Palladium(II)-η3-Allyl Complexes Bearing N-Trifluoromethyl N-Heterocyclic Carbenes: A New Generation of Anticancer Agents that Restrain the Growth of High-Grade Serous Ovarian Cancer Tumoroids

Article abstract of DOI:10.1002/chem.202002199

The first palladium organometallic compounds bearing N-trifluoromethyl N-heterocyclic carbenes have been synthesized. These η³-allyl complexes are potent antiproliferative agents against different cancer lines (for the most part, IC₅₀

Full text of DOI:10.1002/chem.202002199

Accepted Article
Accepted Article
Title: Palladium(II)-η3-allyl complexes bearing N-trifluoromethyl
N-heterocyclic carbenes:a new generation of anticancer
agents which restrain the growth of high grade serous ovarian
cancer tumoroids
Authors: Fabiano Visentin, Thomas Scattolin, Enrica Bortolamiol,
Isabella Caligiuri, Tiziana Perin, Vincenzo Canzonieri, Stefano
Pluda, Alessandro Angelini, Nicola Demitri, Flavio Rizzolio,
Antonio Togni, and Stefano Palazzolo
This manuscript has been accepted after peer review and appears as an
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 published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202002199
Link to VoR: https://doi.org/10.1002/chem.202002199
01/2020

10.1002/chem.202002199
Chemistry - A European Journal
RESEARCH ARTICLE
Palladium(II)-η3-allyl complexes bearing N-trifluoromethyl N-
heterocyclic carbenes: a new generation of anticancer agents
which restrain the growth of high grade serous ovarian cancer
tumoroids
Thomas Scattolin,* [a] Enrica Bortolamiol,[b] Fabiano Visentin,*[b] Stefano Palazzolo,[c] Isabella Caligiuri,[c]
Tiziana Perin,[c] Vincenzo Canzonieri,[c,d] Nicola Demitri[e], Flavio Rizzolio[b,c] and Antonio Togni*[f]
[d]
[e]
[f]
Prof. Dr. V. Canzonieri
Department of Medical, Surgical and Health Sciences
Università degli Studi di Trieste
Strada di Fiume 447, Trieste, Italy
Dr. N. Demitri
Area Science Park
Elettra-Sincrotrone Trieste, S.S. 14 Km 163.5,
Basovizza, 34149, Trieste, Italy.
Prof. Dr. A. Togni
Department of Chemistry and Applied Biosciences
Swiss Federal Institute of Technology, ETH Zꢀrich
Vladimir-Prelog-Weg 2, CH-8093 Zurich, Switzerland
E-mail: antonio.togni@inorg.chem.ethz.ch
[a]
[b]
[c]
Dr. T. Scattolin
Department of Chemistry and Centre for Sustainable Chemistry
Ghent University
Krijgslaan 281,S-3, 9000 Ghent, Belgium
E-mail: thomas.scattolin@ugent.be
E. Bortolamiol, Prof. Dr. F. Visentin, Prof. Dr. F. Rizzolio
Department of Molecular Sciences and Nanosystems
Università Ca’ Foscari
Campus Scientifico Via Torino 155, 30174, Venezia-Mestre, Italy
E-mail: fvise@unive.it
Dr. S. Palazzolo, Dr. I. Caligiuri, Dr. T. Perin, Prof. Dr. V. Canzonieri,
Prof. Dr. F. Rizzolio
Pathology Unit, Department of Molecular Biology and Translational
Research
Centro di Riferimento Oncologico di Aviano (CRO) IRCCS
via Franco Gallini 2, 33081, Aviano, Italy
Supporting information for this article is given via a link at the end of the
document.
Abstract: The first palladium organometallic compounds bearing N-
trifluoromethyl N-heterocyclic carbenes have been synthesized.
These 3-allyl complexes are potent antiproliferative agents against
different cancer lines (the most part of IC50 falls in the range of 0.02-
0.5 M) and by choosing PTA as co-ligand, we can improve the
selectivity toward tumor cells, whereas the introduction of 2-methyl
substituents generally slightly reduces the antitumor activity.
A series of biochemical assays, aimed at defining the cellular targets
of these palladium complexes, has shown that mitochondria are
damaged before DNA, therefore revealing a behavior substantially
different from that of cisplatin and derivatives
transition metals different from platinum and, among these,
palladium complexes have gained increased interest. As a matter
of fact, some derivatives of this metal described in the literature
have shown good antiproliferative activity toward several tumor
cell lines. Moreover, their mode of action appears sometime quite
different from that of cisplatin and analogues.[7]
We assume that the specific mechanism of action of these
organometallic compounds involves the nucleophilic attack on the 3-
allyl fragment.
The effectiveness of the representative complex 4c has been verified
on ovarian cancer tumoroids derived from patients. The results are
promising: contrary to carboplatin, our compound resulted very active
and showed a low toxicity toward normal liver organoids.
Introduction
Figure 1. Some palladium-based complexes as potential anticancer agents.[7]
Cancer represents a widespread and heterogeneous class of
pathologies that in 2018 have caused approximately 10 million
deaths worldwide,[1] despite the different therapeutic approaches
currently available.
Nowadays about half of the patients are treated with
chemoradiotherapy[2] and the most part of protocols requires the
use of platinum-based antineoplastic agents.[3] In spite of this,
cisplatin[4] and its second and third generation derivatives,[5] have
shown some severe limitations ascribable above all to side effects
on kidneys, liver and brain and intrinsic or acquired resistance
observed in some types of cancer.[6] These limitations have
stimulated researchers to develop alternative drugs based on
Some of the most active palladium compounds are equipped with
N-heterocyclic carbenes (NHCs)[7d-e] (Figure 1). These ligands,
widely employed in homogenous catalysis,[8] are able to strongly
stabilize complexes with different transition metals and this
property can be exploited to avoid a fast decomposition of the
metal-based drug in a biological environment. For this reason,
NHCs play now a significant role also in medicinal chemistry, as
highlighted by numerous published papers.[9] The strength of the
metal-carbene bond can be mainly ascribed to the good σ-
electron donating ability of NHCs. However, recent works have
highlighted the importance of metal-carbene π-back-donation in
1
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RESEARCH ARTICLE
the activity of NHC-based transition metal catalysts.[10] This
feature can be enhanced with the introduction of electron-
withdrawing groups in the backbone structure or on the nitrogen
substituents of the NHCs.[11] Trifluoromethyl is an excellent
electron-attracting group because of hyperconjugation between
the nitrogen lone pair and the σ*C-F antibonding orbitals.[11] In this
respect, one of us have recently described the synthesis of N-
trifluoromethyl carbenes and their application as ligands in
efficient ruthenium-based catalysts for olefin metathesis.[12b-c]
Furthermore, it is well-established that the introduction of groups
containing fluorine atoms, especially perfluorinated fragments,
may significantly alter the pharmacological properties of the
compounds under investigation.[13]
In addition to N-trifluoromethyl NHCs, we have adopted PTA
(1,3,5-triaza-7-phosphaadamantane) as further spectator ligand
for our palladium complexes, with the aim to enhance their
compatibility with the biological environment. This phosphine is
well-known for its high water-solubility, a property that is generally
transferred also to its complexes with transition metals.[14]
Moreover, in this paper the biological activity of PTA complexes
has been then compared with that one of the homologous
compounds containing the “classical” triphenylphosphine.
Finally, the two remaining palladium(II) coordination sites were
occupied by the η3-allyl fragment. The reactivity of Pd(η3-allyl)
complexes[15] and their use as excellent catalysts in cross-
coupling reactions[16] have been extensively studied.
The availability of innovative biobanks of tumoroids/organoids
represents a revolutionary tool to study protein function in the
onset and development of cancer[19] or to develop innovative
therapies.[20] In this regard, most researchers are modelling
ovarian cancer utilizing commercially available cancer cell lines
sensitive or resistant to platinum treatments. But innate or
acquired chemoresistance together with tumor heterogeneity are
the causes of therapeutic failure in ovarian cancer[21] and
innovative preclinical models are required to solve this complexity.
A few leading groups in this field are developing animal[22] and ex
vivo organoid[23,24] models of ovarian cancer to better mimic
treatment options.
Finally, a series of experiments were then carried out with the aim
to define the cellular target of the compounds, proving that their
mechanism of action is different from that one of cisplatin.
Results and Discussion
Synthesis of N-trifluoromethyl benzimidazolium salts a-c
The N-trifluoromethyl benzimidazolium salts a-c, which are the
precursors of the carbene ligands studied in this work, were
synthesized by alkylation or arylation of N-trifluoromethyl
benzimidazole. In turn, this reagent can be obtained on a
multigram scale by electrophilic trifluoromethylation of
benzimidazole with trifluoromethyl-1,3-dihydro-3,3-dimethyl-1,2-
benziodoxole as depicted in Scheme 2.[11b]
The anticancer activity of complexes containing the Pd(II)-η3-allyl
organometallic function, was explored for the first time by some of
us in a recent work, in which purine-based NHCs were employed
as spectator ligands.[17]
Their cytotoxicity toward the A2780 and SKOV-3 ovarian cancer
lines, if compared with those obtained for complexes with the
same
spectator
ligands
but
containing
the
palladacyclopentadienyl and Pd(0)-η2-olefin fragments,[18]
appeared very promising both in terms of antiproliferative and pro-
apoptotic activity.
Scheme 2. Synthesis of N-trifluoromethyl benzimidazole
Therefore, hoping that the combination of all these positive factors
allows to give the best result we have synthesized and
exhaustively characterized the novel compounds shown in
Scheme 1 and tested their efficiency and selectivity as anticancer
agents against different human tumor cell lines.
In order to make the comparison among the anticancer activities
of the final cationic allyl palladium species more reliable, it was
decided to maintain in all cases the same counterion
(tetrafluoroborate).
Therefore, the synthesis of compound a was carried out by
reacting, under the conditions reported in Scheme 3, N-
trifluoromethyl benzimidazole with a slight excess of Me3OBF4
(Meerwein's salt). Compound b was obtained by reacting silver
tetrafluoroborate with a stoichiometric amount of 3-isopropyl-1-
(trifluoromethyl)-benzimidazolium iodide, which in turn was
synthesized starting from 2-iodopropane and N-trifluoromethyl
benzimidazole according to the protocol reported in the
literature.[11b] After filtration of the silver iodide, compound b was
isolated by precipitation with diethyl ether.
Finally, the benzimidazolium salt c was obtained, as described in
Scheme 1. η3-allyl Palladium complexes bearing N-trifluoromethyl NHCs
a
previous paper,[11b] by arylation of N-trifluoromethyl
and phosphines (PPh3 or PTA) synthesized in this work
benzimidazole with Ph2IBF4, using copper(II) triflate as catalyst
(Scheme 3).
One of the most promising compounds was evaluated in normal
liver organoids and ovarian cancer organoids (tumoroids) derived
directly from patients. Organoids are lab-built mini-organs that
can act as models to recapitulate cancer development.
2
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10.1002/chem.202002199
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Figure 2. Representation of the two atropoisomers related to compound 1c
(DFT calculations).
1
Moreover, the presence in the H-NMR spectra of five different
signals ascribable to the allyl protons for each atropoisomer
demonstrates that, if active, the typical η3–η1–η3 or syn-syn/anti-
anti rearrangements are slow in the NMR time scale (Figure S6).
The coordination of the triphenylphosphine is demonstrated by
the appearance in the 31P{1H}-NMR spectra of two sharp quartets
(JP-F = 1-4 Hz) at ca. 25 ppm (Figure S7), whereas that one of N-
heterocyclic carbene is confirmed by the presence in the 13C{1H}-
NMR spectra of two multiplets (one for each atropoisomer) at
about 190 ppm attributable to the carbenic carbon. The multiplicity
of the signals is consequence of the simultaneous coupling of the
carbon with phosphorus and fluorine nuclei (Figure S8).
Finally, 19F{1H}-NMR spectra exhibit the two doublets of the CF3
groups at about -55 ppm (JF-P = 1-4 Hz) and the signals of
tetrafluoroborate counterion at about -153 ppm (Figure S9).
The mixed NHC/PTA palladium η3-allyl compounds 3a-c and 4a-
c were prepared in an analogous manner as for the compounds
containing the triphenylphosphine (Scheme 5).
Scheme 3. Synthesis of N-trifluoromethyl benzimidazolium salts (a-c)
Synthesis of η3-allyl palladium complexes stabilized by N-
trifluoromethyl NHCs
The synthesis of the mixed NHC/PPh3 palladium η3-allyl
complexes 1a-c and 2a-c was performed in a one-pot process, by
reacting the starting benzimidazolium salts a-c with 0.5
equivalents of silver oxide in acetonitrile and, after seven hours,
adding the palladium allyl precursor ([Pd(μ-Cl)(η3-C3H5)]2 or
[Pd(μ-Cl)(η3-2-MeC3H4)]2) and PPh3 to the reaction mixture
(Scheme 4).
Scheme 4. Synthesis of η3-allyl Pd complexes bearing N-trifluoromethyl NHCs
and PPh3
This approach was adopted because of the difficulty in isolating
the NHC-silver(I) complexes in pure form and in good yields.
The six new mixed NHC/PPh3 palladium compounds were easily
isolated and exhaustively characterized by IR and NMR
spectroscopies and elemental analysis.
Examination of NMR spectra reveals that every isolated η3-allyl
palladium derivative is composed of a couple of atropoisomers
(exo and endo, see Figure 2), owing to the hindered rotation of
the asymmetric carbenes around the Pd-C bond. This
phenomenon, already observed for similar compounds,[15i,17] is
evidenced by two distinct set of signals in the corresponding NMR
spectra.
Scheme 5. Synthesis of η3-allyl Pd complexes bearing N-trifluoromethyl NHCs
and PTA
Also for each one of these compounds the presence in solution of
a pair of atropoisomers is confirmed. In particular, in the 1H-NMR
spectra (Figure S34), the allyl fragment gives rise to five distinct
allyl signals (for each isomer), whereas the methylene protons of
PTA (NCH2N and NCH2P) bring about two multiplets between 4
and 5 ppm. The coordination of PTA is also corroborated by the
two superimposed quartets (JP-F = 1-5 Hz) at about -58 ppm (Δδ
≈ 50 ppm with respect to the uncoordinated PTA), observed in the
31P{1H}-NMR spectra (Figure S35).
X-Ray Crystal Structure Determination of 1c
Crystalline 1c contains one crystallographically independent
palladium complex (Figure 3). The palladium center adopts a
square planar coordination geometry with bond lengths and
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angles in agreement with literature structural data of similar
complexes (Table S1), such as e.g. triphenylphosphine-(η3-allyl)-
(1,3,7,9-tetramethylxanthinium-8-ylidene)-palladium
the nucleophilic attack by an amino or thiol group on the
coordinated allyl ligand.[15]
On the contrary, under the same conditions the mixed NHC/PTA
complex 3c does not react appreciably with GSH after 24 hours
as confirmed monitoring the process by 1H and 31P NMR
spectroscopies (Figures S56-57).
In order to investigate the potential anticancer activity of the mixed
NHC/PPh3 and NHC/PTA allyl palladium complexes, a panel of
six different human tumor cell lines (ovarian cancers OVCAR5,
A2780, with its cisplatin resistant clone A2780cis, lung cancer
A549, colon cancer DLD1 and malignant melanoma A375) and
MRC-5 normal cells (human lung fibroblasts) were treated for 96
hours with our compounds and cisplatin (positive control). In
Table 1 are reported the resulting half inhibitory concentrations
(IC50) values.
First of all, Table 1 highlights that, with the exception of some rare
cases, the tested compounds are more (and often much more)
cytotoxic than cisplatin in all six tumor cell lines taken into
consideration.
tetrafluoroborate (CCDC 1825948).[17a] The complex bears a
-
positive charge that is balanced by a BF4 counterion, located
close to the allyl ligand, which represent the area where the metal
is more exposed (shortest F···Pd contacts are 3.88(1) Å in 1c and
3.50(1) Å in CCDC 1825948).
Molecular model of 1c is well superimposable with
triphenylphosphine-(η3-allyl)-(1,3,7,9-tetramethylxanthinium-8-
ylidene)-palladium tetrafluoroborate (CCDC 1825948, see Figure
S54) with minimal shifts of atoms directly bound to the metal and
equivalent xanthine and allyl arrangements (R.M.S.D. ~0.14 Å).
This comparison highlights the phosphine ligand conformational
freedom, which is influenced by crystal packing contacts and
relative bulkiness of xanthine substituents. Crystal packing of 1c
shows hydrophobic contacts among neighbour molecules,
involving weak intermolecular interactions: π···π (i.e. dπ···π
=
3.778(1) Å with 0.82 Å slippage between ring centroids) and
CH···π (i.e. dCH···π = 3.663(2) Å with 63° between CH and π-plane), Secondly, the antiproliferative activity exerted by each compound
among neighbour aromatic sidechains.
A
well oriented
on the A2780 (cisplatin-sensitive ovarian cancer) and A2780cis
(cisplatin-resistant ovarian cancer) cell lines is substantially
comparable, therefore suggesting a different mechanism of action
from cisplatin (DNA platination).
intramolecular CH···π contact in 1c (dCH···π = 3.678(2) Å with 45°
between CH and π-plane) involves xanthine lateral phenyl
substituent and one phosphinic phenyl and probably restrains the
conformation adopted by the ligands. The η3-allyl instead can
adopt two nearly equally probable configurations specular with
respect to the Pd coordination plane.
Moreover, it is possible to notice how, with the same ancillary
ligands, the compounds bearing the Pd(II)-2-methylallyl
organometallic fragment (2a-c and 4a-c) are generally less active
than those bearing the Pd(II)-allyl fragment (1a-c and 3a-c). This
evidence seems to suggest that the lower tendency of the Pd(II)-
2-methylallyl substrates compared to the Pd(II)-allyl ones to
undergo nucleophilic attack on the terminal allyl carbons, as
reported in many published papers,[15] might be correlated to their
lower anticancer activity. As a matter of fact, we suppose that a
plausible mechanism of action of our palladium complexes might
involve a nucleophilic attack of some group present in the main
biotarget (i.e. amino or thiol group) to the allyl fragment and the
consequent allylation of the involved biomolecule could
compromise its functionality. This kind of reactivity of palladium
allyl compounds, is well known and exploited in important cross-
coupling processes (i.e. Tsuji-Trost reaction).
It is also noteworthy that, with the same allyl fragment and
carbene ligand, the complexes bearing the 1,3,5-triaza-7-
phosphadamantane (PTA) are generally slightly less active on the
tumor lines than the triphenylphosphine analogues, but the mixed
NHC/PTA derivatives have the great advantage of being poorly
active or inactive against normal cells (MRC-5 fibroblasts) namely
the slight loss of cytotoxicity allows to gain in selectivity against
cancer cells. Moreover, the lower activity of the mixed NHC/PTA
complexes, which is probably due to their lower reactivity toward
nucleophiles present in the biological environment (the strong σ-
electron releasing capability of PTA partially inhibits the
electrophilicity of the allyl fragment), might help such compounds
to reach unaltered the biotarget.
C8_3
P_4
Pd_1
C19_2
C20_2
Figure 3. Ellipsoid representation of 1c crystal ASU content (50% probability).
Atom labels in use for Pd coordination sphere are reported. Disorder omitted
for clarity.
Anticancer activity on human cancer and normal cell lines
In a preliminary phase, we have monitored the stability of the
palladium complexes in 1:1 DMSO-d6/D2O solution by NMR
spectroscopy. After 24 and 48 hours no significant changes of the
spectra are observable, indicating that the complexes retain their
structural integrity.
Nevertheless, the 31P-NMR spectra collected in the presence of
reduced glutathione (GSH) reveal a relevant reactivity of the
mixed NHC/PPh3 complexes with this important biological thiol. In
further detail, the signal of the complex 1c (at concentration 0.2
mM in presence of GSH 1 mM and at pH ≈ 7) disappear
completely within 24 hours, replaced by that one of
triphenylphosphinoxide (Figure S55). Triphenylphosphinoxide
might be one of final products of decomposition of the unstable
complex NHC-Pd0-PPh3, which is normally obtained as a result of
On the other hand, based on the reactivity shown with GSH (vide
supra), the marked activity of the mixed NHC/PPh3 complexes
even on normal cells could be ascribed to their propensity to form
triphenylphosphinoxide, which is known to have a certain intrinsic
toxicity.[25]
4
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Table 1. Effects of the Pd-complexes on the proliferation of several cancer and normal cell lines.[a].
IC50 (μM)
A549
Complex
A2780
A2780cis
43 ± 5
OVCAR-5
5.2 ± 0.8
0.26 ± 0.03
0.30 ± 0.05
0.20 ± 0.06
0.5 ± 0.1
0.10 ± 0.01
0.04 ± 0.01
10 ± 1
A375
DLD1
MRC-5
14 ± 1
Cisplatin
1a
0.81 ± 0.06
0.03 ± 0.01
0.14 ± 0.03
0.024 ± 0.002
0.18 ± 0.05
0.024 ± 0.006
0.043 ± 0.009
0.23 ± 0.07
0.25 ± 0.01
0.30 ± 0.02
0.76 ± 0.01
0.05 ± 0.04
0.02 ± 0.01
6 ± 2
4.7 ± 0.4
19 ± 4
0.28 ± 0.06
0.31 ± 0.04
0.28 ± 0.01
0.50 ± 0.09
0.31 ± 0.01
0.36 ± 0.07
1.11 ± 0.09
48 ± 29
0.051 ± 0.004
0.08 ± 0.02
0.027 ± 0.004
0.12 ± 0.02
0.096 ± 0.005
0.24 ± 0.04
0.9 ± 0.2
0.18 ± 0.09
0.14 ± 0.06
0.17 ± 0.01
0.32 ± 0.05
0.029 ± 0.002
0.030 ± 0.004
10 ± 3
0.041 ± 0.003
0.083 ± 0.009
0.045 ± 0.005
0.24 ± 0.02
0.028 ± 0.004
0.034 ± 0.004
0.286 ± 0.006
1.7 ± 0.3
0.28 ± 0.01
1.8 ± 0.6
0.30 ± 0.05
1.5 ± 0.3
0.25 ± 0.05
0.25 ± 0.03
>100
2a
1b
2b
1c
2c
3a
4a
1.4 ± 0.1
6 ± 1
2.1 ± 0.1
>100
3b
0.22 ± 0.05
1.3 ± 0.1
2.8 ± 0.7
6.3 ± 0.7
1.1 ± 0.2
0.3 ± 0.1
1.37 ± 0.09
2.5 ± 0.1
0.34 ± 0.06
2.7 ± 0.8
3 ± 1
>100
4b
5.4 ± 0.5
0.54 ± 0.08
>100
>100
3c
0.24 ± 0.04
0.44 ± 0.02
0.23 ± 0.05
0.21 ± 0.07
0.21 ± 0.03
14 ± 4
4.6 ± 0.2
>100
4c
[a] Data after 96 h of incubation. Stock solutions in DMSO for all complexes; stock solutions in H2O for cisplatin. A2780 and OVCAR5 (cisplatin-sensitive ovarian
cancer cell lines), A2780cis (cisplatin-resistance ovarian cancer cell line), A549 (lung cancer cell line), A375 (malignant melanoma cell line), DLD1 (colon cancer
cell line), MRC-5 (normal lung fibroblasts).
Antiproliferative activity on mouse liver organoids and
human ovarian cancer tumoroids
Anticancer activity on human ovarian cancer tumoroids
Among ovarian cancers, the high-grade serous subtype
(HGSOC) is the most common with the worst 5-year survival
rate.[21] Current lines of therapies are not effective, demanding for
novel drugs to overcome innate or acquired resistance that limit
treatment efficacy and in turn, increasing toxicity.
Taking advantages from organoid technology, three HGSOC
patients were selected to test the efficacy of compound 4c.
The tumoroids were derived primarily from solid tumors extracted
from primary (OV-A and OV-C) and metastatic (OV-B) sites.
Hematoxylin and eosin (H&E) staining confirm a similarity
between tumoroids and parental tumors at morphologic and
cytologic levels. Typical markers of HGSOC are evident such as
nuclear pleomorphism, prominent nucleoli, and dense chromatin.
IHC analysis confirmed the expression of the Mꢀllerian marker
PAX8 typical of this type of tumor (Figure 4).[23]
Complex 4c was chosen for further studies because of its low
cytotoxicity on human lung fibroblasts and the very high
anticancer activity toward ovarian cancer cells.
It is important to underline that the research on new
chemotherapeutic agents for the treatment of ovarian cancer is
still of fundamental importance, being one of the most lethal
tumors for women worldwide. As a matter of fact, the platinum-
based therapy is currently the main first line treatment, but
unfortunately almost all patients relapse with a 5-year survival rate
of less than 40 percent.
Liver organoid Toxicity
The correlation between hepatotoxicity and chemotherapy is well
documented although the etiology of various agents remains
mostly unexplained.[26] Here we utilized normal liver organoids to
assess their viability at different concentrations of compound 4c.
Organoids are reported in literature as a reliable model to
determine the IC50 of chemical compounds in different cancer
types[27] and to assess liver toxicity.[28-29]
Organoids were characterized by immunohistochemistry for
premature (SOX9 and cytokeratin 19) and mature (HNF4α,
albumin and E-cadherin) hepatocyte markers as previously
published.[29] Cisplatin showed a strong cytotoxicity on organoids
with an IC50 value of 0.03±0.01 µM. Compound 4c had about 100-
fold less toxicity (IC50 = 3±2 µM). The values confirmed the data
obtained on cell lines and strongly support the use of compound
4c for future clinical applications.
Figure 4. Example of characterization by H&E staining and PAX8 marker of
parental tumor and tumoroids (OV-A patient).
Furthermore, the IC50 of carboplatin was evaluated, as in clinical
standard therapy (Table 2).
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Table 2. IC50 (μM) of 4c and carboplatin on ovarian cancer tumoroids after 96
Cytochrome C release
h of incubation.
Mitochondria play a fundamental role in the initiation of apoptosis
and the release of different proteins that are usually present in
their intermembrane space (i.e. cytochrome C and adenylate
kinase-2) has been observed during the early stages of apoptotic
cell death.[31]
Patients
Compound
OV-A
>100
3 ± 2
OV-B
>100
3 ± 2
OV-C
30 ± 8
2 ± 1
Carboplatin
4c
For this reason, the release of cytochrome C is one of the most
widely used assays for assessing damage to mitochondria.
This early event was studied by immunofluorescence and the
results are summarized in Figure 6.
Contrary to carboplatin, compound 4c was very active on
tumoroids with all values below 4 µM. Considering that almost all
patients during the clinical course will not respond to carboplatin,
compound 4c could be considering more than an opportunity for
patients. In addition, the low activity on normal liver organoids
suggest an excellent profile for long term therapy.
Mechanism of cell death
The mixed NHC/PTA complex 4c was also chosen as model
compound for further mechanism studies based on
immunofluorescence techniques.
In particular, DNA damage, cytochrome
C release and
mitochondrial membrane depolarization were analyzed to clarify
the biological target of the palladium allyl complexes reported in
this work. To strengthen the data, the biological experiments were
carried out by a kinetic analysis at different timepoints. With this
approach, it is possible to determine the primary biological effects
generated directly by the compounds.
DNA damage
To evaluate the DNA damage, a well-established approach is
represented by the analysis of the phosphorylation of histone
H2AX (γH2AX).[30]
Figure 6. Immunofluorescence analysis of cytochrome
C release after
Phosphorylation of H2AX occurs immediately after the presence
of a DNA break and involves the formation of clusters of proteins
at the site of damage, called DNA damage response foci.
In this respect, A2780 cells were treated with 4c (10, 20 and 80
nM) and cisplatin (10 μM as positive control) for 3, 6 and 12 hours.
As shown in Figure 5, the cells treated with compound 4c do not
show phosphorylation of Ser139 of histone H2AX after 12 hours.
On the contrary, DNA damage in cells treated with cisplatin is
already evident after 3 hours (green foci in the nucleus).
These results suggest that DNA is unlikely to be a major
molecular target of 4c.
incubation of A2780 cells with 4c (10, 20 and 80 nM) and CisPt (10 µM). Arrows
indicate cytochrome C release.
The collected images have shown the release of cytochrome C
already after 3 hours in the case of A2780 cell line treated with
compound 4c. Moreover, this result is clearly evident even at the
lowest concentration used (10 nM) and decreases starting from
12 hours after treatment.
For cisplatin (10 µM), a slight release of the aforementioned
protein occurs after 3 hours but is clearly evident only after 6
hours from the treatment of the cells.
Mitochondrial membrane depolarization
Loss of mitochondrial membrane potential is another indication of
apoptosis proceeding through events involving mitochondria. In
this context, JC-1 dye is widely used to monitor the integrity of the
mitochondrial membrane as it exhibits a potential-dependent
accumulation.[32]
This molecule is characterized by a red emission when it forms
the aggregates with the mitochondrial membrane and a green
emission when the membrane is depolarized.
Consequently, the mitochondrial depolarization is indicated by a
decrease in the red/green fluorescence intensity ratio.
Based on the timing observed in the cytochrome C release assay,
A2780 cells were treated for 1 and 2 hours with the compound 4c
Figure 5. Assay on DNA damage after incubation of A2780 cell line with 4c (10,
20 and 80 nM) and CisPt (10 µM). Arrows indicate nuclear foci of Ser139-
Histone H2AX.
6
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10.1002/chem.202002199
Chemistry - A European Journal
RESEARCH ARTICLE
(10, 20 and 80 nM), cisplatin (10 μM) and hydrogen peroxide
(1mM). The latter is known to strongly alter the membrane
potential and is therefore used as a positive control.[33]
complexes could be the attack to allyl fragment carried out by a
nucleophile present in the bio-target, which is favored by the
presence of electron-withdrawing substituents (CF3) on the NHC
ligands and penalized (electronically and sterically) by that one of
methyl substituent on allyl group. Therefore “allylation” of a
strategic biomolecule could be one of the most important reasons
of the cytotoxicity of these palladium complexes.
One of the most efficient compounds (4c) has been also assessed
in a series of biological experiments to determine its cellular
targets.
This data, in some way, agrees with the results of biological tests
carried out to determine the primary cellular target of our
compounds. A kinetics analysis at different time point have proved
that the first cellular damage is not on DNA (γH2AX test) but on
mitochondria (JC-1 and cytochrome C tests). This different
mechanism of action in comparison with cisplatin and derivatives
could make these novel compounds suitable to treat those types
of tumors with spontaneous or induced resistance to classical
platinum drugs.
Finally, the effectiveness of the new allyl-palladium compounds
has been verified also on more complex structures as ovarian
cancer tumoroids, obtained by cancer tissues of three HGSOC
patients. The results are promising and in all three cases complex
4c is resulted definitely more active than carboplatin. Moreover,
the low activity on normal liver organoids is a good premise in
view of a therapeutic application.
Figure 7. Effect of 4c (10, 20 and 80 nM), Cisplatin (10 μM) and H2O2 (1 mM)
on mitochondrial potential of A2780 cell line after incubation for 1 and 2 hours
with JC-1 dye.
The results summarized in Figure 7 show the ability of hydrogen
peroxide and compound 4c, even at very low concentrations, to
cause the depolarization of the mitochondrial membrane already
after 1-2 hours. On the other hand, cisplatin shows no
appreciable alteration at a mitochondrial level.
The immunofluorescence experiments reported above prove that,
in a time sequence, mitochondria are damaged before DNA by
our palladium allyl compounds as opposed to what happens for
cisplatin.
This behavior is not general for palladium complexes and in a
previous paper we have shown that in the case of
palladacyclopentadienyl derivatives this progression of events is
inverted. [18b]
This suggests that the nature of organic fragment bonded to the
metal centre plays a crucial role in the biological activity of the
complex.
Acknowledgements
FR was financially supported by Fondazione AIRC per la Ricerca
sul Cancro (Grant AIRC IG23566) and VC from Ministero della
Salute – Ricerca Corrente.
Keywords: N-trifluoromethyl carbenes • Ovarian cancer •
Organoids • Palladium allyl complexes • Allylation
Conclusion
In this paper we have synthesized a new class of palladium
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8
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Entry for the Table of Contents
The first palladium organometallic compounds bearing N-trifluoromethyl N-heterocyclic carbenes have been synthesized. These 3-
allyl complexes are potent antiproliferative agents toward different cancer lines (the most part of IC50 falls in the range of 0.02-0.5 M)
and their primary cellular target is not DNA but mitochondria.
Their effectiveness has been even verified on ovarian cancer tumoroids derived from patients.
9
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