dye (Sigma)
diluted to 0.1% concentration for 2 min at room temperature, washed
with PBS, and fixed using 4% paraformaldehyde. This method relies on
a breakdown in membrane integrity that is determined by the uptake of
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S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
Fig. 4. Crystal structure of yellow monomeric Pd(DECO)2: A – top, and B – side views showing cyanoxime's cis-arrangement in the complex and planar metal center
environment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
a dye, such as Trypan Blue, which is normally membrane impermeable.
Cells were viewed using a phase contrast microscope. Digital pictures of
the cells were analyzed to evaluate compound toxicity. Two fields of
cell views (×40 magnification) from two independent dishes (wells in
24-wells plate) were analyzed for each condition. Random fields of cells
were counted, and any blue/dark cells were recorded as nonviable.
Thus, cells viability was assessed for each prepared compound and for
cisplatin. Cells in pure media and calculated level of DMSO were used
as negative controls. The percent of viable cells for each well were
averaged and standard deviation was calculated for each plate.
2.2.5. Nerve histomorphometry
Sciatic nerves were harvested and stored in 3% glutaraldehyde
(Polysciences Inc.). The tissues were post-fixed in 1% osmium tetroxide
and serially dehydrated in ethanol and propylene oxide. Stained and
dehydrated nerves were then embedded in epoxy (Polysciences), sec-
tioned on an ultramicrotome into 1 μm cross sections, and placed on
microscopy slides. Slides were counter-stained with 1% toluidine blue.
The slides were then analyzed at 1000× on a Leitz Laborlux S micro-
scope. A semi-automated digital image-analysis system linked to mor-
phometry macros developed for peripheral nerve analysis (Clemex
Vision Professional); Clemex Technologies, Longueuil, Québec was
utilized to quantify myelinated axon number, density, and myelin width
as previously described [15,16]. All analysis was done by an observer
blinded to the experimental groups.
2.2.3. In vivo studies
All animal studies were conducted in compliance with the
Washington University Institutional Animal Studies Committee and
NIH guidelines. C57BL/6 male mice age 8–10 weeks old (Charles River)
were randomized to one of three groups: control, 7.5 mg/kg, and
15 mg/kg. Mice were housed in a central animal care facility and
provided with food and water ad libitum. Animals were kept in standard
cages at room temperature and 12 h day/night light cycle.
The mice were injected with freshly prepared Pt(DECO)2 inter-
periotoneally (ip). Control mice were injected with the similar volume
of 5% dextrose solution. Administrations of Pt(DECO)2 were performed
weekly, at the same time of the day to eliminate circadian effects.
2.2.6. Nerve conduction velocity (NCV)
NCV from mice tails were measured in a lab-built instrument inside
a grounded Faraday cage. Mice were anesthetized using a constant flow
of isoflurane. The external body temperature was maintained at 37 °C
with a chargeable warm heating pad (Kent Scientific Inc). The body
temperature was continuously monitored with
a thermal camera
(Micro-Epsilon). Tail skin and stainless steel recording electrodes (~29
ga, World Precision Instruments) were sanitized with 70% isopropanol.
The recording electrodes were inserted 1 cm distal to the base of the
tail. The nerve compound action potential was measured using an ex-
tracellular amplifier (DAM80, World Precision Instruments). Stimu-
lating electrodes were inserted approximately 30 mm distal to the re-
cording electrodes. Voltage pulses (2–5 msec duration, 0.6–4 V) were
delivered to the stimulating electrodes from a battery powered stimulus
2.2.4. Injection preparation
Pt(DECO)2 powder (~1 mg) was first dissolved in DMSO and then
re-dissolved in 5% dextrose prepared by diluting commercial 50%
dextrose solution (Phoenix Pharmaceuticals, Inc.). The injection vo-
lume per mouse was 200–250 μL depending on the weight of the mouse.
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S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
2.2.8. Cytokine analysis
A
100
EDTA-treated tubes of blood collected from mice at week 7 post-
injections were placed on ice and then centrifuged (10,000 rpm, 4 °C,
10 min) to separate the red cells. The supernatant was placed at -80 °C.
The cytokine levels in the supernatant were assessed using a commer-
cially available, customized magnetic bead multiplex kit (Procartaplex
Mouse Simplex beads for TNFa, IL-6, ThermoFisher). Cytokine analysis
was performed using Mouse 10-Plex Panel for the Luminex™ Platform.
The assays were performed according to the manufacturer re-
commended protocol, with the modification of performing an overnight
+4 °C incubation for the multiplex cytokines. Milliplex Analyst 5.1.0.0
software was used to calculate the level of cytokine from the median
fluorescence intensity (MFI) of each bead.
HeLa cells
80
60
40
20
0
2.2.9. Cardiotoxicity
The hearts extracted from euthanized mice were fixed in 10%
neutral buffered formalin. After fixation, the tissues were trimmed,
processed according to standard procedures, paraffin-embedded, and
sectioned at 5-μm thickness. The sections were stained with hematox-
ylin and eosin for histopathologic evaluation and imaged with a mi-
croscope (Olympus BX51) and a colour camera (Infinity 3.0).
B
3. Results and discussion of biological testing
WiDR cells
100
3.1. Pt and Pd- cyanoximes show strong in vitro cytotoxicity
Both platinum complexes Pt(DECO)2 and Pt(PyrCO)2 were highly
toxic to HeLA and WiDR cancer cells (Fig. 5A). Pt(DECO)2 was equally
toxic as cisplatin against HELA cells, but had much higher toxicity
against WiDR cells than did cisplatin.. Low viability of the cells in the
presence of Pt(DECO)2 that destroyed 95% of WiDr cells and > 60% of
the HeLa cells suggested high efficacy of the complex. The Pt(PyrCO)2
showed even higher cytotoxicity destroying 96.4% of WiDr and 95.2%
of HeLa cells.
80
60
40
20
0
Palladium complexes, especially Pd(DECO)2, were also highly cy-
totoxic. After 24 h of treatment, 94% of HeLa and 98%% of WiDr cells
were destroyed with Pd(DECO)2 (Fig. 5). In comparison, the same
concentration of cisplatin destroyed 55% of HeLa and only 12% of
WiDR cells after 24 h. The Pd(PyrCO)2 showed mixed results with
somewhat lower efficacy against WiDR cells. The Pd(PyrCO)2 complex,
precipitates above the cell layer in the culture dish, which, apparently,
explains its low cytotoxic activity.
PyrCO-based metal complexes showed lower water solubility. Both
Pt (II) and Pd (II) complexes were relatively short-lived in aqueous
buffers forming an insoluble layer above the cells by the end of 24 h
incubation period. While being highly potent in vitro, low solubility of
these complexes would lead to poor bioavailability of the complexes in
vivo. For this reason, PyrCO complexes were excluded from in vivo
testing. Pd(II) complexes only recently came into the spotlight as po-
tential chemotherapy drugs [17,18]. Given that most approved metal
based chemotherapy drugs are derived from Pt with its well docu-
mented side effects, we selected Pt(DECO)2 for the in vivo toxicity
evaluation.
Fig. 5. Viability of the prepared complexes and A: HeLa cells and B: WiDr cells.
After 24-h incubation
isolator (Model 2200, A-M Systems). Recorded data from the extra-
cellular amplifier and from the stimulator were collected and analyzed
with PowerLab 8/35 (ADInstruments Inc.) and used to calculate nerve
conduction velocity of the compound action potential after completion
of the experiment. Mice were returned to their home cages for recovery.
2.2.7. Blood analysis
3.2. Pt(DECO)2 treated mice do not show sign of weight change
The blood samples were used to analyze for cytokines, hemoglobin,
white cells, and platelets. Blood was collected from the submandibular
vein with a lancet. Mice were restrained by hand at the nape of the neck
so there is minimum movement of the head and easy access to the
mandibular vein. A one-time blood collection up to 100 μL was per-
formed. The blood was kept in the EDTA-treated tubes on ice for less
than an hour to avoid coagulation, and then transferred to Washington
U. core facility for blood analysis. The whole blood samples were tested
with Hemavet 1700 Veterinary Multispecies Hematology System (Drew
Scientific Group Inc.). Blood smears were examined microscopically for
red and white blood cell counts, hemoglobin, and platelet counts.
Loss of weight is one of the most common side effects of che-
motherapy in humans and may occur as a result of stress, appetite loss,
diarrhea, nausea and dehydration. The severe weight loss accompanied
by rapid depletion of skeletal muscle mass during the chemotherapy
treatment is clinically known as chemotherapy-induced cachexia [19].
This complex metabolic disorder frequently leads to a lethal outcome.
Similar side effects are also observed in mice treated with many
common chemotherapeutics. Thus, loss of weight > 10% has been ob-
served in healthy mice treated with Folfox (a combination of ox-
aliplatin, 5-Fluorouracil, and leucovorin) [4]. Similar effects have been
7

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
8.8 × 103 per μl to 5.8 × 103 per μl after 7 weeks of treatments
(Fig. 7a).
1.25
1.2
Previous studies have shown that a single dose of cisplatin at 12 mg/
kg decreases the white blood cell count to ~ 50% relative to the con-
trols [25]. In the case of Pt(DECO)2, white blood cell counts only show a
marginal decrease following a much higher accumulative dosage
(7 × 15 mg/kg), making Pt(DECO)2 significantly less myelotoxic than
cisplatin. Importantly, even after 7 weeks of weekly high-dosage in-
jections, the number of white blood cells (5500/μL) remain within the
healthy limit (for mice: 2000–10,000/μL [24]).
1.15
1.1
1.05
1
0.95
0.9
0.85
0.8
In addition to the weakening of the immune system, chemotherapy
induced myelotoxicity also leads to anemia in cancer patients due to the
decrease of the red blood cells (RBCs) that carry oxygen from the lungs
to the rest of the body. The decrease of RBCs is often worsened by the
concomitant renal dysfunction that leads to the decline of ery-
thropoietin, a hormone produced by the kidneys that stimulates RBC
production [26]. Almost no changes in the RBC counts were observed
after 7 weeks of treatment (Fig. 7b). The level of RBCs remained within
the normal healthy range 7 × 106 to 13 × 106/μL [27]. In this study
we also used hemoglobin as a marker of anemia. Hemoglobin values
ranging from 13.6 to 16.4 g/dL are generally considered normal for
adult male mice [28]. Mice treated with the high dose of Pt(DECO)2
showed a small decrease in total hemoglobin to 12.5 g/dL (Fig. 7c).
The slightly decreased level of hemoglobin suggests a mild effect of
Pt(DECO)2 on the mice. A similar drop in hemoglobin in humans would
not require any special treatment. Hemoglobin levels from 12.5 to
13.5 g/dL are considered very mild and from 10.7–12.4 g/dL are mild.
The results from our study showed a very mild effect of the Pt(DECO)2
on hemoglobin.
Week, PI
7
0.75
0
1
2
3
4
5
6
8
Control
7.5 mg/kg
15 mg/kg
Fig. 6. Weight change of the mice treated with Pt(DECO)2 All weights are
normalized to the preinjection weight (week zero = 1). Control group was
treated with a 5% dextrose solution.
found on oxaliplatin alone [20], cisplatin [21], and carboplatin treated
mice [22]. In our study, mice treated with Pt(DECO)2 did not show any
change of the weight loss compared with the control, dextrose-treated
group (Fig. 6). Gradual weight increase in the treated mice was similar
to the typical increase of mass in healthy mice characteristic with the
normal aging process.
3.3. Pt(DECO)2 showed negligible myelotoxicity
For many cancer treatments, chemotherapy is used to shrink the
tumor. Then surgery is finally performed to remove the mass. If platelet
counts are too low, surgery cannot be safely performed. The fact that Pt
(DECO)2 doesn't seem to impact platelet counts (Fig. 7d), with the
normal range 3 to 10 × 105/μL [27], is a significant benefit.
Applications of chemotherapy drugs are frequently associated with
a significant degree of myelotoxicity, leading to the decrease of the
white blood cells. Common chemotherapy drugs can damage the red
bone marrow, lowering the production of white cells, resulting in the
decrease in the ability of the body to fight the microbial and fungal
infection [2]. Lowered white blood cell counts accompanied with high
fever, a condition known as febrile neutropenia, occurs in 25 to 40% of
chemotherapy treated patients and reduces patient's quality of life and
leads to hospitalization [23].
3.4. Mice demonstrate no peripheral neurotoxicity
Chemotherapy-induced peripheral neuropathy (CIPN) is the most
common neurological complication of cancer treatment [29]. This
chronic painful condition causes severe debilitating symptoms leading
to reduction of doses to mitigate CIPN or the premature terminations of
treatment. The symptoms of CIPN (sharp and dull pain, loss of sensi-
tivity, motor dysfunction) are varied and related to the specific toxicity
White blood cell counts reflect the ability of the animal to fight
pathogens. Normal mouse white blood cell counts range from 2000 to
10,000/μL [24]. Our measurements showed that the Pt(DECO)2 de-
creases the level of the white blood cells by an average of 35% from
Fig. 7. Level of white blood cells (WBC),
red blood cells (RGB), total hemoglobin
(HGB), and platelets (PLAT) in the mice
treated Pt(DECO)2 at different dosages (ip,
once/weekly). Control group mice were
treatedwith
a 5% dextrose solution (ip,
once/weekly).The blood was collected from
the mice after 7 weeks of weekly drug in-
jections. (For interpretation of the refer-
ences to colour in this figure legend, the
reader is referred to the web version of this
article.)
8

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
NCV is usually seen with many classes of chemotherapeutic drugs both
in human and rodents. For example, cisplatin-treated animals usually
confer a relatively small but significant reduction of the NCV when
N C V
5 0
measured caudally (−9%
3%, p < 0.05) [31]. Relatively low re-
duction in NCV even after a large dose of Pt(DECO)2 (Fig. 8) suggests
the stability of the myelinated fibers in the peripheral nerves.
To confirm the resistance of the fibers to the drug, we conducted
nerve histomorphometry on sciatic nerves extracted from the mice after
8 weeks of treatment. Optical images of the nerves are shown in Fig. 9
and the derived quantitative parameters are shown in Table 1. No ar-
chitectural changes were present in the nerves of the treated mice.
Representative sections demonstrate normal myelinated fiber distribu-
tion with no evidence of myelinated fiber injury or myelinated fiber
debris. The perineurium of the nerve fascicles shows normal archi-
tecture of perineural cells. Nerve endoneurium collagen and micro-
vessels are normal in appearance absent of any edema or inflammatory
cells.
4 0
3 0
2 0
1 0
0
Chemotherapy treatments are also linked to a variety of cognitive
side effects that affecting thinking, learning, processing or remembering
information. These side effects collectively known as “chemo brain”
[32] are characterized by the release of pro-inflammatory cytokines
that facilitate demyelination of neurons in the central nerve system.
While hydrophilic chemotherapeutic agents usually cannot cross the
blood-brain-barrier (BBB), cytokines such as tumor necrosis factor-
alpha (TNF-α) and interleukin-6 (IL-6) can penetrate through the BBB
and induce inflammatory responses in the brain.
Fig. 8. Nerve conduction velocity measured caudally in mice treated with Pt
(DECO)2 for 8 weeks.
While a low level of TNF-α protects the nerves [33], high level of
this cytokine is often associated with demyelination of the neurons in
the central nerve system. TNF-α elevation in plasma has long been
implicated in demyelinating processes in the brain [34] and is con-
sidered as one of the main candidate mechanisms underlying cognitive
of the class of drugs involved. Nerve conduction velocity (NCV) is a
frequent clinically used test for CIPN patients to evaluate both motor
and sensory nerves [30]. NCV studies are considered the gold standard
CIPN test in patients receiving platins family drugs [5]. A decrease in
Fig. 9. Histology of the sciatic nerve. A: Control mice treated
with a 5% dextrose solution; B: Mice treated with (Pt(DECO)2,
ip, 8 weeks, 15 mg/kg once/weekly). Sections were stained
with OsO4, and counterstained with toluidine blue. Each
panel shows images with magnification 200× and 2000×.
(For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this ar-
ticle.)
9

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
3.5. Pt(DECO)2 shows negligible cardiotoxicity
IL-6 Plasma
TNFa Plasma
60
40
20
0
3.3
3.2
3.1
3.0
2.9
Chemotherapy induced cardiotoxicity is one of the hallmarks of
several anticancer drugs, such as doxorubicin [38]. Cardiotoxicity
causes irreversible damage to the myocardium and leads to an im-
portant increase in morbidity and mortality in cancer patients. The
knowledge of the onset of the cardiac dysfunction during chemotherapy
and the susceptibility of patients to develop cardiotoxicity are scarce.
Although several biomarkers have been proposed to predict the cardi-
otoxicity (i.e. serum troponin levels), the histology of the heart tissue
considered to be the gold standard in preclinical studies. The histolo-
gical evaluation of the heart excised from the control and treated mice
showed no gross difference. There were no significant lesions noted in
the heart across all groups. All samples from the mice, including con-
trols showed a few minimal foci of increased interstitial cells in the
myocardium of the left ventricle, with no associated necrosis or in-
flammation (Fig. 11).
Fig. 10. Level of cytokines TNF-α and IL-6 in plasma following 8 weeks of
weekly injections of Pt(DECO)2.
3.6. Pt(DECO)2 induces hepatotoxicity
Among the above-mentioned parameters, we also noted a sizable
increase of the liver in the Pt(DECO)2 treated animals post mortem. The
effect was dose dependent with the greatest increase in mice treated
with the highest dose. The hepatotoxicity occurs frequently in cancer
patients treated with different chemotherapy drugs, including platinum
complexes [39]. Acute and chronic damage to the liver can lead to a
number of serious complications such as sinusoidal obstructive syn-
drome, steatosis, and pseudocirrhosis that are commonly associated
with chemotherapies. These conditions can display clinical signs of
acute hepatitis, liver cirrhosis, and even liver failure [40] and is one of
the largest concerns in clinical oncology. Since the liver is the primary
site of metabolism for many chemotherapy drugs, the interaction be-
tween the liver and Pt(DECO)2 will have to be investigated in more
details. The future study will also identify the mechanism of hepato-
toxicity and whether the liver damage is reversible or can be prevented
with adjuvant therapies.
Fig. 11. Cardiac histology of mice treated with Pt(DECO)2, 15 mg/kg, weekly,
ip suggests low level of cardiotoxicity. A few minimal foci of increased inter-
stitial cells in the myocardium can be seen (arrow). No necrosis of inflammation
was noted. Magnification 400×.
4. Conclusions
impairment. Our study showed that the application of the Pt(DECO)2
did not change the level of TNF-α in plasma (Fig. 10a), even at the
maximum high dosage.
Four new complexes of Pd and Pt with DECO− and PyrCO− cya-
noxime anions were synthesized and characterized using spectroscopic
methods and thermal analysis. Structures of several complexes were
confirmed by the X-ray analysis. Platinum cyanoximates were found to
be highly cytotoxic in vitro against two different types of human cancer
cells, with several of the tested compounds out-performing cisplatin.
Largely, Pt(DECO)2 showed minimal in vivo toxicity. No changes were
observed in the animal weight in the treated mice compared to the
control dextrose-treated group. The levels of erythrocytes, leukocytes,
hemoglobin, and platelets were within the normal level suggesting low
myelotoxicity. NCV and nerve histomorphometry suggested no impact
of Pt(DECO)2 on the peripheral nerve architecture. Negligible cardio-
toxicity was observed from the histological evaluation of the hearts
from the treated animals. The complex, however, induced certain he-
patotoxicity and lead to the elevation of IL-6, a pro-inflammatory cy-
tokine. Overall, cyanoximates present promising candidate for future
testing in the animal models of cancer.
In contrast, the level of IL-6 turned out to be dose dependent
reaching the relatively high level of > 50 pg/mL in one of the mice
(Fig. 10b). IL-6 is a cytokine with complex pleiotropic functions, ex-
erting positive and negative roles in homeostasis of many tissues. In a
recent clinical study, the increased level of IL-6 has been correlated
with a self-perceived cognitive disturbance in doxorubicin treated
breast cancer patients [35]. Similar finding was observed in mice
models [36] where the elevated level of circulating IL-6 has been shown
to exert neurochemical modifications in the mice models of Alzheimer's
disease, causing neuronal degeneration in the brain [36]. IL-6 is usually
non-detectable in normal mice. However, it can reach a really high level
as much as 10 ng/mL in certain animal models [37]. Nevertheless, the
observed increase of circulating IL-6, introduces a potential risk factor
and needs to be considered in greater details.
Table 1
Quantitative parameters from representative sections from sciatic nerves of control and treated mice.
Density fiber, per 100 μm2
Fiber area, μm2
Axon area, μm2
Myelin area, μm2
Fiber width, μm
G-ratio (Eq. diameter)
Control
261.17
266.54
32.14
26.38
12.43
10.21
20.39
16.79
4.92
4.75
0.58
0.61
Pt(DECO)2 15 mg/kg
10

S.D. Dannen, et al.
JournalofInorganicBiochemistry208(2020)111082
Glossary and abbreviations
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H(DECO) 2-oximino-2-cyano-N,N′-diethylacetamide
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Acknowledgment
We are grateful to Prof. Viktor Nemykin (University of Manitoba)
for preliminary structure determination of the red [Pt(PyrCO)2]2 dimer
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Diana Bender from Bursky Center for Human Immunology
&
Immunotherapy Programs at Washington University for cytokine ana-
lysis and Ms. Suellen Greco from Washington University DCM Research
Animal Diagnostic Laboratory for the histology of the heart. We also
thank the Petroleum Research Fund for the award (NG) and the
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