Investigation of photo-activation on ruthenium(II)–arene complexes for the discovery of potential selective cytotoxic agents

Article abstract of DOI:10.1016/j.poly.2019.02.041

Rational design of ruthenium complexes with optimized ligands is a promising approach to modulate their stability and antiproliferative effects in cancerous cells. The release of ligands from the coordination sphere of the ruthenium complexes can lead to

Full text of DOI:10.1016/j.poly.2019.02.041

Accepted Manuscript
Investigation of photo-activation on Ruthenium(II)-arene complexes for the
discovery of potential selective cytotoxic agents
Uttara Basu, Johannes Karges, Florian Chotard, Cédric Balan, Pierre Le Gendre,
Gilles Gasser, Ewen Bodio, Raluca Malacea Kabbara
PII:
S0277-5387(19)30142-1
https://doi.org/10.1016/j.poly.2019.02.041
POLY 13785
DOI:
Reference:
Polyhedron
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Revised Date:
Accepted Date:
15 December 2018
20 February 2019
21 February 2019
Please cite this article as: U. Basu, J. Karges, F. Chotard, C. Balan, P. Le Gendre, G. Gasser, E. Bodio, R. Malacea
Kabbara, Investigation of photo-activation on Ruthenium(II)-arene complexes for the discovery of potential
selective cytotoxic agents, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.02.041
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Investigation of photo-activation on Ruthenium(II)-arene complexes for the
discovery of potential selective cytotoxic agents
Uttara Basu,a Johannes Karges,a Florian Chotard,b Cédric Balan,b Pierre Le Gendre,b
Gilles Gasser,a* Ewen Bodio,b* Raluca Malacea Kabbarab*
a
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and
Health Sciences, Laboratory for Inorganic Chemical Biology, F-75005 Paris,
France. Tel. +33 1 44 27 56 02; Email: gilles.gasser@chimieparistech.psl.eu;
www.gassergroup.com.
b
ICMUB UMR6302, CNRS, Univ. Bourgogne Franche-Comté, F-21000 Dijon,
France. Tel: +33 3 80 39 60 76; Email: ewen.bodio@u-bourgogne.fr,
raluca.malacea@u-bourgogne.fr
ORCID-ID:
Uttara Basu: 0000-0002-0509-2421 uttarabasu.iisc@gmail.com
Johannes Karges: 0000-0001-5258-0260 johannes.karges@chimieparistech.psl.eu
Pierre Le Gendre : 0000-0003-2635-5216 pierre.le-gendre@u-bourgogne.fr
Gilles Gasser: 0000-0002-4244-5097 gilles.gasser@chimieparistech.psl.eu
Ewen Bodio: 0000-0003-2896-113X ewen.bodio@u-bourgogne.fr
Raluca Malacea-Kabbara 0000-0002-6684-1967 raluca.malacea@u-bourgogne.fr
1

Abstract: Rational design of ruthenium complexes with optimized ligands is a
promising approach to modulate their stability and antiproliferative effects in
cancerous cells. The release of ligands from the coordination sphere of the
ruthenium complexes can lead to the formation of biologically active Ru species that
are able to exert cytotoxic effects. Herein, we have studied this approach on four
ruthenium(II) complexes bearing (3,5-cycloheptadienyl)diphenylphosphine or
(cycloheptyl)diphenylphosphine and ethylbenzoate or p-cymene and an unusual
bimetallic analogue. The stability of the complexes was investigated in DMF as well
as DMEM/FBS using 1H and 31P{1H} NMR spectroscopy. We studied the
photocytotoxicities of the complexes in two cell lines – HeLa and RPE-1 – to
understand their behavior and cytotoxicity upon ligand dissociation. All complexes
showed moderate to high cytotoxicity in the two cell lines upon light irradiation.
2

1. Introduction
Despite the success of cisplatin, its heavy off target side-effects and eventual
appearance of resistance prompted numerous chemists to explore other metal
complexes, especially organometallic complexes, for their anticancer properties.[1–8]
Among them, ruthenium complexes appear to be promising alternatives.[9] NAMI-A,
(N)KP-1339 and their derivatives are to date the most well-known representative
complexes since they have reached phase II clinical tests (Figure 1).[10,11] The
exploration of the potential of ruthenium complexes is not limited to its +III oxidation
state; numerous Ru(II) complexes such as those of the RAPTA (Ru(η6-p-
cymene)Cl2(pta)) family have also been investigated (Figure 1). Concerning these
neutral Ru(II)-based-arene-phosphine complexes, they usually displayed poor
cytotoxicity in vitro.
Figure 1. Chemical structures of some ruthenium-based drug candidates and of the
photodynamic therapy photosensitizer TLD-1433.
Organometallic ruthenium(II)–arene complexes have promising potential for cancer
therapy because the coordination ligands in such complexes can be modulated for
their pharmacological properties such as cellular accumulations and kinetic
reactivities. Naphthalimide tagged Ru(II)-arene complexes, which showed moderate
to high toxicity and selectivity towards cancerous cells, were reported by Dyson et
al.[12] and Sadler and coworkers showed that half-sandwich ruthenium(II)-arene
complexes with chelated diamine ligands can undergo reversible oxidation to diimine
complexes and this was accompanied by strong changes in their cytotoxic behavior
in ovarian cancer A2780 cells.[13] A very recent work by Xu et al. reported a few of
ruthenium(II)−arene complexes containing a hypoxia inducible factor-1α inhibitor,
which allowed the organometallic complexes to target hypoxia in cancer cells,
enhancing their anticancer activities.[14] We note that the ruthenium(II) polypyridyl
3

complex of McFarland, namely TLD-1433, has completed phase I clinical trial as a
photodynamic therapy (PDT) photosensitizer (PS).[15]
Very recently, some of us published a study highlighting the cytotoxicity profile of
some Ru(II) arene complexes (Figure 2). Six Ru(II) complexes were designed and
two of them (IV and V) displayed low micromolar IC50 in CT26, 4T1, and LLC1 cancer
cell lines (Figure 1).[16] More interestingly, we were able to link directly the toxicity of
each complex of the family to its ability to be taken up by cells (correlation between
IC50 and ruthenium uptake determined by ICP-MS analysis).
Figure 2: Structures of ruthenium(II) previously prepared by some of us for their
anticancer activity.
More specifically, the complexes bearing an ethylbenzoate as an arene ligand
displayed an uptake up to twenty times higher than the one containing a p-cymene
ligand. These results imply that the ruthenium(II) cation itself is the biologically active
species and that the ligands are used to promote the cellular uptake of the
complexes. For this reason, we looked for a manner of forcing the release of the
ligands, especially of the arene. Being able to do so will either confirm that the role of
the ligands is just for the transport or to give access to activatable ligands (removing
the arene will lead to very active ruthenium species).
With this idea in mind, we capitalized on the work that some of us developed for
catalysis. We have recently described the ruthenium complexes 1-5 bearing either
(3,5-cycloheptadienyl)diphenylphosphine or (cycloheptyl)diphenylphosphine groups
and studied their catalytic activity in ATRA (Atom transfer Radical Addition) reactions
(Figure 3).[17] We showed that upon light irradiation, complexes 1-4 lose their arene
4

ligand. It is worth noting that under the same conditions, the ligand ethylbenzoate in
compounds 3 and 4 is released in 15 minutes, while it took more than two hours for
the p-cymene derivatives (compounds 1 and 2). Consequently, we decided to
investigate the photo-activation of these Ru(II)-based complexes in vitro. Moreover,
we have investigated compound 5 in this study as an important control. As shown
previously, this cationic dinuclear compound is generated upon light treatment of the
(cycloheptadienyl)phosphine-containing complexes 1 and 3 in organic solvents
(dichloromethane, chloroform etc.).
Figure 3: Ruthenium complexes described by some of us for their catalytic activity in
ATRA reactions.
In this article, we describe the study of the photocytotoxicity of these ruthenium
complexes on two cell lines, namely a cancerous one (HeLa) and a model of non-
cancerous one (RPE-1), as well their stability in DMEM-10% FBS solution.
2. Experimental
2.1. Photocytotoxicity of Ruthenium Complexes 1-5
2.1.1. Materials:
Dulbecco’s Modified Eagles Medium (DMEM, catalogue number 11500416),
Dulbecco’s Modified Eagles Medium supplemented with nutrient mixture F-12
(DMEM/ F-12, catalogue number 11580546), fetal bovine serum (FBS, catalogue
number 11573397), Gibco™ Penicillin-Streptomycin-Glutamine (penstrep, catalogue
number 12090216) Dulbecco’s Phosphate-Buffered Saline (DPBS, catalogue number
12559069), Trypsin-EDTA (catalogue number 11560626) and resazurin (catalogue
5

number 10751244 were purchased from ThermoFisher Scientific. N, N-Dimethyl
formamide (DNAse, RNAse and protease free, molecular biology grade, catalogue
number 327175000) was purchased from ACROS Organics. The pooled human
plasma was obtained from Biowest. Complexes 1-5 were prepared as previously
reported.[17] Analytical data matched that previously reported.
2.1.2. Cell culture
HeLa and RPE-1 cells were cultured in DMEM and DMEM/ F-12, respectively
reconstituted with 10 % FBS and 1 % penstrep. Cells were grown in a humidified cell
culture incubator at 37 °C and with 5 % CO2 and passaged three times before being
used for the experiments.
2.1.3. Photocytotoxicity Studies
The experiments were performed using a similar procedure previously used in our
laboratories.[10,18,19] More specifically, HeLa and RPE-1 cells were seeded at a
density of 4000 cells/ well in 100 µL media and grown overnight in a humidified cell
culture incubator at 37 °C and with 5 % CO2. All experiments were performed in
triplicates. The media was removed and replenished with varied concentrations of the
ruthenium complexes diluted in appropriate volume of the culture media in a total
volume of 200 µL. The stock solutions of the Ru complexes were made in DMF and
the final concentration of DMF in culture media was 1% or less. They were incubated
for 4 h and replaced with 200 µL of fresh culture media. Three of the plates were
irradiated at 450 nm (10 mins for HeLa cells, dose: 10 J cm-2 and 2 mins for RPE-1
cells, dose: 2 J cm-2). Irradiation was performed in the 96-well culture plates using a
LUMOS-BIO photoreactor (Atlas Photonics, Switzerland). Each well was individually
illuminated with a 50 mW LED at constant current and the temperature of the plates
was maintained constant at 37 °C with a cooling system. The cells were then allowed
to grow in the incubator for another 44 h. From the three other plates for the dark
cytotoxicity control, the media was removed after 4 h, replaced with 200 µL of fresh
culture media and incubated for another 44 h. After 48 h of the initial treatment, the
media was exchanged for resazurin solution (0.2 mg/ mL) made in the respective
culture media and sterile filtered. After 4 h incubation, the fluorescence of the product
resorufin was measured at 590 nm with excitation at 540 nm using a Spectramax M5
6

UV-visible spectrophotometer. The data was analyzed and plot using Graph Pad
Prism 8.
2.2. Stability studies
2.2.1. Stability in DMF
In NMR tubes, 0.01 mmol (5.8-6 mg) of complexes 1-4 or 0.005 mmol (4.5 mg) of the
dimer 5 were dissolved in 0.5 mL of DMF-H7 (protected from air) to give clear
solutions. NMR experiments were conducted on each compound under light
protection at 10 min, 1.5 h, 2.5 h, 3 h, 4.5 h, 21 h and 45 h (0.01 mmol/0.5 ml = 0.02
mmol/1 ml = 20 mM)
2.2.2. Stability in DMF + DMEM-10% FBS
On the samples at 48 h, 0.1 mL solution (DMEM-10%FBS) was added on each
compound directly in the NMR tube (under air). The samples were light protected. At
the beginning, the solutions were clear and no obvious change was observed. 2 h
after DMEM addition the samples 1-4 presented a precipitate (~ suspension at the
bottom) while sample 5 remain clear. After 5 days, the samples were let to evolve for
24 h under natural light.
2.3. NMR Studies
31
The acquisition of P{1H} NMR spectra DMF-H7 was recorded at 300K using a no-
lock sequence on a Bruker Avance III HD 600 MHz spectrometer (equipped with
1
double resonance broad band probes). Monitoring the reaction by H NMR was
possible as the diene signals (4-6 ppm) do not overlap with the DMF-H7 signals.
3. Results and discussion
3.1. Synthesis of the complexes
The five complexes 1-5 were synthesized in good yield following procedures
previously reported by some of us (Scheme 1).[17] All the products are air-stable.
However, they need to be protected from light, especially when they are in solution.
7

Scheme 1: Synthetic pathways of the ruthenium complexes reported by some of us
earlier.[17]
3.2. Preliminary stability investigation
Since the stability of a compound in a biological environment is a crucial property in
view of its use as a PDT PS or in photo-activated chemotherapy (PACT),[20] this
parameter has been thoroughly investigated in this study. When the biological
studies have been performed, the complexes were first dissolved in DMF since this
solvent was shown to be suitable for biological applications due to its low propensity
for coordination compared to DMSO.[21] These stock solutions were then dissolved
in culture media (DMEM-10% FBS). Thus, the stability of ruthenium complexes 1-5
1
was further evaluated by H and 31P{1H} NMR analysis in pure DMF, then after
dilution with DMEM-10% FBS.
8

Figure 4: 31P{1H} NMR spectra of ruthenium complexes 1-5 after 10 min and 45 h in
DMF-H7.
For this purpose, solutions of complexes 1-5 were prepared in DMF under argon at a
concentration of 20 mM of ruthenium. Clear solutions were obtained, which were
1
analysed by NMR after being kept in the dark from 10 min to 45 h. The H and
31P{1H} NMR analyses showed that complexes 1, 2, 4, 5 are stable over time, no
other signal being detected after 45 h (Figure 4). Only compound 3 is transforming
very slowly into the dimer 5 (after 21 h, 5 % of 5 were detected; after 45 h, 15 % of 5
were detected by 31P{1H} NMR). This result agrees with our previous observation
regarding complex 3, which had a higher sensitivity than complex 1.[17] This stability
in DMF is interesting since a recent investigation of the stability of N-heterocyclic-
[Ru(η6-arene)Cl2] complexes in DMSO, a much more coordinating solvent, concludes
to their instability (the heterocyclic ligand was replaced with DMSO).[22]
We were further interested in the behavior of these complexes in the presence of
DMEM-10% FBS in the dark. For this, in the previous NMR samples, 0.1 mL solution
(DMEM-10%FBS) was added on each compound under an air atmosphere. This
9

rapidly allowed the formation of a suspension in the bottom of samples 1 to 4 (the
high concentration required for NMR investigation are not compatible with the limited
solubility of the complexes in culture medium). Only compound 5 remained as a clear
orange solution. However, we were able to evaluate the evolution of these samples
in the dark by 1H and 31P{1H} NMR analysis (Figure 5).
After 5 days under these conditions, both complexes 1 and 2 were present as the
major compound in solution, even if two new signals slowly appeared on 31P{1H}
NMR spectra (in the case of complex 2 one of the signals corresponds to free
diphenylcycloheptylphosphine). Compared to those two, complex 3 displays lower
31
stability. After 5 days, very low signals were detected by P{1H} NMN. We noticed
that the dimer 5 present in 15 % before adding DMEM-10% FBS disappeared also
very quickly. The only complex, which presents higher stability than complex 2, is
complex 4, which shows lower degradation after 5 days. Concerning dimer 5, the
31P{1H} NMR signal disappeared as soon as DMEM-10% FBS was added. This
suggests that this complex reacts immediately with the biological media.
After 5 days in dark, all solutions were exposed to natural light for 24 h and analyzed
again by NMR. The obtained spectra lead to three conclusions, namely 1) the signals
of the complexes 1, 2 and 4, which were still present as the major compound in
solution, totally disappears. This confirms the light-induced arene released; 2) the
signals of the “degradation products” – at ≈ 70 ppm for complex 1 and at ≈ 40 ppm
for complexes 2 and 4 – are still present after light exposure. This suggests that
these complexes do not any longer bear an arene on their ruthenium; 3) it highlights
the necessity to protect these complexes from light during the studies.
10

31
Figure 5: P{1H} NMR spectra of ruthenium complexes 1-5 with DMEM 10% FBS
(blue line: 45 h in DMF; red line: 5 days under dark; and green line: after additional 1
day of light exposure).
3.3. Photo-cytotoxicity of Ruthenium Complexes 1-5
The photo-cytotoxicity of the ruthenium complexes 1-5 was evaluated in two cell
lines, namely HeLa (human cervical cancer cells) and RPE-1 (human Retinal
Pigmented Epithelial cells). After 4 h incubation in both cell lines with the ruthenium
complexes 1-5, the cells were washed and half of the samples were irradiated at 450
nm (10 mins for HeLa cells, dose: 10 J cm-2 and 2 mins for RPE-1 cells, dose: 2 J
cm-2) and then allowed to grow in the incubator for another 44 h. Longer irradiation
times are not feasible in RPE-1 cells because these cells are not robust enough and
prolonged irradiation leads to profound cell death. Similar experiments were also
conducted without light irradiation to serve as controls. The antiproliferative
properties of the ruthenium complexes on both samples – with and without irradiation
– were determined by the resazurin assay where the fluorescence of resorufin was
measured and the results are presented in Table 1.
11

Table 1: IC50 values of complexes 1-5 in different cell lines with and without light
irradiation.
HeLa - IC50 (µM)
RPE-1 - IC50 (µM)
Complex
Structure
(450 nm, ~
10 J.cm-2)
(450 nm, ~
2 J.cm-2)
In the
dark
In the dark
PI
PI
1
2
11.3 ± 0.9
20.3 ± 2.2
1.8
76.3 ± 2.7
75.7 ± 3.0 1.0
19.9 ± 2.1 1.3
7.6 ± 0.5
11.0 ± 1.6
29.9 ± 2.6
1.4
1.3
15.4 ± 1.7
83.6 ± 4.0
3
22.9 ± 0.7
79.2 ± 2.6 0.9
4
5
14.0 ± 0.4
12.3 ± 1.8
17.9 ± 1.4
30.4 ± 4.6
1.3
2.5
8.1 ± 0.8
8.9 ± 1.5 1.1
28.6 ± 0.4 1.0
27.7 ± 2.7
From these data, we can observe several trends concerning the photo-activation.
First, for RPE-1 cell line, there is no significant difference between irradiated and
non-irradiated cells, which suggests two different possibilities: either the dose used of
2 J.cm-2 is not enough to induce the photo-release of the arene or the arene ligand is
already gone before light irradiation. When the corresponding results with HeLa is
analyzed – this time with a five-time higher dose –, a small difference is observed
when the arene ligand is an ethylbenzoate, but a more significant one appeared for
the complexes bearing a p-cymene ligand. The number of compounds is too low to
draw definitive conclusions. However, it suggests that most of ethylbenzoate is
released before light irradiation and that p-cymene-complexes need more than 2
J.cm-2 to be photo-activated. The case of the complex 5 is even more interesting. It is
clear that it requires at least 10 J.cm-2 to be activated, but this time, the photo-
activation must involve another mechanism.
12

IC50 values themselves indicate that the complexes display interesting properties
against HeLa cells. However, the main point of this study is that cycloheptadienyl
complexes 1 and 3 are more efficient on the cancer cell line than on the healthy one
(3.7 and 2.6 times more efficient, respectively and even 6.7 and 3.7 if we compare
the value after irradiation). These results are very promising for future applications
because they suggest that these products may display an in vivo selectivity for tumor
vs. healthy tissues. Concerning the compound 5, its IC50 needs to be taken with
caution due to the fact that compound 5 contains two ruthenium ions. Thus, it can be
concluded that compounds 1 and 3 display very high antiproliferative activities on the
HeLa cancer cell line along with moderate to low antiproliferative properties on the
non-cancerous RPE-1 cell line. Ruthenium-arene complexes bearing a p-cymene
ligand can be photo-activated in vitro, but require more energy and the unusual
cationic bimetallic complex display interesting antiproliferative properties, especially
when photo-activated. The mechanism of action of the latter is still under
investigation.
4. Conclusions
In this study, we investigated the biological activity of four ruthenium(II) complexes
bearing (3,5-cycloheptadienyl) diphenylphosphine or (cycloheptyl)diphenylphosphine
and ethylbenzoate or p-cymene and an unusual bis-ruthenium complex. All the
complexes were found to be stable in DMF and three of them display good stability in
culture medium in the dark. On the contrary, they rapidly degrade under light
exposure. All complexes display significant antiproliferative properties against the
HeLa cancer cell line. Interestingly, ruthenium-arene complexes bearing (3,5-
cycloheptadienyl) diphenylphosphine present a limited toxicity against the healthy
model cell line RPE-1. We could also demonstrate the possibility to further activate
the different complexes by light irradiation (450 nm), which results in an increase of
the antiproliferative properties by a factor up to 2.5. This photo-activation results in
the formation of an activated ruthenium complex produced by the released of the
arene ligand for complexes 1-4. Concerning the bis-ruthenium, the photoactivation
mechanism is still under investigation.
Acknowledgments
This work was financially supported by an ERC Consolidator Grant PhotoMedMet to
G.G. (GA 681679) and has received support under the program “Investissements d’
13

Avenir” launched by the French Government and implemented by the ANR with the
reference ANR-10-IDEX-0001-02 PSL (G.G.).Support was also provided by the
Conseil Régional de Bourgogne Franche-Comté, the Ministère de l’Enseignement
Supérieur et de la Recherche, the Centre National de la Recherche Scientifique
(CNRS), and the French Research National Agency (ANR) via project JCJC “SPID”
ANR-16-CE07-0020 (E.B.). This work is part of the project PHARMACOIMAGERIE
ET AGENTS THERANOSTIQUES and of the project CHIMIE DURABLE,
ENVIRONNEMENT ET AGROALIMENTAIRE, supported by the Université de
Bourgogne, Conseil Régional de Bourgogne through the plan d'actions régional pour
l'innovation (PARI) and the European Union through the PO FEDER-FSE Bourgogne
2014/2020 programs. FrenchBIC is acknowledged for fruitful discussion.
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Four Ru(II)-arene complexes which are stable in DMF were found to display
significant anti-proliferative properties against HeLa cancer cell line. We could also
demonstrate the possibility to further activate the different complexes by light
irradiation, which results in an increase of the anti-proliferative properties to a factor
up to 2.5.
17