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Ir(III) Diamine Transfer Hydrogenation Catalysts in Cancer Cells
University of Birmingham
Ir(III) Diamine Transfer Hydrogenation Catalysts in
Cancer Cells
Fry, Millie E.; Alsaif, Sitah; Khanom, Yasmin; Keirle, Alice K.; Pheasey, Chloe E.; Song, Ji
Inn; Bedford, Rebecca A.; Romero Canelon, Isolda; Sadler, Peter J; Coverdale, James
DOI:
10.1002/cctc.202401490
License:
Creative Commons: Attribution (CC BY)
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Citation for published version (Harvard):
Fry, ME, Alsaif, S, Khanom, Y, Keirle, AK, Pheasey, CE, Song, JI, Bedford, RA, Romero Canelon, I, Sadler, PJ
& Coverdale, J 2024, 'Ir(III) Diamine Transfer Hydrogenation Catalysts in Cancer Cells', ChemCatChem.
https://doi.org/10.1002/cctc.202401490
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ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
www.chemcatchem.org
Ir(III) Diamine Transfer Hydrogenation Catalysts in Cancer Cells
Millie E. Fry,[a] Sitah A. Alsaif,[a] Yasmin Khanom,[b] Alice K. Keirle,[a] Chloe E. Pheasey,[a]
Ji Inn Song,[b] Rebecca A. Bedford,[a] Isolda Romero-Canelon,[a, b] Peter J. Sadler,[b]
and James P. C. Coverdale*[a, b]
The development of catalytic metallodrugs is an emerging field
that may offer new approaches to cancer chemotherapeutic
design. By exploiting the unique properties of transition metal
complexes, in-cell catalysis can be applied to modulate the
cellular redox balance as part of a multi-targeting mechanism
of action. We describe the synthesis and characterization of
six coordinatively unsaturated iridium(III) diamine catalysts that
are stable at physiological pH in aqueous solution. Reduction
of the colorimetric substrate 2,6-dichlorophenolindophenol by
transfer hydrogenation under biologically compatible conditions
achieved turnover frequencies up to 63 ± 2 h−1 and demonstrated that the source of hydride (sodium formate) is the
limiting reagent, despite being in a 1000-fold excess of the catalyst. The catalyst showed low in vivo acute toxicity in zebrafish
embryos and modest in vitro potency towards cancer cells.
When administered alone, the catalyst generated oxidative stress
in cells (an effect that was conserved in vivo), but co-treatment
with a nontoxic dose of sodium formate negated this effect.
Co-treatment with sodium formate significantly enhanced catalyst potency in cancer cells (A2780 ovarian and MCF7 breast
cancer cells) and drug-resistant cells (A2780cis and MCF7-TAMR1)
but not in non-tumorigenic cells (MRC5), demonstrating that a
redox-targeting mechanism may generate selectivity for cancer
cells.
1. Introduction
fold increase in the potency of a Ru(II) N-tosyldiamine catalyst
was achieved in A2780 ovarian cancer cells by coadministration with sodium formate (a sacrificial source of reducing
hydride). This therapeutic combination was not only synergistic
but demonstrated the first example of in-cell catalytic reduction, converting the coenzyme NAD+ to NADH.[4a] It was since
shown that catalytic performance is highly dependent on both
steric and electronic effects at the N-substituent.[4b] Using a
chiral Os(II) diamine catalyst, we reported the first example
of in-cell asymmetric transfer hydrogenation, catalytically generating unnatural D-lactate from endogenous pyruvate with
high enantioselectivity.[5a] With this system, we demonstrated
the potential of using catalytic metallodrugs to overcome drug
resistance in cisplatin- and tamoxifen-resistant cells, while no
significant catalytic potency modulation was observed in noncancerous cells.[5b,10] More recently, we have shown that the
catalytic generation of lactate can be enhanced as part of a
combination therapy involving a metal catalyst and a monocarboxylate transporter inhibitor,[5b] and have described structural
improvements to the catalyst involving covalent tethering of the
diamine to the η6 -arene ligand.[11]
Inspired by biocompatible Ru(II) and Os(II) catalyst systems,
interests have expanded to include Ir(III) catalysts. Iridium(III)
picolinate catalysts have been shown to catalyze NADH oxidation and increase levels of reactive oxygen species (ROS)
in cancer cells,[12] while iridium(III) 2,2 -bipyridine catalysts
have been shown to catalyze the photooxidation of the coenzyme NADPH, with examples achieving turnover frequencies
up to 308.0 h−1 .[13] While the Ir(III) N-tosyldiamine catalyst
[IrCl(Cp*)(TsDPEN-H)] is well established for conventional chemical catalysis,[14] it has not been explored within the context
of medicinal chemistry. In this work, we report a new family
Modulation of cellular redox using an in-cell catalyst exploits
an inherent vulnerability of cancer cells.[1] Redox disruption
is a multi-targeting approach which impacts various cellular
processes,[2] as opposed to targeted therapeutics, for which
resistance mechanisms may readily develop.[3] Various examples of subcellular redox perturbation have been described,
including nicotinamide adenine dinucleotide (NAD+ /NADH),[4]
pyruvate/lactate,[4e,5] quinone/hydroquinone,[6] and glutathione
(GSH/GSSG).[7] Second and third row transition metal ions offer
the kinetic inertness required to deliver an intact catalyst to a
biological system rich in nucleophiles (e.g., thiols) which would
otherwise poison or deactivate the catalyst. While cyclometalated catalysts and protein scaffold artificial metalloenzymes
have shown promise,[4h,8] metal-arene N-tosyldiamine catalysts
have proven particularly attractive for in-cell applications, since
the chemistry of the catalyst cycle is well defined.[9] A 50-
[a] M. E. Fry, S. A. Alsaif, A. K. Keirle, C. E. Pheasey, R. A. Bedford,
I. Romero-Canelon, J. P. C. Coverdale
School of Pharmacy, School of Health Sciences, College of Medicine and
Health, University of Birmingham, Edgbaston B15 2TT, UK
E-mail: j.p.coverdale@bham.ac.uk
[b] Y. Khanom, J. I. Song, I. Romero-Canelon, P. J. Sadler, J. P. C. Coverdale
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/10.1002/cctc.202401490
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
ChemCatChem 2024, 0, e202401490 (1 of 13)
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
Research Article
doi.org/10.1002/cctc.202401490
ChemCatChem
Figure 1. Synthesis of Ir catalysts 1–6 studied in this work. Ir catalysts 1–3 bear the TsDPEN (N-tosyl diphenyl ethylenediamine) bidentate ligand, while Ir
catalysts 4–6 introduce a methoxy (R2 = OCH3 ) substituent on the chiral diamine ligand to improve aqueous solubility.
Table 1. Physicochemical, in vitro, and in vivo biological properties of Ir complexes 1–6 and cisplatin. Hydrophobicities determined experimentally as
octanol/water partition coefficients (Log PO/W) by ICP-MS in experimental triplicate. Antiproliferative activities (IC50/μM, 24 h exposure + 72 h recovery
time, sulforhodamine B assay, duplicate of triplicate analysis). Cellular Ir accumulation (fg Ir cell-1, 1 × IC50 concentration, 24 h, no recovery time) determined using A2780 cancer cells (independent triplicate experiments, N = 3). In vivo toxicities (LC50/μM, 96 h exposure) determined in Singapore wild-type
zebrafish embryos (Danio rerio) as described by OECD test 236: acute zebrafish embryo toxicity test (two independent duplicate experiments). N.D. = not
determined.
A2780 Ir/fgcell−1
Danio rerio LC50/μM
20.9 ± 0.7
52 ± 1
8±1
14 ± 2
41.7 ± 0.7
1.6 ± 0.3
10.2 ± 0.6
89 ± 4
0.7 ± 0.1
0.03 ± 0.04
17 ± 1
8±1
11 ± 2
OCH3
0.32 ± 0.02
8.0 ± 0.8
13 ± 1
3.6 ± 0.2
OCH3
0.62 ± 0.03
0.7 ± 0.1
5.3 ± 0.2
2.9 ± 0.8
N.D.
1.2 ± 0.3
N/A
0.6 ± 0.2
R1
R2
Log PO/W
1
CH3
H
0.86 ± 0.02
2
Phenyl
H
1.41 ± 0.02
3
Biphenyl
H
2.24 ± 0.02
4
CH3
OCH3
5
Phenyl
6
Biphenyl
Cisplatin
N/A
N/A
of coordinatively unsaturated 16-electron organo-Ir(III) catalysts
and have investigated their mechanism of action and ability to
catalyze transfer hydrogenation reactions inside cells.
2. Results and Discussion
Iridium catalyst 1 [Ir(Cp*)(TsDPEN)] is derived from 18-electron
chlorido pre-catalyst [IrCl(Cp*)(TsDPEN-H)], which has previously
been shown to achieve the rapid conversion of ketones to chiral alcohols with high enantioselectivity in an organic solvent
system within the context of synthetic chemistry.[14] Catalyst 1
is the active catalytic species, which can be readily prepared
by treatment of the pre-catalyst with a suitable base to eliminate HCl. Alternatively, catalyst 1 may be prepared in a one-pot
reaction from a precursor Ir(III) cyclopentadienyl (Cp*) dimer, Ntosyl diphenyl ethylenediamine (TsDPEN), and a base, utilizing
a biphasic reaction optimized for the preparation of structurally
similar Os(II) catalysts (Figure 1, Table 1).[15] Like its osmium(II)
counterpart,[5a] this 16-electron Ir(III) catalyst is highly stable
in air and solution. Time-dependent 1 H-NMR spectra acquired
ChemCatChem 2024, 0, e202401490 (2 of 13)
A2780 IC50/μM
in deuterated dimethyl sulfoxide did not exhibit any new or
shifted peaks after 24 h compared to the initial (t = 0 h)
spectrum (Supporting Information Figure S1). Similarly, UV–vis
spectroscopic measurements showed catalyst 1 to be stable in
phosphate-buffered saline (containing 10% v/v dimethyl sulfoxide to aid solubility) for 24 h (Supporting Information Figure
S2). Though it is possible that modification, ligand substitution,
or degradation may occur on a timescale more rapid than the
experimental design, the chemical species identified in the first
spectrum acquired (ca. 10 min) remained unchanged for the
24 h period investigated and did not undergo further structural
modifications.
Extended arene catalysts 2 (CpxPh ) and 3 (CpxBip ) were prepared in a similar manner to catalyst 1, using extended arene
dimer precursors (R1 = phenyl or biphenyl, for 2 and 3, respectively) and were obtained as purple amorphous solids, which
were recrystallized from dichloromethane/hexane. To improve
aqueous solubility, methoxyphenyl-functionalized catalysts 4–
6 (R2 = OCH3 ) were prepared with equivalent arene ligands
(Cp*, CpxPh , and CpxBip ). This structural modification significantly
increased the hydrophilicity of catalysts 4–6 (Log PO/W = 0.03,
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
0.32, and 0.62, respectively) in comparison to their unfunctionalized analogues 1–3 (Log PO/W = 0.86, 1.41, and 2.24, respectively).
An ideal in-cell transfer hydrogenation (reduction) catalyst should not be significantly toxic when administered
alone to ensure that a significant intracellular concentration can be achieved prior to initiating catalysis by coadministration of a source of reducing hydride. The zebrafish
embryo (Danio rerio) model was used to assess acute toxicity, and its suitability to support small molecule therapeutic development has been extensively reviewed.[16] Remarkably, catalyst 1 (LC50 = 8 ± 1 μM) was threefold less toxic
than its isoelectronic Os(II) analogue [Os(p-cymene)(TsDPEN)]
(LC50 = 2.4 ± 0.4 μM) and over an order of magnitude
less toxic than cisplatin (LC50 = 0.6 ± 0.2 μM) and structurally similar Ir(III) azopyridine piano-stool catalysts.[10,17] While
methoxyphenyl-functionalization did not significantly impact
acute toxicity, toxicity increased with increasing arene extension
(Cp* < CpxPh < CpxBip , Figure 1, Table 1).
With encouraging in vivo data to support the further study of
this catalyst system in living cells, antiproliferative activities were
determined using A2780 ovarian cancer cells. Methoxyphenylfunctionalized catalysts 4–6 were more potent than 1–3, and
the anticancer activity of catalyst 6 exceeded that of cisplatin
(catalyst 6 IC50 = 0.7 ± 0.1 μM, cisplatin IC50 = 1.2 ± 0.3 μM).
Within each series of compounds containing the same bidentate ligand (1–3 and 4–6), IC50 values directly correlated with
lipophilicity (Log P, Figure 1, Table 1). However, ICP-MS analysis of cells treated with equipotent concentrations of Ir catalyst
revealed that intracellular accumulation of Ir in cells treated with
1–3 was an order of magnitude greater (41.7–89 fg Ircell−1 ) than
those treated with equipotent concentrations of 4–6 (5.3–13 fg
Ircell−1 ). Therefore, although methoxy-functionalized catalysts
4–6 achieved high potency at low intracellular Ir accumulation,
in this work we aimed to achieve in-cell catalysis, and improving
catalyst in-cell availability was a critical objective.
Catalysts 1–3 were subsequently screened for activity towards
13 human cell lines (Table 2). Increased potency is correlated
with arene extension (Cp* < CpxPh < CpxBip ) with some examples
exceeding the anticancer activity of cisplatin. A direct correlation
was observed between potency (IC50 ) and Ir cellular accumulation, leading us to focus on ovarian (A2780) and breast (MCF7)
cells, in which Ir accumulation was greatest. Activities were not
significantly different between A2780 (cisplatin-sensitive ovarian cancer cells) and A2780cis (cisplatin-resistant ovarian cancer
cells), suggesting that Ir(III) catalysts 1–3 do not share a common
mechanism of resistance with cisplatin in this cell line. Overcoming cisplatin resistance is frequently observed for Ir piano-stool
catalysts, including those bearing azopyridine and iminopyridine ligands,[17,18] as well as alcohol-functionalized Ir Cp* dimeric
catalysts.[19] Antiproliferative activities determined in HCT116 colorectal cancer cells with either p53 knockout or p21 knockout
were higher (less potent) than those determined in the parental
HCT116 cell line, which might implicate p53 or p21 proteins in
the mechanism of action of 1. While no significant cell cycle
arrest was observed in A2780 cells treated with sodium formate
relative to the untreated control (Figure 2a and Supporting Infor-
ChemCatChem 2024, 0, e202401490 (3 of 13)
mation), a small increase in G2 /M arrest was observed in the
presence of catalyst 1, irrespective of formate co-treatment (G2 /M
population increased from 12.0 ± 0.5% in the untreated control
to 15.4 ± 0.8% in the presence of 1). Nonetheless, this increase
was far less prominent than the S and G2 /M-phase arrest (S
phase = 37 ± 1%, G2 /M-phase = 35.4 ± 0.2%) observed upon
cisplatin treatment (P < 0.001).
Subcellular fractionation of A2780 cancer cells treated with
catalyst 1 revealed Ir to be predominantly located in the membrane/organelle fraction (61 ± 2%) and only a small percentage
of Ir located in the nucleic fraction (1.2 ± 0.6%). This result is
comparable to studies using the osmium(II) p-cymene analogue
of catalyst 1 (membrane/organelle fraction: 48 ± 3%, nucleic fraction: 1.6 ± 0.5%) but contrasts with A2780 cells treated with
cisplatin, where 4.9 ± 0.8% of the total cellular Pt was found
in the nucleic fraction (Figure 2b and Supporting Information).
Measurement of DNA double or single-strand breaks using an
alkaline comet assay showed that cisplatin significantly increased
comet tail moment (P < 0.001), while no significant difference
was observed between A2780 cells treated with Ir catalyst 1
and untreated (negative control) cells (Figure 2c and Supporting Information). As such, while Ir-DNA interactions cannot be
excluded with certainty, it is unlikely that DNA is a primary target
of catalyst 1.
Glutathione conjugation is a commonly identified deactivation pathway for therapeutic metallodrugs, including cisplatin.
Surprisingly, the antiproliferative activity of catalyst 1 was not
significantly affected by coadministration with L-buthionine
sulfoximine, a selective inhibitor of glutamyl cysteine synthetase, which is known to deplete intracellular glutathione
(IC50 = 20.9 ± 0.7 and 21.7 ± 0.9 μM, in the absence and
presence of L-BSO, respectively). The apparent lack of deactivation by glutathione may be attributed to the slower ligand
exchange kinetics of the low-spin d6 Ir(III) metal ion compared to
other transition metal catalysts, for example d8 Pt(II), but further
investigation in this regard is beyond the scope of this study.
When administered alone, catalyst 1 offers modest selectivity
for cancer cells over noncancerous cell lines (MRC5 lung fibroblasts or MCF10-A non-tumorigenic breast cells). Selectivity for
cancer cells over noncancerous cells (usually considered to be
greater than one order of magnitude) is uncommon for Ir(III)
catalysts, though some have been reported to exhibit up to
15 × selectivity towards cancer cells.[21] The modest selectivity
of catalysts 1–3 is similar to other structurally similar Ir pianostool catalysts bearing BINAP (2,2 -bis(diphenylphosphino)-1,1 binaphthyl) and phenyl pyridine ligands.[4g,22] Nonetheless, in
this study we hypothesize that catalytic modulation by coadministration of a hydride source may overcome this limitation.
Further accumulation studies were undertaken with catalyst 1 in A2780 ovarian cancer cells (Supporting Information).
Temperature-dependent accumulation measurements (1 × IC50 ,
3–6 h exposure) revealed lower Ir accumulation at 277 K
compared to 310 K, suggesting the involvement of both energydependent and energy-independent mechanisms of Ir influx.
Concentration-dependent accumulation studies (24 h, 310 K, no
recovery time) revealed a linear relationship between dose and
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
ChemCatChem
Research Article
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Table 2. Antiproliferative activities of Ir TsDPEN catalysts 1–3 and cisplatin towards 13 human cell lines. Cells were exposed to test compounds for 24 h
and allowed 72 h recovery time in drug-free medium. Cell viability was determined using the sulforhodamine B assay.[20] Data are reported as the average of two independent triplicate experiments (duplicate of triplicate) with the associated standard deviation of the average measurement. N.D. = not
determined.
Antiproliferative Activity (IC50 )/μM
Cell Line
1 (Cp*)
2 (CpxPh )
3 (CpxBip )
Cisplatin
A2780 (ovarian cancer)
20.9 ± 0.7
14 ± 2
10.2 ± 0.6
1.2 ± 0.3
A2780cis (ovarian, cisplatin-resistant)
17 ± 2
18 ± 1
6.3 ± 0.8
13.4 ± 0.3
A549 (lung cancer)
38 ± 2
25.2 ± 0.3
17 ± 3
3.2 ± 0.1
HCT116 (colorectal cancer)
40.3 ± 0.7
58.2 ± 0.9
15.0 ± 0.1
5.2 ± 0.3
HCT116-p21-/- (colorectal cancer, p21 knockout)
62.4 ± 0.3
N.D.
N.D.
9.2 ± 0.5
HCT116-p53-/- (colorectal cancer, p53 knockout)
63 ± 2
N.D.
N.D.
36.7 ± 0.3
MCF10-A (breast, non-tumorigenic)
42 ± 3
N.D.
N.D.
6±1
HOF (ovarian, non-tumorigenic)
19.0 ± 0.1
26.8 ± 0.6
15.0 ± 0.7
10.2 ± 0.7
MCF7 (breast cancer)
14.6 ± 0.2
8±1
3.7 ± 0.7
6.6 ± 0.4
MCF7-TAMR1 (breast, tamoxifen-resistant)
17.8 ± 0.6
N.D.
N.D.
6.0 ± 0.7
12.8 ± 0.5
MRC5 (lung, non-tumorigenic)
17.3 ± 0.4
11.9 ± 0.9
5.9 ± 0.2
OE19 (esophageal cancer)
>50
33.2 ± 0.5
12.9 ± 0.3
9±1
PC3 (prostate cancer)
35.1 ± 0.6
18.8 ± 0.2
12 ± 1
4.1 ± 0.5
Figure 2. Mechanistic investigation of A2780 cancer cells treated with Ir catalyst 1. (a) Cell cycle analysis: cells were either untreated or treated with Ir
catalyst 1 (20 μM, 1 × IC50 ) ± 2 mM sodium formate or an equipotent concentration of cisplatin (1 μM, 1 × IC50 ) for 24 h without recovery time. Cells were
fixed, stained using propidium iodide, and analyzed by flow cytometry in technical triplicate (N = 3). (b) Cellular metal distribution (fg Ir/cell) treated with
Ir catalyst 1 ± 2 mM sodium formate, or cisplatin, at 1 × IC50 for 24 h. Cell pellets were fractionated using the FractionPREP fractionation kit (BioVision),
resulting in four fractions: (i) cytosolic, (ii) membrane/organelle, (iii) nucleic, and (iv) cytoskeletal fractions. Metal content in each fraction was determined
using ICP-MS after acidic digestion, and data were reported as % distribution (with associated standard deviation, N = 3) relative to the sum of the metal
content across all four fractions. (c) Comet assay: cells were either untreated, treated with Ir catalyst 1 (20 μM, 1 × IC50 ) or an equipotent concentration of
cisplatin (1 μM, 1 × IC50 ) for 24 h without recovery time. Cells were lysed under alkaline conditions and electrophoresis was carried out, DNA was stained
using propidium iodide, and comet images (N = 20) were acquired by fluorescence microscopy. Statistical significances were tested using a two-tailed
unpaired t-test assuming unequal sample variances (Welch’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001. NS = not statistically significant. Full numerical and
statistical data are available in the Supporting Information.
intracellular Ir accumulation, while time-dependent measurements revealed that maximal Ir accumulation is achieved within
3 h of exposure and does not decrease within the 24 h exposure
time. This supports the further development of catalyst 1 as an
in-cell catalyst and contrasts with the accumulation of a structurally similar isoelectronic Os catalyst [Os(p-cymene)(TsDPEN)]
which reached maximal intracellular Os accumulation after
ChemCatChem 2024, 0, e202401490 (4 of 13)
6 h and then decreased irrespective of the extracellular Os
concentration.[5a]
Catalyst 1 is known to catalyze transfer hydrogenation reactions in organic solvents, using formic acid as a sacrificial
hydride donor (itself becoming irreversibly converted to carbon dioxide).[14] To catalyze transfer hydrogenation reactions in
aqueous media, the sodium salt of formic acid was selected
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
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Research Article
doi.org/10.1002/cctc.202401490
Figure 3. Catalytic reduction of 2,6-dichlorophenolindophenol (2,6-DCPIP, blue) to its colorless reduced form by Ir catalyst 1 and sodium formate (hydride
donor, 0–20 mM) in phosphate buffered saline (containing 1% v/v dimethyl sulfoxide to aid catalyst solubility). 2,6-DCPIP concentration was determined by
UV–vis spectroscopy (λ = 600 nm) using a 3-point calibration (Supporting, Figure S3). TOFmax = maximal turnover frequency. Data are reported as the
average of three independent experiments (N = 3) with an associated standard deviation. Full numerical information can be found in the Supporting
Information.
as a hydride donor since formate is a naturally occurring
metabolite.[23] Determination of the acute toxicity (LC50 ) in the
zebrafish embryo model showed sodium formate to be nontoxic
at millimolar concentrations (LC50 = 19.4 ± 0.6 mM). Formate
is rapidly absorbed in humans and reaches peak plasma levels
within 10–30 min post-administration, with negligible change in
blood pH, followed by rapid elimination (plasma t 1 = 45 min).[24]
2
Nonetheless, formate may be involved in the pathology of
defective one-carbon metabolism,[23] so further clinical safety
evaluation remains necessary.
Catalytic activity was assessed using the water-soluble redox
probe 2,6-dichlorophenolindophenol (2,6-DCPIP) as a substrate
for transfer hydrogenation (Figure 3). 2,6-DCPIP is commonly
used to measure photosynthetic rates: oxidized 2,6-DCPIP is blue
(λmax = 600 nm), while its reduced amino-phenol form is colorless. The reduction of 2,6-DCPIP to its colorless form by catalyst
1 (20 μM, equivalent to 1 × IC50 in A2780 cells) and 2 mM
sodium formate was successful in phosphate buffered saline
solution containing 1% v/v dimethyl sulfoxide to aid catalyst solubility, achieving a maximum turnover frequency (TOFmax ) of
6.8 ± 0.4 h−1 . To ensure 2,6-DCPIP was directly reduced by catalyst 1 and sodium formate (as opposed to indirectly reporting
redox modulation of another reaction component), experiments
were repeated substituting 2,6-DCPIP for two alternative colorimetric redox indicators: Alamar Blue (reduction of blue resazurin
N-oxide to pink resorufin phenoxazine) or 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT – reduction of yellow
tetrazolium to purple formazan), neither of which displayed any
change in absorbance in the presence of catalyst 1 and sodium
formate. Increasing the concentration of sodium formate significantly improved the rate of 2,6-DCPIP reduction (20 μM catalyst
and 20 mM sodium formate TOFmax = 63 ± 2 h−1 , Figure 3a)
while increasing only the catalyst concentration had no effect
on the rate of 2,6-DCPIP reduction (80 μM catalyst and 2 mM
sodium formate TOFmax = 6.85 ± 0.07 h−1 , Figure 3b). Importantly, no reduction in 2,6-DCPIP absorbance (λmax = 600 nm)
was observed in the absence of catalyst, nor upon substitution
of sodium formate for sodium acetate (which cannot donate
ChemCatChem 2024, 0, e202401490 (5 of 13)
hydride) confirming the specific role of both Ir catalyst 1 and
sodium formate (Figure 3c).
With an aqueous-compatible transfer hydrogenation catalyst
in hand, in-cell activity modulation experiments were carried out
by coadministering Ir catalyst 1 with nontoxic concentrations of
sodium formate (0–2 mM) to A2780 ovarian cancer cells (Figure 4
and Supporting Information). This combination of catalyst and
hydride donor resulted in a 75% decrease in cell survival relative
to the sodium formate-free control (P = 0.0040). Measurement of
cofactor-dependent Ir accumulation in cells demonstrated that
potency modulation was not caused by increased Ir uptake (no
cofactor: 33.4 ± 0.5 fgcell−1 , with 2 mM formate: 34.9 ± 0.6
fg cell−1 , P = 0.22). Similarly to 2,6-DCPIP experiments, the specific role of the formate anion for in-cell transfer hydrogenation
was confirmed by substitution for sodium acetate, a chemically
similar carboxylate that cannot act as a suitable hydride donor
and did not significantly reduce A2780 cell survival compared to
the acetate-free control (P = 0.4572). The catalytic modulation of
cell survival by Ir catalyst 1 and sodium formate was conserved
in MCF7 breast cancer cells (60% reduction in cell viability in
the presence of 2 mM formate, P = 0.0027), as well as in two
drug-resistant cell lines A2780cis (cisplatin-resistant) and MCF7TAMR1 (tamoxifen-resistant), achieving 53% (P = 0.0082) and 75%
(P = 0.0012) reductions in viability, respectively, relative to the
sodium formate-free control (Supporting Information Table S9
and Figure S6). As such, in-cell catalysis appears to offer potential
as a novel strategy to overcome drug resistance. Crucially, no catalytic modulation of cell survival was observed in noncancerous
MRC5 cells (Figure 4, P = 0.3827 compared to sodium formatefree control). This suggests that such a catalytic mechanism of
action in cells generates selectivity for cancer cells over noncancerous cells, supporting previous findings using both Ru(II)
and Os(II) in-cell catalysts.[1a,5a]
To develop a more comprehensive understanding of the
mechanism of action of Ir catalyst 1, iridium speciation in human
serum was investigated using anion exchange (AEX) chromatography coupled to offline ICP-MS quantification and SDS-PAGE.
Ir was observed in two distinct regions of the LC-ICP-MS chro-
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
Research Article
doi.org/10.1002/cctc.202401490
100
*
75
50
**
25
0
125
100
75
50
25
0
0.0
0.5
1.0
Acetate / mM
125
100
2.0
*
75
**
**
50
25
0
0.0
2.0
Normalised cell survival / %
Normalised cell survival / %
0.5
1.0
Formate / mM
MRC5
0.5
1.0
Formate / mM
125
100
75
50
25
0
0.0
0.5
1.0
Acetate / mM
125
100
75
50
25
0
2.0
Normalised cell survival / %
125
0.0
Sodium acetate
A2780cis
Normalised cell survival / %
Normalised cell survival / %
Sodium formate
A2780
Normalised cell survival / %
ChemCatChem
2.0
0.0
0.5
1.0
Formate / mM
2.0
0.0
0.5
1.0
Acetate / mM
2.0
125
100
75
50
25
0
Figure 4. Activity modulation of Ir Cp* catalyst 1 in cells: A2780 ovarian cancer cells, A2780cis cisplatin-resistant ovarian cancer cells, and MRC5
noncancerous human lung fibroblasts. Cells were treated in the presence (grey bars) or absence (white bars) of catalyst 1 (1.0 × IC50 ) with coadministration
of a nontoxic concentration of either sodium formate (0–2 mM, upper) or sodium acetate (0–2 mM, lower). Experiments were carried out in triplicate as
part of two independent experiments. Statistical significances (Welch’s t-test) were determined relative to the co-factor free (0 mM) control. *P < 0.05,
**P < 0.01, ***P < 0.001. Full numerical and statistical data can be found in the Supporting Information.
matogram: tR = 1–2 min (4.8% of the total recovered Ir) and
tR = 10.5–12.5 min (75.7% of the total recovered Ir). SDS-PAGE
revealed the latter to contain a highly abundant serum protein
(Rf = 0.517, ∼70 kDa), likely to be albumin (66.5 kDa, pI = 5),[25]
which is well known to transport metal ions and has been shown
to reversibly bind structurally similar half-sandwich Ir(III), Os(II),
Ru(II), and Ru(III) catalysts.[26] Albumin has also attracted interest
for development as a drug delivery vehicle,[27] and its conjugates
with the Ru(III) metallodrug NAMI-A promote prolonged activity and selective accumulation in tumor cells.[26,28] The earlier
iridium-containing fraction (tR = 1–2 min) contained an abundant ∼50 kDa protein (Rf = 0.570), which is likely attributable to
the IgG heavy chain (50 kDa, pI = 6.4–7.6).[29] Though there are
few examples of metallodrug-IgG interactions, a Ru(II) pyrithione
catalyst has been reported to exhibit a similar protein binding
distribution (IgG = 5% Ru, albumin = 70% Ru, unbound = 25%
Ru).[30] Nevertheless, given Ir was quantified at a short retention
time (tR = 1–2 min), it remains unclear whether Ir was bound to
either IgG or another protein of a similar size, nonprotein bound
Ir (for example, elution of the neutral Ir(III) TsDPEN catalyst), or
Ir bound to a protein of different size that was not resolved
by Coomassie staining. Further investigations into Ir extracellular
speciation, while stimulating, were however beyond the scope of
this in-cell catalysis study.
Since the catalyst appeared to be redox-targeting, levels
of intracellular ROS were quantified using confocal fluorescence microscopy using the membrane-permeable probe 2 ,7 dichlorodihydrofluoescein diacetate (H2 -DCFDA) (Figure 5a). This
ChemCatChem 2024, 0, e202401490 (6 of 13)
reagent is oxidized intracellularly to produce dichlorofluorescein
(DCF), which exhibits a green fluorescence. A2780 cells were
co-stained using 4 ,6-diamidino-2-phenylindole (DAPI), which
exhibits blue fluorescence when bound to double-stranded DNA,
providing a marker for cell localization. Assay suitability was confirmed by the positive control (1 μM hydrogen peroxide, 1 h),
which generated a significant increase in green fluorescence
(23 ± 3 RFU, P = 0.0078) compared to the untreated control
(2.7 ± 0.9 RFU). Treatment of cells with Ir catalyst 1 (1 × IC50 ,
24 h) significantly increased green fluorescence (11 ± 3 RFU,
P = 0.0443) relative to the untreated control; however, the combination of Ir catalyst 1 and sodium formate (3 ± 1 RFU) remained
statistically similar to the untreated control (P = 0.7251). When
administered to cells alone, Ir catalyst 1 generated oxidative
stress, which was negated (due to the reduction catalysis mechanism) when co-administered with a suitable hydride source
(Figure 5b).
To explore whether the generation of ROS by Ir catalyst 1
was conserved in vivo, zebrafish embryos were treated with 1
(1 × LC50 , 10 μM) for 96 h and stained using H2 -DCFDA.[10] The
suitability of H2 -DCFDA as a probe for in vivo ROS detection
was confirmed using pyocyanin, a known inducer of ROS in
zebrafish,[31] as a positive control to validate experimental conditions. Basal ROS levels associated with normal physiological
pathways were observed in the untreated (stained) control, while
510 nm fluorescence was significantly enhanced in embryos
incubated in the presence of either pyocyanin or catalyst 1
(Figure 5c). The in vivo distribution of ROS in embryos treated
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
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Research Article
doi.org/10.1002/cctc.202401490
Figure 5. (a) Structure of green-fluorescent ROS probe 2 ,7 -dichlorodihydrofluoescein diacetate (H2 -DCFDA). (b) DCF fluorescence in A2780 cancer cells
which were either untreated (stained), or treated with 1 μM hydrogen peroxide (H2 O2 , positive control), Ir catalyst 1 (1 × IC50 ) or Ir catalyst 1 (1 × IC50 ) +
2 mM sodium formate for 24 h, performed as part of three technical biological replicates (N = 3). (c) Fluorescence microscopy detection of in vivo ROS
production by catalyst 1 in zebrafish (Danio rerio) embryos using H2 -DCFDA. Zebrafish embryos were treated with catalyst 1 (1 × LC50 , 8 μM, 96 h), a
positive control to confirm assay validity (pyocyanin, 100 μM, 30 min) or were untreated control, showing basal signaling levels of ROS. Statistical
significances were testing using a two-tailed unpaired t-test assuming unequal sample variances (Welch’s t-test). *P < 0.05, **P < 0.01, ***P < 0.001.
with Ir catalyst 1 was consistent with previous ROS distribution in zebrafish generated by a structurally similar Os(II) redox
catalyst,[10] localized in the swim bladder of the embryo.
alternative biologically compatible hydride sources that could be
administered at higher concentrations.
4. Materials and Methods
3. Conclusions
Ir(III) tosyl-diamine transfer hydrogenation catalysts with low
in vivo toxicity have been studied for selective targeting of
cancer cells and overcoming drug resistance. These catalysts
exploit a unique redox-targeting mechanism of action that is distinct from traditional DNA-binding therapeutics and may offer
a new approach to chemotherapeutic design. Catalytic performance under biologically relevant conditions revealed that the
amount of the hydride source formate was rate-limiting, challenging the hypothesis that the main barrier to achieving in-cell
transfer hydrogenation is the delivery and availability of the
catalyst itself. Ir catalysts showed lower in vivo acute toxicity
in zebrafish embryos than previously reported Os(II) analogues,
and in-cell catalytic activity was maintained in both cisplatin
(ovarian) and tamoxifen (breast) drug-resistant cells. In vitro generation of oxidative stress was shown to be formate-dependent,
providing further evidence to support the proposed transfer
hydrogenation mechanism in cells. Future work in this field
will involve exploring delivery mechanisms and/or formulation
strategies that enhance hydride source availability, as well as
ChemCatChem 2024, 0, e202401490 (7 of 13)
Synthetic methods to prepare iridium dimer precursors
[Ir(Cp*)Cl2 ]2 , [Ir(CpxPh )Cl2 ]2 , and [Ir(CpxBip )Cl2 ]2 have been previously reported,[32] and are also commercially available.
Iridium CpxPh and CpxBip dimer precursors were kindly provided by Dr Abraha Habtemariam (University of Warwick).
Organic and deuterated solvents, triethylamine, sodium and
potassium hydroxides, propidium iodide, Tris (99.9999% trace
metal basis), ammonium acetate (99.9999% trace metal basis),
2,6-dichlorophenolindophenol, cisplatin, trichloroacetic acid,
sodium formate, and sodium acetate were purchased from
Merck and used as received unless specified. (1R,2R)-1,2bis(4-methoxyphenyl)ethylenediamine dihydrochloride was
purchased from Alfa Aesar. (R,R)-TsDPEN and (S,S)-TsDPEN were
purchased from Arran Chemicals (Ireland). Inorganic Ventures
certified reference materials for ICP-MS analysis (193 Ir and 195 Pt,
1000 mg L−1 ) were purchased from ESS Lab (Essex, UK). Dulbecco’s Modified Eagle Medium (DMEM, high glucose without
sodium pyruvate, D5796), trypsin/EDTA (0.25% in HBSS, T4049),
Dulbecco’s phosphate buffered saline (DPBS without Ca2+ or
Mg2+ , D8537), penicillin-streptomycin (10,000 units penicillin and
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ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
10 mg mL−1 streptomycin in 0.9% NaCl, P0781) and fetal calf
serum (non-USA origin, F9665) were purchased from Merck Life
Sciences and used as received. 4–15% mini-protean TGX precast
gels, Coomassie stain, tris/glycine/SDS running buffer, and Precision Plus unstained protein ladder were purchased from Bio-Rad
Laboratories. Laemmli buffer was purchased from Scientific Laboratory Supplies (Nottingham, UK). Centrifuge tubes (Corning
CentriSTAR 15 and 50 mL tubes) were purchased from Appleton
Wood Limited (Birmingham, UK). Sterile pipette tips were purchased from Starlab (UK). C-Chip disposable hemocytometers
were purchased from Cambridge Biosciences. All other cell culture plasticware (Greiner GmbH) was purchased from Scientific
Laboratory Supplies (Nottingham, UK). All other non-specified
reagents were purchased from Merck and used as received.
A2780, A2780cis, A549, HCT116, HCT116-p21-/-, HCT116-p53-/-,
MCF7, MCF7-TAMR1, MRC5, OE19, and PC3 cells were obtained
from the European Collection of Authenticated Cell Cultures
(ECACC). MCF10-A cells were obtained from LGC Standards Limited (Teddington, UK). Human ovarian fibroblast (HOF) cells were
obtained from Caltag Medsystems (Buckingham, UK).
4.1. Synthesis of Ligand L1 and Ir(III) Catalysts 1–6
Ligand L1: Methoxyphenyl diamine ligand L1 was prepared
from (1R,2R)-1,2-bis(4-methoxyphenyl)ethylenediamine dihydrochloride (380 mg, 1.10 mmol) dissolved in anhydrous
dichloromethane (50 mL) cooled in an ice bath. Triethylamine
(0.23 mL, 1.65 mmol) was added under an inert nitrogen atmosphere with stirring before slowly adding a solution of p-toluene
sulfonyl chloride (210 mg, 1.10 mmol) in dry dichloromethane
(80 mL) dropwise. The ice bath was removed, and the solution
was allowed to warm to ambient temperature for 1 h. The
organic phase was washed with water (3 × 15 mL), dried over
magnesium sulfate, and the solvent removed in vacuo to afford
a light-yellow oil. The product was recrystallized from hot diethyl
ether and washed with additional ice-cold diethyl ether to afford
an amorphous white solid (363 mg, 0.85 mmol, 74%). 1 H NMR
(500 MHz, CDCl3 , TMS): δ = 7.32 (2H, d, 3 J = 8.1 Hz, ArH), 7.04 (2H,
br. s, ArH), 6.98 (4H, d, 3 J = 7.7 Hz, SO2 ArH), 6.67 (4H, d, 3 J = 8.1 Hz,
ArH), 4.34 (1H, br. s, TsNHCH), 4.10 (1H, d, ArCHNH2 ), 3.73 (3H, br.
s, OCH3 ), 3.70 (3H, s, OCH3 ), 2.32 (3H, s, SO2 ArCH3 ), 1.81 (2H, br. s,
NH2 ); 13 C NMR (125 MHz, CDCl3 , TMS): δ = 158.8, 142.4, 137.3, 129.0,
128.6, 127.0, 113.7, 113.6, 55.2, 55.1, 45.8, 21.4; UV–vis: λmax 275 and
285 nm. HRMS (m/z): [M + Na]+ calculated for C23 H26 N2 NaO4 S:
449.1511; found 449.1503; analysis% (calculated for C23 H26 N2 O4 S,
found): C (64.77, 64.46), H (6.14, 6.10), N (6.57, 6.60).
Catalyst 1 [Ir(Cp*)((R,R)-TsDPEN)]: Catalyst 1 has been previously
reported using a different synthetic method.[14] In this work,
catalyst 1 was prepared by combining [Ir(Cp*)Cl2 ]2 (120 mg,
0.15 mmol) and (R,R)-(H)TsDPEN (110.6 mg, 0.30 mmol) in chloroform (10 mL) with freshly ground KOH (168 mg, 3 mmol). A color
change from orange to purple was observed in <1 min. After
5 min, water (10 mL) was added with stirring for a further 10 min.
The organic layer was removed and concentrated in vacuo to
ChemCatChem 2024, 0, e202401490 (8 of 13)
yield a purple oil, which was dissolved in the minimum amount
of dichloromethane, followed by precipitation with n-pentane.
The product was collected as a purple crystalline solid (144 mg,
0.21 mmol, 69%). 1 H NMR (400 MHz, CDCl3 , 25 °C, TMS): δ = 7.55
(d, 3 J(H,H) = 7.4 Hz, 2H), 7.06–7.36 (m, 10H), 6.79 (d, 3 J(H,H) =
8.0 Hz, 2H), 5.34 (s, 1H; CHNTs), 4.15 (br. d, 1H; CHNH), 2.24 (s, 3H;
CH3 ), 1.92 (s, 15H; Cp*); 13 C NMR (100 MHz, CDCl3 , 25 °C, TMS): δ =
128.2, 127.8, 127.6, 126.9, 126.6, 126.5, 126.2, 85.2, 80.3, 74.1, 10.2; UV–
vis: λmax 429 and 536 nm; HRMS (m/z): [M + H]+ calculated for
C31 H36 IrN2 O2 S, 693.2121; found, 693.2118; analysis % (calculated for
C31 H35 IrN2 O2 S, found): C (53.81, 53.80), H (5.10, 5.09), N (4.05, 4.02).
Catalyst 2 [Ir(CpxPh )((R,R)-TsDPEN)]: This catalyst was obtained
following the method described for catalyst 1 using dimer
[Ir(CpxPh )Cl2 ]2 (50.3 mg, 0.055 mmol) and (R,R)-(H)TsDPEN (40 mg,
0.11 mmol). The product was isolated as a dark purple amorphous solid (50 mg, 0.066 mmol, 61%). 1 H NMR (500 MHz, CDCl3 ,
25 °C, TMS): δ = 7.58 (d, 3 J(H,H) = 7.5 Hz, 2H), 7.08–7.53 (m, 15H),
6.79 (d, 3 J(H,H) = 8.0 Hz, 2H), 5.67 (br. d, 3 J(H,H) = 4.0 Hz, 1H; NH),
4.42 (s, 1H; CHNTs), 4.20 (d, 3 J(H,H) = 4.0 Hz, 1H; CHNH), 2.25 (s,
3H; CH3), 2.07 (s, 3H; CpxPh ), 2.02 (s, 3H; CpxPh ), 1.89 (s, 3H; CpxPh ),
1.88 (s, 3H; CpxPh ); 13 C NMR (125 MHz, CDCl3 , 25 °C, TMS) δ = 146.7,
146.5, 141.1, 140.1, 131.5, 130.9, 128.6, 128.2, 127.9, 127.7, 126.9, 126.7,
126.6, 126.3, 89.8, 88.0, 87.5, 84.8, 83.8, 80.5, 74.2, 21.2, 10.7, 10.6,
10.5, 10.3; UV–vis: λmax 432 and 547 nm; HRMS (m/z): [M + H]+
calculated for C36 H38 IrN2 O2 S, 755.2278; found, 755.2268; analysis
% (calculated for C36 H37 IrN2 O2 S, found): C (57.35, 56.94), H (4.95,
4.96), N (3.72, 3.70).
Catalyst 3 [Ir(CpxBip )((R,R)-(TsDPEN)]: This catalyst was obtained
following the method described for catalyst 1 using dimer
[Ir(CpxBip )Cl2 ]2 (58.6 mg, 0.055 mmol) and (R,R)-(H)TsDPEN
(40 mg, 0.11 mmol). The product was isolated as a dark purple
crystalline solid (33 mg, 0.040 mmol, 37%). 1 H NMR (500 MHz,
CDCl3 , 25 °C, TMS): δ = 7.52–7.64 (m, 8H), 7.44–7.50 (m, 2H),
7.35–7.42 (m, 1H), 7.07–7.30 (m, 10H), 6.80 (d, 3 J(H,H) = 8.0 Hz,
2H; ArH), 5.67 (d, 3 J(H,H) = 4.0 Hz, 1H; NH), 4.43 (s, 1H; CHTs),
4.22 (d, 3 J(H,H) = 4.0 Hz, 1H; CHNH), 2.25 (s, 3H; CH3), 2.09 (s, 3H;
CpxBip ), 2.03 (s, 3H; CpxBip ), 1.94 (s, 3H; CpxBip ), 1.93 (s, 3H; CpxBip );
13
C NMR (125 MHz, CDCl3, 25 °C, TMS) δ = 146.7, 146.5, 141.1, 141.0,
140.4, 140.1, 131.3, 128.9, 128.6, 127.9, 127.7, 127.6, 127.3, 127.1, 127.1,
127.0, 126.7, 126.6, 126.3, 89.4, 88.1, 87.6, 84.8, 83.9, 80.6, 74.2, 21.25,
10.8, 10.6, 10.5, 10.3; UV–vis: λmax 430 and 547 nm; HRMS (m/z):
[M + H]+ calculated for C42 H42 IrN2 O2 S, 831.2592; found, 831.2599;
analysis % (calculated for C42 H41 IrN2 O2 S, found): C (60.77, 61.21),
H (4.98, 5.07), N (3.37, 3.34).
Catalyst 4 [Ir(Cp*)((R,R)-TsDPEN)]: This catalyst was obtained
following the method described for catalyst 1 using dimer
[Ir(Cp*)Cl2 ]2 (46.7 mg, 0.06 mmol) and diamine ligand L1 (50 mg,
0.12 mmol). The product was isolated as a dark purple amorphous solid (37.5 mg, 0.05 mmol, 41%). 1 H NMR (500 MHz, CDCl3 ,
25 °C, TMS): δ = 7.44 (d, 3 J(H,H) = 8.4 Hz, 2H; ArH), 7.25 (d, 3 J(H,H)
= 8.2 Hz, 2H; ArH), 7.04 (d, 3 J(H,H) = 8.5 Hz, 2H; ArH), 6.82 (d,
3
J(H,H) = 8.0 Hz, 2H; ArH), 6.79 (d, 3 J(H,H) = 8.5 Hz, 2H; ArH),
6.65 (d, 3 J(H,H) = 8.6 Hz, 2H; ArH), 5.36 (br. s, 1H; NH), 4.18 (s, 1H;
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
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doi.org/10.1002/cctc.202401490
TsNCH), 4.05 (s, 1H; CHNH), 3.78 (s, 3H; OCH3 ), 3.78 (s, 3H; OCH3 ),
2.26 (s, 3H; CH3 ), 1.91 (s, 15H; CH3 ); 13 C NMR (125 MHz, CDCl3 , 25 °C,
TMS) δ = 158.5, 158.1, 140.9, 138.9, 138.7, 128.2, 127.1, 126.7, 113.1, 85.1,
79.8, 73.4, 55.3, 21.2, 10.2; UV–vis: λmax 285, 430, and 538 nm; HRMS
(m/z): [M + H]+ calculated for C33 H40 IrN2 O4 S, 753.2333; found,
753.2336; analysis % (calculated for C33 H39 IrN2 O4 S, found): C (52.71,
53.03), H (5.23, 5.43), N (3.73, 3.65).
Aqueous stability: Stock solutions of catalyst 1–6 (3 mM) were
prepared in phosphate buffered saline (300 μM final working
concentration, 10% v/v DMSO to aid solubility). Samples were
analyzed using a Shimadzu UV-2600 UV–vis Spectrophotometer
(800–250 nm, slow scan speed, 1.0 nm sampling interval) immediately after preparation (t = 0 h) and after incubation at 37 °C (t
= 24 h). Data were analyzed in Microsoft Excel.
Catalyst 5 [Ir(CpxPh )((R,R)-TsDPEN)]: This catalyst was obtained
following the method described for catalyst 1 using dimer
[Ir(CpxPh )Cl2 ]2 (53.9 mg, 0.06 mmol) and diamine ligand L1
(50 mg, 0.12 mmol). The product was isolated as a dark purple
amorphous solid (35.5 mg, 0.04 mmol, 37%). 1 H NMR (500 MHz,
CDCl3 , 25 °C, TMS): δ = 7.51 (d, 3 J(H,H) = 6.9 Hz, 2H; ArH), 7.47 (d,
3
J(H,H) = 8.5 Hz, 2H; ArH), 7.39 (br. s, 1H; NH), 7.32–7.38 (m, 3H;
ArH), 7.28 (d, 3 J(H,H) = 8.2 Hz, 2H; ArH), 7.05 (d, 3 J(H,H) = 8.5 Hz,
2H; ArH), 6.82 (d, 3 J(H,H) = 7.9 Hz, 2H; ArH), 6.77 (d, 3 J(H,H) =
8.5 Hz, 2H; ArH), 6.65 (d, 3 J(H,H) = 8.5 Hz, 2H; ArH), 4.30 (s, 1H;
CHNTs), 4.10 (s, 1H; CHNH), 3.79 (s, 3H; OCH3 ), 3.78 (s, 3H; OCH3 ),
2.26 (s, 3H; CH3 ), 2.07 (s, 3H; CH3 ), 2.02 (s, 3H; CH3 ), 1.89 (s, 3H;
CH3 ), 1.88 (s, 3H; CH3 ); 13 C NMR (125 MHz, CDCl3 , 25 °C, TMS) δ
158.6, 158.2, 141.2, 140.0, 138.8, 131.5, 130.9, 128.6, 128.2, 127.9, 127.2,
126.7, 113.1, 89.8, 87.9, 87.5, 84.6, 84.0, 80.0, 73.5, 55.3, 21.2, 10.7,
10.6, 10.5, 10.4; UV–vis: λmax 278, 433, and 549 nm; HRMS (m/z):
[M + H]+ calculated for C38 H42 IrN2 O4 S, 815.2490; found, 815.2485;
analysis % (calculated for C38 H41 IrN2 O4 S, found): C (56.07, 55.98),
H (5.08, 5.31), N (3.44, 3.31).
Octanol–water partition coefficient (Log P) determination: Partition coefficients were determined by shake-flask method. Solutions of n-octanol-saturated water and water-saturated n-octanol
were prepared by stirring equal volumes of each solvent for 24 h.
To the water-saturated n-octanol was added enough catalyst to
achieve a saturated solution, which was filtered using a 0.2 μm
filter to remove undissolved particulates. This solution was combined with an equal volume of n-octanol-saturated water and
shaken using an IKA Vibrax VXC basic shaker (1000 g min−1 ,
24 h, room temperature). Next, solutions were allowed to separate for 1 h. Metal content (193 Ir) was determined in the aqueous
layer before and after shaking using an Agilent 7900 series ICPMS using an internal 166 Er standard in no-gas mode. Partition
coefficients (Log P) were calculated as part of two independent
experiments, each carried out in triplicate. Data were reported as
the average of the two experiments with the associated standard
deviation of the measurement.
Catalyst 6 [Ir(CpxBip )((R,R)-TsDPEN)]: This catalyst was obtained
following the method described for catalyst 1 using dimer
[Ir(CpxBip )Cl2 ]2 (62.2 mg, 0.06 mmol) and diamine ligand L1
(50 mg, 0.12 mmol). The product was isolated as a dark purple
amorphous solid (30.7 mg, 0.03 mmol, 28%). 1 H NMR (500 MHz,
CDCl3 , 25 °C, TMS): δ = 7.61 (d, 3 J(H,H) = 7.5 Hz, 2H; ArH), 7.57 (d,
3
J(H,H) = 5.1 Hz, 2H; ArH), 7.44–7.51 (m, 4H; ArH), 7.39 (m, 2H; ArH),
7.30 (d, 3 J(H,H) = 8.0 Hz, 2H; ArH), 7.16 (d, 3 J(H,H) = 8.5 Hz, 1H;
ArH), 7.07 (d, 3 J(H,H) = 8.4 Hz, 2H; ArH), 6.96–7.04 (br. s, 1H; NH),
6.84 (d, 3 J(H,H) = 8.0 Hz, 2H; ArH), 6.79 (d, 3 J(H,H) = 8.5 Hz, 2H;
ArH), 6.66 (d, 3 J(H,H) = 8.5 Hz, 2H; ArH), 4.31 (s, 1H; CHNTs), 4.11 (s,
1H; CHNH), 3.79 (s, 3H; OCH3 ), 3.78 (s, 3H; OCH3 ), 2.27 (s, 3H; CH3 ),
2.09 (s, 3H; CH3 ), 2.04 (s, 3H; CH3 ), 1.94 (s, 3H; CH3 ), 1.93 (s, 3H; CH3 );
13
C NMR (125 MHz, CDCl3 , 25 °C, TMS) δ = 158.6, 158.2, 141.2, 141.0
140.4, 140.1, 138.8, 131.3, 128.9, 128.2, 127.9, 127.7, 127.3, 1271, 126.7,
113.6, 113.2, 113.1, 89.2, 88.0, 87.6, 84.7, 84.0, 80.0, 73.5, 61.2, 55.3, 21.2,
10.9, 10.7, 10.5, 10.4; UV–vis: λmax 263, 431, and 548 nm; HRMS (m/z):
[M + H]+ calculated for C44 H46 IrN2 O4 S, 891.2803; found, 891.2800.
4.2. Physicochemical Experimentation
Stability in DMSO: Proton 1 H NMR spectra were obtained using
a Bruker AV400 NMR spectrometer using DMSO-d6 as solvent.
Spectra were acquired after preparation (t = 21 min, allowing
for sample preparation and instrument lock, shim, temperature,
average point of acquisition) and after 24 h incubation at 37 °C.
Data were analyzed using TopSpin version 4.4.0.
ChemCatChem 2024, 0, e202401490 (9 of 13)
Aqueous (cell free) catalysis using 2,6-dichlorophenolindophenol
(DCPIP): Stock solutions of (i) catalyst 1, sodium formate (hydride
source) and 2,6-DCPIP were prepared in phosphate buffered
saline (PBS) solution containing 1% v/v dimethyl sulfoxide to
aid solubility. Solutions were combined to achieve final working concentrations of catalyst: 0–20 μM (equivalent to 1 × IC50
concentration in A2780 cells), sodium formate: 0–20 mM, and
2,6-DCPIP: 0–80 mM. Solutions were added independently to
a 96-well plate, but within 1 min of each other. The substrate
(2,6-DCPIP) was added at t = 0 h and defined the initiation
of the reaction. This experiment included all appropriate control samples (catalyst alone, hydride source alone, 2,6-DCPIP
alone, 2,6-DCPIP plus sodium formate). All experiments were
carried out in triplicate (N = 3). Microplate absorbance was measured using an Omega FLUOstar microplate reader (600 nm) at
ambient temperature (20 °C). UV–vis absorbance data were converted to molar concentrations using a 3-point calibration plot
(Supporting Information. Figure S3). Initial reaction rates were
determined within the first minute of data acquisition using
Microsoft Excel and converted to TOFmax where the catalyst
concentration was known. This experiment was repeated with
the following modifications: (1) specificity of the hydride source:
sodium formate (2 mM) was replaced by sodium acetate (2 mM).
(2) specificity of the substrate: 2,6-DCPIP was replaced by Alamar
Blue (reduction of blue resazurin N-oxide to pink resorufin phenoxazine) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (reduction of yellow tetrazolium to purple formazan).
Iridium extracellular speciation: A 1 mM solution of catalyst 1
was prepared in DMSO, which was diluted 100-fold with human
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Research Article
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serum and incubated at 37 °C for 24 h prior to speciation analysis. After incubation, the sample was further diluted 10-fold
using 50 mM Tris (Buffer A) for HPLC speciation analysis. Speciation was achieved using an Agilent Technologies Infinity
II series HPLC fitted with a TSK gel Q-STAT anion exchange
chromatography column (4.6 mm I.D. x 10 cm, 7 μM) and a
UV–vis DAD detector (280 nm, referenced to 360 nm). After
column equilibration with Buffer A, analysis was carried out
with the following method parameters: 100 μL injection volume,
linear gradient, 0.7 mL min−1 flow rate, 30 min run time, 10 MPa
maximum pressure, pH 7.4, 25 °C. Elution gradient: 0.0–3.0 min,
0% Buffer B; 3.0–9.0 min, 20% Buffer B; 9.0–13.5 min, 50% Buffer
B; 13.5–16.5 min, 100% Buffer B; 16.5–22.5 min, 100% Buffer B; 22.5–
27.0 min, 0% Buffer B (re-equilibration); 27.0–30.0 min, 0% Buffer
B. Eluent fractions (0.35 mL) were collected at 30 s intervals into
centrifuge tubes for the first 20 min of analysis. The procedure
was repeated five times and samples were pooled to obtain
final fraction volumes of 1.75 mL. HPLC data were acquired and
processed using Agilent CDS software version 2.7. Fractions were
subject to two separate analysis methods: (1) ICP-MS quantification of 193 Ir was achieved using freshly prepared calibration
standards in 50 mM Tris base (Buffer A) ranging from 0.1 to
1000 ppb. Data were acquired as instrumental triplicates using
an Agilent 7900 Series ICP-MS with MassHunter 4.4 (Agilent
Technologies) and processed using Microsoft Excel. (2) Protein
identification was achieved using SDS-PAGE. Fifty microliters
of each fraction were diluted two-fold with Laemelli buffer
(50 μL) and vortexed to achieve a homogenous mixture before
heating to 70 °C for 10 min. A Mini-Protean TGX Precast Gel
was loaded with fractions 1–28 (retention time (tR ) = 0–14 min,
10 μL of denatured sample) alongside 10 μL of Precision Plus
protein standard unstained ladder (0–250 kDa). Upon completion of electrophoresis, gels were removed and washed three
times with Type I water, before staining using 50 mL Bio-Safe
Coomassie G-250 stain for 48 h. Stained gels were subsequently
rinsed for 30 min in Type I water. Rf values were calculated, and
a calibration plot was constructed from ladder proteins (Rf as a
function of molecular weight) to estimate the molecular weight
of serum proteins (Supporting Information, Figure S4).
4.3. Biological Experimentation
Cell maintenance: All cell lines were grown as adherent monolayers in 75 cm2 tissue culture flasks in a 5% CO2 humidified
atmosphere at 37 °C and passaged upon reaching 80%–90%
confluence. All cell lines (except MCF10-A and HOF) were maintained in DMEM culture medium supplemented with 10% v/v
fetal calf serum and 1% v/v penicillin/streptomycin antibiotics
at 10,000 units. MCF10-A cells were maintained in DMEM/F12
medium supplemented with 5% v/v horse serum, human epidermal growth factor (20 ng mL−1 ), hydrocortisone (0.5 mg mL−1 ),
cholera toxin (100 ng mL−1 ), insulin (10 μg mL−1 ), and 1%
v/v penicillin/streptomycin antibiotics at 10,000 units. HOF cells
were maintained in fibroblast medium supplemented (Cat. No.
2301, ScienCell) with 2% v/v fetal calf serum, 1% v/v penicillin/streptomycin and 1% fibroblast growth supplement (Cat.
ChemCatChem 2024, 0, e202401490 (10 of 13)
No. 2352, ScienCell). Drug-resistant cell lines (A2780cis and
MCF7-TAMR1) were inoculated with drug (cisplatin or tamoxifen,
respectively; 5 × IC50 , 24 h) every 10 passages to maintain resistance, and resistance was confirmed by antiproliferative activity
screening in comparison to the sensitive parental cell line.
Antiproliferative activity screening: Briefly, 5 × 103 cells (A2780,
A2780cis, A549, HCT116, HCT116-p21-/-, HCT116-p53-/-, HOF, MCF7,
MCF7-TAMR1, MCF10-A, MRC5, OE19, or PC3) were seeded in flatbottom 96-well plate and incubated for 48 h. A stock solution
of test compound was prepared in DMEM, using 5% v/v DMSO
to aid solubilization of the metallodrug. Serial dilutions of the
test compound were performed to obtain six final working
concentrations (typically 100, 50, 25, 10, 1, 0.1 μM, DMSO not
exceeding 5% v/v). The supernatant medium was removed
from wells by aspiration, and cells were treated (200 μL solution per well) for 24 h. After this time, the drug-containing
supernatant was removed by aspiration, cells were washed with
PBS (100 μL) and were allowed 72 h recovery time in drug-free
medium (200 μL per well). Cell viability was determined using
the sulforhodamine B colorimetric assay.[20] Cells were fixed by
addition of 50% w/v trichloroacetic acid (50 μL per well) and
incubated at 4 °C for 1 h. The supernatant was removed, and
cells washed with water, before staining with sulforhodamine
B solution (0.4% w/v sulforhodamine B solution prepared in 1%
v/v acetic acid; 50 μL per well) for 1 h. Excess dye was removed
by sequential washing with 1% v/v acetic acid, and then dye
was re-solubilized by addition of 100 mM Tris base (200 μL per
well). Absorbance was measured using an Omega FLUOstar
microplate reader (492 nm). Data were normalized to untreated
control wells, and sigmoidal dose/response curves were fitted
using GraphPad Prism 10. Exact working concentrations of metal
catalyst were accurately determined by ICP-OES analysis post
biological experimentation. All experiments were carried out in
duplicate of triplicate and final values reported are the average
of each independent duplicate experiment with associated
standard deviation. Statistical significances were evaluated using
a two-tailed t-test assuming unequal samples variances (Welch’s
t-test) at the 95% confidence level.
Activity modulation in cells using sodium formate: Briefly, 5 ×
103 cells (A2780, A2780cis, MCF7, MCF7-TAMR1, or MRC5) were
seeded in flat-bottom 96-well plate and incubated for 48 h. After
this time, cells were treated with catalyst and sodium formate
to give final concentrations equal to 1.0 × IC50 (catalyst 1) and
0–2 mM (sodium formate) respectively, which were added independently but within 5 min of each other, for 24 h. This included
Ir-free controls for each concentration of sodium formate (0.0,
0.5, 1.0, or 2.0 mM) and a positive control of cisplatin (0–100 μM).
After this time, cells were washed with PBS (100 μL) and were
allowed 72 h recovery time in drug-free medium (200 μL per
well). Cell viability was determined using the sulforhodamine
B colorimetric assay as described for Antiproliferative Activity
Screening.[20] Data were normalized relative to the sodium
formate-free Ir control to demonstrate activity modulation by
sodium formate. All experiments were carried out in duplicate
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
of triplicate and final values reported are the average of each
independent duplicate experiment with associated standard
deviation. Statistical significances were evaluated relative to
the sodium formate-free Ir control, using a two-tailed t-test
assuming unequal samples variances (Welch’s t-test) at the 95%
confidence level. This experiment was repeated with the following modification: sodium formate (0–2 mM) was exchanged for
sodium acetate (0–2 mM).
Cellular iridium accumulation: Briefly, 4 × 106 cells (A2780,
A2780cis, A549, HCT116, MCF7, MCF7-TAMR1, OE19, PC3) were
seeded in P100 dishes and incubated for 24 h. After this time,
cells were exposed to 1 × IC50 equipotent concentrations of catalyst 1 (10 mL) for 24 h with no recovery time. The supernatant
was removed by aspiration, cells were washed with PBS (10 mL)
and harvested using trypsin/EDTA. A cell count was performed,
and a cell pellet obtained by centrifugation (1000 rpm, 5 min,
room temperature). The cell pellet was resuspended in PBS,
transferred to an Eppendorf vial, and re-centrifuged under the
same conditions. The supernatant was removed by aspiration
and the cell pellet was digested by addition of 200 μL 72% v/v
ultrapure nitric acid, which was incubated overnight at 80 °C.
Ultrapure milli-Q water (Type I) was added to obtain a final
working acid concentration of 3.6% v/v. Samples were analyzed
using an Agilent 7900 series ICP-MS operated in no-gas and
He-gas modes, using an internal standard of Er (50 ppb) and
freshly-prepared matrix matched calibration standards for 193 Ir
or 195 Pt (0–1000 ppb). Untreated (negative control) samples were
obtained and measured for Ir content in triplicate to ensure
methodological reliability. All experiments were performed in
biological triplicate (N = 3) with further instrumental triplicate
measurement of each biological replicate. Data were normalized by cell count and reported as an average femtograms Ir
(fg Ir cell−1 ) or Pt (fg Pt cell−1 ) per cell with associated standard deviation. Statistical significances were evaluated using a
two-tailed t-test assuming unequal samples variances (Welch’s
t-test) at the 95% confidence level. This experiment was also
carried out with the following modifications: (1) time-dependent
accumulation: cells were incubated in the presence of catalyst
1 (1 × IC50 , 37 °C) for 3, 6, 18, or 24 h, or alternatively were incubated 24 h drug exposure followed by 24 h recovery in drug-free
medium; (2) temperature-dependent accumulation: cells were
incubated in the presence of catalyst 1 (1 × IC50 ) at 4 °C for 3
or 6 h. These experiments were carried out in parallel to 3–6 h
incubation experiments at 37 °C to facilitate direct comparison;
(3) concentration-dependent accumulation: cells were incubated
24 h at 37 °C using 0.25, 0.5, 1, or 1.5 × IC50 concentrations of
catalyst 1; (4) cofactor-dependent accumulation: cells were treated
with catalyst 1 (final concentration: 0.5 × IC50 ) in the presence
or absence of 2 mM sodium formate.
Cellular iridium distribution: Cell pellets were obtained as
described for cellular iridium accumulation (1 × IC50 concentration ±2 mM sodium formate, 24 h, no recovery time)
using either catalyst 1 or cisplatin. Cells were fractioned using
the FractionPREP kit (BioVision) to obtain four fractions per
ChemCatChem 2024, 0, e202401490 (11 of 13)
sample: cytosol, membrane/organelle, nucleus, cytoskeleton;
according to the manufacturer’s instructions. Each fraction was
independently analyzed for Ir using ICP-MS as described above
(digestion with 72% v/v nitric acid, followed by dilution to obtain
final working acid concentrations of 3.6% v/v). All experiments
were performed in biological triplicate (N = 3) with further
instrumental triplicate measurement of each biological replicate.
Data were normalized to the sum of all four compartments and
reported as average percentage distribution per fraction of the
triplicate samples (N = 3) with associated standard deviation.
Statistical significances were evaluated using a two-tailed t-test
assuming unequal samples variances (Welch’s t-test) at the 95%
confidence level.
A2780 cell cycle analysis by flow cytometry: Cell pellets were
obtained as described for cellular iridium accumulation (1 × IC50
concentration ±2 mM sodium formate, 24 h, no recovery time)
using either catalyst 1 or cisplatin. Cell pellets were resuspended
in 1 mL 80% v/v ethanol on ice for 1 h, before centrifugation
(1000 rpm, 5 min). The supernatant was discarded, and cells were
resuspended in a staining buffer containing propidium iodide
(0.1% v/v from a 1 mg mL−1 DMSO stock) and 0.15% w/v ribonuclease A for 30 min on ice. The supernatant was removed by
centrifugation (1000 rpm, 5 min) then the resulting cell suspension was filtered using a 70 μm cell strainer. Data were acquired
using a Beckman Coulter CytoFLEX S flow cytometer using CytExpert software at the Technology Hub Flow Cytometry core
facility (University of Birmingham) with analysis for propidium
iodide using a yellow (561 nm) laser and PC5.5 filter (690/50 nm).
Data were analyzed in FlowJo version 10.8.1 using the Watson
(pragmatic) cell cycle fitting model and were reported as the
average of three technical replicates (N = 3) with associated standard deviations. Statistical significance tested using an unpaired
t-test assuming unequal variances (Welch’s t-test) at the 95%
confidence level.
A2780 reactive oxygen species (ROS) analysis by fluorescence
microscopy: Cell pellets were obtained as described for cellular iridium accumulation (1 × IC50 concentration ± 2 mM
sodium formate, 24 h, no recovery time). Positive control samples were treated with 1 mM hydrogen peroxide for 1 h prior
to analysis to confirm assay suitability and performance. Samples were stained using 2 ,7 -dichlorodihydrofluorescein diacetate (H2 -DCFDA, 0.1% v/v from a 1 mg mL−1 DMSO stock) and
4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution in
serum-free colorless cell culture medium for 45 min. After this
time, cells were washed three times with PBS and data were
acquired using an EVOS XL Core Imaging System at 10× magnification fitted with DAPI (λEx = 357/44 nm, λEm = 447/60 nm)
and GFP (λEx = 470/22 nm, λEm = 510/42 nm) light cubes. Cells
were located using DAPI and light transmission images, then
mean green fluorescence intensity within the defined cell area
was quantified using ImageJ version 1.54 for MacOS. This analysis
was repeated for three independent biological replicates (N = 3).
Statistical significances were evaluated using a two-tailed t-test
assuming unequal samples variances (Welch’s t-test) at the 95%
confidence level.
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
Comet assay: This experiment was adapted from a procedure
described by Olive et al.[33] Briefly, 1 × 106 A2780 cells were
seeded in a 6-well plate and incubated for 24 h, after which time
cells were treated with equipotent concentrations of Ir catalyst
1 or cisplatin, equal to 1 × IC50 , for 24 h without recovery time.
Cells were harvested using trypsin/EDTA and a single cell suspension was obtained at a density of 2 × 104 cells mL−1 , which
was mixed with low gelling temperature agarose (1% prepared
in Type I water, 40 °C) and transferred to pre-prepared microscope slides (glass slides coated and dried with 1% low-gelling
temperature agarose) at a density of 5 × 103 cells mL−1 . Slides
were carefully submerged in basic lysis solution (0.1% sodium
sarcosinate, 0.26 M NaOH, 1.2 M NaCl, and 100 mM Na2 EDTA
in Type I water, 4 °C) for 24 h in the dark, and then washed
in rinse solution (0.03 M NaOH and 2 mM Na2EDTA in Type I
water) for 20 min. Electrophoresis was carried out in a horizontal chamber containing rinse solution at 20 V for 25 min, after
which slides were washed with Type I water, stained with propidium iodide (2.5 μgmL−1 , 20 min, room temperature) and excess
stain was removed by sequential water washing (3 × 10 mL).
Comets were visualized (N = 20 per sample) using an EVOS XL
Core Imaging System at 20× magnification fitted with a Texas
Red (λEx = 585/29 nm, λEm = 628/32 nm) light cube. At least 20
comets (comet head diameter, tail length, comet tail moment)
were measured for each sample. Comets with a head diameter <20 μm were excluded. Data were analyzed using Microsoft
Excel and reported as the average tail moment with associated
standard deviation. Statistical significances were evaluated using
a two-tailed t-test assuming unequal samples variances (Welch’s
t-test) at the 95% confidence level.
Acute in vivo toxicity in zebrafish (Danio rerio) embryos: Acute
toxicities were determined as previously described.[10] Experiments were performed using Singapore wild-type zebrafish
embryos (D. rerio) under 5 days postfertilization, under project
license AWERB.10/16–17 and AWERB.85/21–22. All animals were
maintained in accordance with ASPA 1986. The University of
Warwick is a member of the Institute of Animal Technology
and the Laboratory Animal Science Association, and all animal
work carried out was approved by the University Ethical Review
Committee. Singapore wild-type zebrafish were housed in 3.5 L
tanks, monitored daily, and provided with food (live and powder) four times daily. Regular system checks were carried out
daily to ensure water quality and parameters are maintained.
Light cycle: 14 h day, 10 h night. Fish were mated once a week
using two pairs per 1 L breeding tank. Embryos were collected
and placed into petri dishes with fresh egg water. A stock solution of the catalyst was prepared in egg water, using DMSO
to aid catalyst solubilization (final DMSO concentrations did not
exceed 1% v/v in working solutions). Embryos were exposed to
test solutions in 20 wells of a 24-well plate (one 24-well plate
per concentration), which were seeded using at least one (and
a maximum of three) embryos per well (N = 20). The remaining wells were treated with egg water only (untreated control).
Plates were incubated for 96 h at 28.5 °C. At the end of the assay,
embryo mortality was assessed by microscopy in accordance
ChemCatChem 2024, 0, e202401490 (12 of 13)
with OECD fish embryo acute toxicity text (OECD-236). Embryos
exhibiting any one of the following traits were considered nonviable: (a) embryo coagulation, (b) absence of somite formation, (c)
tail detachment, and (d) absence of a heartbeat. Dose-response
curves were obtained by plotting % viable embryos against
the logarithm of the drug concentration. Sigmoidal curves were
fitted using GraphPad Prism 10. LC50 values were reported as
the average ± standard deviation of two independent experiments (N = 2) each containing 20 replicates per concentration.
Statistical significances were evaluated using a two-tailed t-test
assuming unequal samples variances (Welch’s t-test) at the 95%
confidence level.
Localization of reactive oxygen species in zebrafish (D. rerio)
embryos: Treated zebrafish embryos were obtained as part of
LC50 screening assays (above). After 96 h exposure to catalyst 1
(1 × LC50 , 8 μM), embryos were collected and washed with egg
water to remove excess Ir catalyst. Separately, positive control
embryos were treated with pyocyanin (100 μM, 30 min) a known
in vivo ROS inducer to confirm assay performance or remained
untreated (negative control). All embryos were anaesthetized
using tricaine (0.2mg mL−1 in egg water, 30 min) after which
time excess tricaine was removed by washing and embryos were
stained with 2 ,7 -dichlorofluorescin diacetate (H2 -DCFDA) in the
dark (1 mg mL−1 , 30 min). Excess H2 -DCFDA was removed by
washing with egg water, and embryos were then mounted in
1% low-gelling point agarose and carefully covered with a solution of 0.2 mg mL−1 tricaine to maintain anesthesia. Images were
acquired using an EVOS XL Core Imaging System fitted with a
GFP (λEx = 470/22 nm, λEm = 510/42 nm) light cube.
Author Contributions
Peter J. Sadler and James P. C. Coverdale conceptualized the
work. James P. C. Coverdale and Yasmin Khanom synthesized and
characterized the iridium catalysts. James P. C. Coverdale performed trace elemental analysis. Millie E. Fry, Sitah A. Alsaif, Alice
K. Keirle, Rebecca A. Bedford, Isolda Romero-Canelon, and James
P. C. Coverdale carried out in vitro biological experiments. Millie E. Fry, Alice K. Keirle, Ji Inn Song, and James P. C. Coverdale
carried out in vivo biological experiments Chloe E. Pheasey and
James P. C. Coverdale carried out extracellular speciation analysis.
All authors gave approval for the final version of the manuscript.
Acknowledgements
James P. C. Coverdale thanks the Royal Society of Chemistry
(E22-1637945680) and the University Of Birmingham for financial
support. Peter J. Sadler research is supported by Anglo American
Platinum and the EPSRC (grant no. EP/P030572/1). The authors
thank Dr. Abraha Habtemariam for the kind provision of Ir CpxPh
and CpxBip dimer precursor compounds, and Mr. Ian Bagley for
support with zebrafish in vivo studies.
© 2024 The Author(s). ChemCatChem published by Wiley-VCH GmbH
ChemCatChem
Research Article
doi.org/10.1002/cctc.202401490
Conflict of Interests
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available in
the Supporting Information of this article.
Keywords: Cancer • Catalysis • Formate • Iridium • Redox
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Manuscript received: August 28, 2024
Revised manuscript received: October 03, 2024
Accepted manuscript online: October 04, 2024
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