Synthesis and Mode of Action Studies on Iridium(I)–NHC Anticancer Drug Candidates

Article abstract of DOI:10.1002/ejic.201800225

We report the synthesis, characterization, and biological activity of Ir^I complexes with triazole- (NNHC) and thiazole-based (NSHC) N-heterocyclic carbene ligands. Starting from the dimeric [Ir(COD)Cl]₂, we obtained complexes of composition Ir(COD)(NNHC)Cl (4a), Ir(COD)(NNHC)X (4b: X = Cl; 4bBr: X = Br), [Ir(COD)(NNHC)(NHC)]I (5a), [Ir(COD)(NSHC)₂]Cl (6a), and [Ir(COD)(NSHC)(NNHC)]Cl (6b) by adaptation of established synthetic methods for metal–NHC complexes. Their interactions with model proteins cytochrome c and lysozyme, as well as with the oligonucleotide hexamer (CG)₃ (ODN1), were studied. Although most complexes did not show any strong interactions with these biomolecules, all complexes were active against HT-29 and MCF-7 cancerous cells, with IC₅₀ values ranging between 1 and 60 μm. The most active compounds were the cationic bis(carbene) derivatives 5 and 6. All compounds generated high levels of reactive oxygen species (ROS) after incubation for 48 h in MCF-7 cells, possibly suggesting a redox-mediated mechanism of action. Interestingly, there were distinctive differences in the superoxide/(total ROS) ratios induced by the different groups of compounds.

Full text of DOI:10.1002/ejic.201800225

DOI: 10.1002/ejic.201800225
Full Paper
CLUSTER
ISSUE
Metal-Based Drugs
Synthesis and Mode of Action Studies on Iridium(I)–NHC
Anticancer Drug Candidates
Yvonne Gothe,[a] Isolda Romero-Canelón,[b] Tiziano Marzo,[c,d] Peter J. Sadler,[e]
Luigi Messori,[c] and Nils Metzler-Nolte*[a]
Abstract: We report the synthesis, characterization, and bio- Although most complexes did not show any strong interactions
logical activity of IrI complexes with triazole- (NNHC) and thiaz- with these biomolecules, all complexes were active against
ole-based (NSHC) N-heterocyclic carbene ligands. Starting from HT-29 and MCF-7 cancerous cells, with IC50 values ranging be-
the dimeric [Ir(COD)Cl]2, we obtained complexes of composition tween 1 and 60 μM. The most active compounds were the
Ir(COD)(NNHC)Cl (4a), Ir(COD)(NNHC)X (4b: X = Cl; 4bBr: X = cationic bis(carbene) derivatives 5 and 6. All compounds gener-
Br), [Ir(COD)(NNHC)(NHC)]I (5a), [Ir(COD)(NSHC)2]Cl (6a), and ated high levels of reactive oxygen species (ROS) after incuba-
[Ir(COD)(NSHC)(NNHC)]Cl (6b) by adaptation of established syn- tion for 48 h in MCF-7 cells, possibly suggesting a redox-medi-
thetic methods for metal–NHC complexes. Their interactions ated mechanism of action. Interestingly, there were distinctive
with model proteins cytochrome c and lysozyme, as well as differences in the superoxide/(total ROS) ratios induced by the
with the oligonucleotide hexamer (CG)3 (ODN1), were studied. different groups of compounds.
Introduction
ent on the nature of the other ligands. Some are very stable
under biological conditions and ligand exchange is remarkably
slow, reminiscent of the relatively slow chloride-to-water ex-
change in cisplatin, but others can exchange ligands on a much
faster timescale. Octahedral and pseudo-octahedral IrIII com-
pounds have been comprehensively investigated for their anti-
proliferative activity.[4] IrI, on the other hand, has a d8 electronic
configuration, identical to PtII compounds, with similar square-
planar geometry. Unlike PtII, however, IrI compounds are readily
oxidized to IrIII derivatives. This redox chemistry is elegantly
used in Ir-based catalytic metallodrugs, as investigated by the
groups of Sadler, Liu, and others recently.[5,6] There is, however,
only a handful of studies on the biological activity of IrI com-
pounds.[7]
With respect to IrI-based compounds, most notable is the
lack of knowledge concerning their interactions with biological
systems. We have investigated the antiproliferative activity of
IrI compounds with cyclooctadiene (COD) and N-heterocyclic
carbene (NHC) ligands. In these studies, we found that these
complexes bind to model proteins such as cytochrome c
through an unusual oxidative mechanism, in that IrI becomes
oxidized to IrIII, which causes irreversible binding to protein
targets and might thus be responsible for the good activity of
the IrI compounds.[7c] However later, we discovered that
cationic IrI–bis(carbene) complexes do not readily bind to pro-
teins by this mechanism, but still exhibit even greater antiprolif-
erative activity.[7e]
The field of medicinal inorganic chemistry arguably originates
from the success of the clinical Pt-based anticancer complexes,
most importantly cisplatin. Numerous studies have investigated
the mode of action of this important inorganic compound in
impressive detail.[1] Inspired by this work, complexes from met-
als other than Pt were investigated for their antiproliferative
activity. Many of those are indeed organometallic com-
pounds,[2] and Ir compounds in particular have gained signifi-
cant attention recently. Iridium, in its two biologically-relevant
oxidations states +I and +III, has some attractive features. For
example, IrIII with the d6 electronic configuration has excellent
luminescent properties, which have been exploited for cellular
imaging by Lo and others.[3] Perhaps surprisingly, organometal-
lic IrIII half-sandwich complexes with cyclopentadienyl (Cp) li-
gands exhibit a wide range of ligand substitution rates, depend-
[a] Inorganic Chemistry I – Bioinorganic Chemistry,
Faculty of Chemistry and Biochemistry, Ruhr-University Bochum,
Universitätsstrasse 150, 44801 Bochum, Germany
E-mail: nils.metzler-nolte@rub.de
www.chemie.rub.de/ac1
[b] School of Pharmacy, Institute of Clinical Sciences,
University of Birmingham,
Birmingham B15 2TT, UK
[c] Department of Chemistry, University of Florence,
Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
[d] Department of Chemistry and Industrial Chemistry (DCCI),
University of Pisa,
Via Moruzzi 13, 56124 Pisa, Italy
[e] Department of Chemistry, University of Warwick,
Coventry CV4 7AL, UK
To further investigate these seemingly contradictive findings,
we have now prepared a number of new IrI complexes with
different carbene-type ligands beyond the prototypical N-
heterocyclic Arduengo carbenes, and have investigated their
antiproliferative behavior as well as binding to model proteins
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejic.201800225.
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and DNA. In addition – noting the possibility of catalytic redox 1 mol equivalent of the respective alkyl halide in either THF or
cycling between IrI and IrIII – the possible involvement of ROS acetonitrile for 48–96 h. 1,4-Dimethyl-4H-1,2,4-triazolium iodide
(reactive oxygen species) in their mechanism of action was in- (2c) formed within 5 min in the absence of any solvent, as indi-
vestigated.
cated by precipitation of the compound. NMR spectra of the
ligands showed the expected signals. For all ligands, the frag-
ment [M – X]+ with (X = Cl, Br, I) is observed by mass spectrome-
try (ESI-MS, positive ion detection mode), due to the positive
charge of these organic ligands.
Results and Discussion
Synthesis and Characterization
The monocarbene complexes 4a, b were obtained by a
transmetalation reaction from the corresponding silver carbene
derivatives by a two-step synthesis following previously de-
scribed procedures.[7e] The silver carbene complexes were gen-
erated in situ by treatment of the respective triazolium salts
with half an equivalent of Ag2O. The addition of [Ir(COD)Cl]2 led
to the corresponding iridium complexes 4a and 4b (Scheme 1).
The synthesis of the iridium complexes studied in this work is
summarized in Scheme 1. The compounds consist of different
heterocyclic ligand systems which are imidazole (NHC), triazole
(NNHC) and thiazole (NSHC). In our previous work, we showed
that a high lipophilicity of IrI carbene complexes has a positive
impact on the compounds' antiproliferative activity.[7e] For this
reason, we decided to N-substitute the NHC-type ligands used
in this work with pentamethyl-benzyl side-chains as well.
All ligands were prepared according to well-established pro-
cedures starting from 1-methyl-1,2,4-triazole or thiazole via an
alkylation reaction (Menschutkin reaction) of the second aro-
Compound 4bBr was obtained by simply exchanging the
chlorido ligand in 4b with bromide by stirring 4b with an ex-
cess of KBr in acetonitrile. The successful exchange was proven
by FAB+-MS, which shows the M+ peak in each case. Further-
more, small changes in the chemical shifts of the olefinic COD
matic nitrogen.[8] Compounds 2a, b and 3a were obtained by carbon atoms in the 13C NMR and of the methyl group in the
reacting 1-methyl-1,2,4-triazole (2a, b) or thiazole (3a) with 1H NMR spectrum are observed.
Scheme 1. (a) 0.5 equiv. Ag2O, 0.5 equiv. [Ir(COD)Cl]2, CH2Cl2, room temp. overnight, (b) 3 equiv. KBr, acetonitrile, r.t. overnight, (c) 1/3a respectively,1 equiv.
K2CO3, CH2Cl2, r.t., 2–5 d, (d) NaBF4, acetonitrile, r.t., (e) 3a, KOtBu, r.t., overnight.
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The synthesis of bis(carbene) complexes such as 7 (Figure 1) thesis procedure mentioned above. [Ir(COD)(NCCH3)2]BF4 was
containing two NHC ligands has been described in previous synthesized by treating [Ir(COD)Cl]2 with sodium tetrafluoro-
work.[7e] The bis(carbene) complexes 5a and 6b were prepared borate in acetonitrile resulting in the replacement of both
in the same way as 7 by reacting the monocarbene complex chlorido ligands by the more labile acetonitrile ligand. Subse-
with the corresponding imidazolium/thiazolium salt in the pres- quent addition of thiazolium salt 3a and KOtBu led to the de-
ence of a mild base. Herein, the reaction time strongly depends sired bis(carbene) complex 6a of the type [Ir(NSHC)2(COD)]Cl.
1
This complex was characterized by H/13C NMR and ESI-MS
confirming its identity and purity. The 3JH4-H5 coupling constant
of 3.9 Hz is comparatively large and is attributed to the strong
influence of the sulfur atom. The 13C NMR signal of the carbene
carbon atom is observed at δ = 206.1 ppm and is consequently
strongly shifted to lower field compared to its triazole ana-
logues. The same observation was made for complex 6b. Inter-
estingly, stirring a solution of 6a and triazolium salt 2c led to
the complete exchange of the thiazole carbene ligands by the
triazole carbene ligands. This relatively low stability of the thiaz-
ole carbenes was affirmed by initial stability studies (see below).
on the steric demand of the N-substituent. Eventually, also
these complexes were obtained in high purity and very good
yields. NMR spectroscopy and mass spectrometry confirmed
the successful formation of the complexes including the charac-
teristic appearance of a second carbene signal in the 13C NMR
spectra.
Antiproliferative Activity
To evaluate the suitability of the new iridium complexes as anti-
cancer drug candidates, their in vitro antiproliferative effects
were studied on two well-established cancer cell lines that are
commonly used for a first screening: HT-29, which is a human
colorectal adenocarcinoma cell line and MCF-7, a human cell
line from breast adenocarcinoma with low metastatic potential.
These studies were performed with an incubation time of 48 h
using the cell-based MTT assay. Stock solutions of the com-
pounds were prepared in DMSO, due to poor water solubility
of the monocarbene complexes. Since DMSO itself may impact
on the proliferation, final DMSO concentrations of 0.5 % were
Figure 1. Chemical structures of the investigated Ir complexes.
used for concentrations up to 100 μM. In addition, the use of a
The synthesis of a bis(carbene) complex of the type
[Ir(NSHC)2(COD)]X was challenging since all efforts to synthesize
the IrI-NSHC monocarbene complexes remained ineffective. A
first test reaction for a monocarbene iridium complex com-
posed of a thiazole ligand was undertaken with thiazolium salt
3a under the very same conditions as for the monocarbene
complexes 4a, b using Ag2O and [Ir(COD)Cl]2. After stirring for
24 h followed by the established work-up procedure, the target
complex could not be obtained, neither by changing solvent,
temperature or increasing reaction time. The most obvious ex-
planation is a reaction of silver with sulfur, since this problem
did not occur for the respective iridium complexes with imidaz-
ole- and triazole ligands. Consequently, a second attempt was
made by using [Ir(COD)OMe]2 as iridium precursor and base
at the same time, thus avoiding the presence of silver ions.
Unfortunately, the desired product was not obtained. The syn-
thesis of thiazole-based IrI-carbene complexes appears to be a
prevalent problem, corroborated by a few reports in the litera-
ture.[9] In 2006, Huynh et al. published a synthesis for IrI-
carbenes with a benzothiazolin-2-ylidene ligand yielding the
desired IrI-NSHC monocarbene complex. Therein, the successful
formation was achieved via precoordination of an N-allyl sub-
stituent of the ligand to the [Ir(COD)(NCCH3)2]BF4 precursor and
subsequent deprotonation at the C2 position by addition of a
base. The successful synthesis of the IrI-NSHC bis(carbene) com-
plex 6a was finally accomplished by slightly changing the syn-
concentration of 0.5 % DMSO was extended to all complexes
even with higher water solubility, in order to guarantee a valid
comparison between the different classes of compounds. Fig-
ure 1 gives an overview of the investigated complexes.
All complexes show antiproliferative effects against the two
representative cancer cell lines. The antiproliferative activity of
the bis(carbene) complexes 5a, 6a and 6b and 7[7e] in the high
nanomolar range is notably higher (Table 1), whereas the anti-
proliferative activity of the monocarbene complexes lies in the
low micromolar range with similar activities for MCF-7 and
HT-29 cells ranging from 10.3 to 63 μM. This behavior is in ac-
cordance with previous results that we obtained for a series of
mono- and bis(carbene) complexes with imidazole-based li-
Table 1. Antiproliferative effects of the complexes [μ
M
SD]. Numbers are
reported to two relevant digits in all cases for consistency, thus resulting in
apparently different precision.
Complex
MCF-7
HT-29
7
0.54 0.27[7e]
10.3 2.9
0.470 0.063[7e]
17.2 2.8
63 10
4a
4b
4bBr
5a
6a
6b
35
6
44 10
53
9
1.5 0.2
0.92 0.06
1.0 0.3
1.5 0.3
0.9 0.2
0.9 0.2
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gands where the introduction of a second carbene ligand sig- in the metalation behavior were observed for the different
nificantly increased the antiproliferative activity.[7e] It is none- classes of compounds.
theless remarkable, as the neutral monocarbene complexes
tend to be more lipophilic than the cationic biscarbenes, and
thus their cellular uptake can be assumed to be higher.
Among the monocarbene complexes 4a, 4b and 4bBr, the
highest antiproliferative activity is observed for 4a with three
times lower IC50 values. This could be due to the more lipophilic
ligand side chain of 4a which might be related to a better cellu-
lar uptake. Different from other compound classes, the effect of
the substitution of the chlorido ligand by an bromido ligand in
the case of 4b/4bBr is negligible.
For complexes 4 (Figure 1), we could detect ions that corre-
spond to Ir(NHC) metal fragments binding to the respective
proteins, after loss of the COD and halide ligands and oxidation
to IrIII (see Supporting Information for examples of spectra), as
observed in our previous work. The HR-ESI-MS of the bis-
carbene 5a did not show any peaks with masses higher than
lysozyme or cytochrome c, respectively, that might have been
assigned to metal complex adducts. Again, this is in line with
previous observations where cationic IrI complexes did not
show any protein binding. However unlike all previous bis-
(carbene) complexes, compounds 6 with thiazole ligands did
show clearly discernible interactions with the two model pro-
teins (Figure 2).
Complex 7 possesses the highest activity among all bis-
(carbene) complexes. However, only small differences are no-
ticed between IC50 for members of this compound class, in the
For the interaction of 6a with lysozyme only one relevant
peak is observed at 15062.6 Da, while for 6b, two additional
peaks at 14915.0 and 15025.9 Da appear after incubation of the
metal complex with lysozyme. The formation of additional
peaks was also observed after treatment of cytochrome c with
these complexes. Unfortunately, it was not possible to identify
any reasonable fragments of the complexes that would fit to
the observed m/z peaks in the mass spectra.
range of 1 μM. With regard to the cell lines used, no general
selectivity toward MCF-7 or HT-29 cells is observed.
Reactivity with Proteins
In order to investigate the interactions of the compounds with
proteins, metalation studies were systemically carried out on
horse heart cytochrome c and hen egg white lysozyme as two
model proteins. The interaction between these proteins and the
metal complexes has been studied using high-resolution elec-
trospray ionization mass spectrometry (HR-ESI-MS), a widely
used tool for studying the interactions of metallodrugs with
biomolecules.[10]
Interactions with Short Oligonucleotides
Since the iridium bis(carbene) complexes showed no or rather
very few identifiable interactions with our model proteins cyto-
chrome c and lysozyme, we sought to gain more information
about other possible targets for these complexes.
The complexes were generally dissolved in ammonium acet-
ate buffer [10 % DMSO for the bis(carbene) complexes (30 %
DNA is a confirmed target for Pt-based anticancer drugs.
DMSO for 6a) and 50 % DMSO for all monocarbene complexes] Their mode of action has been linked to their ability to crosslink
and incubated with the protein (3:1 and 10:1 complex/protein purine bases on the DNA which can lead to a significant dam-
ratio) at 37 °C. At selected time points (24 h, 48 h and 72 h), age of the DNA resulting in the subsequent induction of apop-
aliquots were analyzed by HR-ESI MS. Unambiguous differences tosis.
Figure 2. HR-ESI mass spectrum of cytochrome c treated with 1 m
M of 6a (left) or 6b (right) (complex/protein = 10:1) in 20 mM ammonium acetate buffer
(30 % DMSO for 6a, 10 % DMSO for 6b), recorded after 72 h of incubation at 37 °C.
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To probe DNA as a potential target for the iridium bis-
(carbene) complexes, their reactivity with DNA was investigated
with high resolution electrospray ionization mass spectrometry
(HR-ESI-MS) using an oligonucleotide as DNA model system.
Oligonucleotides are short nucleic acid polymers, which are
used for studying DNA-metal interactions to identify potential
nucleobase binding sites. The oligonucleotide used in this study
consists of six nucleotides of the bases guanine and cytosine
with the sequence 5′-CGCGCG-3′, and is abbreviated ODN1 (Fig-
ure 3).
Figure 4. Multicharged ESI mass spectrum of ODN1 incubated with 6a (1:3)
for 24 h at 37 °C.
correct assignment of this signal. Hence it is confirmed that
6a interacts with ODN1 after or with concomitant loss of its
cyclooctadiene ligand. The same binding motif was confirmed
for 6b where the main adduct at m/z = 774.48708 with z = 3
was assigned to the ODN1-[IrI(NSHC)(NNHC)] adduct (see Sup-
porting Information).
Interestingly, from the exact mass and charge of these peaks
it can be concluded that the oxidation state of iridium remains
unchanged with +I, which is different from the observation of
the interactions of monocarbene complexes with proteins from
our previous work,[7e] wherein binding to proteins goes in par-
allel with an oxidation of the iridium center from +I to +III.
Generation of Reactive Oxygen Species (ROS)
Figure 3. Chemical structure of oligonucleotide ODN1 of sequence 5′-
CGCGCG-3′.
It has previously been shown that redox active metal complexes
may be involved in the generation of reactive oxygen species
(ROS). ROS are produced in all aerobic cells in a wide range of
physiological processes, in particular by mitochondria, playing
important roles in signaling transduction.[11] ROS refer to a
group of reactive oxygen radicals, ions or molecules, and they
can be categorized into two groups: free oxygen radicals and
non-radical ROS. Compared to normal cells, cancer cells already
show an increased level of oxidative stress expressed by ROS,
partly due to abnormal mitochondrial activity,[12] in other
words, their redox equilibrium is already slightly off balance. It
has been shown that an additional increase in ROS levels in
cancer cells, through treatment with ROS producing agents, can
lead to cell death.[11c,13a–13d] Therefore, an emerging anticancer
The bis(carbene) complexes were incubated with ODN1 in a
3:1 ratio (complex/ODN1) at 37 °C in H2O and 30 % DMSO for
6a and 10 % DMSO for all other bis(carbene) complexes. After
an incubation time of 24 and 48 h, adduct formation was de-
tected by HR-ESI MS in negative ion mode and with charge
states between –2 and –4. In general, no adduct formation was
evident for the imidazole and triazole-based complexes 5a and
7 whereas the thiazole-containing complexes 6a and 6b show
a well-defined interaction with ODN1. Figure 4 shows the result-
ing HR-ESI MS spectrum of ODN1 after incubation with com-
pound 6a for 24 h, the respective data for 6b are shown in the
Supporting Information.
The two signals (blue) at m/z = 596.43882 with z = 3 and at strategy is the generation of reactive oxygen species in cancer
m/z = 895.16226 with z = 2 originate from the [ODN1 – 2H]2– cells. It is further supposed, that such oxidative stress would
and [ODN1 – 3H]3– species, respectively. Two additional signals have a smaller effect in normal cells, which have an intact redox
(green) at m/z = 823.83981 with z = 3 and at m/z = 1236.26342 balance.[14] In this way, higher cancer selectivity could be
were obtained and identified as one species. The signals of achieved with ROS-inducing drug candidates such as redox-
ODN1 and the adduct differ by 683.2 g/mol. This mass differ- active metal complexes.
ence fits the molecular mass of 6a after loss of the cyclooctadi-
ene ligand.
To see whether the antiproliferative activities of the cytotoxic
complexes presented in this work is associated with a ROS-me-
For verification, the spectrum resulting from a potential diated mechanism of action, selected complexes were tested
ODN1-[Ir(NSHC)2] adduct was simulated. Both the experimental for their ability to produce ROS in MCF-7 cells. A frequently
and calculated isotope patterns for the signal at m/z = used method for detecting ROS in living cells is the application
823.83981 with z = 3 are presented in the Supporting Informa- of fluorescent probes, which are excellent sensors of ROS since
tion. Comparing the isotopic patterns confirms the origin and they provide a high sensitivity.[15] In this work, the total ROS
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production as well as the production of superoxide has been Afterwards, cells were stained with the fluorescent dyes and
investigated with a ROS/Superoxide detection kit (Enzo Life Sci- analyzed by flow cytometry. Table 2 shows the normalized pop-
ences) using flow cytometry. Superoxide is a precursor of most ulation numbers, and Figure 5 shows selected flow cytometry
other reactive oxygen species, and further involved in the prop- dot plots, as well as histograms with the different populations
agation of oxidative chain reactions.[16] Two different fluores- observed for compounds 4a (left), 4bBr (middle), and 6b
cent dyes were used; one for total ROS detection [Oxidative (right).
Stress Detection Reagent (Green)] and the other one for the
detection of superoxide [Superoxide Detection Reagent (Or-
ange)]. The combination of these dyes allows the specification
and differentiation of reactive oxygen species (ROS) and super-
oxide in living cells. Positive controls were obtained using
pyocyanin, while negative controls included cells treated only
with medium and buffer, and are thus expected to show “nor-
mal” signaling levels of ROS/superoxide.
Additional plots for the other compounds in this study are
available in the Supplementary Information. For compound 4a,
a substantial increase in total ROS levels is observed after treat-
ment of the cells compared to untreated cells. Interestingly, the
chloride to bromide substitution in 4bBr leads to a significantly
different behavior, in that 4bBr produces very high amounts of
superoxide, with no additional ROS species detectable com-
pared to the control. The bis(carbene) complexes (see Figure 5,
In this work, MCF-7 cells were incubated with equipotent right, for 6b as an example) again show very similar behavior
concentrations of our compounds, corresponding to the IC50. to the monocarbene complex 4a. In this case, only 1–2 % of
Table 2. Normalized population numbers (%), obtained for Q1–Q4 of the negative control and the complexes 4a, 4bBr, 6a, b and 7. Q1: FL1–/FL2+ (high
superoxide), Q2: FL1+/FL2+ (high total oxidative stress and high superoxide), Q3: FL1+/FL2– (high total oxidative stress), Q4: FL1–/FL2– (low oxidative stress
and low superoxide). The numbers are reported to two relevant digits (related to the error) in all cases for consistency, thus resulting in apparently different
precision.
Sector
Neg. control
4a
4bBr
7
6a
6b
Q1
Q2
Q3
Q4
0.0081 0.0038
0.026 0.020
0.0022 0.0039
99.965 0.021
5.0 0.14
95.0 0.14
0.0250 0.0026
0.035 0.014
99.810 0.069
0.0102 0.0031
0
1.720 0.036
92.28 0.52
4.94 0.59
7.83 0.32
0.86 0.14
91.26 0.36
0.693 0.041
0.220 0.070
98.08 0.20
0.970 0.061
0.096 0.043
0.180 0.066
1.060 0.076
Figure 5. Flow cytometry dot plots and histograms of the different populations observed for 4a (left), 4bBr (middle) and 6b (right).
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the total ROS production is related to superoxide (ca. 5 % for the nature of our Ir compounds in cancerous cells. With regard
4a). Of all compounds tested in this work, the bis(carbene) to target elucidation, IrI compounds with NHC-type ligands
complex 6a produces the highest amount of total ROS.
show promising features, and this class of compounds certainly
merits further investigation.
Conclusions
Experimental Section
In this work, the synthesis, characterization and biological activ-
ity of Ir complexes in the +I oxidation state are reported. In
comparison to the many IrIII compounds reported in the litera-
ture, biological investigations on IrI compounds are still
relatively rare. The compounds are stabilized by the use of N-
heterocyclic carbene-type ligands, namely triazole (NNHC) and
thiazole-derived (NSHC) ligands. The synthesis of all com-
pounds follows standard routes, however care has to be taken
with the order of addition of carbene ligands in bis(carbene) Ir
complexes as not all synthetic routes will lead to the desired
products. Namely, thiazole-derived ligands seem to be more
labile, and should be added in the last steps only.
General: Reactions were carried out under N2 by using standard
Schlenk techniques. All solvents were of analytical grade. Chemicals
were obtained from commercial sources and used without further
purification. 1H and 13C NMR spectra were recorded at room tem-
perature on Bruker DPX 200, DPX 250 and DRX 400 spectrometers,
respectively, in deuterated solvents which were used as internal
reference. The chemical shifts are reported in ppm (parts per mil-
lion) relative to TMS. Coupling constants J, are reported in Hz, multi-
plicities being marked as: singlet (s), doublet (d), triplet (t) or multi-
plet (m). IR spectra were measured on a Bruker Tensor 27 instru-
ment equipped with an attenuated total reflection (ATR) unit at
4 cm–1 resolution. ESI mass spectra were measured on a Bruker
Esquire 6000 mass spectrometer. FAB and EI high resolution mass
spectrometry were performed with a Fisons VG Instruments Auto-
spec spectrometer. High-resolution mass spectra were recorded
In line with this observation about the relative lability of the
NSHC ligand in those complexes, significant interactions be-
tween the model proteins lysozyme and cytochrome c and the with an LTQ Orbitrap high-resolution mass spectrometer (Thermo
Scientific, San Jose, CA, USA) equipped with a conventional ESI
source. The mass-to-charge relation (m/z) is given as a dimension-
less number. UV/Vis absorption spectra were recorded on a Varian
Cary 50 spectrophotometer. Absorption maxima λmax are given in
nm. Elemental analyses were carried out on a Vario EL (Elementar
Analysensysteme GmbH, Hanau, D) in C, H, N mode.
bis(carbene) complexes were only observed for the thiazole-
containing complexes 6a, b. Still, the intensity and quantity of
the observed peaks was much lower than the monocarbene
complexes studied earlier,[7c,7e] indicating weak protein-metal-
ating ability for the compounds under investigation herein.
Among the cationic complexes, only compounds 6 showed in-
teraction with the hexameric oligonucleotide ODN1. Concern-
ing a possible mode of action, it should be noted that Cyt c and
lysozyme are just two model proteins, similar to ODN1 being a
model nucleotide. While the fact that our compounds do not
strongly interact with either model protein or ODN1 seems like
an indication against proteins or DNA as the primary target, the
possibility that a specific target interaction causes the antiprolif-
erative activity cannot be ruled out completely, e.g. selective
inhibition of one particular vital enzyme.
In general, treatment of MCF-7 cells with each of the Ir–NHCs
described in this work increased ROS production significantly.
Most compounds show a very similar pattern of ROS produc-
tion with high total amounts of ROS, but only small amounts of
superoxide. However, with more than 99 % of total ROS being
superoxide, 4bBr presents a notable exception among all tested
compounds. This is even more notable in that 4bBr does not
show any different overall activity in MCF-7 cells (or HT-29 for
that matter) from the other related monocarbene compounds.
Electrochemistry gives no hint of reversible redox activity of the
IrI carbene complexes studied herein (see Supporting Informa-
tion for cyclic voltammograms of two representative examples),
so ROS production is likely not caused by a direct ROS-produc-
ing catalytic mechanism involving the metal complexes, but
rather a downstream cellular response.
Cell Culture and Cytotoxicity: Dulbecco's Modified Eagle's Me-
dium (DMEM), containing 10 % fetal calf serum, 1 % penicillin and
streptomycin, was used as growth medium. MCF-7 and HT-29 cells
were detached from the wells with trypsin and EDTA, harvested by
centrifugation and resuspended again in cell culture medium. The
assays were carried out on 96-well plates with 6000 cells per well
for both cell lines, MCF-7 and HT-29. After 24 h of incubation at
37 °C and 10 % CO2, the cells were treated with the compounds
(with DMSO concentrations of 0.5 %) with a final volume of 200 μL
per well. For a negative control, one series of cells was left un-
treated. The cells were incubated for 48 h followed by adding 50 μL
MTT (2.5 mg/mL). After an incubation time of 2 h, the medium was
removed and 200 μL DMSO were added. The formazan crystals were
dissolved and the absorption was measured at 550 nm, using a
reference wavelength of 620 nm. Each test was repeated in tripli-
cate or quadruplicate in at least three independent experiments for
each cell line.
Interaction with Cytochrome c (Lysozyme): Solutions of the com-
plexes (100 μ
M) with cytochrome c (lysozyme) (3:1 or 10:1 complex/
protein molar ratio) in ammonium acetate buffer (20 μ
M, pH = 6.8)
and a compound-specific percentage of DMSO were incubated at
37 °C. After 24 h (48 h, 72 h) and 20-fold dilution with LC-MS
grade water, ESI MS spectra were recorded by direct injection at
5 μL min–1 flow rate in an Orbitrap high-resolution mass spectrome-
ter (Thermo, San Jose, CA, USA), equipped with a conventional ESI
source. The working conditions were as follows: spray voltage
3.1 kV, capillary voltage 45 V, capillary temperature 220 °C, tube
lens voltage 230 V. The sheath and the auxiliary gases were set,
respectively, at 17 (arbitrary units) and 1 (arbitrary units). For acqui-
sition, Xcalibur 2.0. software (Thermo) was used and monoisotopic
and average deconvoluted masses were obtained by using the inte-
grated Xtract tool. For spectrum acquisition a nominal resolution
We thus conclude that despite the electronic analogy be-
tween the d8-PtII and d8-IrI systems, at least for the organome-
tallic, NHC-derived metal complexes studied in this work, very
different mechanisms seem to be responsible for their antipro-
liferative activity. A first indication might be the fact that ROS
production increases dramatically, and is indeed dependent on (at m/z 400) of 100,000 was used.
Eur. J. Inorg. Chem. 2018, 2461–2470
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Full Paper
Interaction with Oligonucleotide ODN1: Solutions of the com-
plexes (100 μ
8.46 mmol) were dissolved in dry THF (5 mL) and stirred under
M
) were incubated with ODN1 (3:1 ratio complex/ reflux for 48 h. The precipitated solid was filtered and washed two
ODN1) in H2O and a compound-specific percentage of DMSO at
37 °C. After 24 h (48 h, 72 h) and 20-fold dilution with LC-MS grade
water, HR-ESI MS spectra were recorded in negative mode by direct
times with THF to obtain 3a as white solid (1.72 g, 72.3 %). 1H NMR
(250 MHz, CD3OD): δ = 8.28 (s, 2 H, S-CH=CH-N), 5.90 (s, 2 H,
N-CH2), 2.30 (s, 3 H, Me), 2.27 (s, 6 H, Me), 2.25 (s, 6 H, Me) ppm.
MS (ESI+): m/z = 245.8 [M – Cl]+.
injection at a flow rate 5 μL min–1
.
4a: A Schlenk flask was charged with 2a (70.0 mg, 250 μmol) and
Ag2O (29.0 mg, 125 μmol) in dry dichloromethane (4 mL) and stirred
at r.t for 1 h. Following, 0.5 equiv. of [Ir(COD)Cl]2 (84.0 mg, 125 μmol)
was added and the resulting mixture was stirred for 24 h. The result-
ing suspension was filtered through Celite, and the filtrate was con-
centrated to dryness. The crude product was purified by column
chromatography on silica using mixtures of dichloromethane and
ROS and Superoxide Determination: Flow cytometry analyses of
ROS/superoxide generation by exposure to the complexes was car-
ried out using the Total ROS/Superoxide detection kit (Enzo life
sciences) according to the supplier's instructions. Briefly, 1.0 × 106
MCF-7 cells per well were seeded in a 6-well plate. Cells were pre-
incubated in drug-free media at 37 °C for 24 h in a 5 % CO2 humidi-
fied atmosphere, and then drugs were added at equipotent concen-
trations equal to the IC50 value. After 48 h of drug exposure, super-
natants were removed by suction and cells were washed and har-
vested. Staining was achieved in the dark by re-suspending the cell
pellets in buffer containing the orange/green fluorescent reagents.
Cells were analyzed in a Becton Dickinson FACScan Flow Cytometer
using Ex/Em: 490/525 nm for the oxidative stress and Ex/Em: 550/
620 nm for superoxide detection. Data were processed using the
Flowjo software. Negative controls included untreated cells. Positive
controls were obtained using pyocyanin.
1
methanol to obtain 4a (102 mg, 70.4 %) as a yellow solid. H NMR
(400 MHz, CD2Cl2): δ = 7.20 (s, 1 H, N-C3H), 5.76 (d, J = 14.5 Hz, 1
H, N-CH2), 5.35 (d, J = 14.7 Hz, 1 H, N-CH2), 4.69–4.60 (m, 2 H, COD),
4.10 (s, 3 H, HNMe), 3.17–3.06 (m, 2 H, COD), 2.35–2.26 (m, 4 H, COD),
2.28 (s, 3 H), 2.24 (s, 6 H), 2.22 (s, 6 H), 1.89–1.76 (m, 2 H, COD),
1.76–1.64 (m, 2 H, COD) ppm. 13C NMR (101 MHz, CD2Cl2): δ = 183.6
(NCN), 141.3 (C3), 137.0 (CAr-1), 134.2 (CAr-2), 134.1 (CAr-3), 127.1 (CAr-
4), 86.7 (COD), 86.5 (COD), 52.7 (COD), 52.1 (COD), 49.0 (N-CH2), 40.2
(CNMe), 34.3 (COD), 34.0 (COD), 30.2 (COD), 30.0 (COD), 17.5 (Me),
17.2 (Me), 17.1 (Me) ppm. MS (FAB+): m/z = 579.1 [M]+, 544.1 [M –
Cl]+, 471.0 [M – COD]+.
Synthesis
The synthesis of compound 7 has been reported previously.[7e]
4b: A Schlenk flask was charged with 2b (170.0 mg, 765 μmol) and
Ag2O (88.9 mg, 383 μmol) in dry dichloromethane (10 mL) and
stirred at r.t for 1 h. Following, 0.5 equiv. of [Ir(COD)Cl]2 (257.8 mg,
383 μmol) was added and the resulting mixture was stirred for 12 h.
The resulting suspension was filtered through Celite, and the filtrate
was concentrated to dryness. The crude product was purified by
1: In a flame-dried Schlenk flask 1-methylimidazole (971 μL,
12.2 mmol) and iodomethane (1.35 mL, 14.6 mmol) were dissolved
in dry THF (15 mL) and stirred at room temperature for 2 h. The
precipitated solid was washed three times with THF to obtain 1 as
a white solid (2.55 mg, 93.4 %). 1H NMR (200 MHz, CD2Cl2): δ = 9.08
(s, 1 H, N-CH=N), 7.70 (d, J = 1.6 Hz, 2 H, N-CH=CH-N), 3.85 (s, 6 H, column chromatography on silica using mixtures of dichloro-
H
NMe) ppm. 13C NMR (50 MHz, DMSO): δ = 136.9 (N-CH=N), 123.3
methane and methanol to obtain 4b (348 mg, 90.0 %) as yellow
solid. 1H NMR (400 MHz, CD2Cl2): δ = 8.03 (s, 1 H, N-C3H), 4.68 (ddd,
J = 14.3, 5.1, 3.1 Hz, 1 H, N-CH2), 4.59 (ddd, J = 10.6, 7.1, 4.1 Hz, 2
H, COD), 4.43 (ddd, J = 14.3, 7.8, 3.6 Hz, 1 H, N-CH2), 4.08 (s, 3 H,
(NCH=CHN), 35.8 (CNMe) ppm. MS (ESI+): m/z = 96.8 [M – I]+.
2a: 1-(Chloromethyl)-2,3,4,5,6-pentamethylbenzene (804.9 g,
4.09 mmol) and 1-methyl-1,2,4-triazole (365 μL, 4.09 mmol) were
dissolved in dry THF (10 mL) and stirred under reflux for 48 h. The
precipitated solid was filtered and was washed three times with
THF to obtain 2a as a white solid (843.4 mg, 73.7 %). 1H NMR
(250 MHz, CD2Cl2): δ = 12.33 (s, 1 H, N-C5H), 7.98 (s, 1 H, N-C3H),
5.79 (s, 2 H, N-CH2), 4.27 (s, 3 H, HNMe), 2.27 (s, 9 H, Me), 2.24 (s, 6
H, Me) ppm. 13C NMR (63 MHz, CD2Cl2): δ = 144.9 (N=CH-N), 142.7
(N-CH=N), 138.2 (CAr-1), 134.6 (CAr-2), 134.1 (CAr-3), 125.1 (CAr-4), 48.9
(N-CH2), 40.0 (CNMe), 17.6 (Me), 17.3 (Me), 17.2 (Me) ppm. MS (FAB+):
m/z = 244.1 [M – Cl]+.
HNMe), 3.83–3.71 (m, 2 H, O-CH2), 3.35 (s, 3 H, HOMe), 3.02–2.96 (m,
1 H, COD), 2.86 (td, J = 7.1, 3.3 Hz, 1 H, COD), 2.29–2.15 (m, 4 H,
COD), 1.82–1.64 (m, 4 H, COD) ppm. 13C NMR (101 MHz, CD2Cl2):
δ = 183.3 (NCN), 143.6 (C3), 86.7 (COD), 86.6 (COD), 71.6 (O-CH2),
59.4 (COMe), 52.9 (COD), 52.5 (COD), 48.9 (N-CH2), 39.8 (CNMe), 34.1
(COD), 34.0 (COD), 30.0 (COD), 30.0 (COD) ppm. MS (FAB+): m/z =
476.8 [M]+. C14H23ClIrN3O (477.03): calcd. C 35.25, H 4.86, N 8.81;
found C 35.00, H 4.60, N 8.58.
4bBr: Compound 4b (22.9 mg, 48 μmol) was stirred with 3 equiv.
of KBr (17.1 mg, 144 μmol) in acetonitrile for 72 h. The solution was
filtered through Celite and evaporated to give 4bBr (22.1 mg,
2b: To a flame-dried Schlenk flask 2-bromoethyl methyl ether
(498 μL, 5.30 mmol) and 1-methyl-1,2,4-triazole (200 μL, 2.65 mmol)
were dissolved in dry acetonitrile (5 mL) and stirred under reflux
for 96 h. After removal of the solvent under reduced pressure, 2b
was obtained as a colorless liquid (335.5 mg, 57 %). 1H NMR
(200 MHz, D2O): δ = 8.86 (s, 1 H, N-C3H), 4.52 (t, J = 4.9 Hz, 2 H, N-
CH2), 4.14 (s, 3 H, HNMe), 3.85 (t, J = 4.9 Hz, 2 H, O-CH2), 3.40 (s, 3
H, HOMe) ppm. 13C NMR (50 MHz, D2O): δ = 144.7 (N=CH-N), 144.6
(N-CH=N), 69.0 (O-CH2), 58.2 (COMe), 47.7 (N-CH2), 38.6 (CNMe) ppm.
1
88.4 %) as a yellow solid. H NMR (400 MHz, CD2Cl2): δ = 8.04 (s, 1
H, N-C3H), 4.72–4.61 (m, 3 H, N-CH2, COD), 4.41 (ddd, J = 14.3, 8.3,
3.2 Hz, 1 H, N-CH2), 4.05 (s, 3 H, HNMe), 3.81 (ddd, J = 10.4, 8.2,
2.9 Hz, 1 H, O-CH2), 3.73 (ddd, J = 10.4, 5.2, 3.2 Hz, 1 H, O-CH2), 3.35
(s, 3 H, HOMe), 3.01 (td, J = 7.2, 3.6 Hz, 1 H, COD), 2.87 (td, J = 7.0,
2.7 Hz, 1 H, COD), 2.30–2.07 (m, 4 H, COD), 1.85–1.73 (m, 2 H, COD),
1.61–1.54 (m, 2 H, COD) ppm. 13C NMR (101 MHz, CD2Cl2): δ = 183.3
(NCN), 143.7 (C3), 86.1 (COD), 86.0 (COD), 71.4 (O-CH2), 59.4 (COMe),
53.8 (COD), 53.3 (COD), 48.8 (N-CH2), 39.8 (CNMe), 33.8 (COD), 33.7
(COD), 30.4 (COD), 30.2 (COD) ppm. MS (FAB+): m/z = 520.9 [M]+.
C14H23BrIrN3O (521.48): calcd. C 32.25, H 4.45, N 8.06; found C 32.33,
H 4.16, N 7.94.
2c: 1-Methyl-1,2,4-triazole (200 μL, 2.65 mmol) and iodomethane
(165 μL, 2.65 mmol) were stirred in a flame-dried Schlenk flask. A
white solid formed within 5 min. The reaction was continued for
another 2 h to ensure complete conversion to the product to give
2c as white solid (584.4 mg, 98 %). The product was used for the
next reaction without further purification and characterization.
4c: In a flame-dried Schlenk flask [Ir(COD)Cl]2 (248 mg, 368 μmol)
and NaBF4 (80.7 mg, 735 μmol) were dissolved in acetonitrile (2 mL)
and stirred for 30 min at room temperature. Subsequently, 2c
(165 mg, 735 μmol) and KOtBu (82.5 mg, 735 μmol) were added to
3a: In
a flame-dried Schlenk flask 1-(chloromethyl)-2,3,4,5,6-
pentamethylbenzene (1.66 g, 8.46 mmol) and thiazole (600 μL,
Eur. J. Inorg. Chem. 2018, 2461–2470
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2468
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
the suspension and it was stirred overnight. After removal of the
solvent, the crude product was purified by column chromatography
on silica using mixtures of dichloromethane and methanol to give
4c as yellow solid (270 mg, 70.0 %). 1H NMR (200 MHz, CD2Cl2): δ =
7.88 (s, 1 H, N-C3H), 4.87–4.69 (m, 2 H, COD), 3.99 (s, 3 H, HNMe),
3.84 (s, 3 H, HNMe), 3.09–2.90 (m, 2 H, COD), 2.23–2.08 (m, 4 H, COD),
1.89–1.74 (m, 2 H, COD), 1.47–1.33 (m, 2 H, COD) ppm. 13C NMR
(50 MHz, CD2Cl2): δ = 183.9 (NCN), 143.5 (C3), 84.7 (COD), 84.6 (COD),
55.9 (COD), 55.4 (COD), 39.7 (CNMe), 35.2 (COD), 33.4 (COD), 33.3
(COD), 30.8 (COD) ppm.
(CNMe), 36.1 (CNMe), 32.5 (COD), 32.4 (COD), 31.6 (COD), 31.6 (COD),
17.6 (Me), 17.2 (Me), 17.1 (Me) ppm. MS (ESI+): m/z = 642.9 [M –
Cl]+, 534.9 [M – Cl – COD]+.
Acknowledgments
Y. G. thanks Elena Michelucci for measurements and simulations
of the ESI-MS spectra. T. M. thanks AIRC-FIRC (Fondazione
Italiana per la Ricerca sul Cancro, 3-year Fellowship for Italy
Project Code: 18044). The authors also gratefully acknowledge
financial support from the European Cooperation in Science
and Technology (COST) (COST) action CM1105 (STSM to Y. G.),
the Beneficentia Stiftung (Vaduz) and CIRCMSB (Bari, Italy). Fur-
ther, financial support to Y. G. by the Ruhr University Research
School PLUS, funded by Germany's Excellence Initiative [DFG
GSC 98/3], the ERC (grant no. 247450) and EPSRC (grant no. EP/
F034210/1) for P. J. S. are gratefully acknowledged. Dr. Matthew
Reback and Isabelle Daubit (RUB) helped measuring the cyclic
voltammograms during the revision of this paper.
5a: Compound 4a (20 mg, 34.5 μmol), 1 (7.7 mg, 34.5 μmol) and
K2CO3 (4.9 mg, 34.5 μmol) were stirred in dry dichloromethane
(3 mL) at r.t for 48 h. The resulting suspension was filtered through
Celite and the filtrate was concentrated to dryness. The remaining
orange oil was purified by column chromatography on silica using
mixtures of dichloromethane and methanol to give 5a (21.9 mg,
82.8 %) as orange solid. 1H NMR (400 MHz, CD2Cl2): δ = 7.28 (s, 1
H, N-C3H), 7.18 (d, J = 3.3 Hz, 2 H, N-CH=CH-N), 5.34 (d, J = 13.9 Hz,
1 H, N-CH2), 5.07 (d, J = 13.9 Hz, 1 H, N-CH2), 4.31–4.23 (m, 1 H,
COD), 4.22 (s, 3 H, HNMe), 4.21–4.17 (m, 1 H, COD), 4.10 (s, 3 H, HNMe),
3.87 (d, J = 7.6 Hz, 3 H, HNMe), 3.87–3.81 (m, 1 H, COD), 3.80–3.72
(m, 1 H, COD), 2.48–2.37 (m, 2 H, COD), 2.35–2.30 (m, 2 H, COD),
2.27 (s, 3 H, Me), 2.23 (s, 6 H, Me), 2.20–2.15 (m, 2 H, COD), 2.04 (s,
6 H, Me), 1.99–1.91 (m, 2 H, COD) ppm. 13C NMR (101 MHz, CD2Cl2):
δ = 182.0 (NCNTriaz), 176.2 (NCNImidaz), 142.1 (C3), 137.9 (CAr-1), 134.6
(CAr-2), 133.6 (CAr-3), 125.8 (CAr-4), 124.4 (N-CH=CH-N), 123.6 (N-CH=
CH-N), 81.5 (COD), 78.3 (COD), 77.8 (COD), 76.9 (COD), 48.9 (N-CH2),
41.4 (CNMe), 39.1 (CNMe), 38.6 (CNMe), 33.5 (COD), 30.4 (COD), 30.3
(COD), 17.5 (Me), 17.2 (Me), 16.8 (Me) ppm. MS (FAB+): m/z = 640.1
[M]+.
Keywords: Carbenes · Iridium · Medicinal chemistry ·
Metal-based drugs · Protein interactions
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1
(72 mg, 44.8 %). H NMR (400 MHz, CD2Cl2): δ = 7.49 (d, J = 3.8 Hz,
2 H, N-CH=CH-S), 7.12 (d, J = 3.9 Hz, 2 H, N-CH=CH-S), 5.69 (s, 4 H,
N-CH2), 4.17 (s, 4 H, COD), 2.55–2.34 (m, 8 H, COD), 2.30 (s, 6 H, Me),
2.25 (s, 12 H, Me), 2.07 (s, 12 H, Me) ppm. 13C NMR (101 MHz,
CD2Cl2): δ = 206.1 (NCN), 138.0 (CAr-1), 135.7 (N-CH=CH-S), 134.5
(CAr-2), 133.9 (CAr-3), 127.2 (CAr-4), 123.4 (N-CH=CH-S), 87.9 (COD),
56.6 (N-CH2), 32.5 (COD), 32.4 (COD), 17.6 (Me), 17.2 (Me), 17.0 (Me)
ppm. MS (ESI+): m/z = 682.9 [M – COD – Cl]+, 790.9 [M – Cl]+. MS
(FAB+): m/z = 682.9 [M – COD – Cl]+, 790.9 [M – Cl]+.
6b: A Schlenk flask was charged with 4c (40.0 mg, 76.3 μmol), 3a
(21.5 mg, 76.3 μmol) and K2CO3 (10.6 mg, 76.3 μmol) and the mix-
ture was stirred in dry dichloromethane (4 mL) at r.t for 120 h. The
resulting suspension was filtered through Celite and the filtrate was
concentrated to dryness. The remaining orange oil was purified by
column chromatography on silica using mixtures of dichloro-
methane and methanol to give 6b (43.9 mg, 84.8 %) as orange
1
solid. H NMR (400 MHz, CD2Cl2): δ = 8.66 (s, 1 H, N-C3H), 7.53 (d,
J = 3.9 Hz, 1 H, N-CH=CH-S), 7.07 (d, J = 3.9 Hz, 1 H, N-CH=CH-S),
5.52 (d, J = 14.7 Hz, 1 H, N-CH2), 5.43 (d, J = 14.6 Hz, 1 H, N-CH2),
4.14 (s, 3 H, HNMe), 4.22–3.95 (m, 4 H, COD), 4.09 (s, 3 H, HNMe), 2.47–
2.33 (m, 4 H, COD), 2.29 (s, 3 H, Me), 2.24 (s, 6 H, Me), 2.22–2.11 (m,
4 H, COD), 2.07 (s, 6 H, Me) ppm. 13C NMR (101 MHz, CD2Cl2): δ =
207.9 (NCNThiaz), 179.2 (NCNTriaz), 145.7 (C3), 138.0 (CAr-1), 135.2 (N-
CH=CH-S), 134.5 (CAr-2), 133.9 (CAr-3), 127.0 (CAr-4), 123.9 (N-CH=CH-
S), 83.6 (COD), 81.8 (COD), 81.1 (COD), 80.7 (COD), 56.7 (N-CH2), 40.6
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Received: February 16, 2018
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