Thiabendazole-based Rh(III) and Ir(III) biscyclometallated complexes with mitochondria-targeted anticancer activity and metal-sensitive photodynamic activity

Article abstract of DOI:10.1016/j.ejmech.2018.07.065

Two pairs of Rh(III) and Ir(III) biscyclometallated complexes with thiabendazole (L¹), named [Ir-a]Cl and [Rh-a]Cl, and N-benzyl-thiabendazole (L²), named [Ir-b]Cl and [Rh-b]Cl, have been designed and synthesized to explore the photophysical and biological effects that arise from changing both the metal center and the ancillary ligand. In the dark, the four metal complexes exhibit greater cytotoxicity than cisplatin against human colon (SW480) and human lung (A549) adenocarcinoma cell lines. Moreover, the pair of complexes bearing the ligand L² is markedly more cytotoxic and present higher uptake values than complexes with L¹, thereby their biological properties were studied further to determine their mechanism of action. Interestingly, in spite of the different metal center both the [Ir-b]Cl and [Rh-b]Cl complexes are responsible for the loss of mitochondrial functionality and the activation of apoptotic cell death pathways. Moreover, the photodynamic activity of the four complexes, [Ir-a,b]Cl and [Rh-a,b]Cl, was tested using visible blue light (460 nm) under soft irradiation conditions (20 min, 5.5 mW cm^?2). While the Rh complexes are not photopotentiated, the phototoxicity index (IC₅₀ non-irradiated/IC₅₀ irradiated) of [Ir-a]Cl and [Ir-b]Cl complexes was 15.8 and 3.6, respectively. We also demonstrate that only the Ir derivatives are capable of photocatalyzing the oxidation of S-containing L-amino acids under blue light irradiation, [Ir-a]Cl being more active than [Ir-b]Cl, which provides a reasonable mechanism for their biological action (oxidative stress could be selectively promoted through a photocatalytic action) upon irradiation. This different PDT behaviour depending on the metal center and the ancillary substituent may be useful for future rational design of metal-based photosensitizers.

Full text of DOI:10.1016/j.ejmech.2018.07.065

Accepted Manuscript
Thiabendazole-based Rh(III) and Ir(III) biscyclometallated complexes with
mitochondria-targeted anticancer activity and metal-sensitive photodynamic activity
Cristina Pérez-Arnaiz, María Isabel Acuña, Natalia Busto, Igor Echevarría, Marta
Martínez-Alonso, Gustavo Espino, Begoña García, Fernando Domínguez
PII:
S0223-5234(18)30630-5
10.1016/j.ejmech.2018.07.065
EJMECH 10600
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 May 2018
Revised Date: 26 July 2018
Accepted Date: 28 July 2018
Please cite this article as: C. Pérez-Arnaiz, Marí.Isabel. Acuña, N. Busto, I. Echevarría, M.
Martínez-Alonso, G. Espino, Begoñ. García, F. Domínguez, Thiabendazole-based Rh(III) and
Ir(III) biscyclometallated complexes with mitochondria-targeted anticancer activity and metal-
sensitive photodynamic activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.07.065.
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ACCEPTED MANUSCRIPT
Thiabendazole-based Rh(III) and Ir(III) Biscyclometallated Complexes
with Mitochondria-Targeted Anticancer Activity and Metal-Sensitive
Photodynamic Activity
a,b
b
a
a
a
Cristina Pérez-Arnaiz, María Isabel Acuña, Natalia Busto, Igor Echevarría, Marta Martínez-Alonso,
a,*
a,*
b,*
Gustavo Espino, Begoña García, Fernando Domínguez
a
Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Bañuelos s/n,
9001, Burgos, Spain.
0
b
CIMUS, Universidad de Santiago de Compostela, Avenida Barcelona s/n, 15782, Santiago de
Compostela, Spain.
Keywords: Biscyclometalated Iridium(III) and Rhodium(III) Complexes / thiabendazole / Anticancer /
Photodynamic Therapy / O photosensitizers.
2
TOC GRAPHICS
ABSTRACT
1
Two pairs of Rh(III) and Ir(III) biscyclometallated complexes with thiabendazole (L ), named
2
[
Ir-a]Cl and [Rh-a]Cl, and N-benzyl-thiabendazole (L ), named [Ir-b]Cl and [Rh-b]Cl, have
been designed and synthesized to explore the photophysical and biological effects that arise
from changing both the metal center and the ancillary ligand. In the dark, the four metal
complexes exhibit greater cytotoxicity than cisplatin against human colon (SW480) and human
2
lung (A549) adenocarcinoma cell lines. Moreover, the pair of complexes bearing the ligand L
1
is markedly more cytotoxic and present higher uptake values than complexes with L , thereby
their biological properties were studied further to determine their mechanism of action.
Interestingly, in spite of the different metal center both the [Ir-b]Cl and [Rh-b]Cl complexes
are responsible for the loss of mitochondrial functionality and the activation of apoptotic cell
death pathways. Moreover, the photodynamic activity of the four complexes, [Ir-a,b]Cl and
[
Rh-a,b]Cl, was tested using visible blue light (460 nm) under soft irradiation conditions
-2
(
20 min, 5.5 mW cm ). While the Rh complexes are not photopotentiated, the phototoxicity
index (IC non-irradiated/IC irradiated) of [Ir-a]Cl and [Ir-b]Cl complexes was 15.8 and 3.6,
50
50
1

ACCEPTED MANUSCRIPT
respectively. We also demonstrate that only the Ir derivatives are capable of photocatalyzing the
oxidation of S-containing L-amino acids under blue light irradiation, [Ir-a]Cl being more active
than [Ir-b]Cl, which provides a reasonable mechanism for their biological action (oxidative
stress could be selectively promoted through a photocatalytic action) upon irradiation. This
different PDT behaviour depending on the metal center and the ancillary substituent may be
useful for future rational design of metal-based photosensitizers.
1
. Introduction
A great deal of effort has been invested over the past few decades to develop non-platinum
metal complexes with anticancer activity by mechanisms different from those of cisplatin to
circumvent the platinum resistance and its undesired side effects. In line with this,
cyclometalated complexes of various metal centres such as palladium(II), gold(III),
ruthenium(II), platinum(II), rhodium(III) and iridium(III) constitute an interesting field of
research due to their variety of cellular targets and mechanisms of action [1]. Among the
former, mitochondria stands out, as it plays a key role in the cell metabolism. It is well known
that central metabolic pathways operating in malignant cells are different from those in normal
cells [2,3]. Cancer cells exhibit various degrees of alterations in the mitochondria function, such
as higher mitochondrial membrane potential (MMP) and increased oxidative stress [4]. Because
of this, recent studies point to the potential benefits of targeting mitochondria metabolism for
anticancer therapy[5] and several mitochondria-targeted drugs are currently in clinical trials [6].
These drugs often act by directly disturbing mitochondrial function, which triggers the
activation of mitochondrial cell death pathways via apoptosis.
+
Biscyclometalated complexes of the type [Ir(C^N) (N^N)] present a wide variety of cellular
2
targets depending on the ancillary ligands. Indeed, mitochondria [7–12], lysosomes[13–15],
endoplasmic reticulum [16,17], endosomes[18] or nucleus[19,20] have all been recognized as
target organelles for different Ir(III) biscyclometalated derivatives. Nonetheless, cyclometalated
Rh(III) complexes have not received much attention so far, probably due to the lack of the
exceptional photophysical properties typical of analogous Ir(III) complexes. However, Rh
complexes could also be a potential alternative to platinum metallodrugs [21], and their
biological properties deserve further study.
In addition, Photodynamic Therapy (PDT) has emerged as a prominent chemotherapeutic anti-
cancer treatment, as it renders feasible local and transitory activation of drugs to achieve spatio-
temporal control over its biological action and therapeutic effects [22]. The resulting selective
activity over target (tumor) tissues eventually contributes to reducing systemic toxicity. PDT
requires the combined action of three individually nontoxic elements: light, oxygen and a
photosensitizer (PS), in such a way that the excited PS agent (PS*) can generate reactive oxygen
2

ACCEPTED MANUSCRIPT
1
3
species (ROS) and specifically singlet oxygen ( O ) from triplet oxygen ( O ) through energy
2
2
transfer (ET) upon excitation with UV, visible or even near-infrared (NIR) light of specific
1
wavelengths [23]. O is a very reactive species and can damage biomolecules such as DNA and
2
1
proteins through redox reactions, which ultimately triggers cancer cell death. Alternatively, O2
can evolve to other cytotoxic ROS. Cyclometalated Ir(III) complexes are currently being studied
as potential PDT agents due to their outstanding photophysical properties [23,24]. The crucial
advantage over organic fluorescent molecules is the high spin-orbit coupling constant of the
-1
iridium metal core (ξ = 3909 cm ) that favors the singlet-to-triplet intersystem crossing (ISC)
25]. This peculiarity is responsible for other features of these Ir(III) compounds such as their
[
long PS triplet excited state lifetimes, their appropriate energy gap that exceeds the excitation
3
energy of O and, overall, their high capability as O photosensitizers [26].
2
2
In this context, arylazoles are attractive ancillary N^N ligands, as they can be prepared and
modified readily and can offer a remarkable structural and chemical diversity. Therefore, they
can be used to fine-tune the photophysical and chemical properties of this type of
organometallic compounds [27]. Specifically, the imidazole ring can be easily functionalized
through the reactive N−H bond [28]. In this work, we have replaced the N-H function of
1
2
thiabendazole (L ) with a benzyl group N-CH Ph (L ) in an attempt to increase their
2
lipophilicity, which could improve their cellular internalization properties [29], and their chemo-
stability as well, since the N-H group can exhibit both acid-base and redox reactivity. As a
result, we have prepared two pairs of Rh(III) and Ir(III) biscyclometallated complexes of
general formula [M(ppy) (N^N)]Cl (M = Rh, Ir; ppy = 2-phenylpyridinate) bearing N^N ligands
2
1
2
based on the thiabendazole scaffold (L and L ), and we have studied their potential use as
anticancer and PDT agents as well. First, we demonstrate that actually the rational benzyl
2
modification in L yields an appreciable improvement in the cytotoxic activity in the dark. A
2
deeper biological study demonstrates that both complexes bearing the L ligand induce
apoptosis via mitochondrial perturbation. Secondly, as there is major interest in finding
sensitizers capable of potentiating their cytotoxic activity upon irradiation [27], we also tested
the PDT behavior of the four complexes under soft irradiation conditions, using blue visible
light (λmax = 460 nm) and minimizing the exposure time of cells to light. Interestingly, only the
1
2
Ir complexes, bearing either the ligand L or L , present a remarkable photopotentiation of their
cytotoxic activity and are capable of photocatalyzing the oxidation of S-containing L-amino
acids.
2
. Results and discussion
3

ACCEPTED MANUSCRIPT
2
.1. Synthesis of Ligands and Complexes
2
The ancillary ligand L (see Scheme 1) was prepared by reacting 2-(4-thiazolyl)benzimidazole
1
(
thiabendazole, TBZ, L ) with benzyl bromide in the presence of K CO as the base and using
2
3
DMF as the solvent, according to a method adapted from the literature [30,31].
The complexes [Rh-a,b]Cl and [Ir-a,b]Cl depicted in Scheme 1 were synthesized following the
classical two-steps procedure reported by Nonoyama et al.[32] and the experimental details are
given in the ESI. These complexes were isolated as white (Rh) or yellow (Ir) solids, consisting
of racemic mixtures of ∆ and Λ enantiomers with good yields and purities according to
analytical and spectroscopic data. Moreover, they are soluble in common organic solvents such
as DMSO, acetone, CH CN, methanol and CH Cl and only slightly soluble in water. The
3
2
2
structure and composition of all the new derivatives were determined by multinuclear NMR, IR,
and mass spectroscopy and by elemental analysis. 2D NMR experiments were conducted to
1
13
1
partially assign the signals in the H and C{ H} NMR spectra (Figures S1-S6).
2
Scheme 1. Synthesis of ligand L as well as Rh(III) and Ir(III) complexes
.
2
.2. Characterization of Complexes
1
13
1
The H and C{ H} NMR spectra of complexes [Rh-a,b]Cl and [Ir-a,b]Cl showed resonances
for the coordinated ancillary (N^N) and cyclometalated (C^N) ligands with the following
relevant features: (1) Two different sets of peaks were observed for the two inequivalent 2-
phenylpyridinate (ppy⁻) ligands of every complex due to their C -asymmetric nature; (2) Broad
1
4

ACCEPTED MANUSCRIPT
1
singlets around 15 ppm were recorded for the N-H protons of [Rh-a]Cl and [Ir-a]Cl in the H
2
NMR spectra; (3) Two mutually coupled doublets ( JHH = 17.5 Hz) were detected at 6.2 and 6.1
1
ppm for the diastereotopic protons of the –CH groups in the H NMR spectra of [Rh-b]Cl and
2
2
[
(
[
Ir-b]Cl owing to the helical chirality implicit in trischelate octahedral complexes, while free L
1
3
1
achiral) gave a singlet for the –CH ; (4) The C{ H} NMR spectra of complexes [Rh-b]Cl and
2
Ir-b]Cl exhibited a singlet at around 48 ppm for the –CH group, whereas the spectra of both
2
1
103
13
Rh derivatives showed two doublets ( J( Rh- C) ≈ 33 Hz) above 165 ppm, for the metallated
C atoms of ppy‾.
The ESI+ MS spectra display peaks whose m/z values and isotopic distribution are congruent
n
+
with those calculated for the cationic metal complexes with formula [M(ppy) (L )] (M = Rh, Ir;
2
n
1
2
L = L , L ). Molar conductivity measurements carried out on solutions of complexes [Rh-b]Cl
-3
and [Ir-b]Cl in acetonitrile (10 M) are consistent with a 1:1 electrolyte nature. However,
complexes [Rh-a]Cl and [Ir-a]Cl provided very low values of ΛM likely due to ion pairing
through N-H---Cl hydrogen bonding interactions (see ESI).
2
.3. Crystal Structures
Attempts to crystalize the chloride salts [Rh-b]Cl and [Ir-b]Cl were unsuccessful. However,
single crystals of the respective PF ‾ salts, [Rh-b]PF and [Ir-b]PF ·CH COCH suitable for
6
6
6
3
3
X-ray diffraction analysis were obtained by slow diffussion of a saturated solution of NH PF in
4
6
water into a solution of either [Rh-b]Cl in methanol or [Ir-b]Cl in methanol/acetone. The
ORTEP diagrams for (∆)-[Rh-b]PF and (∆)-[Ir-b]PF are depicted in Figure 1. Selected bond
6
6
lengths and angles and crystallographic refinement parameters are given in the ESI (Tables S1
and S2). The corresponding unit cells show two pairs of the enantiomers (Λ) and (∆) resulting
-
from helical chirality. Furthermore, the unit cell contains four PF6 counterions and four
CH COCH molecules in the case of [Ir-b]PF . The metal centers display a slightly distorted
3
3
6
octahedral coordination geometry with the expected cis-C,C and trans-N,N mutual disposition
for the C^N ligands.
The M-Cppy and M-Nppy (M = Rh, Ir) bond distances lie within the expected ranges for the
2
-phenylpyridanate ligands, that is, very close to 2 Å [33–38]. The M-N bond lengths for the
ancillary ligand are significantly longer due to the strong trans influence attributed to the C
atoms of the C^N ligands [24,39], and the effect is even more pronounced in the Rh complex
(
Table S1) [40]. The bond angles are comparable to those previously reported for similar
derivatives [15,41]. Furthermore, the three chelate rings are essentially planar.
5

ACCEPTED MANUSCRIPT
The 3D crystal networks of [Rh-b]PF and [Ir-b]PF ·CH COCH are mainly held down by
6
6
3
3
intermolecular hydrogen bonding contacts with involvement of PF ‾ anions as acceptors (Tables
6
S3 and S4 and Figures S7-a and S7-b). Remarkably, the crystal structure of [Ir-a]PF6,
previously reported by some of us [42], features dimeric entities as a result of π-π interactions
between the benzimidazole units of two neighbour complexes. However, similar contacts have
not been found either in [Rh-b]PF nor in [Ir-b]PF . Consequently, we surmise that the
6
6
bulkiness of the benzyl group works against these intermolecular interactions, preventing
[
Ir-b]PF from aggregation in highly concentrated solutions or cellular environments [43]. The
6
2
crystal structure of free L has also been determined by X-diffraction (see Figure S8).
Figure 1. ORTEP diagrams for (∆)-[Rh-b]PF and (∆)-[Ir-b]PF forming part of the asymmetric unit of
6
6
racemic [Rh-b]PF and [Ir-b]PF ·CH COCH . Thermal ellipsoids are shown at the 30% probability
6
6
3
3
level. The molecule of CH COCH has been omitted in the diagram of [Ir-b]PF ·CH COCH for the
3
3
6
3
3
sake of clarity.
2
.4. pH Stability
The pH effect in aqueous media was evaluated for the four metal complexes. As expected,
1
[
Rh-a]Cl and [Ir-a]Cl undergo deprotonation of the reactive N-H group in L upon increasing
the pH, and display pK values of 7.30 ± 0.01 and 6.76 ± 0.03 (see Figure S9), respectively,
a
which implies that they would be partially deprotonated at physiological pH. On the other hand,
[
Rh-b]Cl and [Ir-b]Cl do not undergo acid-base processes over the whole pH range tested
(
pH = 3–12).
2
.5. Photophysical Properties
The UV-Vis absorption spectra of complexes [Rh-a,b]Cl and [Ir-a,b]Cl were recorded in air-
saturated aqueous buffer and acetonitrile (See Figure 2.A and Figure S10). All the complexes
6

ACCEPTED MANUSCRIPT
-1
-1
exhibit intense (ε > 30000 M cm ), broad absorption bands in the UV region below 340 nm,
1
1
attributed to spin-allowed intra-ligand transitions ( IL) ( π → π*) within the cyclometalated
N^C) and ancillary (N^N) ligands. In the case of the Rh(III) complexes, the band at about
70 nm is attributed to a MLCT transition. On the other hand, the absorption of the Ir(III)
(
3
complexes (MLCT) spreads into the visible region of the spectrum.
The emission spectra of the four complexes were also recorded in air-saturated aqueous buffer
(
Figure 2.B) and deoxygenated acetonitrile (Figure S11) and the photophysical parameters are
collected in Table S5. The Ir complexes present excitation bands at λexc = 305 nm and
λexc = 385 nm and exhibit broad emission bands with one maximum for [Ir-a]Cl at 500 nm and
two maxima for [Ir-b]Cl at 480 nm and 506 nm. Both Ir complexes display large Stokes shifts,
moderate quantum yields (φPL) and long emission lifetimes. On the contrary, the Rh complexes
displayed no emission bands at any of the tested excitation wavelengths (λexc = 290 - 500 nm).
The emission spectra of the four metal complexes are collected in Figure 2.B, revealing the
noticeable dependence of the photophysical properties on the nature of the metal center.
A
B
7
5
2
5
0
5
0
[
[
[
Ir-a]Cl
Ir-b]Cl
Rh-a]Cl
[Ir-a]Cl
[Ir-b]Cl
[Rh-a]Cl
[Rh-b]Cl
0
0
0
0
.6
.4
.2
.0
[Rh-b]Cl
3
00
400
500
450
525
600
675
λ
,
nm
λ, nm
Figure 2. Absorption (A) and emission (B) spectra of the four metal complexes at a concentration
Ccomplex = 15 µM in aqueous air-saturated buffer (2.5 mM NaCaC, pH = 7.0) at λexc = 385 nm and
o
T = 25 C.
2
.6. Biological Properties
2
.6.1. In Vitro Cytotoxicity. The in vitro antiproliferative activity of [Ir-a,b]Cl and [Rh-a,b]Cl
was evaluated against human lung carcinoma (A549) and human colon adenocarcinoma
SW480) cell lines to compare the cytotoxic activity of the four metal complexes. The IC (half
(
5
0
maximal inhibitory concentration) values are summarized in Table 1. The four metal complexes
exhibit lower IC50 values than cisplatin against both cell lines. Moreover, the Ir and Rh
2
complexes bearing the ligand L , [Ir-b]Cl and [Rh-b]Cl, exhibit greater cytotoxic activity than
1
their analogues bearing the ligand L .
7

ACCEPTED MANUSCRIPT
Table 1. IC50 (µM) of [Ir-a,b]Cl, [Rh-a,b]Cl and cisplatin against A549 (human lung) and SW480
(
human colon) cancer cell lines after 24 h of treatment. Data were obtained from quadruplicates of three
independent experiments and expressed as mean values ± standard deviation (SD).
IC (µM)
5
0
Complex
A549
SW480
10.0 ± 0.1
7.7 ± 0.4
2.9 ± 0.1
3.3 ± 0.1
37 ± 6
[
[
[
[
Ir-a]Cl
Rh-a]Cl
Ir-b]Cl
Rh-b]Cl
18.9 ± 0.2
10.3 ± 0.1
4.0 ± 0.4
4.1 ± 0.2
50 ± 3
Cisplatin
2
.6.2. Cellular Uptake. The cellular uptake of the two pairs of complexes was evaluated to
2
assess if the benzyl modification in L , which provides the complexes with stability against
acid-base reactions at physiological pH, succeeded in increasing the cellular internalization of
the complexes. A549 cells were treated with each complex at a concentration of 5 µM for 4
hours and then harvested and analyzed by ICP-MS. Indeed, the uptake of [Ir-a]Cl was a
5
0.9 % (± 5) lower than that of [Ir-b]Cl, and the uptake of [Rh-a]Cl was a 23.3 % (± 2) lower
than that of [Rh-b]Cl (Figure S12). For this reason, the pair of complexes formed by [Ir-b]Cl
and [Rh-b]Cl, which are also the most cytotoxic (see Table 1), were selected to carry out a
deeper biological study and determine their mechanism of action.
2
.6.3. Cell Cycle Arrest. The cytotoxicity of many anticancer drugs often is associated with
DNA damage and cell cycle perturbation [44]. For instance, although cisplatin interacts with
several cell components, its primary biological target is DNA [45]. As a result, this antitumour
complex causes arrest at the G2-phase [46]. The effect of [Ir-b]Cl and [Rh-b]Cl on cell cycle
distribution was investigated by flow cytometry and propidium iodide (PI) staining (Figure 3).
Treatment of A549 cells with the complexes at a concentration close to their IC values (5 µM)
5
0
caused a marked accumulation of cells in the G0/G1 phase. Compared with the vehicle-treated
DMSO) control, the percentage of cells in the G0/G1 phase increased by 32.8% and 33.3% at
4 h and 35.9% and 36.9% at 48 h for [Ir-b]Cl and [Rh-b]Cl, respectively. Accordingly, the
(
2
number of cells in the S-phase strongly decreased (Table 2). These results point out that both
complexes can act through mechanisms of action different from cisplatin [46]. Although some
previously reported mitochondria-targeted compounds display no effects on the cell cycle
distribution [47,48], our results concur with recent studies on other Ir(III) complexes that also
can induce dose-dependent G0/G1 cell cycle arrest [7].
8

ACCEPTED MANUSCRIPT
A)
[Ir-b]Cl
[Rh-b]Cl
Fluorescence Intensity
Ir-b]Cl
B)
[Rh-b]Cl
[
Fluorescence Intensity
Figure 3. Effect of [Ir-b]Cl and [Rh-b]Cl on A549 cell cycle distribution analysed by PI staining with
flow cytometry after treatment at Ccomplex = 5 µM for 24 h (A) and for 48 h (B). Vehicle-treated cells are
represented by the grey area and [Ir-b]Cl and [Rh-b]Cl-treated cells by the black line.
Table 2. Percentage (%) of cells in the G0/G1, S and G2-M phases of the cell cycle upon treatment with
[
Ir-b]Cl and [Rh-b]Cl at 5 µM for 24 h and 48 h. Differences between % of cells in G0/G1 phase for
vehicle-treated cells and cells treated with [Ir-b]Cl or [Rh-b]Cl are significant at p = 0.0009 after 24 h
and at p = 0.002 at 48 h. Differences between % of cells in S phase for vehicle-treated cells and cells
treated with [Ir-b]Cl or [Rh-b]Cl are significant at p = 0.0004 after 24 h and at p = 0.0008 at 48 h. Data
were obtained from duplicates of two independent experiments and expressed as mean values ± standard
deviation (SD).
2
4 h
48 h
S
G0/G1
S
G2/M
G0/G1
G2/M
Vehicle
49.7 ± 0.9 27.6 ± 0.1 22.7 ± 0.1
52.8 ± 0.8 25.5 ± 0.7 21.7 ± 0.5
[
[
Ir-b]Cl
82 ± 1
Rh-b]Cl 83.0 ± 0.3
7.4 ± 0.6
7.9 ± 0.6
10.6 ± 0.5
9.1 ± 0.9
89 ± 1
89.7 ± 0.7
4.7 ± 0.3
1.8 ± 0.2 8.5 ± 0.5
6.3 ± 0.1
2
.6.4. Induction of apoptosis. The evolution of cell morphology was followed for 48 h upon
treatment with 5 µM of [Ir-b]Cl or [Rh-b]Cl to seek for morphological hallmarks of apoptosis.
After 48 h, a net fall in the number of cells was observed compared to the vehicle-treated cells.
Morphological changes compatible with apoptosis: cell shrinkage, rounded form, membrane
blabbing and formation of apoptotic bodies were seen and the number of cells affected increased
9

ACCEPTED MANUSCRIPT
with time (Figure S13). Staining with Hoechst and propidium iodide (PI) can help to
differentiate between apoptotic and necrotic cells. After 24 h of treatment with each complex
few PI positive cells were found, and they were associated with secondary necrosis with a
balloon-like morphology (Figure 4). Thus, necrosis can be excluded as the principal cause of
dead.
BF
Hoechst
PI
[
Ir-b]Cl
[
Rh-b]Cl
Vehicle
Figure 4. Morphological changes induced by [Ir-b]Cl, [Rh-b]Cl and DMSO (vehicle) in A459 cells after
treatment at Ccomplex = 5 µM for 24 h. Bright field (BF), Hoechst 33342 nuclear staining (Hoechst) and
Propidium Iodide necrotic cells staining (PI) (Magnification 20X).
[
Ir-b]Cl treated cells show an apoptotic morphology (red arrows): rounding, membrane babbling,
retraction of cytoplasm, nuclear fragmentation, formation of apoptotic bodies. Some cells presented
secondary necrosis (white arrows).
[
Rh-b]Cl treatment induced cytoplasmic vacuole, rounding cells and emission of cytoplasmic
prolongations. No necrotic cells were seen with PI staining.
To further confirm that the cell death occurred via apoptosis, time-lapse fluorescence
microscopy was used to detect activation of caspase-3/7. A549 cells were treated with [Ir-b]Cl
or [Rh-b]Cl (5 µM) and images were recorded every 30 min for 24 h to monitor the process.
o
During this procedure, the cells remained continuously under culture conditions at 37 C and
5
% CO . In the case of [Ir-b]Cl, its intrinsic green emission interfered with the fluorogenic
2
probe used to detect the activation of caspase-3/7. However, this activation was clearly observed
for [Rh-b]Cl, with the bulk amount of activation events starting after 12 h the treatment
(
Supplemental video).
1
0

ACCEPTED MANUSCRIPT
Additionally, A549 cells were treated with [Ir-b]Cl or [Rh-b]Cl for 24 h in the presence or
absence of 20 µM of the Z-VAD-FMK caspase inhibitor. Figure 5 shows that caspase inhibition
increases the survival rate after treatment with both [Ir-b]Cl and [Rh-b]Cl in a significant
manner (p < 0.03) by 17.4 % and 18.1 %, respectively. This confirms the role of apoptosis in the
cell death induced by these complexes [49].
Figure 5. A549 cells were treated with 5 µM of [Ir-b]Cl or [Rh-b]Cl and co-treated (+ Z-VAD-FMK) or
not (- Z-VAD-FMK) with the caspase inhibitor Z-VAD-FMK for 24 h. Cell survival was assessed by
flow cytometry through the Guava ViaCount Assay (Millipore). Difference significant at *p < 0.03
compared to cells not treated with the caspase inhibitor.
2
.6.5. Cellular Localization. The subcellular localization of [Ir-b]Cl can be determined by
confocal microscopy thanks to its intrinsic emission properties. The colocalization experiments
were performed after treatment with a low dose of [Ir-b]Cl (1.5 µM), in which cell survival was
nearly 100 %. As shown in Figure 6, [Ir-b]Cl displays an intense and punctate staining pattern
as a result of its compartmentalization. Colocalization experiments of [Ir-b]Cl with the
sensitive probe for mitochondrial membrane potential, TMRM (Figure 6.A) and with the
lysosome dye LysoTracker (Figure 6.B) demonstrates that [Ir-b]Cl selectively accumulates in
mitochondria. In fact, Pearson’s colocalization coefficient with TMRM is 0.80, whereas with
LysoTracker the value is 0.09.
1
1

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A
Bright Field
TMRM
Merged
[Ir-b]Cl
B
Bright Field
LysoTracker
Merged
[Ir-b]Cl
Figure 6. A) Representative confocal images of A549 cells exposed to [Ir-b]Cl (1.5 µM, 1h) and to
TMRM (100 nM, 30 min). B) Representative confocal images of A549 cells exposed to [Ir-b]Cl (1.5 µM,
1
h) and to LysoTracker (75 nM, 30 min).
1
2

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2
.6.6. Effect on the Mitochondrial Membrane Potential. To assess if the accumulation of
complex [Ir-b]Cl in mitochondria leads to an actual mitochondrial dysfunction, mitochondrial
membrane potential (MMP, ∆Ψ ) was analysed after staining living cells with the fluorescent
m
cationic red dye TMRM, which accumulates only in active, polarized mitochondria due to their
relatively negative charge. Depolarized or inactive mitochondria displays decreased membrane
potential and fail to sequester TMRM. Treatment with 5 µM of [Ir-b]Cl or [Rh-b]Cl leads to
quick loss of TMRM fluorescence in less than 2 h (Figure 7). These results indicate that
[
Ir-b]Cl and [Rh-b]Cl cause mitochondrial depolarization.
[Ir-b]Cl
[Rh-b]Cl
Vehicle
A
B
Figure 7. Effect of [Ir-b]Cl and [Rh-b]Cl treatment on the mitochondrial membrane potential observed
by TMRM fluorescence (red). Representative images of A549 cells treated with DMSO (vehicle),
[
Ir-b]Cl (5 µM) and [Rh-b]Cl (5 µM) for 2 h under the same experimental conditions. A) Phase contrast
image. B) Red fluorescence emission.
2
.6.7. Intracellular ROS detection. It is well known that mitochondrial depolarization is
followed by other mitochondrial and non-mitochondrial perturbations. Mitochondria are the
major source of intracellular reactive oxygen species (ROS), and mitochondrial dysfunction is
closely related to ROS accumulation in the process of apoptosis [50]. To elucidate whether the
observed depolarization of mitochondria (Figure 7) led to ROS accumulation in the cell, the
intracellular ROS levels were examined by using DHE (dihydroethidium) and H2DCFDA
(
(
2',7'-dichlorodihydrofluorescein diacetate). DHE allows one to measure cytosolic superoxide
•–
O ) and H2DCFDA other radical species including hydrogen peroxide (H O ), in intact
2
2
2
adherent cells [51]. These probes are oxidized by the reactive species to generate the
corresponding fluorescent products, which then can be measured by flow cytometry. [Ir-b]Cl
could not be included in this assay due to its green emission that partially overlapped with the
fluorescent probes. A549 cells were treated with [Rh-b]Cl (5 µM) for 2 or 4 h. Treatment with
1
3

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H O (0.25 mM) was used as a positive control, as it causes oxidative stress that generates ROS
2
2
overproduction in the cell. Figure 8.A shows that the population of cells with enhanced levels
of superoxide after treatment with [Rh-b]Cl is even higher than after treatment with H O . For
2
2
each experiment, the fluorescence of non-treated cells (negative control) is shown in grey. The
generation of other radical species was also detected (Figure 8.B), but this time the treatment
with H O induced a greater effect than the [Rh-b]Cl treatment. These results entail that
2
2
superoxide is probably the main radical species generated in the cell by [Rh-b]Cl.
H O (0.25 mM, 2 h)
H O (0.25 mM, 4 h)
2 2
A
2
2
[Rh-b]Cl (5 µM, 2 h)
[Rh-b]Cl (5 µM, 4 h)
Fluorescence Intensity
H O (0.25 mM, 2 h)
H O (0.25 mM, 4 h)
B
2
2
2
2
[Rh-b]Cl (5 µM, 2 h)
[Rh-b]Cl (5 µM, 4 h)
Fluorescence Intensity
Figure 8. The level of intracellular ROS was detected by DHE (A, yellow) and H2DCFDA (B, green)
assays using flow cytometric analysis in A549 cells treated with [Rh-b]Cl (5 µM) or H O (0.25 mM) for
2
2
2
or 4 h. Grey: control cells; coloured: treated cells.
2
.6.8. Oxidized Cardiolipin Study. Because of depolarization, mitochondria overproduce ROS
which, in turn, oxidize mitochondrial cardiolipins. The damage in mitochondria produced by
1
4

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ROS can be determined indirectly by assessing the oxidation state of cardiolipin, a molecule
restricted to the inner mitochondrial membrane that plays a key role in the apoptotic cell death
pathway [52]. The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for
location and quantitative determination of cardiolipin in living cells. NAO interacts with intact,
nonoxidized cardiolipin forming NAO dimers, which can be detected based on their red
emission [53]. Therefore, a reduction in NAO fluorescence indicates a decrease in cardiolipin
content [54]. A549 cells were preincubated with NAO and then treated for 4 h in the presence of
different concentrations of [Rh-b]Cl. The fluorescence of NAO in the cells was then measured
by flow cytometry (Figure 9). [Rh-b]Cl featured a great dose-dependent impact on NAO
fluorescence intensity in agreement with cardiolipin oxidation. Again, [Ir-b]Cl could not be
included in this study due to its emission features.
Vehicle
[Rh-b]Cl (2.5 µM)
[Rh-b]Cl (5 µM)
[Rh-b]Cl (7.5 µM)
NAO Fluorescence Intensity
Figure 9. The decrease of NAO fluorescence upon treatment for 4 h with [Rh-b]Cl at different
concentrations (2.5, 5 and 7.5 µM) was analysed by flow cytometry. Grey: control cells; coloured: treated
cells.
2
.6.9. Photodynamic activity. The PDT effect of the [Ir-b]Cl and [Rh-b]Cl was first examined
on A549 cells irradiated in the UV region at 365 nm (where all the complexes present strong
-2
absorption) for a short period of 5 min at 20 mW cm . Interestingly, the cytotoxicity of [Ir-b]Cl
increased significantly, displaying a phototoxicity index (IC non-irradiated/IC irradiated) of
5
0
50
1
8 ± 2, whereas no significant changes were observed for the [Rh-b]Cl complex that displayed
a photoindex of 1.1 ± 0.1 (Figure S14). Although the phototoxicity index quoted for [Ir-b]Cl is
a promising result, the use of visible light and nontoxic compounds is preferable in PDT
therapy. Hence, irradiation of cells treated with the four metal complexes included in this work
with blue light (λmax = 460 nm) was tested. Figure 10 summarizes the cytotoxic activity of
complexes [Ir-a,b]Cl and [Rh-a,b]Cl with and without irradiation for 20 min with blue LED
1
5

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-2
light at 5.5 mW cm . Control experiments were carried out to assess that irradiation alone under
these experimental conditions did not affect cell survival. After 20 min of irradiation with blue
-
2
LED light at 5.5 mW cm the survival rate obtained for non-treated A549 cells (control) was
9
7.7 % (± 0.8 %). The phototoxicity index values obtained for complexes [Ir-a]Cl and [Ir-b]Cl
were 15.8 ± 0.7 and 3.6 ± 0.3, respectively, whereas [Rh-a]Cl and [Rh-b]Cl did not display
significant difference in their IC values with and without irradiation. It is worth noting that the
5
0
IC for [Ir-a]Cl and [Ir-b]Cl after blue light irradiation is nearly the same for both complexes
5
0
(
1.2 ± 0.1 µM and 1.1 ± 0.1 µM, respectively). However, the phototoxicity index of [Ir-a]Cl is
considerably higher due to its lower cytotoxicity in the dark.
Thus, despite the close biological properties of the Ir and the Rh complex in the dark, the
different photophysical features give rise to very distinct photodynamic activity. The
explanation could lie on the heavy atom effect provided by the Ir(III) core within complexes
[
Ir-a,b]Cl which favours an intersystem crossing process and long-lived triplet excited states,
which in turn promotes the excitation of the triplet molecular oxygen molecule to generate
singlet oxygen. This circumstance is absent for the [Rh-a,b]Cl complexes.
Figure 10. IC50 (µM) values obtained for A549 cells treated with [Ir-a,b]Cl and [Rh-a,b]Cl for 24 h with
-2
(
grey columns) and without (black columns) irradiation for 20 min with blue LED light at 5.5 mW cm .
Difference significant at **p = 0.0019 and ****p = 0.00001.
2
.7. Ir(III)-promoted singlet oxygen photo-generation and photooxidation of sulfides
In view that the use of visible light presents a number of advantages in photodynamic therapy,
the results obtained for complexes [Ir-a,b]Cl on A549 cells irradiated with blue light prompted
us to assess the potential ability of the Ir complexes to photo-catalyze the generation of ROS
under irradiation with blue light. The results could provide a reasonable mechanism for the
1
6

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biological action of these Ir(III) complexes under irradiation. For comparison purposes, the
Rh(III) complexes were also included in this study.
2
.7.1. Photo-stability of complexes [Ir-a]Cl and [Ir-b]Cl. The photo-stability of the complexes
1
[
Ir-a]Cl and [Ir-b]Cl was studied by H NMR over a period of 24 h of irradiation (blue LED
light, λmax = 460 nm) in an NMR tube under air atmosphere (Figures S15 and S16). Both
complexes undergo slight photo-decomposition to give the same degradation product which we
attribute to [IrCl(ppy) (CD SOCD )]. The conversion values were calculated from the
2
3
3
corresponding integration values as 1.6 and 1.3% after 6 h and 6.0 and 4.7% after 24 h for
[
[
Ir-a]Cl and [Ir-b]Cl, respectively, suggesting that the photolysis process is slightly slower for
Ir-b]Cl. Conversely, equivalent solutions of [Ir-a]Cl and [Ir-b]Cl are stable in the dark after 5
days.
2
.7.2. Photo-oxidation of thioanisole. To properly analyze the ability of the new complexes to
1
photo-catalyze the potential generation of O or other ROS, we evaluated the oxidation of
2
1
thioanisole by H NMR in the presence of the [Rh-a,b]Cl and [Ir-a,b]Cl complexes under
irradiation at room temperature with a blue LED strip (blue LED light, λmax = 460 nm, 24W).
1
This model reaction (eq (1)) was reported previously as an illustrative O -mediated photo-
2
oxidation process [55]. The respective sulfoxide (methyl phenyl sulfoxide, MPS) was obtained
in a chemo-selective manner in the presence of both Ir(III) PS, although the conversion values
determined experimentally demonstrate certain degree of dependence on the coordinated
1
ancillary ligand. Thus, the complex [Ir-a]Cl with L gave full conversion after 12 h, whereas
2
[
Ir-b]Cl with L gave 82% conversion under the same conditions (entries 1 and 2, Table 3 and
Figure S17). On the other hand, negligible conversions were detected in the presence of either
the free ligands or the Rh(III) derivatives (entries 3 to 6 in Table 3).
To gain some insight into the reaction mechanism, several control experiments were performed.
Thus, the oxidation of thioanisole was inhibited in the absence of light (entry 7 in Table 3) or in
the absence of the Ir(III) PS (entry 8, Table 3), which proved the Ir(III)-induced photocatalytic
nature of the reaction. On the other hand, the low conversion detected under nitrogen
atmosphere (N bubbling) supported the role of O as the oxidant source (compare entries 2 and
2
2
9
in Table 3). Nonetheless, two mechanisms have been described in the literature for the
photocatalytic oxidation of sulfides with O under visible light [56]. The first one put forward
2
1
the formation of O , by means of an ET process and its involvement as the actual oxidant,
2
whereas the second involves the formation of superoxide via electron-transfer processes. In
1
particular, in our photocatalytic oxidation system the participation of O was established by
2
1
using NaN (11 mM), a specific O scavenger, which dramatically inhibited the formation of
3
2
the sulfoxide (entry 10, Table 3). Moreover, the presence of a scavenger of the superoxide
1
7

ACCEPTED MANUSCRIPT
•
radical anion O ‾, such as 1,4-benzoquinone, also led to a relative decrease in the conversion
2
•
(
entry 11, Table 3), suggesting that also O ‾ could contribute to some degree to the oxidation of
2
organo-sulfides as well. Hence, both the energy transfer and electron transfer pathways seem to
play a role in this reaction [57].
Table 3. Catalyst screening and control experiments in the photo-oxidation of
thioanisole under an O atmosphere and blue-LED irradiation
.
2
a
b
Entry
Conditions (Cat / Light / Gas)
Conv (%, 12 h)
1
2
3
4
5
6
7
8
9
[Ir-a]Cl / Yes / O2
[Ir-b]Cl / Yes / O2
[Rh-a]Cl / Yes / O2
100
82
1.1
1.3
6.7
3.3
0
1.7
8.3
0.2
23.6
[Rh-b]Cl / Yes / O2
1
L / Yes / O
2
2
L / Yes / O2
[Ir-b]Cl / No / O2
No / Yes / O2
[Ir-b]Cl / Yes / N2
c
1
1
0
1
[Ir-b]Cl / Yes / O / NaN
2
3
d
[Ir-b]Cl / Yes / O / bzq
2
a
-2
b
Thioanisole (10 mM), PS (10 mM), see experimental part. Conversion yields were
determined by H NMR analysis of the crude mixture as average values of two independent
experiments. NaN (11 mM). p-benzoquinone (bzq, 11 mM).
1
c
d
3
2
.7.3. Photo-oxidation of L-cysteine and L-methionine. In addition, the photo-oxidation of
L-cysteine (eq (2)) and L-methionine (eq (3)) was proved under similar conditions in the
presence of [Ir-a]Cl and [Ir-b]Cl. Both PSs produced the disulfide derivative of L-cysteine
(
cystine) in a quantitative manner, whereas the control experiments in the absence of light or PS
gave only partial conversions (Table 4, entries 1 to 4 and Figure S18). The oxidation of
L-methionine afforded methionine sulfoxide very efficiently with both PS, while the control
experiments without PS or in the dark did not produce the sulfoxide (Table 4, entries 5 to 8 and
Figure S19). The irreversible oxidation of exposed L-cysteine and L-methionine residues in
tissue enzymes can alter the weak interactions responsible for their secondary and tertiary
structure leading to misfolding and consequently rendering these enzymes dysfunctional [26].
Hence, the aforementioned results suggest a possible mechanism for the potential photo-
cytotoxic activity of the Ir(III) complexes [Ir-a]Cl and [Ir-b]Cl.
1
8

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Table 4. Photo-oxidation of L-cysteine and L-methionine under an O atmosphere and
2
blue-LED irradiation.
a
b
Entry
Substrate
Conditions
%Conv, 12 h (18 h)
1
2
3
4
5
6
7
8
Cys
Cys
Cys
Cys
Met
Met
Met
Met
[Ir-a]Cl / Blue light / O2
[Ir-b]Cl / Blue light / O2
[Ir-b]Cl / No light / O2
No Cat / Blue light / O2
[Ir-a]Cl / Blue light / O2
[Ir-b]Cl / Blue light / O2
[Ir-b]Cl / No light / O2
No Cat / Blue light / O2
> 99
> 99
45
44
100
79 (100)
0
0
a
-2
b
L-cysteine/L-methionine (10 mM), PS (10 mM, 0.1 mol %), see experimental part. Conversion
1
yields were determined by H NMR analysis of the crude mixture as average values of two
independent experiments.
3
. Conclusions
Two pairs of Rh(III) and Ir(III) biscyclometalated complexes of general formula
1
[
M(ppy) (N^N)]Cl (M = Rh, Ir) bearing N^N ligands based on the thiabendazole scaffold (L
2
2
and L ) have been designed, synthesized and their potential as anticancer and PDT agents has
been evaluated. In the dark, the Rh and Ir benzyl-derivatives are one order more cytotoxic
against human lung carcinoma (A549) and human colon carcinoma (SW480) cell lines than
cisplatin. Additional biological studies with the Rh and Ir benzyl-derivatives show that both
complexes interfere with mitochondria functionality, causing the activation of apoptotic cell
death pathways. Moreover, the [Ir-a]Cl and [Ir-b]Cl complexes exhibit enhanced cytotoxic
activity after irradiation with visible blue light and photo-oxidation of sulphur-containing
aminoacids was successfully demonstrated, suggesting that under irradiation conditions
oxidative stress could be selectively promoted through a photocatalytic action. On the contrary,
the non-photoluminescent Rh complexes, which are unable to induce this photo-oxidation, do
not exhibit enhanced cytotoxicity upon irradiation with neither UV nor visible light.
1
9

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4
. General methods and starting materials
4
.1. Starting materials. RhCl ·xH O and IrCl ·xH O were purchased from Johnson Mattey and
3
2
3
2
used as received. [Rh(µ-Cl)(ppy) ] and [Ir(µ-Cl)(ppy) ] (Hppy = 2-phenylpyridine) were
2
2
2 2
prepared according to literature procedures.[58] 2-(4'-thiazolyl)benzimidazole (thiabendazole,
tbz) was purchased from Sigma-Aldrich and used without further purification. The ligand N-
benzyl-2-(4-thiazolyl)benzimidazole was prepared according to a procedure adapted from the
literature (vide infra). Deuterated solvents were obtained from SDS (CD SOCD ) and Carlo
3
3
Erba or Euriso-top (CDCl3).
Other reagents were purchased from commercial sources and used without further purification.
Deionized water was used to prepare all aqueous solutions for the photophysical measurements.
Cell culture reagents and fetal bovine serum (FBS) were purchased from Sigma-Aldrich. Stock
solutions of [Rh-b]Cl and [Ir-b]Cl were prepared in DMSO at 20 mM at -20 °C, aliquoted in
small volumes to avoid freeze/thaw cycles.
4
.2. Synthesis method and complexes characterization. All synthetic manipulations were
carried out under, oxygen-free, dry nitrogen atmosphere using standard Schlenk techniques. The
solvents were dried and distilled under nitrogen atmosphere before use. Elemental analyses
were performed with a LECO CHNS-932 Elemental Microanalyzer. The analytical analysis for
the new compounds was obtained from the powder solids. As the agreement between calculated
and found values for carbon was >0.4%, solvent molecules were introduced in the molecular
formulae to improve agreement. IR spectra were recorded on a Jasco FT/IR-4200
–1
spectrophotometer (4000–400 cm range) with Single Reflection ATR Measuring Attachment.
ESI+ Mass spectra (position of the peaks in DA) were recorded with an Autospec spectrometer.
The isotopic distribution of the heaviest set of peaks matched very closely in every case that
calculated for the formulation of the complex cation. NMR samples were prepared under
nitrogen atmosphere by dissolving the suitable amount of compound in 0.5 mL of pre-dried
(
Molecular Sieves 4A) oxygen-free deuterated solvent and the spectra were recorded at 298 K
1
13
1
on a Varian Unity Inova-400 (399.94 MHz for H; 100.6 MHz for C). Typically, 1D H NMR
1
spectra were acquired with 32 scans into 32 k data points over a spectral width of 16 ppm. H
1
3
1
1
and C{ H} chemical shifts were internally referenced to TMS via the residual H signal of
13
CHD SOCD (δ = 2.50 ppm) and the C signal of (CD ) SO (δ = 39.52 ppm), according to the
2
3
3 2
values reported by Fulmer et al.[59] Chemical shift values are reported in ppm and coupling
1
constants (J) in Hertz. The splitting of proton resonances in the reported H NMR data is
defined as s = singlet, d = doublet, t = triplet, st = pseudotriplet, q = quartet, sept = septet, m =
1
1
1
1
1
13
multiplet, bs = broad singlet. 2D NMR spectra such as H- H gCOSY, H- H NOESY, H- C
1
13
gHSQC and H- C gHMBC were recorded using standard pulse sequences. The probe
2
0

ACCEPTED MANUSCRIPT
temperature (± 1 K) was controlled by a standard unit calibrated with methanol as a reference.
All NMR data processing was carried out using MestReNova version 10.0.1.
4
.3. X-ray Crystallography. A summary of crystal data collection and refinement parameters
for [Rh-b]PF and [Ir-b]PF ·CH COCH are given in Table S2. Single crystals of both
6
6
3
3
complexes were coated in high-vacuum grease, mounted on a glass fiber and transferred to a
Bruker SMART APEX CCD-based diffractometer equipped with a graphite monochromated
Mo-Kα radiation source (λ = 0.71073 Å). The highly redundant datasets were integrated using
SAINT[60] and corrected for Lorentz and polarization effects.
The software package SHELXTL (version 2016/6)[61] was used for space group determination,
2
structure solution, and refinement by full-matrix least-squares methods based on F . A
successful solution by direct or Patterson methods for [Rh-b]PF and [Ir-b]PF ·CH COCH
3
6
6
3
2
and intrinsic phasing for L provided most non-hydrogen atoms from the E-map. The remaining
non-hydrogen atoms were located in an alternating series of least-squares cycles and difference
2
Fourier maps. In L a hexagon was fitted and refined as a free rotating group. Moreover, rigid
bond restraints (RIGU) were used for the thermal displacement. All non-hydrogen atoms were
refined with anisotropic displacement coefficients. Hydrogen atoms were placed using a “riding
model” (with free rotation for methyl groups) and included in the refinement at calculated
2
positions. CCDC reference numbers for L , [Ir-b]PF ·CH COCH and [Rh-b]PF6 are
6
3
3
1
829160, 1829161 and 1829162, respectively.
.4. Photooxidation of thioanisole and L-aminoacids. Aliquots of stock solutions of the Ir(III)
4
PS in DMSO-d and the S-containing substrate in D O are added to a 5 mL test tube to achieve
6
2
-2
final concentrations of 10 mM, (0.1 mol %) and 10 mM respectively in a mixture DMSO-
d /D O (0.5 mL, 3:2 or 1:10 for cysteine). The test tube is capped with a septum and the mixture
6
2
is saturated with an atmosphere of either O or N using a needle and a balloon and then stirred
2
2
at room temperature under irradiation with blue light (LED, λ = 460 nm, 24 W). Conversion
1
yields were determined by H NMR analysis (see Figure S16) of the crude mixture as average
values of two independent experiments. ESI MS spectra were recorded to confirm the identity
of all the products (see Figures S20 and S21). Methyl phenyl sulfoxide was obtained as a
racemic mixture, (enantiomers S and R ), cystine was obtained as the (R,R) diastereomer and
S
S
methionine sulfoxide was obtained as a 1:1 mixture of diastereoisomers which are epimers at
the sulfur atom, that is (S ,S ) and (R ,S ). Overoxidation to the corresponding sulfone products
S
C
S
C
(
thioanisole or methionine) or cysteine sulfenic, sulfinic or sulfonic acids were not detected in
any experiment. Either NaN3 or p-benzoquinone were added in the respective control
experiments to achieve final concentration of 11 mM in both cases. A turbid suspension was
obtained after stopping the reaction for cystine.
2
1

ACCEPTED MANUSCRIPT
4
.5. Photophysical Measurements. Photophysical measurements were carried out in aqueous
buffer under physiological conditions (PBS or sodium cacodylate buffer, pH 7.4) or in
o
acetonitrile at T = 25 C. Emission lifetime was determined by time-correlated single photon
counting using a FLS980 Edinburgh Instrument following excitation with a pulsed laser diode at
3
75 nm. Quantum yield was measured with an integrating sphere in a FLS980 Edinburgh
Instrument.
4
.6. Cell culture. Human lung carcinoma-derived A549 cells were cultured using Dulbecco’s
modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM glutamine and
% antibiotic. Colorectal adenocarcinoma SW480 cells were cultured using Dulbecco’s
1
modified Eagles Medium (DMEM) with 10% FBS and 1% antibiotic. All cells were incubated
o
at 37 C under 5% CO .
2
4
.7. Cytotoxicity assays. Cells were seeded in 96 well plates at 5000 cells/well and allowed to
o
proliferate for 24 h at 37 C and 5 % CO before treatment. Cells were then incubated with
2
different concentrations of the complexes for another 24 h period. For the PDT studies, cells
were incubated for 30 min with the complexes, then irradiated at 365 nm for 5 min at
2
2
2
0 mW/cm or with blue LED light for 20 min at 5.5 mW/cm and allowed to incubate in the
-1
dark for another 23.5 h. After that, 100 µL of 0.5 mg mL of MTT (thiazolyl blue tetrazolium
bromide) dissolved in DMEM was added to each well for 4 h and the formazan product eluted
by adding 100 µL of solubilizing solution (10% SDS in 0.01 M HCl). Formazan absorption was
read at 590 nm in a Cytation 5 Imaging Reader (BioTek) and cell survival was calculated as
follows: cell viability (%) = (mean of Abs. value of treated cells/mean Abs. value of
control) × 100 %. IC50 values correspond to the concentration for which cell survival is 50 %
after 24 h of treatment.
5
4
.8. Cellular uptake studies. A549 cells were seeded in 6 well plates at 1.5 × 10 cells/well and
o
incubated for 24 h at 37 C and 5 % CO . After that, cells were treated with the indicated
2
concentrations of the Ir and Rh complexes for 4 h and harvested in cold PBS. The samples were
digested with 65% HNO at room temperature for 24 h and then diluted with Milli-Q water to
3
obtain 2% HNO solutions to be analyzed by ICP-MS.
3
4
.9. Cell cycle analysis. Cell cycle distribution was analysed by flow cytometry and PI
(
propidium iodide) staining as previously described [62]. Briefly, A549 cells were exposed to
[
Ir-b]Cl or [Rh-b]Cl at 10 µM for 24 or 48 h, harvested in cold PBS, centrifuged, resuspended,
o
fixed in 70% EtOH and storage overnight at 4 C. The same procedure was carried out with
vehicle-treated cells (0.025 % DMSO). Cells were then washed twice with cold PBS,
resuspended in 500 µL of PI staining solution (10 µg/mL) and DNAse-free RNase (100 µg/mL)
2
2

ACCEPTED MANUSCRIPT
and analysed by flow cytometry with a Guava®easyCyte Flow Cytometer. Cell cycle
distribution was determined by analysis with the GuavaSoft™ 3.3 Software.
4
.10. Colocalization experiments. Cells were seeded in glass bottom 35 mm petri dishes
purchased from MatTek Corporation and allowed to adhere for 24 h. Cells were then incubated
o
with MitoTracker (100 nM) or LysoTracker (75 nM) for 30 min at 37 C and 5% CO , as
2
specified by the manufacturer. After that cells were treated with 1.5 µM of [Ir-b]Cl for another
3
0 min. Finally, cells were washed twice with PBS and visualized in fresh DMEM medium
without phenol red with a confocal laser microscopy (TCS-SP2; Leica, Heidelberg, Germany).
4
.11. ROS detection. Cells were seeded in 6 well plates at 125.000 cells/well and allow to
o
adhere for 24 h at 37 C and 5 % CO . Then cells were stained with DHE (6 µM) or H2DCDF
2
(
10 µM) for 30 minutes in HBSS supplemented with 1% FBS. Then the cells were treated with
[
Ir-b]Cl, [Rh-b]Cl, DMSO as the vehicle control or H O as the positive control for 2 h or 4 h.
2
2
Cells were washed with PBS, trypsinized and analyzed by flow cytometry with a Guava®
easyCyte Flow Cytometer at the excitation and emission wavelengths specified by the
manufacturer.
4
.12. Analysis of mitochondrial membrane potential (MMP) [63]. Cells were seeded in 12
o
well plates and allow to adhering for 24 h at 37 C and 5 % CO . Then, cells were incubated
2
with TMRM (75 nM) and afterwards they were treated with [Ir-b]Cl or [Rh-b]Cl as a positive
control for 2 h or 4 h. Cells were washed twice with PBS and visualized in fresh DMEM
medium without phenol red.
4
.13. Cardiolipin oxidation study. Cells were seeded in 6 well plates at 150.000 cells/well and
o
allow to adhering for 24 h at 37 C and 5 % CO . Cells were then stained with 10-N-nonyl
2
acridine orange NAO (100 nM in HBSS) for 15 min at 37 ºC and then washed twice with PBS
prior to be treated with [Rh-b]Cl at different concentrations for another 4 h. Finally, cells were
collected in cold PBS and analyzed by flow cytometry.
Conflicts of interest
There are no conflicts of interest to declare
Acknowledgements
We gratefully acknowledge the financial support provided “la Caixa” Foundation
(
(
LCF/PR/PR12/11070003), the Spanish Ministerio de Economía y Competitividad - FEDER
CTQ2014-58812-C2-2-R, CTQ2014-58812-C2-1-R and CTQ2015-70371-REDT), Consejería
2
3

ACCEPTED MANUSCRIPT
de Educación – Junta de Castilla y León - FEDER (BU042U16). C. P-A is grateful for the FPU
grant from Ministry of Education, Culture and Sports, Madrid, Spain (FPU13/00180). I.
Echevarría wants to acknowledge his fellowship to both the European Social Fund and
Consejería de Educación de la Junta de Castilla y León.
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