(Pyridin-2-yl)-NHC Organoruthenium Complexes: Antiproliferative Properties and Reactivity toward Biomolecules

Article abstract of DOI:10.1021/acs.organomet.8b00153

Organoruthenium compounds have been widely investigated for their anticancer activity. Here we use one of the classic ligand classes found in organometallics, i.e., N-heterocyclic carbenes (NHC), and coordinate them to the Ru(η⁶-p-cymene) scaff

Full text of DOI:10.1021/acs.organomet.8b00153

Article
Cite This: Organometallics XXXX, XXX, XXX−XXX
(Pyridin-2-yl)-NHC Organoruthenium Complexes: Antiproliferative
Properties and Reactivity toward Biomolecules
Sanam Movassaghi,† Sukhjit Singh,† Aewan Mansur,† Kelvin K. H. Tong,† Muhammad Hanif,†
Hannah U. Holtkamp,† Tilo Sohnel,† Stephen M. F. Jamieson,‡ and Christian G. Hartinger*,†
̈
†School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
‡Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
S
* Supporting Information
ABSTRACT: Organoruthenium compounds have been widely investigated
for their anticancer activity. Here we use one of the classic ligand classes
found in organometallics, i.e., N-heterocyclic carbenes (NHC), and coor-
dinate them to the Ru(η6-p-cymene) scaffold as N,C-bidentate ligands sub-
stituted with a pyridyl moiety. Introduction of different substituents gave
compounds with a wide variety of properties. We investigated their stability in
solution and in the presence of biomolecules, in vitro anticancer activity, and
cellular uptake to rationalize their biological properties in dependence on the
structure. A clear effect of their structure on the stability in water and DMSO
was found for some derivatives, which was reflected in the reactivity to bio-
molecules that was determined with selected representatives of the compound
classes. The antiproliferative activity of the compounds was widely dependent
on the lipophilicity of the N,C-bidentate ligand, but as cellular accumulation studies revealed, lipophilicity does not provide the
full picture and additional effects must be responsible for the anticancer activity.
INTRODUCTION
■
N-Heterocyclic carbenes (NHC) are currently receiving much
attention for their wide application in coordination chemistry
and catalysis.1,2 In the latter area, NHC ligands are considered
an alternative to phosphanes,1−5 and a large number of different
monodentate NHC ligands derived from imidazole, imidazo-
lidine, benzimidazole, and triazole6 have been synthesized.6,7
They have been used in a number of chemical transformations,8
including palladium-catalyzed C−N coupling reactions,9,10 rho-
dium-catalyzed hydroformylation,11 ruthenium-catalyzed olefin
metathesis reactions,12 and Heck- and Suzuki-type C−C coupling
reactions.13 NHCs can also be part of larger, multidentate ligand
systems, such as in combination with pyridyl groups, where for
Figure 1. Exemplary structures for metal−NHC complexes with
example a tridentate pincer framework gave remarkable catalytic
promising properties for medicinal applications.
activities in the Heck reaction.1,8,14,15
More recently, NHC ligands have entered the area of medic-
from imidazolium salts as anticancer agents.16,24 The lipophilicity
inal inorganic chemistry.16−19 For example, metal−NHC com-
of linear AuI complexes led to the triggering of Ca2+-sensitive
plexes (Figure 1) have been investigated for the treatment of
mitochondrial swelling.25 The primary mode of action for the
cancer and infectious diseases. The earliest documentation of
NHC−AuI complexes is thought to involve targeting the enzyme
metal−NHC complexes with promise for medicinal applications
thioredoxin reductase (TrxR),26 which plays an important role in
was on RuII and RhI organometallics which expressed antibacterial
properties.19−22 Later, Youngs and co-workers reported Ag−NHC
sustaining the intracellular redox homeostasis, promotes cell growth
and survival, and is upregulated in some cancer types.24,26
compounds with outstanding antimicrobial activity against both
RuII(arene) complexes of the general formula [RuII(arene)-
(X)(Y)(Z)] are well-established bioactive compounds.27−31
The ligands coordinated to the Ru center can be mono- or
Gram-positive and/or Gram-negative bacteria.23 The effective
antimicrobial properties of the Ag−NHC complexes were con-
cluded to be a result of the slow release of Ag+ ions from the
stabilizing ligands into the culture medium.23 Inspired by the
use of gold(I) complexes in the therapy of rheumatoid arthritis,
researchers reported a series of NHC−AuI complexes derived
Received: March 12, 2018
© XXXX American Chemical Society
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a
Scheme 1. Preparation of the Ru Complexes 1a−10a
a
The counterions to the imidazolium compounds 1−10 were bromide (3−5, 7, 9, 10) or iodide (1, 2, 8). The numbering scheme was used to assign
the peaks observed in the NMR spectra.
multidentate and influence the chemical and biological prop-
erties of the compounds.32−37 NHC ligands coordinated to bio-
active metals such as Ru afforded remarkably stable complexes
and some (benz)imidazole-derived NHC−Ru complexes exhib-
ited strong antiproliferative effects possibly through inhibition
of TrxR.38 In comparison to the number of monodentate NHC
ligands used in complexes with biological properties, the lit-
erature is scarce for bioactive complexes that feature bidentate
ligands containing an NHC group.16,19 In this paper, we aim to
fill this gap with the introduction of N,C-coordinating pyridyl-
functionalized NHC ligands and studies on the effect of such
modifications on stability and reactivity toward biomolecules as
well as in vitro cytotoxicity and cellular uptake.
of AgPF6 to the imidazolium halides leads to the formation of
AgX (X = Br, I).41 A solvent combination of dichloromethane
(DCM) and 1,2-dichloroethane (DCE) (3/1) provided the best
results to afford the bidentate NHC complexes. The imidazolium
carbene precursor was dissolved in DCE before DCM was added.
Addition of AgPF6 to the mixture caused immediate precipitation
of AgX, indicating halide/PF6 metathesis. The reaction mixture
was then kept in darkness and stirred at 55 °C for 3 h before
Ag2O was added, and the temperature was increased to 65 °C
to produce the AgI(NHC-pyridin-2-yl) intermediate. A reaction
time of 24 h was identified as the optimal time to allow for
complete conversion. Then, [RuII(η6-p-cymene)X2]2 was added
and the reaction mixture was stirred overnight. The stoichiometry
of Ag2O and NHC-pyridin-2-yl precursor was identified as crucial
for successful conversion, and the highest yield and purity were
obtained when ratios of around 2:2.5:1.5:1.5 of NHC pre-
cursor, AgPF6, Ag2O, and [RuCl2(η6-p-cymene)]2 were used
(see the Experimental Section for the exact ratios used for the
individual complexes). This synthetic approach allowed the
isolation of the novel compounds 4a−6a, 9a, and 10a, while
1a−3a, 7a, and 8a have been reported earlier.15,47−49
RESULTS AND DISCUSSION
■
The precursors 2-(1H-imidazol-1-yl)pyridine (I), 2-(1H-benzimi-
dazol-1-yl)pyridine (II), 1-(2-pyridinylmethyl)-1H-imidazole
(III), and 1-(2-pyridinylmethyl)-1H-benzimidazole (IV) were
prepared using a modified Ullman-type CuI coupling reaction
between 2-bromopyridine derivatives and the chosen azole in
DMSO instead of DMF and by replacement of K2CO3 with
K3PO4.39−43 The subsequent N-alkylation of the second imid-
azole nitrogen atom in I−IV afforded 1−10 in mostly high
yields (Table S1), following literature procedures for 1−5 and
7−10.39−43 The identity of precursors I−IV and ligands 1−10
was confirmed by NMR spectroscopy and electrospray ioniza-
tion mass spectrometry (ESI-MS; Table S2 in the Supporting
Information). In the case of the ethyl ester derivative 5, the ESI
mass spectra showed a transesterification with methanol during
the analysis of the compounds.
Characterization of the complexes by NMR, ESI-MS, elemental
analysis, and X-ray diffraction (for the new derivatives 4a−6a
and 10a) indicated successful formation of 1a−10a, in which
the ligands can form five- or six-membered rings with the Ru
1
center. In the H NMR spectra, the absence of a signal assign-
able to the pro-carbenic proton H-3 (see Scheme 1 for the atom
labeling) and the downfield shift (from 8.7 to 9.4 ppm) of H-13
suggested that the RuII center coordinated to the bidentate
ligand through its C-3 and N-12 atoms. Introduction of the
RuII(η6-p-cymene) moiety resulted in several distinct signals
in the aromatic and aliphatic regions. In addition, the protons
of the methylene linker between the (benz)imidazole and/or
of the CH2 group of the benzyl or of the 2-acetyl substituents
became diastereotopic upon metal coordination (see Figure S1).
The p-cymene aromatic protons H-25a/b and H-26a/b were
observed in the range of 5.76−6.45 ppm as four individual
doublets, which is common for compounds featuring bidentate
coligands.
The synthesis of the RuII(arene) compounds was initially
attempted using the classic silver-mediated transmetalation with
Ag2O to form an Ag(NHC) intermediate, to which [RuII(η6-p-
cymene)Cl2]2 was added.44−46 However, this strategy was unsuc-
cessful and instead a route starting with first AgPF6 and sub-
sequently adding Ag2O and [RuII(η6-p-cymene)X2]2 was used
(Scheme 1). The presence of the halide in the complexation
reaction led to the formation of an insoluble product and
caused difficulties in the isolation of the products, and addition
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Table 1. Key Bond Lengths (Å) and Angles (deg) for 4a, 5a, 7a, 10a, and 12a
a
4a
5a
6a
8a
10a
Bond Length/Å
Ru−Cl
Ru−CNHC
Ru−NPy
2.4012(6)
1.997(2)
2.080(2)
2.3999(6)
2.017(3)
2.100(2)
2.4152(5)
2.012(2)
2.395(3), 2.404(3)
2.022(9), 2.013(9)
2.133(8), 2.134(7)
2.414(1)
2.039(6)
2.114(2)
2.1079(17)
Bond Angle/deg
76.28(10)
CNHC−Ru−NPy
Cl−Ru−NPy
Cl−Ru−CNHC
76.09(10)
85.73(6)
84.20(8)
76.44(7)
87.60(5)
82.87(6)
84.7(4), 85.1(3)
86.3(3), 86.5(3)
86.5(3), 87.7(3)
85.31
84.69
87.80
86.52(6)
83.00(8)
a
Two crystallographically independent molecules.
The NMR data were supported by ESI-MS studies, providing
further confirmation of the identity of the complexes synthe-
sized. The ESI-mass spectra contained mainly the pseudomo-
lecular ions [M − PF6]+ (Table S3). In addition, for 5a trans-
esterification to the methyl ester was detected, most probably
again due to MeOH being used as the solvent for the MS
experiments. The compounds were found to be hygroscopic,
and the elemental analyses indicated for several compounds the
presence of solvent molecules. Furthermore, the X-ray crystal-
lographic data for 4a indicated the presence of small amounts
of bromido instead of chlorido ligands (see below).
Single crystals suitable for X-ray diffraction analysis of 4a−6a,
8a, and 10a were grown via slow diffusion of diethyl ether into
a saturated solution of the complex in acetone. The X-ray
crystallographic data for all complexes are given in Table S4
and the key bond parameters in Table 1. Complexes 5a and 6a
crystallized in the triclinic space group P1, while 4a, 8a, and 10a
̅
crystallized in the monoclinic, orthorhombic, and monoclinic
space groups P21/n, Pna21, and I2/c, respectively. As expected,
the complexes adopted a pseudo-octahedral piano-stool config-
uration (Figure 2 and Figure S2), with the p-cymene acting as
the seat and the bidentate NHC ligand and chlorido group as
the legs. In all cases the complexes crystallized as a mixture of
enantiomers with the chiral center located at the metal. The struc-
tures of 8a and 10a feature a methylene spacer between the NHC
and the pyridyl groups, which is absent in 4a−6a. Therefore,
the latter structures form a virtually planar five-membered ring
with the ruthenium center. In the structures of 8a and 10a, the
metal center is part of a six-membered ring system and the
planarity is absent. This makes the latter compounds less rigid.
For complex 4a the Ru−CNHC bond length is the shortest of
the structures analyzed (1.997(3) Å), while 10a features the
longest bond (2.039(6) Å). The Ru−Cl bonds were, as expected,
in the range of 2.4 Å. Note that the structure of 4a was refined
with 11.4(3)% Br replacing the chlorido ligands. The Ru−N bond
in 8a is notably longer than in the other complexes (Table 1).
Inspection of the structures of 8a and 10a revealed that the
methylene protons are in different chemical environments, explain-
Figure 2. ORTEP representations of the molecular structure of one of
the enantiomers of 4a (top) and of 10a (bottom) drawn at 50%
probability ellipsoid levels. The counterions and any solvent molecules
were omitted for clarity.
Stability in DMSO and Aqueous Solution. Representa-
tive examples of complexes where the Ru center is part of five
(1a−3a)- or six-membered (8a and 10a) rings as well as 5a and
6a bearing an ester moiety were selected for stability studies in
DMSO-d6 and/or 20% DMSO-d6/80% D2O by 1H NMR
spectroscopy (Table S5). In general, the compounds were fairly
stable in DMSO-d6; however, over time a slow release of the
p-cymene ligand was observed, as indicated by the appearance
1
1
ing the diastereotopic behavior in the H NMR spectroscopy
of signals at around 7 ppm in the H NMR spectra (compare
experiments. For complexes 4a, 8a, and 10a π-stacking interactions
were observed to occur between the benzimidazole moieties of
the NHC ligands of the two enantiomers found in the molec-
ular structures (Figure S3 for 4a). In contrast, in the structures
of 5a and 6a (Figure S2), the π-stacking interactions were
formed between the pyridyl rings of the two enantiomers. The
shortest distances were found for 4a at 3.357 Å, followed by
10a (3.376 Å), 6a (3.378 Å), 8a (3.473 Å), and 5a (3.510 Å).
Notably, for 5a a mixture of the methyl and ethyl derivatives
(36(1):64(1)) was identified in the single crystal, confirming
the propensity of transesterification of the compound class.
Figure 3 for 1a). Compound 1a was the least stable of the com-
plexes investigated in terms of p-cymene release, while bulkier
groups around the metal center were found to give higher sta-
bility. The presence of the methylene linker did not influence
the stability in DMSO significantly (compare Figure S4 for 8a).
In the case of the ester derivatives 5a and 6a, minor arene
ligand cleavage was observed. It appears that the ethyl ester
moiety of 5a reacted slowly with residual water in the NMR
solvent, yielding a carboxylic acid, while 6a did not seem to
undergo such a hydrolysis process. The 1H NMR spectra of 5a
featured increasing signals at about 1.1 ppm which can be
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1
Figure 3. Time-dependent H NMR spectroscopic stability study for
1a in DMSO-d6 over 72 h. The box denotes peaks assigned to the slow
release of the p-cymene ligand.
assigned to the CH3 group of ethanol (Figure S5), while the
signal of the CH2 group was covered by the water peak.
For the investigation of aqueous stability, studies were con-
ducted for 2a and 8a, which were dissolved in DMSO-d6, fol-
lowed by immediate dilution with D2O to give a DMSO-d6:D2O
ratio of 1:5 and analysis by 1H NMR spectroscopy. Compound
8a was very stable with no signs of chlorido/aqua ligand
exchange observed, even after 72 h of incubation at room tem-
perature (Figure S6). In contrast, 2a underwent a ligand exchange
reaction to form the respective aqua complex 2aH2O with the first
1
Figure 4. Time-dependent H NMR spectroscopic stability study for
2a in DMSO-d6/D2O over 72 h and for comparison a spectrum
recorded 72 h after addition of AgNO3. The gray shaded box denotes
peaks assigned to the slow release of the p-cymene ligand. The boxes
with the dashed lines indicate the peaks assigned to the aqua complex
formed over time.
1+ to 2+ in case of Cl−/H2O exchange but remains constant
for Cl−/OH−. Both complexes gave very similar results in these
calculations, and the energetically favorable formation of hydroxido
complexes will likely affect the pKa values of their aqua com-
plexes in solution. When the pH values of aqueous solutions of
both 1a and 7a (0.7 μM) were measured, values of 4.9 and 4.2,
respectively, were found. Analysis of the frontier molecular orbitals
of [1a]+ and [7a]+ and their respective aqua and hydroxido
1
signs of a second species appearing in the H NMR spectrum
recorded after about 12 h of dissolution. The formation of the
aqua complex was indicated by additional peaks appearing in
both the aromatic and aliphatic regions of the 1H NMR spectra.
Very notable changes were observed for H-13, which shifted
upfield to about 9.2 ppm. Furthermore, the p-cymene protons
were detected downfield of those assigned to the chlorido com-
plex (Figure 4). The assignment of these peaks as the respective
aqua species was achieved by experiments in which AgNO3 was
added to the reaction mixture (Figure S7), and comparison of
analogues [1a/7aH2O 2+ and [1a/7aOH]+ (Figures S8 and S9)
]
revealed that the LUMO orbitals of [1a]+ and its derivatives are
delocalized over the metal center and ligands with the Ru d
orbitals and π electrons of the pyridyl group contribute
extensively, while the π electrons of the carbene moiety only
1
the H NMR spectra showed large overlaps with the signals
partially contribute. The HOMOs of [1a]+ and [1aH2O 2+
]
obtained in DMSO-d6/D2O (Figure 4). In addition, p-cymene
cleavage was observed to occur after around 1 day of dissolu-
tion (gray shaded box in Figure 4). Similar observations were
made in the reaction of 5a with AgNO3 in DMSO-d6/D2O,
while 8a could not be activated under these conditions (data
not shown). This behavior for 8a also confirms the observa-
tions made in pure DMSO-d6.
In general it appears that complexes with ligands which form
six-membered rings with the metal center are more stable than
five-membered metallacycles, especially if they contain bulky
groups on the NHC. However, the stability studies indicate that
the compounds have sufficient stability for the preparation of
samples for biological assays.
showed similar Ru d orbital contribution and involve π elec-
trons of the imidazolium moiety rather than of the pyridyl group.
The HOMOs of [1aOH]+ are delocalized over the Ru center and
the OH− ligand. For [7a]+, the HOMO and LUMO orbitals are
similar to each other, with the orbitals delocalized over the
metal center and ligands. In contrast to the LUMOs of [7a]+
and its derivatives, the pyridyl group did not contribute to the
LUMOs or HOMOs.
Interactions with Biological Molecules. Metallodrugs
are exposed to a variety of biomolecules once administered to a
living organism. Their pharmacological properties depend on
the nature of these interactions. In order to investigate the
NHC complexes for their reactivity with biomolecules, the five-
and six-membered metallacycles 1a and 7a were selected for
studies with the small biological molecules L-cysteine (Cys),
L-methionine (Met), L-histidine, and 9-ethylguanine (EtG) in 10%
The exchange of the chlorido ligands of 1a and 7a was also
investigated by density functional theory (DFT) calculations in
water and the gas phase using GAUSSIAN 09W. In both cases
the calculated energy difference for a Cl−/H2O ligand exchange
1
D6-DMSO/D2O. The reactions were monitored by H NMR
was slightly positive (Table S6; ΔE1a = 4.5 kcal/mol, ΔE7a
=
spectroscopy for up to 2 days.
7.9 kcal/mol). However, the calculations suggest that the for-
mation of hydroxido complexes is energetically favored (ΔE1a =
−59.5 kcal/mol, ΔE7a = −66.0 kcal/mol). This may be related
to the charge of the complex cation, which changes from
The reaction of 7a with His at a molar ratio of 1:1 was
monitored over a period of 24 h and resulted in the immediate
formation of a dative bond between the imidazole-N and the
ruthenium center by exchange of a chlorido ligand (Figure 5).
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1
Figure 6. H NMR spectroscopic study of the reaction between 7a
and EtG at molar ratios of 1:1 and 1:2 in 10% DMSO-d6/90% D2O
analyzed after 3 and 24 h. The box indicates the region that features
the signal for H-8 of Ru-coordinated EtG.
1
Figure 5. H NMR spectroscopic study of the reaction between 7a
and His in 10% DMSO-d6/D2O, monitored for a period of 24 h. The
new peaks assigned to the complex reacted with His are highlighted in
a box.
unreacted EtG was detected (Figure 6). The reaction occurred
very quickly, as demonstrated by a significant downfield shift of
the H8 signal of EtG from ca. 7.8 to 8.3 ppm, indicating
coordination of Ru to N7 of guanine. Integration of the signals
at about 8.3 and 9.2 ppm showed after 3 h for both incubation
mixtures a ratio of 0.6:1.0 (EtG:7a), which changed after 24 h
to 1:0.8 and 1:1.3 for the equimolar and 1:2 reaction mixtures,
respectively. Addition of another 1 equiv of EtG did not affect
the signal at 8.3 ppm but led to an increase in intensity of the
peak assigned to H8 of unreacted EtG at ca. 7.8 ppm.
In Vitro Anticancer Activity and Cell Uptake. The in
vitro antiproliferative activity of all complexes was studied in
HCT116 human colorectal, NCI-H460 non-small-cell lung, SiHa
cervical carcinoma, and SW480 colon adenocarcinoma cells.
The compounds resulted in a wide range of cytotoxic activity
varying from IC50 values as low as 6.2 μM to not even reaching
the IC50 value in the concentration range used (Table 2).
In general, the compounds were slightly more active in SiHa cells
This was indicated by the appearance of a new signal in the
1H NMR spectra at about 8.5 ppm which was assigned to
H-2imidazole of His. A significant downfield shift of about 0.8 ppm
clearly indicates coordination of Ru to the N-imidazole moiety
of His. The five-membered metallacycle 1a also initially reacted
with His in a similar fashion. However, over the course of the
experiment, several sets of signals were detected in the 1H NMR
spectra, which may be a sign of further reactions taking place,
possibly with His acting as a bidentate ligand (Figure S10).
This clearly shows that there are differing degrees of reactivity
between the two classes of compounds, with the six-membered
metallacycle being more stable than the five-membered analogues.
Neither 1a nor 7a reacted with Cys (molar ratio of 1:1) to
form defined products (Figures. S11 and S12). While 7a was
remarkably stable, given that many other Ru(arene) compounds
decompose within minutes when reacted with Cys,50−52 1a under-
went slow decomposition and after 24 h a significant amount of
the compound had decomposed. However, interestingly also for
this compound type the most abundant signals were still present.
Some of the changes in the spectra recorded for 7a may be due
to oxidation of Cys itself (Figure S9). The reactions under the
same conditions but with Met are in line with these observa-
tions. While 7a did not react with Met, 1a slowly decomposed
over time (Figures S13 and S14).
Table 2. In Vitro Cytotoxic Activity of Compounds 1a−10a
in the Human Cancer Cell Lines HCT116 (Colon), NCI-
H460 (Non-Small-Cell Lung), SiHa (Cervix), and SW480
(Colon) As Determined by SRB Assay after 72 h Incubation
IC50 values (μM)
compound
HCT116
NCI-H460
SiHa
SW480
A similar set of experiments was conducted for 1a and 7a
with the DNA model 9-ethylguanine (EtG) by H NMR spec-
1
five-membered
1a
>250
>250
>250
209 13
>250
>215
troscopy. DNA is considered as the main cellular target of the
Ru compound RAED, bearing a bidentate 1,2-ethylenediamine
ligand and therefore being structurally related to 1a. Incubation
mixtures at molar ratios of approximately 1:1 and 1:2 (metal
complex:EtG) were analyzed 3 and 24 h after mixing by 1H NMR
spectroscopy (Figure 6 and Figure S15). Metallacycle 1a only
formed EtG adducts to a minor extent independent of the molar
ratio of the incubation mixture, as indicated by the appearance
of a signal at around 8.3 ppm which can be assigned to H-8 of
Ru-coordinated EtG (Figure S15). After 24 h, the spectra markedly
changed and featured several species, again supporting the low
stability of 1a. In contrast, 7a formed in all cases a defined
product with EtG coordinated to the Ru center, while still some
2a
201 22
174
48
9
3a
51
3
1
3
44
1
1
58
5
1
4a
16
16 0.3
>428
14
17
5a
>428
>428
>428
6a
199 19
202 23
159 13
223 17
six-membered
7a
>244
>244
>244
>244
8a
69 15
149
130 11
12
7
57
91
4
3
107 11
9a
107
5
130
12
8
10a
8.3 0.6
1
6.2 0.02
2
a
RAED-C
5.9 0.7
a
Taken from ref 53, determined by MTT assay with 96 h incubation.
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than in the other cell lines tested. Considering the structures of
the complexes, the cytotoxicity appeared to be dependent upon
the lipophilicity as well as the stability of the complexes. The
most lipophilic and stable complexes were potent antiprolifer-
ative agents, with IC50 values in the low micromolar range
(see Table S7 for calculated octanol−water partition coefficients
clogP for the imidazolium salts as the pro-carbene ligands). Both 4a
and 10a are based on benzimidazole and feature a benzyl group
rather than a methyl group as in their derivatives 2a and 8a, and
they were the most cytotoxic compounds of the series tested.
While the clogP value for pro-carbene 4 is higher than that for
10, the greater stability of 10a may explain the slightly higher
antiproliferative potency. replacing benzimidazole was replaced
with imidazole as in 3a and 9a, the activity markedly dropped,
which was more pronounced in the case of 9a, for which the
antiproliferative activity was 1 order of magnitude lower than
that for 10a. Making the compounds even more hydrophilic by
the introduction of ester groups (5a and 6a) caused another
decrease in cytotoxicity, with the IC50 values increasing signif-
icantly. The compound most active in SW480 cells was 10a,
and it showed activity similar to that for the widely studied Ru
complex RAED-C, though the conditions used to determine the
antiproliferative activity were different.53
CONCLUSIONS
■
Organoruthenium complexes with monodentate NHC ligands
have been demonstrated to possess tunable cytotoxic activity
depending on the substituents.21 Herein, we report bidentate
NHC-pyridyl ligands and the preparation of the respective
Ru(cym) complexes to study the effect of both hydrophobic and
hydrophilic groups on their biological activity. The compounds
were characterized by standard methods and were found to
crystallize as enantiomeric mixtures. In the case of the ester com-
pound 5a, transesterification was observed in methanol solution.
Stability studies in DMSO with the complexes featuring five
(1a−3a)- or six-membered (8a and 10a) rings as well as the
ester-functionalized 5a and 6a revealed that 1a was the least
stable compound. In water, 8a was very stable while 2a under-
went a chlorido/aqua ligand exchange, which was further sup-
ported by studies using AgNO3 to abstract the chlorido ligand
from the Ru center. We used the five- and six-membered metalla-
cycles 1a and 7a, respectively, to investigate the compounds’
reactivity with biological molecules. Compound 7a was stable
in the presence of Cys, while 1a slowly decomposed. This is
remarkable, given that many other organoruthenium compounds
decompose rapidly in the presence of Cys. In general, both com-
pounds behaved differently; while 7a reacted with His and EtG,
1a slowly degraded with His and EtG.
The in vitro anticancer activity studies showed that 10a was
the most active compound in all cell lines investigated, while
the more hydrophilic compounds were least active. The effect
of the lipophilicity is reflected in the clogP values found for the
pro-carbenes and taking into consideration the stability of the
complexes and therewith the retained +1 charge of the com-
plex cation, while chlorido/aqua exchange would give a +2
charge state. This was confirmed to some extent by the higher
cellular accumulation for 10a in comparison to its closest ana-
logue in the series, 8a. However, additional factors, such as tar-
get interaction, which depends on the substituents, must con-
tribute to the overall biological properties.
With the aim of explaining the low antiproliferative activity of
8a in comparison to 10a, we determined the cellular accumula-
tion of these complexes in HCT116. The structural difference is
that 8a features a methyl substituent, which in 10a is replaced
with a benzyl group. The cellular content of the complexes was
measured through the Ru levels in HCT116 cells after incu-
bations for 4, 24, and 48 h by ICP-MS. Within the first 24 h
there was hardly any difference in the intracellular Ru amounts
detectable (Figure 7). Incubation experiments for 48 h showed
EXPERIMENTAL SECTION
■
General Considerations. All air-sensitive reactions were carried
out under an N2 flow in standard Schlenk or round-bottom flasks. The
preparation of RuII(NHC-pyridin-2-yl) complexes was done in dark-
ness by covering the flask with aluminum foil to prevent photolytic
degradation. Solvents such as acetonitrile (MeCN) and dichloro-
methane (DCM) were dried using a solvent purification system (LC
Technology Solutions., SP-1 solvent purifier). Purified solvents were
transferred into flasks that were dried under vacuum and purged with
N2 prior to use.
Chemicals and Reagents. The chemicals and solvents were
purchased from commercial suppliers. 1-Methylimidazole (99%) was
obtained from Arcos Organic, and 2-(bromomethyl)pyridine hydro-
bromide (98%), imidazole (99%), potassium phosphate tribasic (98%),
silver hexafluorophosphate (98%), α-terpinene (89%), and 2-bromopyr-
idine (99%) were purchased from Sigma-Aldrich. Benzimidazole (98%),
L-proline (98%), and copper(I) iodide (98%) were obtained from AK
Scientific. Ethyl 2-bromoacetate (98%) and iodomethane (99%) were
products of Merck. Ruthenium(III) chloride trihydrate (99%) was
obtained from Precious Metals Online and silver(I) oxide (AR) from
Oakwood Products. Compounds 1−10, 1a−3a, 9a, and 10a were syn-
thesized following literature procedures with slight modifications.39−43
Physical Measurements. Elemental analyses were conducted on a
Vario EL cube (Elementar Analysensysteme GmbH, Hanau, Germany).
1D and 2D (1H−13C HSQC, and HMBC) NMR spectra were recorded
on Bruker Avance AVIII 400 MHz NMR spectrometers at ambient
temperature at 400.13 MHz (1H) or 100.57 MHz (13C{1H}). Acetone-d6,
CDCl3, MeOD-d4, and DMSO-d6 were used as NMR solvents.
Figure 7. Cellular uptake of compounds 8a and 10a in HCT116 cells
after incubation for 4, 24, or 48 h. Statistical analysis was carried out
using the t test (* p < 0.05).
that 10a is taken up into the cells slightly more effectively in
comparison to its methyl counterpart 8a and significantly higher
Ru levels for both compounds were measured in comparison to
those at the first two time points. Some of the behavior of the
complexes may be explained by their lipophilicity (Table S7),
which is considerably lower for pro-carbene 8 than for 10.
However, the effect of lipophilicity on cellular uptake was less
than expected and cannot fully explain the difference in IC50
values between these compounds. The higher cytotoxic activity
of 10a is probably related to additional factors and may be the
result of a differing interaction with biological targets due to the
hydrophobic benzyl residue.
F
DOI: 10.1021/acs.organomet.8b00153
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
High-resolution mass spectra were recorded on a Bruker micrOTOF-
Q II ESI-MS instrument in positive ion mode.
suitable for X-ray diffraction analysis were grown from acetone/diethyl
ether. Anal. Calcd for C28H27ClF6N3PRu·0.5H2O: C, 48.32; H, 4.05;
N, 6.04. Found: C, 48.45; H, 3.94; N, 6.21. HRMS (ESI+): m/z
X-ray diffraction measurements of single crystals of 4a−6a and 10a
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 X-ray
single-crystal diffraction measurement of 8a was performed on a
Siemens/Bruker SMART APEX II single-crystal diffractometer with a
CCD area detector using graphite-monochromated Mo Kα radiation
(λ = 0.71073 Å). The data were processed with the SHELX2016 and
Olex2 software packages.54,55 All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms were inserted at calculated positions
and refined with a riding model or without restrictions. Mercury 3.956
was used to visualize the molecular structures.
1
556.1076 [M − PF6]+ (mcalc = 556.1094). H NMR (400.13 MHz,
3
acetone-d6): δ (ppm) 9.48−9.51 (m, 1H, H-13), 8.61 (d, J = 8 Hz,
1H, H-6), 8.49 (d, 3J = 8 Hz, 1H, H-16), 8.34 (td, 3J = 8 Hz, 4J = 2 Hz,
1H, H-15), 7.54−7.24 (m, 9H, H-7, H-8, H-9, H-19, H-20, H-21,
H-22, H-23), 6.42 (dd, 3J = 7 Hz, 4J = 1.9 Hz, 1H, H-26a/b), 6.31 (dd,
3J = 7 Hz, 4J = 2 Hz, 1H, H-25a/b), 6.21 (d, 2J = 17 Hz, 1H, H17a/b),
6.16 (dd, 3J = 7 Hz, 4J = 2 Hz, 1H, H-25a/b), 5.99 (d, 2J = 16 Hz, 1H,
3
4
H17a/b), 5.91 (dd, J = 7 Hz, J = 2 Hz, 1H, H-26a/b), 2.35 (sept,
3J = 7 Hz, 1H, H-28), 2.11 (s, 3H, H-30), 0.83 (d, J = 7 Hz, 3H,
3
3
H-29a/b), 0.81 (d, J = 7 Hz, 3H, H-29a/b). 13C{1H} NMR (100.57
MHz, acetone-d6): δ (ppm) 183.1 (C-3), 156.5 (C-13), 152.6 (C-11),
142.0 (C-15), 135.6 (C-18), 135.2 (C-5), 130.7 (C-1), 129.0 (C-20,
C-22), 128.3 (C-21), 127.4 (C-19, C-23), 125.4 (C-8, C-7), 122.9
(C-14), 113.5 (C-16), 113.1 (C-9), 113.0 (C-6), 109.7 (C24), 107.2
(C27), 92.0 (C-25a/b), 88.0 (C-26a/b), 52.8 (C-17), 30.4 (C-24),
22.0 (C-29a/b), 21.8 (C-29a/b), 18.6 (C-30).
Syntheses. 1-(Pyridin-2-yl)-3-(tert-butylacet-2-yl)imidazolium
Bromide (6).
[Chlorido(1-{ethylacet-2-yl}-3-{pyridin-2-yl}imidazol-2-ylidene)-
(η6-p-cymene)ruthenium(II)] Hexafluorophosphate (5a).
Compound 6 was prepared by refluxing I (100 mg, 0.44 mmol) with
tert-butyl bromoacetate (0.10 mL, 0.63 mmol) in acetonitrile (40 mL)
for 24 h. The solvent was removed under reduced pressure, and the
crude product was purified by trituration with acetone, affording the
pure product as a pale brown solid (0.12 g, 81%). HRMS (ESI+): m/z
1
260.1389 [M − Br]+ (mcalc = 260.1394). H NMR (400.13 MHz,
DMSO-d6): δ (ppm) 10.14 (s, 1H, H-3), 8.65−8.68 (m, 1H, H-13),
3
3
8.58 (t, J = 2 Hz, 1H, H-5), 8.24 (m, 1H, H-15), 8.08 (d, J = 9 Hz,
The synthesis of 5a was performed according to the general procedure
using 5 (150 mg, 0.48 mmol), silver hexafluorophosphate (160 mg,
0.65 mmol), silver oxide (75 mg, 0.32 mmol), and [RuCl2(η6-p-
cymene)]2 (202 mg, 0.33 mmol) in dry acetonitrile to afford a yellow
powder (120 mg, 74%). Single crystals suitable for X-ray diffraction
analysis were grown from acetone/methanol/diethyl ether. Anal. Calcd
for C22H27ClF6N3O2PRu·H2O·1.5CH2Cl2·0.6MeCN: C, 36.31; H,
4.17; N, 6.17. Found: C, 36.04; H, 4.00; N, 6.56. HRMS (ESI+): m/
3
1H, H-16), 8.04 (t, J = 2 Hz, 1H, H-1), 7.65−7.69 (m, 1H, H-14),
5.27 (s, 2H, H-17), 1.48 (s, 9H, H-20, H-21, H-22). 13C{1H} NMR
(100.57 MHz, DMSO-d6): δ (ppm) 165.3 (C-18), 149.3 (C-13),
146.43 (C-11), 140.9 (C-15), 136.1 (C-3), 125.5 (C-14), 125.1 (C-1),
119.0 (C-5), 114.5 (C-16), 83.1 (C-19), 50.3(C-17), 27.6 (C-20, C-21,
C-22).
General Procedure for the Synthesis of the Ru Complexes.
Compounds 1−10 (2.0 mol equiv) and silver hexafluorophosphate
(2.5−3.0 mol equiv) were added to dry acetonitrile or THF (Table S1)
and methanol (1 mL) in a dry Schlenk flask under N2. The flask was
sealed, and the mixture was stirred in darkness at 55 °C for 3 h. Ag2O
(1.4−1.5 mol equiv) was added and the mixture stirred in darkness for
a further 24 h at 65 °C. A solution of [RuCl2(η6-p-cymene)]2 (1.4−
1.5 mol equiv) in dry 1,2-dichloroethane and dichloromethane was added
and stirred at 70 °C in darkness overnight. The resultant suspension
was filtered over Celite and the filtrate collected and reduced to
dryness. Purification was performed via column chromatography over
silica (MeOH/CH2Cl2 1/25). The solvent of the collected fractions
was evaporated, and the crude compound was dissolved in a minimal
amount of MeOH and precipitated by addition of diethyl ether.
The precipitate was dried in vacuo to afford the Ru(η6-p-cymene)
complexes.
1
z 502.0826 [M − PF6]+ (mcalc = 502.0832). H NMR (400.13 MHz,
acetone-d6): δ (ppm) 9.42 (d, 3J = 6 Hz, 1H, H-13), 8.33 (d, 1H, 3J =
3
3
2 Hz, H-1), 8.30 (t, J = 7 Hz, 1H, H-15), 8.16 (d, J = 8 Hz, 1H,
H-16), 7.83 (d, 3J = 2 Hz, 1H, H-5), 7.57 (t, 3J = 7 Hz, H, H-14), 6.31
3
(d, J = 6 Hz, 1H, H-25a/b), 6.22−6.25 (m, 2H, 26a/b, 25a/b), 5.73
3
(d, J = 6 Hz, 1H, H-25a/b), 5.37−5.48 (m, 2H, 17a/b), 4.39 (quart,
3J = 7 Hz, 2H, H-19), 2.53 (sept, J = 7 Hz, 1H, H-28), 2.21 (s, 3H,
3
3
3
H-30), 1.35 (t, J = 6 Hz, 3H, H-20), 0.98 (d, J = 7 Hz, 6H, H-29a,
29b).13C{1H} NMR (100.57 MHz, acetone-d6): δ (ppm) 169.4
(C-18), 166.3 (C-3), 156.7 (C-13), 152.9 (C-11), 142.5 (C-15), 127.2
(C-5), 124.2 (C-14), 117.9 (C-1), 113.5 (C-16), 109.3 (C-24), 92.1
(C-25a/b), 91.9 (C-26a/b), 87.8 (C-25a/b), 83.1 (C-26a/b), 63.2
(C-19), 53.1 (C-28), 52.9 (C-17a/b), 31.8 (C-28), 22.7 (C-29a/b),
22.3 (C-29a/b), 19.1 (C-30), 14.4 (C-20a/b).
[Chlorido(1-benzyl-3-{pyridin-2-yl}benzimidazol-2-ylidene)(η6-p-
cymene)ruthenium(II)] Hexafluorophosphate (4a).
[Chlorido(1-{tert-butylacet-2-yl}-3-{pyridin-2-yl}imidazol-2-
ylidene)ruthenium(II)] Hexafluorophosphate (6a).
The synthesis of 6a was performed according to the general procedure
using 6 (150 mg, 0.44 mmol), silver hexafluorophosphate (150 mg,
0.58 mmol), silver oxide (100 mg, 1.7 mol equiv, 0.43 mmol), and
[RuCl2(η6-p-cymene)]2 (180 mg, 0.29 mmol) in dry dichloromethane
and 1,2-dichloroethane to afford an orange powder (310 mg, 79%).
Anal. Calcd for C24H31ClF6N3O2PRu·H2O·2CH2Cl2: C, 36.19; H,
4.32; N, 4.87. Found: C, 36.17; H, 4.26; N, 5.33. HRMS (ESI+): m/z
The synthesis of 4a was performed according to the general procedure
using 4 (100 mg, 0.21 mmol), silver hexafluorophosphate (80 mg,
0.32 mmol), silver oxide (48 mg, 0.21 mmol), and [RuCl2(η6-p-cymene)]2
(110 mg, 1.7 mol equiv, 0.18 mmol) in dry dichloromethane and 1,2-
dichloroethane to afford an orange powder (120 mg, 88%). Single crystals
G
DOI: 10.1021/acs.organomet.8b00153
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1
530.1147 [M − PF6]+ (mcalc = 530.1144). H NMR (400.13 MHz,
DMSO-d6): δ (ppm) 9.33 (d, 3J = 6 Hz, 1H, H-13), 8.48 (d, 3J = 2 Hz,
H-5), 8.29 (td, 3J = 8 Hz, 4J = 2 Hz, 1H, H-15), 8.22 (d, 3J = 8 Hz, 1H,
H-14), 7.84 (d, 3J = 3 Hz, 1H, H-1), 7.56 (td, 3J = 7 Hz, 4J = 2 Hz, 1H,
(ESI+): m/z 570.1267 [M − PF6]+ (mcalc = 570.1245). 1H NMR
3
(400.13 MHz, DMSO-d6): δ (ppm) 9.25 (d, J = 6 Hz, 1H, H-13),
3
4
3
8.13 (td, J = 8 Hz, J = 2 Hz, 1H, H-15), 8.08 (d, J = 8.4 Hz, 1H,
H-6), 7.96 (dd, 3J = 8 Hz, 4J = 2 Hz, 1H, H- 16), 7.61 (td, 3J = 7 Hz, 4J
= 2 Hz, 1H, H-14), 7.44−7.42 (m, 8H, H-19, H-21, H-23, H-20, H-22,
3
3
H-14), 6.28 (d, J = 7 Hz, 1H, H-25a/b), 6.21 (d, J = 6.4 Hz, 1H,
2
3
3
3
H-7, H-8, H-9), 6.27 (d, J = 17 Hz, 1H, H-10a/b), 5.96 (dd, J =
6 Hz, J = 2 Hz, 1H, H-26a/b), 5.93 (s, 2H, H-17), 5.82 (dd, J =
H-26a/b), 6.04 (d, J = 7 Hz, 1H, H-25a/b), 5.75 (d, J = 6 Hz, 1H,
4
3
H-26a/b), 5.33 (d, 2J = 17 Hz, 1H, H-17a/b), 5.15 (d, 2J = 18 Hz, 1H,
4
3
4
3
6 Hz, J = 2 Hz, 1H, H-25a/b), 5.77 (dd, J = 6 Hz, J = 2 Hz, 1H,
H-17a/b), 2.68 (sept, J = 7 Hz, 1H, H-28), 2.11 (s, 3H, H-30), 1.55
H-25a/b), 5.67 (dd, 3J = 7 Hz, 4J = 2 Hz, 1H, H-26a/b), 5.08 (d, 2J =
3
3
(s, 9H, H-20a/b/c), 0.86 (d, J = 3 Hz, 3H, H-29a/b), 0.84 (d, J =
3 Hz, 3H, H-29a/b). 13C{1H} NMR (100.57 MHz, DMSO-d6):
δ (ppm) 185.6 (C-18), 167.2 (C-3), 155.7 (C-13), 150.8 (C-11),
141.8 (C-15), 126.2 (C-1), 123.1 (C-14), 117.0 (C-5), 115.9 (C-24),
114.7 (C-27), 112.6 (C-16), 90.8 (C-25a/b), 90.6 (C-26a/b), 86.3
(C-26a/b), 83.1 (C-19), 81.8 (C-25a/b), 52.4 (C-17), 30.5 (C-28),
27.7 (C-20a/b/c), 27.6 (C-22a/b), 21.9 (C-29a/b), 18.5 (C-30).
[Chlorido(1-benzyl-3-{pyridin-2-ylmethyl}imidazol-2-ylidene)(η6-
p-cymene)ruthenium(II)] Hexafluorophosphate (9a).
3
16 Hz, 1H, H-10a/b), 2.73 (sept, J = 7 Hz, 1H, H-27), 2.02 (s, 3H,
3
3
H-30), 1.13 (d, J = 7 Hz, 3H, H-29a/b), 1.05 (d, J = 7 Hz, 3H,
H-29a/b). 13C{1H} NMR (100.57 MHz, DMSO-d6): δ (ppm) 190.7
(C-11), 156.5 (C-13), 151.7 (C-3), 139.9 (C-15), 136.4 (C-18), 134.2
(C-5), 133.8 (C-1), 128.7 (C- 19/23), 127.8 (C-21), 126.5 (C-20/22),
125.1 (C-7), 124.7 (C-8/14),123.5 (C-8/14), 111.7 (C-6), 111.1
(C-27), 110.6 (C-9), 101.45 (C-24), 88.8 (C-25a/b), 87.8 (C-26a/b),
85.8 (C-25a/b), 85.2 (C-26a/b), 51.4 (C-10a/b), 49.7 (C-17), 30.7
(C-28), 22.7 (C-30), 21.1 (C-29a/b), 17.9 (C-29a/b).
Stability Studies. For the stability studies in DMSO, 1a−3a, 5a, 6a,
1
8a, and 10a (1−2 mg) were dissolved in in DMSO-d6 and H NMR
spectra were recorded after 0, 1, 3, 12, 24, 48, and 72 h. To determine
their stability in aqueous solution, 2a and 8a (1−2 mg) were dissolved
1
in DMSO-d6 and diluted with D2O (1/5) and H NMR spectra were
collected over 3 days. The same experiment was conducted but with
1 equiv of AgNO3 added to induce the exchange of the chlorido with
an aqua ligand.
DFT Calculations. GAUSSIAN 09W57 was used to calculate the opti-
mized ground-state structures and frequencies for the different molecules
by density functional theory (DFT) with the B3LYP hybrid exchange
functional and a split basis set for C, H, N, O, and Cl (6-31G(d,p))
and the transition metal ruthenium (SDDAll) under vacuum. The
SCRF (self-consistent reaction field) keyword was implemented for
the optimization of aqueous simulation of the molecules. This method
is the integral equation formalism variant of the polarizable continuum
model (IEFPCM).58 The frontier orbitals were viewed and obtained
through the Avogadro software (version 1.2.0).59
The synthesis of 9a was performed according to the general procedure
using 9 (150 mg, 0.46 mmol), silver hexafluorophosphate (150 mg,
0.60 mmol), silver oxide (75 mg, 0.33 mmol), and [RuCl2(η6-p-cymene)]2
(194 mg, 0.32 mmol) in dry dichloromethane and 1,2-dichloroethane
to afford an orange powder (70 mg, 46%). Anal. Calcd for
C26H29ClF6N3PRu·0.2CH3(CH2)4CH3: C, 47.88; H, 4.70; N, 6.16.
Found: C, 47.57; H, 4.34; N, 5.85. HRMS (ESI+): m/z 520.1075
1
[M − PF6]+ (mcalc = 520.1092). H NMR (400.13 MHz, DMSO-d6):
δ (ppm) 9.22 (d, 3J = 6 Hz, 1H, H-13), 8.08 (td, 3J = 8 Hz, 4J = 2 Hz,
1H, H-15), 7.71 (d, 3J = 8 Hz, 1H, H-16), 7.66 (d, 3J = 8 Hz, 1H, H-1)
Biomolecule Interactions. The biomolecule interactions of 1a
and 7a were studied by 1H NMR spectroscopy. Complexes 1a and 7a
were dissolved in DMSO-d6 and diluted with D2O to obtain a 20%
DMSO-d6/80% D2O solution. Equimolar amounts of the amino acids
Met, Cys, and His were added to each complex, and 1H NMR spectra
3
7.56 (t, J = 7 Hz, 1H, H-5), 7.34−7.45 (m, 5H, H-19, H-23, H-21,
3
3
H-22, H-20), 7.24 (d, J = 8 Hz, 1H, H-1), 5.87 (d, J = 6 Hz, 1H,
H-26a/b), 5.82, (dd, 3J = 17 Hz, 4J = 6 Hz, 1H, H-25a/b), 5.66 (d, 3J =
16 Hz, 1H, H-10a/b), 5.56−5.63 (m, 2H, H-23, H26a/b), 5.44 (d, 3J =
1
3
were collected over periods of up to 24 h. The H NMR spectra for
15 Hz, 2H, H-17a/b), 4.98 (d, J = 16 Hz, 1H, H-10a/b), 2.69−2.77
3
the reactions of each complex and 9-ethylguanine at equimolar and 1:2
ratios were recorded 3 and 24 h after mixing.
(m, 1H, H-28), 2.06 (s, 3H, C30), 1.10 (d, J = 7 Hz, 6H, H-29a,
H-29b). 13C{1H} NMR (100.57 MHz, acetone-d6): δ (ppm) 176.2
(C-11), 159.9 (C-13), 157.5 (C-3), 140.6 (C-15), 137.7 (C-18), 129.7
(C- 23/19), 129.4 (C-22/20), 129.1 (C-21), 125.9 (C-1), 125.6 (C-5),
123.9 (C-16), 123.6 (C-14), 112.4 (C-27), 103.3 (C-24), 90.1 (C-25a/b),
87.2 (C-26a/b), 85.7 (C-25a/b), 85.6 (C-26a/b), 55.0 (C-710a/b), 54.5
(C-17), 32.3 (C-28), 24.0 (C-30), 21.4 (C-29a/b), 18.7 (C-29a/b).
[Chlorido(1-benzyl-3-{pyridin-2-ylmethyl}benzimidazol-2-
ylidene)(η6-p-cymene)ruthenium(II)] Hexafluorophosphate (10a).
Sulforhodamine B Cytotoxicity Assay. HCT116, SW480, and
NCI-H460 cells were supplied by ATCC, while SiHa cells were obtained
from Dr. David Cowan, Ontario Cancer Institute, Canada. The cells
were grown in αMEM (Life Technologies) supplemented with 5%
fetal calf serum (Moregate Biotech) at 37 °C in a humidified incubator
with 5% CO2.
The cells were seeded at 750 (HCT116, NCI-H460), 4000 (SiHa),
or 5000 (SW480) cells/well in 96-well plates and left to settle for 24 h.
The compounds were added to the plates in a series of 3-fold dilutions,
containing a maximum of 0.5% DMSO at the highest concentration.
The assay was terminated after 72 h by addition of 10% trichloroacetic
acid (Merck Millipore) at 4 °C for 1 h. The cells were stained with
0.4% sulforhodamine B (Sigma-Aldrich) in 1% acetic acid for 30 min
in the dark at room temperature and then washed with 1% acetic acid
to remove unbound dye. The stain was dissolved in unbuffered Tris
base (10 mM; Serva) for 30 min on a plate shaker in the dark and
quantified on a BioTek EL808 microplate reader at an absorbance
wavelength of 490 nm with 450 nm as the reference wavelength to
determine the percentage of cell growth inhibition by determining the
absorbance of each sample relative to a negative (no inhibitor) and a
no-growth control (day 0). The IC50 values were calculated with SigmaPlot
12.5 using a three-parameter logistic sigmoidal dose−response curve
between the calculated growth inhibition and the compound concen-
tration. The presented IC50 values are the mean of at least 3 inde-
pendent experiments, where 10 concentrations were tested in dupli-
cate for each compound.
The synthesis of 10a was performed according to the general proce-
dure using 10 (150 mg, 0.40 mmol), silver hexafluorophosphate (130 mg,
0.51 mmol), silver oxide (90 mg, 1.96 mol equiv, 0.39 mmol), and
[RuCl2(η6-p-cymene)]2 (180 mg, 0.30 mmol) in dry dichloromethane
and 1,2-dichloroethane to afford an orange powder (75 mg, 42%).
Single crystals suitable for X-ray diffraction analysis were grown from
acetone/diethyl ether. Anal. Calcd for C30H31ClF6N3PRu·0.3H2O: C,
50.01; H, 4.42; N, 5.83. Found: C, 50.28; H, 4.79; N, 5.72. HRMS
H
DOI: 10.1021/acs.organomet.8b00153
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Cellular Accumulation. The cellular uptake experiments were
carried out as described previously. HCT116 cells (4 × 105 /well)
were seeded into 6-well plates and allowed to settle for 24 h at 37 °C
and 5% CO2. Compounds 8a and 10a were dissolved in DMSO
(6900 and 824 μM, respectively) and diluted with media to a con-
centration of 1% DMSO to reach their previously determined IC50 values.
The cells were incubated with metal complexes for 4, 24, and 48 h,
after which the medium was removed and the cells were washed twice
with 1 mL of ice-cold PBS buffer. The cells were lysed with 2 mL of
(5) Charra, V.; de Fremont, P.; Braunstein, P. Coord. Chem. Rev.
2017, 341, 53−176.
(6) Farrell, J. M.; Posaratnanathan, R. T.; Stephan, D. W. Chem. Sci.
2015, 6, 2010−2015.
(7) Nair, A. G.; McBurney, R. T.; Walker, D. B.; Page, M. J.; Gatus,
M. R. D.; Bhadbhade, M.; Messerle, B. A. Dalton Trans. 2016, 45,
14335−14342.
(8) Herrmann, W. A. Angew. Chem., Int. Ed. 2002, 41, 1290−1309.
(9) Viciu, M. S.; Germaneau, R. F.; Navarro-Fernandez, O.; Stevens,
E. D.; Nolan, S. P. Organometallics 2002, 21, 5470−5472.
(10) Ruiz-Castillo, P.; Buchwald, S. L. Chem. Rev. 2016, 116, 12564−
12649.
concentrated nitric acid (containing 0.1 μL of a 1000
3 μg/mL
thulium standard as internal standard) and digested with an Ethos Up
microwave digestion system (Milestone). After the solutions were diluted
with 10 mL of H2O, the ruthenium content was determined by ICP-MS
(Agilent 7700) with an ASX-500 autosampler (CETAC Technologies)
in a Serie SuSi laminar flow hood (SPECTEC) equipped with a MicroMist
nebulizer and a Scott double-pass spray chamber. The carrier gas flow
rate was 1 mL min−1. The instrument was tuned for cerium, cobalt,
lithium, magnesium, thallium, and yttrium. The reported values are the
mean of at least three independent uptake experiments conducted with
blank wells for each substance to account for unspecific binding to the
plastic of the well plates.
(11) Poyatos, M.; Uriz, P.; Mata, J. A.; Claver, C.; Fernandez, E.;
Peris, E. Organometallics 2003, 22, 440−444.
(12) Do, J. L.; Mottillo, C.; Tan, D.; Strukil, V.; Friscic, T. J. Am.
Chem. Soc. 2015, 137, 2476−2479.
(13) Kantchev, E. A. B.; O’Brien, C. J.; Organ, M. G. Angew. Chem.,
Int. Ed. 2007, 46, 2768−2813.
(14) Bernet, L.; Lalrempuia, R.; Ghattas, W.; Mueller-Bunz, H.;
Vigara, L.; Llobet, A.; Albrecht, M. Chem. Commun. 2011, 47, 8058−
8060.
(15) Leigh, V.; Ghattas, W.; Lalrempuia, R.; Muller-Bunz, H.; Pryce,
M. T.; Albrecht, M. Inorg. Chem. 2013, 52, 5395−5402.
(16) Liu, W. K.; Gust, R. Chem. Soc. Rev. 2013, 42, 755−773.
(17) Chan, C. Y.; Barnard, P. J. Dalton Trans. 2015, 44, 19126−
19140.
(18) Medici, S.; Peana, M.; Crisponi, G.; Nurchi, V. M.; Lachowicz, J.
I.; Remelli, M.; Zoroddu, M. A. Coord. Chem. Rev. 2016, 327-328,
349−359.
(19) Liu, W. K.; Gust, R. Coord. Chem. Rev. 2016, 329, 191−213.
(20) Cetinkaya, B.; Cetinkaya, E.; Kucukbay, H.; Durmaz, R.
Arzneim.-Forsch. 1996, 46, 821−823.
(21) Oehninger, L.; Rubbiani, R.; Ott, I. Dalton Trans. 2013, 42,
3269−3284.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.organomet.8b00153.
■
S
Preparation and characterization of all organic and
organometallic compounds not given in the Experimental
Section, X-ray crystallographic data, and additional
figures, as well as time-dependent NMR spectra recorded
for the stability studies and biomolecule interaction
experiments (PDF)
Accession Codes
CCDC 1826988−1826992 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(22) Oehninger, L.; Stefanopoulou, M.; Alborzinia, H.; Schur, J.;
Ludewig, S.; Namikawa, K.; Munoz-Castro, A.; Koster, R. W.;
Baumann, K.; Wolfl, S.; Sheldrick, W. S.; Ott, I. Dalton Trans. 2013,
42, 1657−1666.
(23) Melaiye, A.; Simons, R. S.; Milsted, A.; Pingitore, F.;
Wesdemiotis, C.; Tessier, C. A.; Youngs, W. J. J. Med. Chem. 2004,
47, 973−977.
(24) Berners-Price, S. J.; Filipovska, A. Metallomics 2011, 3, 863−873.
(25) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.; Baker, M. V.;
Berners-Price, S. J.; Filipovska, A. J. Am. Chem. Soc. 2008, 130, 12570−
1.
(26) Pellei, M.; Gandin, V.; Marinelli, M.; Orsetti, A.; Del Bello, F.;
Santini, C.; Marzano, C. Dalton Trans. 2015, 44, 21041−21052.
(27) Hartinger, C. G.; Dyson, P. J. Chem. Soc. Rev. 2009, 38, 391−
401.
AUTHOR INFORMATION
Corresponding Author
* C.G.H.: e-mail, c.hartinger@auckland.ac.nz; web, http://
hartinger.auckland.ac.nz; tel, +64 9 3737 599 ext 83220.
■
ORCID
Muhammad Hanif: 0000-0002-2256-2317
Christian G. Hartinger: 0000-0001-9806-0893
Notes
(28) Murray, B. S.; Babak, M. V.; Hartinger, C. G.; Dyson, P. J.
Coord. Chem. Rev. 2016, 306, 86−114.
The authors declare no competing financial interest.
(29) Zhang, P.; Sadler, P. J. J. Organomet. Chem. 2017, 839, 5−14.
(30) Thota, S.; Rodrigues, D. A.; Crans, D. C.; Barreiro, E. J. J. Med.
Chem. 2018, DOI: 10.1021/acs.jmedchem.7b01689.
(31) Meier-Menches, S. M.; Gerner, C.; Berger, W.; Hartinger, C. G.;
Keppler, B. K. Chem. Soc. Rev. 2018, 47, 909−928.
(32) Hanif, M.; Nazarov, A. A.; Legin, A.; Groessl, M.; Arion, V. B.;
Jakupec, M. A.; Tsybin, Y. O.; Dyson, P. J.; Keppler, B. K.; Hartinger,
C. G. Chem. Commun. 2012, 48, 1475−1477.
ACKNOWLEDGMENTS
■
We thank the University of Auckland (University of Auckland
Doctoral Scholarship to H.U.H. and K.K.H.T.) for financial
support. We are grateful to Tanya Groutso for collecting the
single-crystal X-ray diffraction data and Tony Chen for ESI-MS
analyses.
(33) Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.;
Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H. F.; Ang, W. H.;
REFERENCES
■
Droge, P.; Rothlisberger, U.; Dyson, P. J.; Davey, C. A. Nat. Commun.
̈
(1) Peris, E. Chem. Rev. 2017, DOI: 10.1021/acs.chemrev.6b00695.
(2) Janssen-Muller, D.; Schlepphorst, C.; Glorius, F. Chem. Soc. Rev.
2017, 46, 4845−4854.
(3) Nasr, A.; Winkler, A.; Tamm, M. Coord. Chem. Rev. 2016, 316,
68−124.
(4) Johnson, C.; Albrecht, M. Coord. Chem. Rev. 2017, 352, 1−14.
2014, 5, 3462.
(34) Soldevila-Barreda, J. J.; Romero-Canelon
́
, I.; Habtemariam, A.;
Sadler, P. J. Nat. Commun. 2015, 6, 6582.
(35) Kubanik, M.; Holtkamp, H.; Sohnel, T.; Jamieson, S. M. F.;
̈
Hartinger, C. G. Organometallics 2015, 34, 5658−5668.
I
DOI: 10.1021/acs.organomet.8b00153
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
(36) Aman, F.; Hanif, M.; Kubanik, M.; Ashraf, A.; Sohnel, T.;
̈
Jamieson, S. M. F.; Siddiqui, W. A.; Hartinger, C. G. Chem. - Eur. J.
2017, 23, 4893−4902.
(37) Kubanik, M.; Lam, N. Y. S.; Holtkamp, H. U.; Sohnel, T.;
Anderson, R. F.; Jamieson, S. M. F.; Hartinger, C. G. Chem. Commun.
2018, 54, 992−995.
(38) Xi, Z. X.; Liu, F. H.; Zhou, Y. B.; Chen, W. Z. Tetrahedron 2008,
64, 4254−4259.
(39) Jahnke, M. C.; Pape, T.; Hahn, F. E. Eur. J. Inorg. Chem. 2009,
2009, 1960−1969.
(40) Lv, R.; Wang, Y. X.; Zhou, C. S.; Li, L. Y.; Wang, R. H.
ChemCatChem 2013, 5, 2978−2982.
(41) Barbante, G. J.; Francis, P. S.; Hogan, C. F.; Kheradmand, P. R.;
Wilson, D. J. D.; Barnard, P. J. Inorg. Chem. 2013, 52, 7448−7459.
(42) Chan, C. Y.; Pellegrini, P. A.; Greguric, I.; Barnard, P. J. Inorg.
Chem. 2014, 53, 10862−10873.
(43) Barbante, G. J.; Doeven, E. H.; Francis, P. S.; Stringer, B. D.;
Hogan, C. F.; Kheradmand, P. R.; Wilson, D. J. D.; Barnard, P. J.
Dalton Trans. 2015, 44, 8564−8576.
(44) Grundemann, S.; Kovacevic, A.; Albrecht, M.; Faller, J. W.;
Crabtree, R. H. J. Am. Chem. Soc. 2002, 124, 10473−10481.
(45) Chiu, P. L.; Lai, C. L.; Chang, C. F.; Hu, C. H.; Lee, H. M.
Organometallics 2005, 24, 6169−6178.
(46) Oehninger, L.; Stefanopoulou, M.; Alborzinia, H.; Schur, J.;
Ludewig, S.; Namikawa, K.; Munoz-Castro, A.; Koster, R. W.;
̃
̈
Baumann, K.; Wolfl, S. Dalton Trans. 2013, 42, 1657−1666.
̈
(47) Fernandez, F. E.; Puerta, M. C.; Valerga, P. Organometallics
2012, 31, 6868−6879.
(48) Tay, B. Y.; Wang, C.; Phua, P. H.; Stubbs, L. P.; Huynh, H. V.
Dalton Trans. 2016, 45, 3558−63.
(49) Viji, M.; Tyagi, N.; Naithani, N.; Ramaiah, D. New J. Chem.
2017, 41, 12736−12745.
(50) Kandioller, W.; Kurzwernhart, A.; Hanif, M.; Meier, S. M.;
Henke, H.; Keppler, B. K.; Hartinger, C. G. J. Organomet. Chem. 2011,
696, 999−1010.
(51) Hanif, M.; Meier, S. M.; Adhireksan, Z.; Henke, H.; Martic, S.;
Movassaghi, S.; Labib, M.; Kandioller, W.; Jamieson, S. M. F.; Hejl, M.;
Jakupec, M. A.; Kraatz, H. B.; Davey, C. A.; Keppler, B. K.; Hartinger,
C. G. ChemPlusChem 2017, 82, 841−847.
(52) Movassaghi, S.; Hanif, M.; Holtkamp, H. U.; Sohnel, T.;
̈
Jamieson, S. M. F.; Hartinger, C. G. Dalton Trans. 2018, 47, 2192−
2201.
(53) Babak, M. V.; Meier, S. M.; Legin, A. A.; Razavi, M. S. A.; Roller,
A.; Jakupec, M. A.; Keppler, B. K.; Hartinger, C. G. Chem. - Eur. J.
2013, 19, 4308−4318.
(54) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.;
Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341.
(55) Sheldrick, G. M. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71,
3−8.
(56) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.;
Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. J. Appl.
Crystallogr. 2006, 39, 453−457.
(57) Frisch, M.; Trucks, G.; Schlegel, H. B.; Scuseria, G.; Robb, M.;
Cheeseman, J.; Scalmani, G.; Barone, V.; Mennucci, B., et al. Gaussian
09W; Gaussian, Inc., Wallingford, CT, 2009.
(58) Scalmani, G.; Frisch, M. J. J. Chem. Phys. 2010, 132, 114110.
(59) Hanwell, M. D.; Curtis, D. E.; Lonie, D. C.; Vandermeersch, T.;
Zurek, E.; Hutchison, G. R. J. Cheminf. 2012, 4, 17.
J
DOI: 10.1021/acs.organomet.8b00153
Organometallics XXXX, XXX, XXX−XXX