Triazolyl-Functionalized N-Heterocyclic Carbene Half-Sandwich Compounds: Coordination Mode, Reactivity and in vitro Anticancer Activity

Article abstract of DOI:10.1002/cmdc.202100311

We report investigations on the anticancer activity of organometallic [M^II/III(η⁶-p-cymene/η⁵-pentamethylcyclopentadienyl)] (M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes (NHCs) substituted with a triazolyl moiety. Depending on the precursors, the NHC ligands displayed either mono- or bidentate coordination via the NHC carbon atom or as N,C-donors. The metal complexes were investigated for their stability in aqueous solution, with the interpretation supported by density functional theory calculations, and reactivity to biomolecules. In vitro cytotoxicity studies suggested that the nature of both the metal center and the lipophilicity of the ligand determine the biological properties of this class of compounds. The Ir^III complex 5 d bearing a benzimidazole-derived ligand was the most cytotoxic with an IC₅₀ value of 10 μM against NCI-H460 non-small cell lung carcinoma cells. Cell uptake and distribution studies using X-ray fluorescence microscopy revealed localization of 5 d in the cytoplasm of cancer cells.

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Triazolyl-Functionalized N-Heterocyclic Carbene Half-
Sandwich Compounds: Coordination Mode, Reactivity and
in vitro Anticancer Activity
Kelvin K. H. Tong,[a, b] Muhammad Hanif,[a, b] Sanam Movassaghi,[a] Matthew P. Sullivan,[a, b, c]
James H. Lovett,[d] Katja Hummitzsch,[e] Tilo Söhnel,[a] Stephen M. F. Jamieson,[b, f]
Suresh K. Bhargava,[g] Hugh H. Harris,[d] and Christian G. Hartinger*[a, b]
We report investigations on the anticancer activity of organo-
metallic
[MII/III(η6-p-cymene/η5-pentamethylcyclopentadienyl)]
biomolecules. In vitro cytotoxicity studies suggested that the
nature of both the metal center and the lipophilicity of the
ligand determine the biological properties of this class of
compounds. The IrIII complex 5d bearing a benzimidazole-
derived ligand was the most cytotoxic with an IC50 value of
10 μM against NCI-H460 non-small cell lung carcinoma cells.
Cell uptake and distribution studies using X-ray fluorescence
microscopy revealed localization of 5d in the cytoplasm of
cancer cells.
(M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes
(NHCs) substituted with a triazolyl moiety. Depending on the
precursors, the NHC ligands displayed either mono- or
bidentate coordination via the NHC carbon atom or as N,C-
donors. The metal complexes were investigated for their
stability in aqueous solution, with the interpretation supported
by density functional theory calculations, and reactivity to
Introduction
More recently, metalÀ NHC complexes have been inves-
tigated as potential drug candidates, including as anticancer
and antibacterial agents.[3] The NHC scaffold derived from
azolium pro-carbenes, most commonly imidazolium derivatives,
offers facile routes for modification, allowing the fine-tuning of
the physicochemical properties and biological activity of
metalÀ NHC complexes.[3c,d] AgI(NHC) compounds were among
the first metalÀ NHC complexes found to exhibit biological
activity.[4] In addition to their antimicrobial activity, some
examples showed promising anticancer properties comparable
Upon coordination to transition metals N-heterocyclic carbene
(NHC) ligands offer σ-donating as well as π-accepting properties
to form strong MÀ L bonds.[1] The inferred stability of MÀ NHC
complexes is advantageous for a variety of applications,
including in homogeneous catalysis.[2]
to that of cisplatin.[4b,5] AuI(NHC) complexes induced Ca2+
-
[a] K. K. H. Tong, Dr. M. Hanif, Dr. S. Movassaghi, Dr. M. P. Sullivan,
Prof. T. Söhnel, Prof. C. G. Hartinger
sensitive mitochondrial swelling, and the onset of mitochondrial
swelling was dependent on the lipophilicity of the compound,
which was influenced by the N-substituents of the NHC
moiety.[6] RuII(arene) complexes and their isostructural OsII-
School of Chemical Sciences, University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
E-mail: c.hartinger@auckland.ac.nz
[b] K. K. H. Tong, Dr. M. Hanif, Dr. M. P. Sullivan, Dr. S. M. F. Jamieson,
Prof. C. G. Hartinger
Maurice Wilkins Centre, University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
[c] Dr. M. P. Sullivan
School of Biological Sciences, University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
[d] J. H. Lovett, Prof. H. H. Harris
(arene),
IrIII(Cp*),
and
RhIII(Cp*)
(Cp*=η5-pentameth-
ylcyclopentadienyl) analogs showed anticancer activity in some
cases, seemingly dependent on the lipophilicity of the com-
plexes as evoked by the substitution pattern of the NHC
moiety.[3b,d,7] Furthermore, surprising reactivity was observed,
particularly in the presence of proteins.[8] Some examples
exhibited antiproliferative effects and inhibited thioredoxin
reductase,[3d,7b,9] similar to AuI(NHC) complexes.[10]
Department of Chemistry, The University of Adelaide
Adelaide, SA 5005 (Australia)
[e] Dr. K. Hummitzsch
Discipline of Obstetrics and Gynecology
The University of Adelaide
By incorporating a chelating NHC ligand, additional func-
tionality can be introduced into metalÀ NHC complexes, as well
as enhancing their stability. Several IrIII(Cp*) complexes with
C,C-, C,N-, and C,O-coordinating NHC ligands exhibited moder-
ate to high antitumor activity against cancer cells.[11] The
cytotoxic activity of the C,N-coordinated complexes was further
improved by altering the substituents of the Cp* and chelating
NHC ligands, to become 3-times more potent than cisplatin.
Robinson Research Institute, Adelaide, SA 5005 (Australia)
[f] Dr. S. M. F. Jamieson
Auckland Cancer Society Research Centre
University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
[g] Prof. S. K. Bhargava
Centre for Advanced Materials and Industrial Chemistry
School of Science, RMIT University, Melbourne (Australia)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100311
Furthermore,
a a chelating
RuII(cym) complex featuring
This article belongs to the joint Special Collection with the European Journal
of Inorganic Chemistry, “Metals in Medicine”.
pyrazole-NHC ligand was a highly efficient catalyst for alcohol
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oxidation as well as displaying significant cytotoxicity against
cisplatin-resistant gastric cancer cells.[12]
carbenic carbon of the imidazole moiety (Scheme 1), yielding
1a and 1b in low yields (28 and 30%, respectively).
One method to introduce new functionality into imidazol-
ium-derived NHC complexes is to alkylate an endocyclic nitro-
gen atom of the imidazole precursor with an alkyne followed
by click chemistry using the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC). The resultant triazolyl moiety converts
the pro-carbene into a chelating ligand, which coordinates to
metal centers in C,C- or C,N-bidentate fashion.[13] While the
catalytic properties of a diversity of complexes with such
ligands were studied for different catalytic applications,[13a,c] the
bioactivity was rarely investigated. We have previously reported
a series of cationic RuII(cym) complexes (cym=η6-p-cymene)
featuring C,N-coordinating NHC-pyridyl ligands that formed 5-
and 6-membered metallacycles upon coordination to the RuII
center.[14] Some of these complexes showed potent antiprolifer-
ative activity with IC50 values in the low μM range. Herein, we
expand on the compound type and explore the impact of the
metal center and denticity of the ligand on the antiproliferative
activity. These investigations are complemented by studies on
the aqueous stability and reactivity toward biomolecules as well
as their intracellular distribution.
Messerle and co-workers[13a,c] reported C,N-coordinated
NHC-triazolyl half-sandwich complexes including the Rh- and
Ir(Cp* compounds 4c and 5c (Scheme 1). Using the same
ligand type, we expanded on the series and prepared
complexes from the corresponding [MII/III(cym/Cp*)Cl2]2 dimers
(M=Ru, Os, Rh, Ir) and the respective pro-carbenes (Scheme 1).
NH4PF6 was added to facilitate counterion metathesis of ClÀ
À
with PF6 . In initial attempts, nearly stoichiometric amounts of
Ag2O, pro-carbene, and [RuII(cym)Cl2]2 were used for the
preparation of 1a and 1b. However, analysis of the products by
1H NMR spectroscopy revealed the formation of only small
amounts of the desired product and traces of unreacted
AgI(NHC) intermediate and dimeric metal precursors. The
stoichiometry of the reaction mixture was optimized and
identified as 2.25:1.375:1.0 (pro-carbene:Ag2O:[MII/III(cym/Cp*)
Cl2]2) for preparation of the desired RuII- and OsII(NHC)
complexes 2c and 3c, respectively, at high purity although low
to moderate yields. The same synthetic approach was unsuc-
cessful in the preparation of the Rh- and IrIII(Cp*) derivatives 4c
and 5c. The ESI-mass spectra indicated the presence of the
bromido complex (Supporting Information) as both pro-
carbenes a and b were prepared as the bromide salts. To
eliminate the bromide ions in subsequent syntheses, a and b
were converted into the hexafluorophosphate salts c and d,
Results and Discussion
À
CuAAC was utilized to prepare the 1,2,3-triazolyl-NHC precur-
sors a and b from 1-methyl-3-propargylimidazolium bromide
and 1-methyl-3-propargylbenzimidazolium bromide.[15] Success-
respectively. The counterion metathesis of bromide to PF6 was
confirmed by 31P NMR spectroscopy (data not shown). This
process allowed the subsequent reaction steps to proceed
successfully to yield complexes 2c–5d (17–66%) in high purity.
The synthesized metal complexes were characterized by
NMR spectroscopy, ESI-MS, and elemental analysis. In the 1H
NMR spectra of 1a and 1b, the absence of the pro-carbene
proton signal and the slight upfield shift of the triazolyl proton
signal from 8.45 to 8.40 ppm and 8.60 to 8.40 ppm, respectively,
was considered indicative of NHC complex formation. In the
case of 2c–5d, the triazolyl proton signals experienced
significant upfield shifts and were detected in the range of
7.68–8.07 ppm. Furthermore, the absence of the pro-carbene
proton signal of the imidazolium and benzimidazolium precur-
1
ful conversion was confirmed by H and 13C{1H} NMR spectro-
scopy, electrospray ionization mass spectrometry (ESI-MS), and
elemental analysis. The carbenic carbon and the triazolyl
nitrogen atoms enable a and b to act either as mono- or
bidentate ligands to metal centers. [RuII(cym)(H2O)(oxalato)] was
used as a metal precursor to synthesize complexes with the
NHC ligand acting as a monodentate ligand and the remaining
coordination sites of the RuII center occupied by the bidentate
oxalato group. Formation of the carbene by addition of Ag2O
directs the coordination of a or b to the metal center via the
Scheme 1. Preparation of the [RuII(cym)(NHC)(oxalato)] complexes 1a and 1b, and of the [M(cym/Cp*)(triazolyl-NHC)Cl]PF6 complexes 2c–5d.
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sors suggested that both c and d coordinated to the metal
centers through the carbenic carbon and the triazolyl nitrogen
atoms to establish a six-membered metallaring. Upon metal
coordination, the signals of the two sets of bridging methylene
protons in the range of 4.83–5.75 ppm became diastereotopic
and showed geminal coupling. Furthermore, the differences in
the chemical shifts of the diastereotopic methylene proton
signals were higher for those being part of the metallaring, with
2JH’À H’’ values ranging from 14 to 17 Hz. The latter were difficult
to assign due to overlapping of these signals with the cym
resonances. In the 13C{1H} NMR spectra, the carbenic carbon
atom resonated in complexes at significantly lower field (153–
188 ppm) compared to the pro-carbenes (138–142 ppm). In
both 4c and 4d, the signals corresponding to the carbon atoms
bonded to the Rh center were detected as doublets (RhÀ CNHC
,
1J=50 Hz; RhÀ CCp*, J=7 Hz). ESI-MS studies further confirmed
the identity of the NHC complexes. The RuII(oxalato) complexes
1a and 1b were detected as [M+Na]+ and 2c–5d as [MÀ PF6]+
pseudomolecular ions (Table S1). The elemental analysis results
indicated the presence of small amounts of solvent for some
complexes, and these solvents were evident in the 1H NMR
spectra.
1
Single crystals suitable for X-ray diffraction analysis of 1b,
2cCl, and 2dCl were grown via slow diffusion of toluene into
saturated solutions in acetonitrile. In contrast, crystals of 5c
were obtained from a reaction mixture containing l-histidine,
water, methanol, and 5d in chloroform via slow evaporation.
Note that the single crystals of 2cCl and 2dCl were obtained from
reactions between [Ru(cym)Cl2]2 and c and d. Complexes 1b
and 5c crystallized in the triclinic space group P-1, while 2cCl,
2dCl, and 5d were detected in the monoclinic space groups
P21/c, C2/c, and P21, respectively (Table S2). Complexes 2cCl,
2dCl, and 5c featuring non-symmetric bidentate ligands crystal-
lized as enantiomeric mixtures with a prochiral metal center.
The complexes adopted a pseudo-octahedral configuration,
where both the cym (1b, 2cCl, 2dCl) and Cp* ligands (5c, 5d)
occupied three coordination sites (Figures 1, S1 and S2). In the
case of 1b, the coordination sphere about the Ru center was
filled with an oxalato and the NHC ligand, and for the latter π-
stacking interactions were observed between two independent
molecules of 1b (3.375 Å). The molecular structures of 2cCl, 2dCl,
5c, and 5d demonstrated the expected bidentate coordination
of the triazole-functionalized NHC ligand to the metal center via
the MÀ CNHC and MÀ N3 bonds. This resulted in the formation of
6-membered metallacycles with distorted boat conformation.
Complexes 2cCl, 2dCl, 5c, and 5d showed shorter MÀ CNHC bonds
compared to 1b (Table 1). In isostructural 2cCl, 2dCl, 5c, and 5d,
the MÀ Cl bond length in 2dCl was significantly longer than for
the other compounds, while the MÀ CNHC and MÀ N3 were the
shortest of the series and similar to related metal-NHC
complexes.[14]
Figure 1. ORTEP representations of the molecular structures of 1b and one
of the enantiomers of 2dCl (bottom), drawn at 50% ellipsoid levels. Any
counterions and solvent molecules were omitted for clarity.
Table 1. Key bond lengths for 1b, 2cCl, 2dCl, 5c, and 5d.
Complex
1b
2cCl
2dCl
5c
5d
Bond length [Å]
MÀ Cl
–
2.4287(7)
2.062(3)
2.084(2)
1.706
2.4435(9)
2.035(3)
2.068(3)
1.698
2.409(2)
2.026(9)
2.087(7)
1.825
2.412(1)
2.035(8)
2.077(5)
1.814
MÀ CNHC
MÀ N3
2.072(3)
–
1.690
MÀ R1,centroid
the 5-membered metallacycle as well as involving S or Se as the
other donor atom of the bidentate ligand.[16]
In 5c, protons of the imidazole moiety, methylene linker,
and triazole moiety showed interactions with a PF6À counterion.
À
Interactions with PF6 ions were also observed for 5d but
involving triazole, methylene, and benzyl protons. The distances
of the observed CÀ H···F interactions ranged from 2.3 to 2.8 Å,
which is in accordance with values reported for a series of
imidazole-thione complexes (Supporting Information, Fig-
ure S2).[17]
Organometallics with metal-halido bonds may undergo
ligand exchange reactions in biologically relevant environ-
ments, such as in water or DMSO, the latter commonly used to
prepare stock solutions for compounds with low aqueous
solubility. Therefore, it is essential to investigate the stability of
The RuÀ N3 bond length of 2cCl and 2dCl was compared to a
series of RuII(η6-benzene) complexes featuring a similar biden-
tate ligand with a triazolyl moiety coordinating to the Ru center
through the N3 atom.[16] The distance of RuÀ N3 of these
complexes is on average 2.09 Å, which is longer than that of
2cCl and 2dCl. The bond length differences may be attributed to
1
the metal complexes in various solvents. H NMR spectroscopic
studies were conducted for 2c–5c in 15% DMSO-d6/D2O for
120 h (Figures S3–S6, Table S3). The formation of aqua species
was confirmed experimentally by adding 1.5 molar equivalents
of AgNO3 to the reaction mixtures and comparing the
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1
respective H NMR spectra. Compounds 2c and 3c underwent
immediate ClÀ /H2O ligand exchange to form the respective
aqua complexes (Figures S3 and S4). Dissolution of 4c in 15%
DMSO-d6/D2O results in the formation of several sets of signals,
presumably a mixture of the chlorido 4c, aqua 4cH2O, and DMSO
4cDMSO complexes, which form an equilibrium rapidly after
dissolution (Figure S5). The addition of AgNO3 changed the
distribution of the signal sets by converting the chlorido into an
aqua complex. In contrast to the other imidazole-based
complexes, gradual ClÀ /H2O ligand exchange was observed for
the Ir derivative 5c, where about 18% of 5cH2O was formed
upon dissolution and went to completion within 3 h (Figure S6),
as confirmed by the addition of AgNO3 (Figure 2). Furthermore,
an aged solution of 5c in DMSO-d6 was diluted with D2O and
analyzed which also demonstrated that aqua species are
formed rather than the DMSO complex (Figure S7). This was
Metal complexes are prone to form dative covalent bonds
to donor atoms of biomolecules. Investigating the interactions
between metallodrugs and biomolecules can provide valuable
data to help determine possible modes of action responsible
for any antiproliferative effects. To study the interactions with
biomolecules, 2c–5c were dissolved in 20% MeOd4/D2O and
one equivalent of l-cysteine (Cys), l-methionine (Met), l-
histidine (His), or 9-ethylguanine (EtG) was added. The reaction
1
mixtures were analyzed by H NMR spectroscopy over a period
of 48 h. Complex 2c showed only minor reactions with the
amino acids and EtG (Figures S13–16), while the Os analog 3c
did not form adducts with any of the nucleophiles (Figur-
es S17–20). In contrast, the Cp* derivatives 4c and 5c reacted
with all the biomolecules (Figures S21–28), the latter however
at a slower rate. In particular with His, several sets of signals
formed, possibly due to different coordination modes (Figur-
es S23 and S27). Notably, addition of another equivalent of His
resulted in significant upfield shifts of the peaks assigned to
unreacted His due to pH changes in the sample. Analysis of an
incubation mixture of 5c with Cys by ESI-MS supported the
formation of a 5c–Cys adduct, which was detected as the [5c–
ClÀ H+Cys]+ ion at m/z 702.2324 (m/ztheory 702.2322), while the
formation of a 5c–EtG adduct was indicated by a signal
1
supported by a H NMR spectroscopic study in CDCl3 to which
several equivalents of DMSO were added and no DMSO
complexes were detectable after 4 h (Figure S8).
The ClÀ /H2O ligand exchange of 2c–5c was investigated
with density functional theory (DFT) calculations using GAUS-
SIAN 09 W.[18] For all cases, the calculated energy difference was
slightly positive for ClÀ /H2O ligand exchange (ΔE range=2.5–
4.6 kcal/mol) and negative for ClÀ /OHÀ ligand exchange (ΔE
ranging from À 55.0 to À 60.3 kcal/mol). This result suggested
that the formation of the hydroxido complexes is more
energetically favorable, which would give a singly charged
rather than a doubly charged complex cation. Similar calculated
energy differences were observed in a series of structurally
related Ru(NHC) complexes.[14] The frontier molecular orbitals of
2c–5c and their respective aqua and hydroxido analogs were
analyzed (Figures S9–12). While in the respective chlorido and
aqua species the HOMOs and LUMOs are located mainly on the
metal and its π-bound ligands, the LUMO in 3c is distributed
over the metal center and the triazole moiety. In general, the π
orbitals of the triazolyl group contributed more extensively to
the LUMOs of the compounds than to the HOMOs, independent
of the nature of the monodentate ligand.
1
emerging at around 8.0 ppm in the H NMR spectrum, which
can be assigned to H8 of EtG coordinated to the Ir center
(Figure 3). The EtG adduct formation was confirmed by ESI-MS
and the pseudomolecular ion [5c–ClÀ H+EtG]+ with m/z
759.2781 (m/ztheory 759.2859) was detected.
The in vitro anticancer activity of the complexes and pro-
NHCs was studied against human cervical carcinoma (SiHa),
colon adenocarcinoma (SW480), colorectal (HCT116), and non-
small-cell lung (NCI-H460) cancer cells (Table 2). The com-
pounds demonstrated variable cytotoxic activity with IC50 values
as low as 10 μM for some derivatives, while others were non-
cytotoxic at the concentrations investigated. The pro-NHC c was
inactive and d only showed minor activity in NCI-H460 and SiHa
cells. The IrIII compounds 5c and 5d demonstrated significantly
Figure 3. Time-dependent 1H NMR spectroscopic stability study of the
reaction between equimolar amounts of 5c and EtG in 20% MeOD/D2O
solution over 48 h. An additional molar equivalent of EtG was added after
48 h to indicate unreacted EtG in solution. Grey boxes denote the change in
the chemical shifts of the proton signals of 5c as the result of reacting with
EtG in solution.
Figure 2. Time-dependent 1H NMR spectroscopic stability study for 5c in
15% DMSO-d6/D2O over 120 h.
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nuclei. Therefore, it is unlikely that nuclear DNA is the molecular
target of 5d.
Table 2. Antiproliferative activity (SRB assay, 72 h treatment) expressed as
the IC50 values (μM) for pro-carbenes c and d, and complexes 1a–5d
against HCT116 (human colorectal carcinoma), NCI-H460 (human non-small
cell lung carcinoma), SiHa (human cervical carcinoma), and SW480 (human
colon adenocarcinoma) cancer cells expressed as mean�standard error
(n=3).
Conclusion
IC50 values [μM]
Compound
HCT116
NCI-H460
SiHa
SW480
>100
NHC complexes have found widespread application and more
recently have attracted interest in anticancer metallodrug
research. We report here investigations on ligands that feature
a bidentate chelation motif composed of an NHC and a triazole
moiety, and their use in different synthetic strategies to prepare
half-sandwich organometallic compounds with the ligands in
either mono- or bidentate coordination modes. Whereas the Ru
and Os complexes underwent rapid chlorido-aqua ligand
exchange resulting in stable hydrolysis products, the Rh and Ir
derivatives gave more complicated 1H NMR spectra after
dissolution in water/DMSO mixtures. The Ir compound con-
verted into the aqua complex more slowly, while for the Rh
derivative a mixture of adducts, most likely with water and
DMSO, was observed. This pattern also translated to the
biomolecule binding studies with the Ru and Os compounds
undergoing only slow or no reaction with biological nucleo-
philes, while the Rh and Ir compounds reacted with the amino
acids and EtG. The Ir derivative proceeded at a slower rate than
the Rh compound, owing to the different levels of inertness.
Moreover, DFT calculations suggested the formation of hydrox-
ido complexes, which may explain both the different behavior
upon dissolution in water and in terms of reactivity to
biomolecules. The anticancer activity of the compounds was
investigated, and the complexes in which the ligands were
coordinated in a monodentate manner were inactive against
human cancer cell lines. Of the complexes featuring a bidentate
NHC-triazole coordination motif, the Ir(Cp*) derivatives were the
only examples that showed anticancer activity with IC50 values
as low as 10 μM for 5d in NCI-H460 cells. The imidazole-derived
complexes were less potent than the benzimidazole analogs,
suggesting that lipophilicity may impact cellular accumulation.
X-ray fluorescence microscopy revealed that the most potent
compound 5d was uptaken by the cell uptake and became
localizated in the cytoplasm in cancer cells.
c
>100
>100
>100
>100
>100
36�1
>100
>100
>100
>100
91�8
17�2
>100
>100
>100
>100
>100
21�6
78�6
>100
>100
>100
66�18
10�1
>100
1a
2c
3c
4c
5c
d
1b
2d
3d
4d
5d
>100
80�48
81�5
>100
12�2
78�15
>100
>100
>100
64�1
13�1
>100
>100
>100
>100
70�19
>100
>100
>100
>100
95�6
26�4
higher cytotoxicity in all cell lines than the compounds with the
other metal centers, with benzimidazole-derived 5d being the
most toxic compound against all cell lines (Figure S29). This
may be due to the relatively slower ClÀ /H2O ligand exchange
kinetics of Ir complexes, as demonstrated for 5c.
The most cytotoxic compound 5d was selected for further
investigations on its uptake and localization in human lung
cancer A549 and ovarian cancer SKOV-3 cells by X-ray
fluorescence microscopy (XFM) (Figure 4). After treating the
cells with 5d (30 μM in 1% DMSO) for 4 h, the confocal images
confirmed that the cells remained intact indicating that the
uptake of the metal complex occurred while the cells were
alive. The high quantities of zinc in cell nuclei enable it to be
easily identified in XFM studies.[19] The Ir complexes were taken
up into the cells, while control experiments showed no signal
for Ir in untreated cells (data not shown). The cellular uptake of
5d was consistently observed for both cell lines as indicated by
the relatively similar Zn/Ir elemental concentration ratio. By
overlaying the distribution of Zn and Ir in the cells, no co-
localization was observed which suggests that 5d is taken up
into the cytoplasm of SKOV-3 and A549 cells rather than the
Figure 4. The distribution of Zn and Ir (heatmap color scale) in SKOV-3 and A549 cancer cells after treatment with 5d as determined by X-ray fluorescence
microscopy. The overlays of Zn and Ir distributions indicate that 5d is accumulated in the cytoplasm instead of the cell nucleus (Zn, green; Ir, red). Elemental
concentrations are given in units of μgcmÀ 2
.
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dichloromethane (ca. 25 mL) was added. The resultant suspension
Experimental Section
was filtered through a celite pad (ca. 5 cm) and flushed with
dichloromethane (ca. 200 mL) followed by a mixture of acetonitrile
and methanol (ca. 200 mL, 8:2) as eluent which was collected
containing the compound. The solvent was evaporated, the crude
product was dissolved in a minimal amount of dichloromethane
and the compound was precipitated by addition of n-hexane. The
precipitate was dried in vacuo to obtain the pure RuII(η6-p-
cymene)(oxalato) complexes.
Materials and Methods
All air-sensitive reactions were carried out under N2 in standard
Schlenk or round-bottom flasks. The complexes were prepared in
darkness by covering the flask with aluminum foil to prevent
photolytic degradation. Acetonitrile and dichloromethane were
dried prior to use with Na2SO4, while all other solvents purchased
from commercial suppliers were used without further purification.
[(Oxalato-k2O,O’){1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylimidazole-2-ylidene-kC}(η6-p-cymene)ruthenium(II)] 1a
1-Methylbenzimidazole (AK-Scientific, 98%), 1-methylimidazole
(Acros Organic, 99%), 1,2,3,4,5-pentamethylcyclopentadiene (Merck,
�88%), 9-ethylguanine (Sigma-Aldrich, 98%), ammonium hexa-
fluorophosphate (Acros Organic, 99%), α-terpinene (Sigma-Aldrich,
89%), benzyl bromide (Merck, �98%), celite (Sigma-Aldrich),
copper sulfate pentahydrate (ECP, �98.0%), l-cysteine (AK Scien-
tific, 98%), l-histidine (AK Scientific, 98%), l-methionine (AK
Scientific, 98%), iridium(III) chloride hydrate (Precious Metals On-
line, 99%), osmium(III) chloride hydrate (Heraeus South Africa, 55%
metal content), propargyl bromide (Aldrich, 80 wt. % in toluene),
rhodium(III) chloride hydrate (Precious Metals Online, 99%),
ruthenium(III) chloride hydrate (Precious Metals Online, 99%), silver
nitrate (Chem-supply, 99%), silver oxide (Sigma-Aldrich, 99%),
sodium azide (Sigma-Aldrich, �99%), sodium l-ascorbate (Sigma-
Aldrich, �98%), sodium oxalate (BDH), and sodium sulfate
anhydrous (ECP, �98%) were from commercial suppliers and used
as received. [Ru(cym)(H2O)(oxalato)][20] and the dimeric precursors
The synthesis of 1a was performed according to the general
procedure using a (115 mg, 0.34 mmol), silver(I) oxide (49 mg,
0.21 mmol), and [RuII(oxalato)(cym)(H2O)] (104 mg, 0.30 mmol) to
afford
a
brown powder (59 mg, 30%). Calcd. for
C26H29N5O4Ru·0.2 C6H14 ·0.5H2O: C, 54.19; H, 5.48; N, 11.62%. Found:
C, 54.49; H, 5.16; N, 11.32%. MS (ESI+): m/z 600.1171 [M+Na]+
1
(mcalc =600.1161). H NMR (399.89 MHz, CDCl3); δ (ppm) 8.40 (s, 1H,
H-6), 7.37–7.28 (m, 5H, H-8, H-9, H-10, H-11, H-12), 7.14 (s, 1H, H-3),
2
3
6.88 (s, 1H, H-2), 5.61 (d, JH’À H’’ =14 Hz, 1H, H-4a/b), 5.57 (dd, J=
4
3
4
6 Hz, J=1.5 Hz, 1H, H-19/20), 5.55 (dd, J=6 Hz, J=1.3 Hz, 1H, H-
19/20), 5.51 (d, 2JH’À H’’ =14 Hz, 1H, H-7a/b), 5.36 (d, 2JH’À H’’ =14 Hz, 1H,
2
H-7a/b), 5.28 (m, 2H, H-21/22), 5.18 (d, JH’À H’’ =14 Hz, 1H, H-4a/b),
3.76 (s, 1H, H-1), 2.82 (sept, J=7 Hz, 1H, H-24), 2.11 (s, 3H, H-15),
3
1.28 (d, J=6 Hz, 3H, H-25/26), 1.26 (d, J=5.5 Hz, 3H, H-25/26). 13C
{1H} NMR (100.57 MHz, CDCl3); δ (ppm) 174.1 (C-14), 165.8 (C-15/C-
16), 165.3 (C-15/C-16), 142.8 (C-5), 134.7 (C-13), 129.1 (C-8/C-9/C-11/
C-12), 128.7 (C-10), 128.5 (C-8/C-9/C-11/C-12), 125.8 (C-6), 124.2 (C-
2), 122.0 (C-3), 108.7 (C-23), 97.9 (C-18), 84.7 (C21/C22), 84.1 (C21/
C22), 82.1 (C19/C20), 81.6 (C19/C20), 54.5 (C-7), 45.1 (C-4), 37.6 (C-
1), 31.6 (C-24), 22.9 (C25/C26), 22.5 (C25/C26), 18.8 (C-17).
3
3
[24]
[Os(cym)Cl2]2,[21] [Ru(cym)Cl2]2,[22] [Ir(Cp*)Cl2]2,[23] and [Rh(Cp*)Cl2]2
were synthesized following literature procedures. Benzyl azide was
prepared according to a literature procedure using ethanol:H2O=
8:1 as the solvent mixture instead of acetone.[25] 1-[(1-Benzyl-1,2,3-
triazol-4-yl)methyl]-3-methylimidazolium bromide
a and 1-[(1-
benzyl-1,2,3-triazol-4-yl)methyl]-3-methylbenzimidazolium bromide
b were prepared in THF:H2O=1:1 as the solvent mixture instead
of DMSO, as reported by others.[13a]
[(Oxalato-k2O,O’){1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylbenzimidazole-2-ylidene-kC}(η6-p-cymene)ruthenium(II)] 1b
Elemental analyses were conducted on a Vario EL cube (Elementar
Analysensysteme GmbH, Hanau, Germany). Both 1D (1H and 13C{1H}
1
DEPTÀ Q)and 2D (1HÀ H COSY, 1H-13C HSQC, and 1H-13C HMBC)
The synthesis of 1b was performed according to the general
procedure using b (128 mg, 0.33 mmol), silver(I) oxide (47 mg,
0.20 mmol), and [RuII(oxalato)(cym)(H2O)] (101 mg, 0.30 mmol) to
afford a light brown powder (58 mg, 28%). Single crystals suitable
for X-ray diffraction analysis were grown from acetonitrile/toluene.
Calcd. for C30H31N5O4Ru·1.55H2O·0.25 C6H14: C, 55.96; H, 5.61; N,
10.36%. Found: C, 56.10; H, 5.24; N, 10.00%. MS (ESI+): m/z
NMR spectra were recorded on Bruker Avance AVIII 400 MHz NMR
spectrometers at 399.89 MHz (1H) or 100.57 MHz (13C{1H}) at
ambient temperature in CDCl3, DMSO-d6, or D2O. ESI-mass spectra
were recorded on a Bruker micrOTOF-Q II ESI-MS in positive ion
mode.
1
650.1301 [M+Na]+ (mcalc =650.1317). H NMR (399.89 MHz, CDCl3);
X-ray diffraction measurements of single crystals of 1b, 2c, 2d, 5c,
and 5d were performed on a Rigaku Oxford Diffraction XtaLAB-
Synergy-S single-crystal diffractometer with a PILATUS 200 K hybrid
pixel array detector using Cu Kα radiation (λ=1.54184 Å; Table S2)
The data sets were processed with the SHELX2016[26] and Olex2[27]
software packages. All non-hydrogen atoms were refined aniso-
tropically. Hydrogen atoms were inserted at calculated positions
and refined with a riding model or without restrictions. Mercury
2020.3.0 was used to visualize and generate figures of the
molecular structures.[28]
3
δ (ppm) 8.40 (s, 1H, H-10), 7.70 (d, J=8 Hz, 1H, H-3/H-6), 7.33–7.24
(m, 7H, H-3/H-6, H-4/H-5, H-12, H-13, H-14, H-15, H-16), 7.19 (td, 3J=
8 Hz, 2J=1.3 Hz, 1H, H-4/H-5), 5.87 (d, 2JH’À H’’ =14 Hz, 1H, H-8a/b),
5.69–5.62 (m, 3H, H-8a/b, H-23/H-24/H-25/H-26), 5.48–5.33 (m, 4H,
H-11a, H-11b, H-23/H-24/H-25/H-26), 3.97 (s, 3H, H-1), 2.86 (sept,
3J=7 Hz, 1H, H-28), 2.11 (s, 3H, H-21), 1.28 (d, 3J=7 Hz, 6H, H-29, H-
30). 13C{1H} NMR (100.57 MHz, CDCl3); δ (ppm) 188.0 (C-18), 165.8 (C-
19/C-20), 165.1 (C-19/C-20), 142.2 (C-9), 135.6 (C-2), 134.7 (C-7/C-
17), 134.3 (C-7/C-17), 129.1 (C-12/C-13/C-15/C-16), 128.6 (C-14),
128.4 (C-12/C-13/C-15/C-16), 125.9 (C-10), 124.0 (C-4, C-5), 112.5 (C-
3/C-6), 110.1 (C-3/C-6), 109.7 (C-27), 98.2 (C-22), 85.4 (C-25/C-26),
85.1 (C-25/C-26), 82.5 (C-23/C-24), 82.4 (C-23/C-24), 54.4 (C-11), 44.9
(C-8), 34.4 (C-1), 31.6 (C-28), 22.7 (C-29, C-30), 18.8 (C-21).
Syntheses
General procedure for the syntheses of compounds 1a and 1b
Silver(I) oxide (1.00–1.50 mol equiv.) was added to a solution of
compounds a or b (1.13–1.50 mol equiv.) dissolved in dichloro-
General procedure for the syntheses of compounds 2c–5d
°
Silver(I) oxide (1.38 mol equiv.) was added to a solution of
methane and the mixture was stirred at 40 C for 4 h in darkness. A
solution of [RuII(oxalato)(cym)(H2O)] (1.00–1.50 mol equiv.) dissolved
in dichloromethane was added and the mixture was allowed to stir
compounds c or d (2.25 mol equiv.) dissolved in dichloromethane
and stirred at 40 C for 4 h in darkness. A solution of [MII/III(cym/
°
°
Cp*)Cl2]2 (1.00 mol equiv.) in dichloromethane was added and the
at 40 C for 24 h in darkness. The solvents were evaporated and
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°
mixture was stirred at 40 C for 24 h in darkness. A solution of
ammonium hexafluorophosphate (6.75–7.86 mol equiv.) dissolved
in methanol (ca. 15 mL) was added to the mixture and allowed to
stir for a further 3 h in darkness. The solvents were evaporated and
dichloromethane (ca. 25 mL) was added. The resultant suspension
was filtered through a celite pad (ca. 10 cm) and fractions were
collected after elution with either dichloromethane, acetonitrile, or
methanol depending on the compound. The solvent was evapo-
rated, the crude product was dissolved in a minimal amount of
dichloromethane and the compound was precipitated by addition
of n-hexane. The precipitate was dried in vacuo to obtain the pure
complexes.
3H, H-23/H-24), 1.17 (d, 3J=7 Hz, 3H, H-23/H-24). 13C{1H} NMR
(100.57 MHz, CDCl3); δ (ppm) 157.9 (C-14), 140.5 (C-5), 132.9 (C-13),
129.5 (C-9, C-10, C-11), 128.8 (C-8, C-12), 123.3 (C-6), 122.8 (C-2/C-3),
122.2 (C-2/C-3), 102.4 (C-21), 93.2 (C-15), 78.9 (C-17/C-18/C-19/C-20),
78.5 (C-17/C-18/C-19/C-20), 78.1 (C-17/C-18/C-19/C-20), 74.9 (C-17/
C-18/C-19/C-20), 55.9 (C-7), 44.7 (C-4), 38.3 (C-1), 31.4 (C-22), 24.0
(C-23/C-24), 21.5 (C-23/C-24), 19.1 (C-15).
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylimidazole-2-ylidene-kC}(η5-pentameth-
ylcyclopentadienyl)rhodium(III)] hexafluorophosphate 4c
The synthesis of 4c was performed according to the general
procedure using silver(I) oxide (50 mg, 0.22 mmol),
c (118 mg,
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
2-methylimidazole-ylidene-kC}(η6-p-cymene)ruthenium(II)]
hexafluorophosphate 2c
0.30 mmol), [Rh(Cp*)Cl2]2 (97 mg, 0.16 mmol), and ammonium hexa-
fluorophosphate (173 mg, 1.06 mmol). The celite pad was washed
with dichloromethane (ca. 100 mL), followed by a mixture of dichloro-
methane and methanol (ca. 100 mL, 9:1). The latter fraction was
collected and evaporated to afford an orange powder (21 mg, 22%).
Calcd. for C24H30ClF6N5PRh·0.45H2O·0.25 C6H14: C, 43.66; H, 4.94; N,
9.98%. Found: C, 43.60; H, 4.89; N, 9.97%. MS (ESI+): m/z 526.1249
[MÀ PF6]+ (mcalc =526.1245). 1H NMR (399.89 MHz, CDCl3); δ (ppm)
7.89 (s, 1H, H-6), 7.38–7.34 (m, 3H, H-9, H-10, H-11), 7.34–7.30 (m, 2H,
H-8, H-12), 7.26 (d, 4J=2 Hz, 1H, H-2/H-3), 7.06 (d, 4J=2 Hz, 1H, H-2/H-
3), 5.63 (d, 2JH’À H’’ =14 Hz, 1H, H-7a/b), 5.58 (d, 2JH’À H’’ =14 Hz, 1H, H-7a/
b), 5.44 (d, 2JH’À H’’ =17 Hz, 1H, H-4a/b), 4.96 (d, 2JH’À H’’ =17 Hz, 1H, H-4a/
b), 3.92 (s, 3H H-1), 1.66 (s, 15H, H-15). 13C{1H} NMR (100.57 MHz,
The synthesis of 2c was performed according to the general
procedure using silver(I) oxide (38 mg, 0.16 mmol), c (106 mg,
0.26 mmol), [Ru(cym)Cl2]2 (72 mg, 0.12 mmol), and ammonium
hexafluorophosphate (151 mg, 0.93 mmol). The celite pad was
washed with dichloromethane (ca. 100 mL) and acetonitrile (ca.
250 mL) was used as an eluent to collect the fraction to afford a
yellow powder (27 mg, 17%). Single crystals suitable for X-ray
diffraction analysis were grown from acetonitrile/toluene after
performing a reaction between [Ru(cym)Cl2]2 and c to yield the
complex with
a
chloride counterion (2cCl). Calcd. for
1
C24H29ClF6N5PRu: C, 43.09; H, 4.37; N, 10.47%. Found: C, 43.47; H,
CDCl3); δ (ppm) 168.1 (d, J(RhÀ C)=50 Hz, C-14), 141.7 (C-5), 133.0 (C-
3.98; N, 10.11%. MS (ESI+): m/z 524.1155 [MÀ PF6]+ (mcalc
=
13). 129.5 (C-9, C-10, C-11), 128.7 (C-8, C-12), 124.4 (C-6), 124.1 (C-2/C-
3), 124.0 (C-2/C-3), 98.6 (d, 1J(Rh-CCp)=7 Hz, C-16), 56.0 (C-7), 44.2 (C-4),
38.1 (C-1), 9.6 (C-15).
1
524.1151). H NMR (399.89 MHz, CDCl3); δ (ppm) 7.69 (s, 1H, H-6),
7.41–7.34 (m, 3H, H-9, H-10, H-11), 7.34–7.29 (m, 2H, H-8, H-12), 7.17
4
4
3
(d, J=2 Hz, 1H, H-2), 7.05 (d, J=2 Hz, 1H, H-3), 5.67 (d, J=6 Hz,
1H, H-17/H-18), 5.60–5.55 (m, 4H, H-7a, H-7b, H-17/H-18, H-19/H-
3
2
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylimidazole-2-ylidene-kC}(η5-pentameth-
ylcyclopentadienyl)iridium(III)] hexafluorophosphate 5c
20), 5.33 (d, J=6 Hz, 1H, H-19/H-20), 5.29 (d, JH’À H’’ =16 Hz, 1H, H-
4a/b), 4.98 (d, 2JH’À H’’ =16 Hz, 1H, H-4a/b), 3.96 (s, 3H, H-1), 2.78
(sept, 3J=7 Hz, 1H, H-22), 2.12 (s, 3H, H-15), 1.22 (d, 3J=7 Hz, 3H, H-
23/H-24), 1.20 (d, 3J=7 Hz, 3H, H-23/H-24). 13C{1H} NMR
(100.57 MHz, CDCl3); δ (ppm) 173.0 (C-14), 141.9 (C-5), 132.8 (C13),
129.5 (C-9, C-10, C-11), 128.8 (C-8, C-12), 123.8 (C-2/C-3), 123.6 (C-6),
123.3 (C-2/C-3), 111.9 (C-21), 101.6 (C-16), 87.4 (C-17/C-18/C-19/C-
20), 87.3 (C-17/C-18/C-19/C-20), 86.7 (C-17/C-18/C-19/C-20), 83.8 (C-
17/C-18/C-19/C-20), 56.0 (C-7), 44.5 (C-4), 38.4 (C-1), 31.4 (C-22), 23.5
(C-23/C-24), 21.4 (C-23/C-24), 19.1 (C-15).
The synthesis of 5c was performed according to the general
procedure using silver(I) oxide (43 mg, 0.19 mmol),
c (121 mg,
0.30 mmol), [Ir(Cp*)Cl2]2 (108 mg, 0.13 mmol), and ammonium hexa-
fluorophosphate (173 mg, 1.06 mmol). The celite pad was washed
with dichloromethane (ca. 100 mL), followed by a mixture of dichloro-
methane and methanol (ca. 100 mL, 9:1). The latter fraction was
collected and evaporated to afford a yellow powder (97 mg, 47%).
Single crystals suitable for X-ray diffraction analysis were grown from
water/methanol. Calcd. for C24H30ClF6IrN5P: C, 37.87; H, 3.97; N, 9.20%.
Found: C, 38.13; H, 3.90; N, 8.92%. MS (ESI+): m/z 616.1811 [MÀ PF6]+
(mcalc =616.1819). 1H NMR (399.89 MHz, CDCl3); δ (ppm) 7.92 (s, 1H, H-
6), 7.40–7.34 (m, 3H, H-9, H-10, H-11), 7.34–7.30 (m, 2H, H-8, H-12), 7.24
(d, 4J=2 Hz, 1H, H-2/H-3), 7.04 (d, 4J=2 Hz, 1H, H-2/H-3), 5.64 (d,
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylimidazole-2-ylidene-kC}(η6-p–cymene)osmium(II)]
hexafluorophosphate 3c
The synthesis of 3c was performed according to the general
procedure using silver(I) oxide (42 mg, 0.18 mmol), c (118 mg,
0.30 mmol), [Os(cym)Cl2]2 (104 mg, 0.13 mmol), and ammonium
hexafluorophosphate (169 mg, 1.04 mmol). The celite pad was
washed with dichloromethane (ca. 100 mL) followed by a mixture
of dichloromethane and methanol (ca. 100 mL, 9:1). The latter
fraction was collected and evaporated to afford a yellow powder
(77 mg, 38%). Calcd. for C24H29ClF6N5OsP·0.2H2O: C, 37.84; H, 3.89;
N, 9.19%. Found: C, 38.00; H, 4.03; N, 9.01%. HRMS (ESI+): m/z
2
2JH’À H’’ =15 Hz, 1H, H-7a/b), 5.59 (d, JH’À H’’ =15 Hz, 1H, H-7a/b), 5.42 (d,
2
2JH’À H’’ =16 Hz, 1H, H-4a/b), 4.86 (d, JH’À H’’ =16 Hz, 1H, H-4a/b), 3.89 (s,
3H, H-1), 1.69 (s, 15H, H-15). 13C{1H} NMR (100.57 MHz, CDCl3); δ (ppm)
153.2 (C-14), 140.8 (C-5), 132.9 (C-13), 129.5 (C-9, C-10, C-11), 128.7 (C-
8, C-12), 124.2 (C-6), 123.1 (C-2, C-3), 91.2 (C-16), 56.1 (C-7), 44.5 (C-4),
37.5 (C-1), 9.4 (C-15).
1
614.1722 [MÀ PF6]+ (mcalc =614.1726). H NMR (399.89 MHz, CDCl3);
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylbenzimidazole-2-ylidene-kC}(η6-p-cymene)ruthenium(II)]
hexafluorophosphate 2d
δ (ppm) 7.74 (s, 1H, H-6), 7.41–7.35 (m, 3H, H-9, H-10, H-11), 7.35–
4
4
7.30 (m, 2H, H-8, H-12), 7.11 (d, J=2 Hz, 1H, H-2/H-3), 6.98 (d, J=
3
2
2 Hz, 1H, H-2/H-3), 5.72 (d, J=6 Hz, 1H, H-17/H-18), 5.66 (dd, J=
The synthesis of 2d was performed according to the general
3
3
13 Hz, J=5.5 Hz, 2H, H-17/H-18, H-19/H-20), 5.59 (d, J=4 Hz, 2H,
H-7a, H-7b), 5.51 (d, 3J=6 Hz, 1H, H-19/H-20), 5.33 (d, 2JH’À H’’ =16 Hz,
1H, H-4a/b), 4.98 (d, 2JH’À H’’ =16 Hz, 1H, H-4a/b), 3.92 (s, 3H, H-1),
procedure using silver(I) oxide (47 mg, 0.20 mmol),
d (150 mg,
0.33 mmol), [Ru(cym)Cl2]2 (91 mg, 0.15 mmol), and ammonium hexa-
fluorophosphate (163 mg, 1.00 mmol). The celite pad was washed
with dichloromethane (ca. 100 mL) and acetonitrile (ca. 250 mL) was
3
3
2.71 (sept, J=7 Hz, 1H, H-22), 2.24 (s, 3H, H-15), 1.23 (d, J=7 Hz,
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used as an eluent to collect the fraction to afford an orange powder
(107 mg, 50%). Single crystals suitable for X-ray diffraction analysis
were grown from acetonitrile/toluene after performing a reaction
between [Ru(cym)Cl2]2 and d to yield the complex with a chloride
counterion (2dCl). Calcd. for C28H31ClF6N5PRu: C, 46.48; H, 4.39; N,
9.68%. Found: C, 46.36; H, 4.47; N, 9.82%. MS (ESI+): m/z 574.1301
[MÀ PF6]+ (mcalc =574.1311). 1H NMR (399.89 MHz, CDCl3); δ (ppm) 7.79
(s, 1H, H-10), 7.57–7.54 (m, 1H, H-3), 7.46–7.42 (m,1H, H-6), 7.40–7.34
(m, 5H, H-4, H-4, H-13, H-14, H-15), 7.34–7.30 (m, 2H, H-12, H-16), 5.79
2JH’À H’’ =14 Hz, 1H, H-11a/b), 5.07 (d, JH’À H’’ =17 Hz, 1H, H-8a/b), 4.13
(s, 3H, H-1), 1.71 (s, 15H, H-19). 13C{1H} NMR (100.57 MHz, CDCl3); δ
(ppm) 181.2 (d, 1JRh-C(NHC) =50 Hz, C-18), 141.5 (C-9), 135.2 (C-2),
134.3 (C-7), 132.7 (C-17), 129.6 (C-13, C-14, C-15), 128.8 (C-12, C-16),
125.0 (C-10), 124.6 (C-4, C-5), 111.0 (C-3), 110.6 (C-6), 99.3 (d, J(Rh-
CCp)=7 Hz, C-20), 56.2 (C-11), 40.7 (C-8), 35.2 (C-1), 9.7 (C-19).
1
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylbenzimidazole-2-ylidene-kC}(η5-pentameth-
ylcyclopentadienyl)iridium(III)] hexafluorophosphate 5d
3
2
(d, J=6 Hz, 1H, H-21/H-22), 5.72 (d, JH’À H’’ =16 Hz, 1H, H-8a/b), 5.69–
5.66 (m, 1H, H-21/H-22), 5.65–5.62 (m, 2H, H-11a/b, H-23/H-24), 5.56 (d,
3
2JH’À H’’ =15 Hz, 1H, H-11a/b), 5.44 (d, J=6 Hz, 1H, H-23/H-24), 5.08 (d,
The synthesis of 5d was performed according to the general
procedure using silver(I) oxide (47 mg, 0.20 mmol),
2JH’À H’’ =16 Hz, 1H, H-8a/b), 4.17 (s, 3H, H-1), 2.52 (sept, 3J=7 Hz, 1H, H-
d (149 mg,
4
4
26), 2.13 (s, 3H, H-19), 1.25 (d, J=2 Hz, 3H, H-27/H-28), 1.24 (d, J=
2 Hz, 3H, H-27/H-28). 13C{1H} NMR (100.57 MHz, CDCl3); δ (ppm) 187.3
(C-18), 141.7 (C-9), 135.3 (C-2), 134.0 (C-7), 132.7 (C-17), 129.5 (C-13, C-
14, C-15), 128.9 (C-12, C-16), 124.4 (C-4/C-5), 124.2 (C-4/C-5), 124.0 (C-
10), 113.4 (C-25), 110.4 (C-6), 110.1 (C-3), 102.0 (C-20), 88.4 (C-21/C-22),
88.0 (C-21/C-22), 87.8 (C-23/C-24), 84.3 (C-23/C-24), 56.0 (C-11), 40.8 (C-
8), 35.5 (C-1), 31.4 (C-26), 23.6 (C-28), 21.3 (C-27), 19.2 (C-19).
0.33 mmol), [Ir(Cp*)Cl2]2 (117 mg, 0.15 mmol), and ammonium hexa-
fluorophosphate (189 mg, 1.16 mmol). The celite pad was eluted with
dichloromethane (ca. 200 mL) and the fraction was collected to afford
a yellow powder (158 mg, 66%). Single crystals suitable for X-ray
diffraction analysis were grown from chloroform. Calcd. for
C28H32ClF6IrN5P·0.25 C6H14: C, 42.55; H, 4.30; N, 8.41%. Found: C, 42.69;
H, 4.20; N, 8.26%. HRMS (ESI+): m/z 666.1963 [MÀ PF6]+ (mcalc
=
666.1970). 1H NMR (399.89 MHz, CDCl3); δ (ppm) 8.07 (s, 1H, H-10), 7.69
(d, 3J=8 Hz, 1H, H-3), 7.46–7.40 (m, 2H, H-4, H-6), 7.40–7.32 (m, 6H, H-
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylbenzimidazole-2-ylidene-kC}(η6-p-cymene)osmium(II)]
hexafluorophosphate 3d
2
5, H-12, H-13, H-14, H-15, H-16), 5.89 (d, JH’À H’’ =17 Hz, 1H, H-8a/b),
2
2
5.66 (d, JH’À H’’ =15 Hz, 1H, H-11a/b), 5.60 (d, JH’À H’’ =15 Hz, 1H, H-11a/
b), 4.96 (d, 2JH’À H’’ =17 Hz, 1H, H-8a/b), 4.09 (s, 3H, H-1), 1.74 (s, 15H, H-
19). 13C{1H} NMR (100.57 MHz, CDCl3); δ (ppm) 165.5 (C-18), 140.7 (C-9),
135.0 (C-2), 133.8 (C-7), 132.7 (C-17), 129.7 (C-13/C-14/C-15), 129.6 (C-
13/C-14/C-15), 128.8 (C-12, C-16), 125.0 (C-4/C-5), 124.8 (C-10), 124.6
(C-4/C-5), 111.0 (C-3/C-6), 110.8 (C-3/C-6), 92.0 (C-20), 56.2 (C-11), 40.9
(C-8), 34.7 (C-1), 9.4 (C-19).
The synthesis of 3d was performed according to the general
procedure using silver(I) oxide (47 mg, 0.20 mmol), d (150 mg,
0.33 mmol), [Os(cym)Cl2]2 (117 mg, 0.15 mmol), and ammonium
hexafluorophosphate (163 mg, 1.00 mmol). The celite pad was
washed with dichloromethane (ca. 100 mL) and acetonitrile (ca.
250 mL) was used as an eluent to collect the fraction to afford a
light brown powder (124 mg, 52%). Calcd. for C28H31ClF6N5OsP: C,
41.61; H, 3.87; N, 8.67%. Found: C, 41.39; H, 3.59; N, 8.51%. MS
(ESI+): m/z 664.1860 [MÀ PF6]+ (mcalc =664.1883). 1H NMR
Stability studies
For stability studies in DMSO, 5c (1–2 mg) was dissolved in DMSO-
d6 and H NMR spectra were recorded for 120 h. To determine the
stability in aqueous solution, 2c–5c (1–2 mg) were dissolved in
DMSO-d6, diluted with D2O to reach a DMSO-d6 content of 15%,
3
(399.89 MHz, CDCl3); δ (ppm) 7.83 (s, 1H, H-10), 7.56 (d, J=8 Hz,
1
1H, H-3), 7.42–7.29 (m, 8H, H-4, H-5, H-6, H-12, H-13, H-14, H-15, H-
3
16), 5.82 (d, J=6 Hz, 1H, H-23/H-24), 5.79–5.69 (m, 3H, H-8a/b, H-
2
21/H-22, H-23/H-24), 5.63 (d, JH’À H’’ =23 Hz, 2H, H-11a, H-11b), 5.58
1
and H NMR spectra were recorded for 120 h. For 5c, 1 equiv. of
(d, 3J=9 Hz, 1H, H-21/H-22), 5.07 (d, 2JH’À H’’ =16 Hz, 1H, H-8a/b), 4.12
(s, 3H, H-1), 2.80 (sept, 3J=7 Hz, 1H, H-26), 2.26 (s, 3H, H-19), 1.27 (d,
AgNO3 was added after 120 h to confirm the completion of the
chlorido/aqua exchange. To determine the possible formation of a
DMSO complex of 5c, the compound (2 mg) was dissolved in
DMSO-d6 and its H NMR spectrum was recorded. After 2 h, a small
portion was taken from the sample and diluted with D2O to reach a
3
3J=7 Hz, 3H, H-27/H-28), 1.19 (d, J=7 Hz, 3H, H-27/H-28). 13C{1H}
NMR (100.57 MHz, CDCl3); δ (ppm) 171.1 (C-18), 140.3 (C-9), 134.9
(C-2), 133.4 (C-7), 132.7 (C-17), 129.5 (C-13, C-14, C-15), 128.9 (C-12,
C-16), 124.5 (C-4, C-5), 124.1 (C-10), 110.7 (C-6), 110.3 (C-3), 104.2 (C-
25), 93.6 (C-20), 79.9 (C-23/C-24), 79.5 (C-23/C-24), 79.2 (C-21/C-22),
75.5 (C-21/C-22), 56.0 (C-11), 41.2 (C-8), 35.4 (C-1), 31.3 (C-26), 23.9
(C-28), 21.3 (C-27), 19.2 (C-19).
1
1
DMSO-d6 content of 15%, and H NMR spectra were recorded for
4 h. AgNO3 (2 eq.) was added after 4 h. Furthermore, a 1H NMR
spectrum of 5c (2 mg) dissolved in CDCl3 was recorded. DMSO
(2 eq.) was then added to the sample and 1H NMR spectra were
recorded after 0, 2, and 4 h. After 4 h, more DMSO (8 eq.) was
added and the 1H NMR spectrum was recorded.
[Chlorido{1-[(1-benzyl-1,2,3-triazol-4-yl-kN)methyl]-3-
methylbenzimidazole-2-ylidene-kC}(η5-pentameth-
ylcyclopentadienyl)rhodium(III)] hexafluorophosphate 4d
To evaluate the ligand exchange reaction over longer periods, 2c–
4c were dissolved in DMSO-d6, diluted with D2O to reach a DMSO-
d6 content of 15%, and 1.5 equiv. of AgNO3 were added immedi-
ately. An additional 1.5 equiv. of AgNO3 were added after 24 h and
the reaction was monitored by 1H NMR spectroscopy for 30 d.
The synthesis of 4d was performed according to the general
procedure using silver(I) oxide (33 mg, 0.14 mmol), d (105 mg,
0.23 mmol), [Rh(Cp*)Cl2]2 (64 mg, 0.10 mmol), and ammonium
hexafluorophosphate (133 mg, 0.82 mmol). The celite pad was
washed with dichloromethane (ca. 100 mL), followed by a mixture
of dichloromethane and methanol (ca. 100 mL, 9:1). The latter
fraction was collected and evaporated to afford an orange powder
(47 mg, 31%). Calcd. for C28H32ClF6N5PRh·0.4H2O: C, 46.12; H, 4.53;
N, 9.61%. Found: C, 46.29; H, 4.62; N, 9.42%. MS (ESI+): m/z
Biomolecule interaction studies
The biomolecule interactions of 2c–5c were studied by 1H NMR
spectroscopy. All complexes were initially dissolved in MeOD-d4
and diluted with D2O to obtain solutions in 20% MeOD-d4/D2O. l-
Methionine, l-cysteine, l-histidine, and 9-ethylguanine were dis-
solved in MeOD-d4 and diluted with D2O to obtain solutions in 20%
MeOD-d4/D2O. Equimolar amounts of the biomolecule solutions
1
576.1406 [MÀ PF6]+ (mcalc =576.1396). H NMR (399.89 MHz, CDCl3);
3
δ (ppm) 8.03 (s, 1H, H-10), 7.70 (d, J=8 Hz, 1H, H-3), 7.48–7.32 (m,
2
8H, H-4, H-5, H-6, H-12, H-13, H-14, H-15, H-16), 5.92 (d, JH’À H’’
=
16 Hz, 1H, H-8a/b), 5.65 (d, 2JH’À H’’ =14 Hz, 1H, H-11a/b), 5.59 (d,
1
were added to each complex and H NMR spectra were recorded
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ChemMedChem
for 48 h. After 48 h, an additional equivalent of the respective
biomolecule solution was added to each complex and 1H NMR
spectra were recorded.
Source (Argonne National Laboratory, Lemont, IL) with modifica-
tions to the protocol described previously.[30] A double multilayer
monochromator and a gold “high flux” zone plate setup was used
to focus a monochromatic beam into a spot of 300–400 nm FWHM
in diameter. Cells treated with 5d were imaged with an incident X-
ray energy of 13.1 keV to excite at the L-edge of Ir; the P and Zn
maps for these cells were generated from K-edge fluorescence of
these elements. An energy-dispersive silicon drift detector (Vortex
EM, SII Nanotechnology, Northridge, California, USA) was used to
collect the X-ray fluorescence spectra from the sample, which was
X-ray fluorescence microscopy (XFM)
Sample preparation. Silicon nitride membranes (Silson Ltd, Warwick-
shire, England) were washed for 2 min each in Milli-Q water, 70%
ethanol, and 100% ethanol in a small Petri dish. The membranes
were air-dried under sterile conditions and transferred into wells of
12-well culture plates, which were exposed to UV-light overnight.
A549 cells were kindly provided by Dr. Judy Li and Dr. Alex
Staudacher from the Hanson Institute at University of South
Australia (passage 32). SKOV-3 cells were kindly provided by Dr.
Carmela Ricciardelli from the Robinson Research Institute at The
University of Adelaide, Australia. The cells were cultured in a T75
flask and were collected after trypsinizing (0.25% trypsin and
ethylenediaminetetraacetic acid) for 3 min. The cells were spun
down at 1,200 rpm for 5 min. Cell supernatant was discarded and
the pellet was suspended in 1 mL culturing media (A549: RPMI
1640, 5% FCS, and 1% penicillin/streptomycin; SKOV-3: DMEM/F12,
10% FCS, l-glutamine, 1% penicillin/streptomycin and 0.1%
fungizone). Cell suspensions were mixed with trypan blue solution
°
placed in a He environment at an angle of 75 to the incident
beam. All elemental maps were recorded in fly-scan mode, with a
0.5 μm step-size in x and y direction, and a 150 ms dwell time for
the 5d-treated cell maps. Elemental maps were generated with the
MAPS software package[31] by Gaussian fitting of the raw emission
spectra for each image pixel. The Gaussian peaks were matched to
characteristic X-ray emission lines to determine the fluorescence
signal for each element. Quantification of the data (in μg/cm2) was
performed by comparing the X-ray fluorescence intensities to those
from National Bureau of Standards thin film standards NBS-1832,
NBS-1833 (National Bureau of Standards, Gaithersburg, MD, USA).
DFT calculations
(0.4%) and transferred into
a cell counting chamber slide
(Invitrogen). The cells were counted with Countess II (Life
Technologies). The cell solutions were prepared with culturing
media, and A549 (2,450) and SKOV-3 cells (2,000) were seeded onto
each membrane without making contact with the pipette tip. The
GAUSSIAN 09 W[18] was used to calculate the optimized ground
state structures and frequencies for the different molecules by
density functional theory (DFT) with the B3LYPÀ D3 hybrid ex-
change functional and a split basis set for C, H, N, Cl (6-31G(d,p))
and the transitional metal iridium, osmium, rhodium, and ruthe-
nium (SDDAll) in vacuum. The SCRF (self-consistent reaction field)
keyword was implemented for the optimization of the molecules in
aqueous environment. This method is the integral equation formal-
ism variant of the Polarizable Continuum Model (IEFPCM).[32] The
EmpiricalDispersion=GD3 keyword was implemented for the
empirical dispersion correction for the optimization of the
molecules.[33] The frontier orbitals were viewed and obtained
through the Avogadro software (version 1.2.0).[34]
°
cells were incubated for 3 h at 37 C and 5% CO2 atmosphere for
attachment. Culturing media (1.4 mL) was added carefully to each
°
well, and the cells were incubated overnight at 37 C, 5% CO2.
Metal complex incubation. 5d (30 μM, 1% DMSO in media) was
added to the wells carefully in 2 separate portions of 0.7 mL,
enough to soak the cell-coated silicon nitride membrane in
solution. The cells were incubated for 4 h. The compound solution
was aspirated from the well carefully, avoiding contact with the
membrane, then the membrane was washed with PBS (0.7 mL) and
aspirated. The cells were fixed onto the membrane with 4%
buffered paraformaldehyde (0.7 mL). After 5 min, the paraformalde-
hyde solution was aspirated. The membrane was washed with PBS
(0.7 mL) and aspirated. A solution of ammonium acetate (100 mM,
0.7 mL) was added to the membrane carefully and soaked for
2 min, after which ammonium acetate was aspirated. The latter
step was repeated before Milli-Q water (0.7 mL) was added to cover
the surface of the membrane and to wash out excess ammonium
acetate. The membrane was then allowed to dry slowly while
covered. The membrane was stored at room temperature for
subsequent experimentation.
Acknowledgements
We thank the University of Auckland (University of Auckland
Doctoral Scholarship to K.K.H.T. and the Faculty of Science
Research and Development Fund) and the Cancer Research Trust
New Zealand for financial support. K.K.H.T. is grateful for a
Maurice Wilkins Centre COVID-19 PhD Student Stipend Extension.
M. H. is supported by a Sir Charles Hercus Health Research
Fellowship through the Health Research Council of New Zealand.
We are grateful to Tanya Groutso for collecting the single crystal
X-ray diffraction data, and Tony Chen and Mansa Nair for
collecting the ESI-MS data. This research was undertaken in part
using the XFM beamlines at the Australian Synchrotron, part of
ANSTO. This research used resources of the Advanced Photon
Source, a U.S. Department of Energy (DOE) Office of Science User
Facility operated for the DOE Office of Science by Argonne
National Laboratory under Contract No. DE-AC02-06CH11357.
Travel funding to perform experiments at the Advanced Photon
Source was provided by the International Synchrotron Access
Program (ISAP) managed by the Australian Synchrotron, part of
ANSTO, funded by the Australian Government.
Australian Synchrotron instrumentation and operating conditions.
The distributions of Zn and Ir in A549 cells incubated with 5d were
mapped at the XFM beamline at the Australian Synchrotron
(Clayton VIC, Australia) with modifications to the protocol described
previously.[29] The beam was tuned to an incident energy of
15.8 keV using a Si(111) monochromator and focused to ~1.5 μm
using a Kirkpatrick-Baez mirror pair. A 384-element silicon array
detector (Maia 384; Brookhaven National Laboratory, Upton, NY
°
and CSIRO, Clayton, Victoria, Australia) in 180 backscatter geome-
try was used to collect the fluorescence signal from the samples
with a dwell time of 80–200 ms/point. Analysis of XRF data was
performed using the Dynamic Analysis method as implemented in
the GeoPIXE software package.
Advanced Photon Source instrumentation and operating conditions.
The distributions of Zn, and Ir in SKOV-3 cells incubated with 5d
were mapped at the 2-IDÀ D beamline at the Advanced Photon
ChemMedChem 2021, 16, 1–11
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The authors declare no conflict of interest.
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Manuscript received: May 5, 2021
Revised manuscript received: June 16, 2021
Accepted manuscript online: July 1, 2021
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FULL PAPERS
Coordinated efforts against cancer:
Organometallic Ru, Os, Rh, and Ir
complexes of mono- or bidentately
coordinating N-heterocyclic carbenes
showed metal center-dependent cy-
totoxicity, with the IrIII derivatives
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agents in vitro. The biological activity
seems related to the stability of the
compounds in aqueous solution,
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vassaghi, Dr. M. P. Sullivan, J. H.
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Hartinger*
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