Imine-N-Heterocyclic Carbenes as Versatile Ligands in Ruthenium(II) p-Cymene Anticancer Complexes: A Structure–Activity Relationship Study

Article abstract of DOI:10.1002/asia.201801058

A family of novel imine-N-heterocyclic carbene ruthenium(II) complexes of the general formula [(η⁶-p-cymene)Ru(C^N)Cl]PF₆^? (where C^N is an imine-N-heterocyclic carbene chelating ligand with varying substituents) have been prepared and characterized. In this imine-N-heterocyclic carbene chelating ligand framework, there are three potential sites that can be modified, which distinguishes this class of ligand and provides a body of flexibilities and opportunities to tune the cytotoxicity of these ruthenium(II) complexes. The influence of substituent effects of three tunable domains on the anticancer activity and catalytic ability in converting coenzyme NADH to NAD⁺ is investigated. This family of complexes displays an exceedingly distinct anticancer activity against A549 cancer cells, despite their close structural similarity. Complex 9 shows the highest anticancer activity in this series against A549 cancer cells (IC₅₀=14.36 μm), with an approximately 1.5-fold better activity than the clinical platinum drug cisplatin (IC₅₀=21.30 μm) in A549 cancer cells. Mechanistic studies reveal that complex 9 mediates cell death mainly through cell stress, including cell cycle arrest, inducing apoptosis, increasing intracellular reactive oxygen species (ROS) levels, and depolarization of the mitochondrial membrane potential (MMP). Furthermore, lysosomal damage is also detected by confocal microscopy.

Full text of DOI:10.1002/asia.201801058

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Imine-N-Heterocyclic Carbene as Versatile Ligands in
Ruthenium(II) p-Cymene Anticancer Complexes: A Structure-
Activity Relationship Study
Authors: Yuliang Yang, Guo Lihua, Zhenzhen Tian, Xicheng Liu,
Yuteng Gong, Hongmei Zheng, Xingxing Ge, and Zhe Liu
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10.1002/asia.201801058
Chemistry - An Asian Journal
Imine-N-Heterocyclic
Carbene
as
Versatile
Ligands
in
Ruthenium(II) p-Cymene Anticancer Complexes: A Structure-Activity
Relationship Study
Yuliang Yang, Lihua Guo,* Zhenzhen Tian, Xicheng Liu, Yuteng Gong, Hongmei Zheng, Xingxing
Ge, Zhe Liu*
Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of
Life-Organic Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural
Medicine, Department of Chemistry and Chemical Engineering, Qufu Normal University, Qufu
273165, China.
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Abstract: A family of novel imine-N-heterocyclic carbene ruthenium(II) complexes of the general
-
formula [(η6-p-cymene)Ru(C^N)Cl]PF6 (where C^N is imine-N-heterocyclic carbene chelating
ligand with varying substituents) have been prepared and characterized. In this imine-N-heterocyclic
carbene chelating ligand framework, there are three potential sites that can be modified, which
distinguishes this class of ligand and provides a body of flexibilities and opportunities to tune the
cytotoxicity of these ruthenium(II) complexes. The influence of substituents effects of three tunable
domains on anticancer activity and catalytic ability in converting coenzyme NADH to NAD+ is
investigated. This family of complexes displays an exceedingly distinct anticancer activity against
A549 cancer cells, despite their close structural similarity. Complex 9 shows the highest anticancer
activity in this series against A549 cancer cells (IC50 = 14.36 μM), whose has ca. 1.5-fold better
activity than the clinical platinum drug cisplatin (IC50 = 21.30 μM) in A549 cancer cells. Mechanistic
studies reveal that complex 9 mediates cell death mainly through cell stress, including cell cycle
arrest, inducing apoptosis, increasing intracellular reactive oxygen species (ROS) levels and
depolarization of the mitochondrial membrane potential (MMP). Furthermore, lysosomal damage is
also detected by confocal microscopy.
Keywords: imine-N-heterocyclic carbene; ruthenium(II) complexes; anticancer; structure-activity
relationship
Introduction
Although the research of anticancer agents has achieved remarkable progress over the past five
decades, cancer remains one of the leading causes of death worldwide.[1] Given the rapid increase in
cancer cases worldwide, the development of novel anticancer drugs with high performance and low
toxicity has become an indispensable need. Organometallic complexes have found wide applications
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in various fields, particularly as catalysts[2] and anticancer agents.[3] At present, the most effective
and well-studied class of metal-based anticancer agents, cisplatin and its derivatives, have been
successfully applied in clinical treatment and shown high efficacy against lung, ovarian, neck,
esophageal and head cancers.[4] However, the undesirable side effects and easily acquired drug
resistance of cisplatin still hindered its clinical applications and future development.[5] Therefore, the
obtaining of new metal-based anticancer drugs, which could broader spectrum of treatable cancers,
diminish side effects, and overcome platinum resistance, has attracted tremendous attention.[6]
Scheme 1. The structure of relevant ruthenium(II) complexes and our current work.
Ruthenium complexes have great potential as anticancer agents, as they are usually less toxic than
cisplatin and hence better tolerated in vivo.[7] For example, a host of ruthenium(II) p-cymene
complexes containing a wide range of ligands including α-diimine ligands, imine-pyridine ligands
and pyridine-NHC ligands has been developed with the aim of improving their anticancer properties
(Scheme 1).[8] Marchetti et al. developed a series of water-soluble ruthenium(II) p-cymene
complexes containing different α-diimine ligands (I, Scheme 1).[8a] The cytotoxicity of these
complexes strongly depended on the nature of the α-diimine N-substituents resulting. Our group has
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designed a kind of half-sandwich iridium(III) and ruthenium(II) complexes with imine-pyridyl
chelating ligands and achieved good selectivity and cytotoxicity (II, Scheme 1).[8b,8c] Overall, their
metal ions and bidentate ligands around the metal mainly determined the cancer cell cytotoxicity and
selectivity of these complexes. More recently, Hartinger et al. reported some pyridyl-NHC p-cymene
ruthenium(II) anticancer complexes (III, Scheme 1)[8d] and investigated their anticancer properties
and reactions with biomolecules. In this system, introduction of varying substituents gave complexes
with different properties, including stability in aqueous solution, reactivity toward biomolecules, in
vitro cytotoxicity and cellular uptake. These results prompted us to further investigate the effect of
varying substituents of imine-N-heterocyclic carbene chelating ligands on chemical and biological
reactivity of the ruthenium(II) complexes.
Herein, a series of structurally similar half-sandwich ruthenium(II) complexes bearing versatile
imine-N-heterocyclic carbene ligands have been synthesized and systematically investigated for their
chemical reactivity, biological reactivity, catalytic ability in transfer hydrogenation converting
coenzyme NADH into NAD+ and in vitro cytotoxicity against A549 cancer cells. To the best of our
knowledge, this form of imine-N-heterocyclic carbene half-sandwich ruthenium(II) complexes seems
to be first time used as anticancer agents. The effects of the substituents on anticancer activity and
catalytic ability in converting coenzyme NADH to NAD+ of the complexes were fully investigated.
This family of half-sandwich ruthenium(II) complexes exhibits uncommonly different in vitro
cytotoxicity, which is significantly correlated with the structure of the ligands. Initial cell death
mechanistic insights, including cell cycle, apoptosis induction, mitochondrial membrane potential,
ROS level and lysosomal damage, are also discussed. All these studies point out that these novel
ruthenium(II) organometallic complexes possess a variety of interesting biological effects that make
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them attractive as a potentially promising candidate for the development of anticancer agents.
Results and Discussion
Synthesis and characterization
-
Scheme 2. Synthetic routes for imine-N-heterocyclic carbene ligands L1-L10 and [(η6-p-cymene)Ru(C^N)Cl]PF6
complexes 1-10.
The imine-N-heterocyclic carbene ligands (L1-L10) and novel ruthenium(II) complexes (1-10)
were prepared according to well-established procedures, depicted in Scheme 2. A series of versatile
imine-N-heterocyclic carbene ligands were synthesized via a coupling reaction of the corresponding
imidoyl chlorides with N-substituted imidazole. Imine-N-heterocyclic carbene silver complexes were
usually used as effective transfer reagents to obtain other transition-metal complexes.[9] Novel
ruthenium(II) complexes 1-10 were synthesized in high yields (60-87%) from the corresponding
(NHC)AgCl complex with [(η6-p-cymene)RuCl2]2 by stirring at ambient temperature overnight, and
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-
the corresponding ruthenium(II) complexes were isolated as PF6 salts. All the synthesized
1
ruthenium(II) complexes were fully characterized by H and 13C NMR spectroscopy (Figures
S1-S36), CHN elemental analysis, and mass spectrometry (Figures S37-S54). The characteristic
peaks for the central imidazolium carbon of these complexes were at δ 186.34-192.68 ppm in the 13C
NMR spectra. Additionally, the molecular structure of complex 9 was unambiguously confirmed by
the X-ray crystallographic study (Figure 1 and Tables S1-S2).
-
Figure 1. X-ray crystal structures of compound of [(η6-p-cymene)Ru(L9)Cl]PF6 (9) with the thermal ellipsoids
-
drawn at the 50% probability level. The hydrogen atoms and PF6 counterions have been omitted for clarity.
Selected bond lengths (Å) and angles (deg): Ru-C (centroid) = 1.7398, Ru-C1 = 2.022(6), Ru-N3 = 2.132(5),
Ru-Cl1 = 2.4026(16), C1-Ru-N3 = 76.0(2), C1-Ru-Cl1 = 78.6(2), N3-Ru-Cl1=87.53(14).
X-ray crystal structures
Single crystal suitable for X-ray diffraction analysis was obtained from the slow diffusion of
petroleum ether into a nearly saturated solution of complex 9 in CH2Cl2/ethyl acetate. Their
structures and atom numbering schemes are shown in Figure 1. Crystallographic data are shown in
Table S1, and selected bond lengths and angles are listed in Table S2. As shown in Figure 1, complex
9 adopts the expected half-sandwich pseudo octahedral three-legged piano-stool arrangement, and
hence, the arene ring displays the common π-bonded η6-coordination mode, whereas the
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imine-N-heterocyclic carbene-type ligand assumes a bidentate chelate coordination mode (κ2-C,N).
The two rings of five-membered chelate ring and the imidazole ring are approximately coplanar. The
Ru-Cl bond distance is 2.4026(16) Å. The Ru-η6-p-cymene ligand (centroid) distance is 1.7398 Å.
The orthometalated Ru-C distance [Ru-C = 2.022(6) Å] is shorter than Ru-N [Ru-N = 2.132(5) Å].
Stability studies.
For the investigation of aqueous stability, studies were conducted by 1H NMR spectroscopy at 310
K for complexes 1-10, which were dissolved in 30% DMSO-d6/70% D2O (v/v). The presence of
DMSO-d6 ensured the solubility of the complex. As shown in Figures S55-S64, no additional peaks
were observed in the 1H NMR spectra after 24 h. In addition, the 35Cl NMR spectra for complexes 4
and 9, recorded on solutions in 60% DMSO-d6/40% D2O (v/v) after 24 h, showed no evidence of
free Cl- ions (Figures S65-S66). It should be noted that some previously reported half-sandwich
metal complexes may undergo Cl-/H2O exchange more easily in diluted solutions with a higher
relative content of water, as is the case of cell culture.[10] As a result, complex 9 was added in water
and stirred at 310 K for 24 h. Subsequently, the solid sample of complex 9 was recovered and
dissolved in CDCl3 to repeat the 1H NMR spectra (Figure S67). There was also no change in the 1H
NMR spectra, which indicated that the hydrolysis also did not occur when high content of water was
employed. Complex 9 was also monitored by UV/vis spectroscopy in 5% MeOH/95% H2O (v/v)
solution (Figure S68) in order to further estimate the stability of these complexes. The results
obtained by UV/vis spectroscopy in high content of water were consistent with the NMR analysis.
Overall, the stability studies suggest that the complexes have sufficient stability for the preparation
of samples for biological assays.
The structure-activity relationship study
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Figure 2. Inhibition of the growth of A549 cells by complexes 1-10 and cisplatin.
The in vitro cytotoxicity of the ligands L1-L10, complexes 1-10 and cisplatin against A549 cancer
cells was examined after 24 exposure period using the
a
h
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The widely used
clinical platinum drug cisplatin was included as a control. All of the imine-N-heterocyclic carbene
ligands showed very low cytotoxicity against A549 cells (>100 µM ) and they were thus deemed as
inactive. On the other hand, although these ruthenium(II) complexes possessed structurally
analogous, they had different anticancer activities. Subtle structural change in ligand substituents can
dramatically alter the cytotoxic activities of these complexes. As depicted in Table 1 and Figure 2,
complexes 1, 2, 3, 4 and 6 were inactive (>100 µM). However, other five complexes displayed
anticancer activity toward A549 cancer cells comparable to or even higher than cisplatin. Notably,
complex 9, the most cytotoxic one against the A549 cancer cells, exhibited approximately 1.5-fold
greater activity than cisplatin. Overall, the in vitro anticancer activity of the test complexes was in
the following order: 9 > cisplatin > 8 > 7 > 10 > 5.
Substituents perturbations in this system can result in significant variation of anticancer activities.
First, the length of the alkyl substitutions on the imidazole ring showed a significant effect on the
cytotoxicity of complexes. When tether length on the imidazole ring increased from methyl- to butyl-
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Table 1. Inhibition of the growth of A549 cancer cells by ligands L1-L10, complexes 1-10 and Cisplatin.a
Ligand IC50 (µM )
Complex
IC50 (µM )
>100
L1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
[(η6-p-cymene)Ru(L1)Cl]PF6 (1)
[(η6-p-cymene)Ru(L2)Cl]PF6 (2)
[(η6-p-cymene Ru(L3)Cl]PF6 (3)
[(η6-p-cymene)Ru(L4)Cl]PF6 (4)
[(η6-p-cymene)Ru(L5)Cl]PF6 (5)
[(η6-p-cymene)Ru(L6)Cl]PF6 (6)
[(η6-p-cymene)Ru(L7)Cl]PF6 (7)
[(η6-p-cymene)Ru(L8)Cl]PF6 (8)
[(η6-p-cymene)Ru(L9)Cl]PF6 (9)
L2
L3
L4
L5
L6
L7
L8
L9
L10
>100
>100
>100
52.16 ± 2.4
>100
38.33 ± 2.3
25.21 ± 1.1
14.36 ± 2.4
[(η6-p-cymene)Ru(L10)Cl]PF6 (10) 46.80 ± 3.7
Cisplatin 21.30 ± 1.7
aIC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations
and cell viability is assessed after 24 h of incubation.
group, the complexes exhibited increased cytotoxicity (complex 6: >100 µM vs complex 9: 14.36
µM). The cytotoxicity followed a rule in the order of butyl- > isopropyl- > ethyl- >
methyl-substituted NHCs. It seems reasonable that, in accordance with the behavior of some
previously reported potent anticancer agents,[11] the increased length of the alkyl substituents may
increase the lipophilicity of these complexes and thus lead to the enhanced cytotoxicity. Next,
maintaining the imidazole ring substituent unchanged and increasing the size of ortho-substituents in
the aniline gradually, the cytotoxicity also increased and followed a rule in the order of isopropyl- >
methyl- > H-substituted aniline. For example, complexes 7, 8, 9 and 10, whose IC50 values were
38.33, 25.21, 14.36 and 46.8 μM, respectively, exhibited in vitro anticancer activity significantly
superior to complexes 2 (>100 μM), 3 (>100 μM), 4 (>100 μM) and 5 (52.16 μM) containing the
same substituents (R1 and R2) against A549 cancer cell lines. Finally, the influence of the substituent
at the imine carbon on anticancer activity of this class of complexes was further investigated.
Replacement of the methyl group on the imine carbon by a more lipophilic phenyl ring lead to
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enhanced anticancer efficacy (complex 3: IC50 > 100 μM vs complex 10: IC50 = 46.8 μM). Pervious
work has shown that the lipophilicity and cytotoxicity of the similar half-sandwich C,N and
N,N-chelating iridium(III) complexes increased by the incorporation of phenyl substituents on
η5-C5Me5.[12] In this system, anticancer activity of such complexes could also be tuned by regulating
the lipophilicity of the substituents on the imine carbon. Overall, small structural changes on three
positions of chelating ligand of these complexes can significantly alter their biological properties.
These results may guide the development of the structure-activity relationships (SARs) for
ruthenium-based anticancer agents and provide a rational strategy for improving their toxicological
properties.
Interaction with nucleobases
The binding studies of anticancer metallodrugs with model nucleobase 9-ethylguanine (9-EtG) and
9-methyladenine (9-MeA) provided insights into their intracellular fate.[13] The reactions of complex
9 with model nucleobase 9-EtG or 9-MeA were monitored using the 1H NMR technique. Solutions of
complex 9 (ca. 1 mM) and 2.0 molar equiv of 9-EtG or 9-MeA in 50% CD3OD-d4/50% D2O (v/v)
1
were prepared, respectively, and H NMR spectra were recorded at different time intervals at 310 K.
1
1
According to H NMR spectra at different time intervals, no additional H NMR peaks were
observed over a period of 24 h (Figures S69-S70). These results suggested that no reaction with
model nucleobase occurred for complex 9. Also, the formation of nucleobase adducts by these
ruthenium(II) complexes were not detected by mass spectrometry. Thus, DNA may not be the major
target for this type of ruthenium(II) complexes.
Reaction with NADH
Coenzyme NADH and NAD+ play a key role in numerous bio-catalyzed processes. Our previous
work has reported that half-sandwich iridium(III) and ruthenium(II) anticancer complexes can
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oxidize NADH to generate reactive oxygen species (ROS) H2O2, which provided a pathway to an
oxidant mechanism of action.[8b 14] To investigate the impact of this family of complex containing
,
three modifiable potential sites on the catalytic ability, the reactions of complexes 3, 5, 8, 9 and 10
(ca. 1 µM) with NADH (100 µM) in 5% MeOH/95% H2O (v/v) were monitored employing
ultraviolet-visible (UV-vis) spectrophotometer at 298 K (Figure 3a and Figure S71). The conversion
of NADH to NAD+ was detected by measuring the number of UV absorption at 339 nm using a
spectrophotometer, as NADH exhibits an absorption peak at 339 nm while NAD+ does not. The
turnover numbers (TONs) of complexes 3 (6.0), 5 (8.1), 8 (13.1), 9 (8.5) and 10 (7.8) were calculated
by measuring the absorption difference at 339 nm (Figure 3b). The size of ortho-substituents on the
aniline moiety, the substituent perturbations on the imine carbon and the length of the alkyl
substitutions on the imidazole ring appear to exhibit little variation on the catalytic activity of this
class of complexes.
Figure 3. (a) UV-vis spectra of the reaction of NADH (100 µM) with complex 8 (1 µM) in 5% MeOH/95% H2O
(v:v) at 298 K for 8 h; (b) The turnover numbers (TONs) of complexes 3, 5, 8, 9 and 10.
Cell cycle arrest
As most metal-based chemotherapeutic agents could disrupt the regulated cell cycle distribution,
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the effect of the most active complex 9 on cell cycle perturbation by flow cytometry analysis in A549
cancer cells was examined. As shown in Figure 4, Figure S72 and Table S3, after treatment of A549
cells with complex 9 at 0.5, 1 and 2 equipotent concentrations of IC50 for 24 h, the percentage of
cells at the S phase increased markedly from 22.52% to 33.25%. Meanwhile, the percentage of the
cells at the G0/G1 phase decreased from 64.00% to 46.82%. The cells at the G2/M phase only slightly
changed. These results indicated that complex 9 can induce perturbations of cell-cycle progression
and effectively stall cells in the S phase of cell cycle in a dose-dependent manner.
Figure 4. Flow cytometry data for cell cycle distribution of A549 cancer cells exposed to complex 9 for 24 h.
Concentrations used were 0.5, 1 and 2 equipotent concentrations of IC50. Cell staining for flow cytometry was
carried out using PI/RNase. Data are quoted as mean ± SD of three replicates.
Apoptosis assay.
To assess whether these ruthenium(II) complexes induce A549 cells death by apoptosis or necrosis,
A549 cells were dual-stained with annexin V-FITC/propidium iodide (PI) reagents and analyzed by
flow cytometry. As shown in Figure 5, Figure S73 and Table S4, upon incubating A549 cells with
complex 9 at 0.5, 1, 2 and 3 equipotent concentrations of IC50 for 24 h, a dose-dependent apoptosis
population was detected. At a maximum concentration (3 × IC50), a total of 82.15% (early apoptotic
+ late apoptotic) of cells were undergoing enhanced apoptosis compared with the untreated group
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(6.21%). In addition, no strikingly increased necrotic population was detected. These results
suggested that complex 9 can induce A549 cells death via a high incidence of apoptosis, and cell
necrotic was not responsible for A549 cells death.
Figure 5. Apoptosis analysis of A549 cells after 24 h of exposure to complex 9 at 310 K determined by flow
cytometry using annexin V-FITC vs PI staining. Populations for cells in four stages treated by complex 9. Data are
quoted as mean ± SD of three replicates.
Figure 6. Changes in mitochondrial membrane potential of A549 cancer cells induced by complex 9.
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Mitochondrial membrane potential (MMP)
The change of mitochondrial membrane potential (MMP, Δψm), which is a significant indicator of
cell health, were detected by 5,5',6,6'-tetrachloro-1,1'-3,3'-tetraethyl-benzimidazolyl carbocyanine
iodide (JC-1) staining and analyzed using flow cytometry. JC-1 can be aggregated in mitochondria in
a potential-dependent manner indicated by a marked red-to-green color shift.[15] Treatment of A549
cells with complex 9 resulted in a dose-dependent increase in the red fluorescence and decrease in
green fluorescence of JC-1 (Figure 6 and Table S5). After 24 h treatment, the percentage of cells
with mitochondrial membrane depolarization was 71.35% at a concentration of 2 × IC50, being
elevated from the vehicle-treated group (10.12%). In addition, representative JC-1 red/green ratio
signals were shown in Figure S74 and Table S6. Treatment of A549 cells with complex 9 at
concentrations of 2 × IC50 values significantly decreased the JC-1 red/green fluorescence ratios
(control: 8.94 ± 1.1; 2 × IC50: 0.40 ± 0.1). This observation suggested that complex 9 can cause
cancer cell death through the dysfunction of the mitochondrial membrane potential.
Figure 7. Analysis of ROS levels by flow cytometry after A549 cells were treated with complex 9 at the 0.25 and
0.5 equipotent concentrations of IC50 for 24 h and stained with H2DCFDA.
ROS determination.
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Perturbation of mitochondrial functions, such as the reduction of mitochondrial membrane
potential (MMP), may result in over-generation of intracellular reactive oxygen species (ROS).[16]
The impact of complex 9 on the number of intracellular ROS was quantified by flow cytometry with
2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The non-fluorescent H2DCFDA can
be converted to the highly bright 2',7'-dichlorofluorescein (DCF) by intracellular ROS.[17] Compared
with the untreated group, a dose-dependent manner in the DCF fluorescence signals was observed
after a treatment period 24 h with test agent at concentrations of 0.25 × IC50 and 0.5 × IC50 (Figure 7
and Figure S75), indicating that complex 9 can result in disruption of mitochondrial function via
production of reactive oxygen species (ROS).
Figure 8. Observation of lysosomal disruption in A549 cells loaded with complex 9 for 6 h at 37 ºC, then stained
with acridine orange (AO) (5 µM) at 37 °C for 15 min. Emission was collected at 510 ± 20 nm (green) and 625 ±
20 nm (red) upon excitation at 488 nm. Scale bar: 20 µm. The cells were treated with (a) only acridine orange (AO);
(b) acridine orange (AO) and complex 9 (1× IC50); (c) acridine orange (AO) and complex 9 (3× IC50).
Lysosomal damage.
Lysosomes play important roles in many physiological processes and cell signaling pathways.
Acridine orange (AO) as an integrity indicator can be used to evaluate the dysfunction of lysosomes
at subcellular level.[18] AO is a useful probe employed to assess the lysosomal functional state at
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subcellular level, because it emits a concentration-dependent red/green fluorescence.[19] As shown in
Figure 8, A549 cells only treated with acridine orange (AO) (5 µM) displayed distinct red
fluorescence in lysosomes, indicating that the lysosomes of A549 cells under such conditions were
intact. However, as the agent concentration increased, the red fluorescence of AO gradually
decreased, indicating that lysosomal integrity was jeopardized via treatment of complex 9. Thus,
complex 9 can induce cell death through lysosomal damage.
Conclusions
In summary, a series of versatile half-sandwich ruthenium(II) p-cymene complexes, which
contained different imine-N-heterocyclic carbene ligands, were explored as promising anticancer
agents. The stability studies revealed that these complexes had sufficient stability for the preparation
of samples for biological assays. This class of ruthenium(II) p-cymene complexes showed anticancer
activity comparable to or even higher than cisplatin toward A549 cancer cells. The structure-activity
relationship study revealed that the longer length of the alkyl substitutions on the imidazole ring, the
larger size of ortho-substituents in the aniline and the more lipophilic substituent on the imine carbon
resulted in the higher anticancer activity of these ruthenium(II) complexes.
No nucleobase binding was detected for complex 9, suggesting that DNA may not be a possible
target. This type of complexes was effective catalysts in transfer hydrogenation converting coenzyme
NADH into NAD+. The effect of substituents in the three positions of the ligands on catalytic ability
seemed to be insignificant. Further mechanistic studies showed that complex 9 can trigger the arrest
of cell growth at S phase and efficiently induce early- and late-stage apoptosis in A549 cells.
Simultaneously, depolarization of the mitochondrial membrane potential (MMP) and the increase
intracellular levels of the ROS were also observed. Interestingly, lysosomal damage was detected in
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A549 cancer cells by confocal microscopy, suggesting that these complexes may mediate cell death
through lysosomal damage.
Experimental Section
General information
RuCl3·nH2O, α-terpinene, 1-methylimidazole, 1-ethylimidazole, 1-isopropylimidazole,
1-butylimidazole,
acetyl
chloride,
benzoyl
chloride,
aniline,
2,6-dimethylaniline,
2,6-diisopropylaniline, triphosgene, thionyl dichloride, silver(I) oxide, 9-ethylguanine and
9-methyladenine were purchased from Sigma-Aldrich and used directly as such. Tetrahydrofuran
was dried over sodium/benzophenone for 24 h, and dichloromethane was dried over phosphorus
pentoxide for 8 h before being used. [(η6-p-cymene)RuCl2]2 was prepared using literature
procedure.[20]
The
intermediate
products
N-(2,6-diisopropylphenyl)acetamide,
N-(2,6-dimethylphenyl)benzamide,
N-(2,6-dimethylphenyl)acetamide,
N-phenylbenz-amide,
N-(2,6-dimethylphenyl)-acetimidoyl chloride, N-(2,6-diisopropylphenyl)-acetimidoyl chloride,
N-(2,6-dimethylphenyl)benzenecarbox-imidoyl chloride, N-phenylbenzenecarboximidoyl chloride
were prepared according to previously reported procedure.[21] The imine-N-heterocyclic carbene
ligands L1-L4[21a], L5[21b-e], L6-L9[21a] and L10[21b-e] were prepared according to slightly modified
procedure reported previously. For the biological experiments, DMEM, fetal bovineserum,
penicillin/streptomycin mixture, trypsin/EDTA, phosphate-buffered saline (PBS), MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), PI (propidium iodide), JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolyl carbocyanine iodide), and H2DCFDA
(2',7'-dichlorodihydrofluorescein diacetate) were purchased from Sangon Biotech. Tested complexes
were dissolved in DMSO just before the experiments, and the concentration of DMSO was 1% (v/v).
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13
1H and C NMR spectra were recorded in 5 mm NMR tubes an ambient temperature on a Bruker
Avance 500 spectrometer or a Bruker Ascend 400 spectrometer using TMS as an internal standard
and CDCl3 or DMSO as solvent. Mass spectra of the L2-L5 and L7-L10 were recorded on a Thermo
LTQ Orbitrap XL (ESI+). Mass spectra of the complexes 1-10 were recorded on a Atouflex Speed
MALDI-TOF MS. Microanalysis (C, H, and N) was carried out using a Carlo Erba model EA 1108
microanalyzer. X-ray Diffraction data were collected at 298(2) K on a Bruker Smart CCD area
detector with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The supplementary
crystallographic data for this paper has been deposited with the Cambridge Crystallographic Data
Centre as CCDC-1848902.
Synthesis and characterizations
Synthesis of the ligands
General method: the limidazole with varying substituents was added dropwise to a solution of the
corresponding iminochloride in dry THF (20 mL) over a period of 5 min. The mixture was stirred at
ambient temperature for 20 h (L1-L4 and L6-L9) or 4 day (L5 and L10), and the product slowly
precipitated as a white solid (L1-L4 and L6-L9) or yellow solid (L5 and L10). The crude product
was obtained by filtration, washed with dry THF (3 × 15 mL), and dried under reduced pressure.
1
The H NMR showed the presence of two geometric isomers (E/Z), which is similar with the
compound reported previously.[21c]
[3-Me-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+Cl- (L1) 1-methylimidazole (0.86 g, 10.47 mmol), the
N-(2,6-dimethylphenyl)-acetimidoyl chloride (1.89 g, 10.40 mmol), Yield: 82% (2.26 g, 8.57 mmol).
This ligand is previously known.[21a]
[3-Et-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+Cl- (L2) 1-ethylimidazole (0.69 g, 7.20 mmol), the
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N-(2,6-dimethylphenyl)-acetimidoyl chloride (1.31 g, 7.21 mmol), Yield: 92% (1.83 g, 6.59 mmol).
1H NMR (400 MHz, CDCl3) isomer 1: isomer 2 = 1:0.37 (molar ratio). isomer 1: δ 11.82 (s, 1H,
NCHN), 8.24 (s, 1H, imidazole-H), 7.49 (s, 1H, imidazole-H), 7.09 (m, 3H, Ar-H), 4.67 (q, J = 7.3
Hz, 2H, N-CH2Me), 2.55 (s, 3H, imine-CH3), 2.02 (s, 6H, o-Ar-CH3), 1.70 (t, J = 7.3 Hz, 3H,
N-CH2CH3). isomer 2: δ 9.48 (s, 1H, NCHN), 7.38 (s, 1H, imidazole-H), 7.19 (s, 1H, imidazole-H),
7.04-7.00 (m, 3H, Ar-H), 4.37 (q, J = 7.3 Hz, 2H, N-CH2Me), 2.28 (s, 3H, imine-CH3), 2.24 (s, 6H,
o-Ar-CH3), 1.58 (t, J = 7.3 Hz, 3H, N-CH2CH3). 13C NMR (101 MHz, CDCl3) δ 173.16, 169.33,
143.19, 135.76 (NCHN), 134.65 (NCHN), 134.46, 128.56, 128.35, 127.99, 127.13, 126.14, 124.83,
122.18, 120.38, 119.94, 117.97, 45.98, 44.69, 23.08, 18.46, 17.98, 16.47, 15.64, 15.55. ESI-MS
(m/z):
calcd
for
C15H20N3:
242.16572,
found:
242.16255,
[3-Et-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+.
[3-iPr-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+Cl- (L3) 1-isopropylimidazole (0.43 g, 3.90 mmol),
the N-(2,6-dimethylphenyl)-acetimidoyl chloride (0.70 g, 3.85 mmol), Yield: 85% (0.96 g, 3.29
mmol). 1H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:0.81 (molar ratio). isomer 1: δ 11.95 (s,
1H, NCHN), 8.25 (s, 1H, imidazole-H), 7.45 (s, 1H, imidazole-H), 7.08 (d, J = 3.4 Hz, 2H, Ar-H),
i
7.04 – 6.98 (m, 1H, Ar-H), 5.34 – 5.23 (m, 1H, Pr-CH3), 2.59 (s, 3H, imine-CH3), 2.03 (s, 6H,
o-Ar-CH3), 1.72 (d, J = 6.5 Hz, 6H, N-iPr-CH3). isomer 2: δ 9.37 (s, 1H, NCHN), 7.38 (s, 1H,
i
imidazole-H), 7.21 (s, 1H, imidazole-H), 7.18-7.05 (m, 3H, Ar-H), 4.84 – 4.76 (m, 1H, Pr-CH3),
2.28 (s, 3H, imine-CH3), 2.24 (s, 6H, o-Ar-CH3), 1.61 (d, J = 6.5 Hz, 6H, N-iPr-CH3). 13C NMR (101
MHz, CDCl3) δ 173.16, 169.25, 143.24, 137.29, 135.76 (NCHN), 133.72 (NCHN), 128.57, 128.35,
128.02, 127.16, 126.18, 124.81, 120.12, 119.83, 118.46, 118.15, 54.24, 52.62, 23.18, 23.01, 18.47,
18.44, 18.00, 16.64. ESI-MS (m/z): calcd for C16H22N3: 256.18137, found: 256.17816, [3-iPr-1-(2,6-
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dimethylphenyl)iminyl-C3H3N2]+.
[3-nBu-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+Cl- (L4) 1-butylimidazole (0.48 g, 3.87 mmol), the
N-(2,6-dimethylphenyl)-acetimidoyl chloride (0.70 g, 3.85 mmol), Yield: 79% (0.93 g, 3.04 mmol).
1H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:0.52 (molar ratio). isomer 1: δ 12.02 (s, 1H,
NCHN), 8.23 (s, 1H, imidazole-H), 7.39 (s, 1H, imidazole-H), 7.07 (d, J = 9.4 Hz, 2H, Ar-H), 7.05 –
n
6.97 (m, 1H, Ar-H), 4.59 (t, J = 6.3 Hz, 2H, Bu-CH2), 2.55 (s, 3H, imine-CH3), 2.02 (s, 6H,
o-Ar-CH3), 2.07 – 1.97 (m, 2H, nBu-CH2), 1.50 – 1.46 (dq, J = 14.3, 7.2 Hz, 2H, nBu-CH2), 1.01 (t, J
= 7.2 Hz, 3H, nBu-CH3). isomer 2: δ 9.38 (s, 1H, NCHN), 7.37 (s, 1H, imidazole-H), 7.12 – 7.18 (m,
2H, imidazole-H and Ar-H), 7.04 – 7.01 (d, J = 9.4 Hz, 2H, Ar-H), 4.29 (t, J = 6.3 Hz, 2H, nBu-CH2),
2.28 (s, 3H, imine-CH3), 2.24 (s, 6H, o-Ar-CH3), 1.86 – 1.89 (m, 2H, nBu-CH2), 1.41 – 1.33 (dq, J =
n
n
13
14.3, 7.2 Hz, 2H, Bu-CH2), 0.97 (t, J = 7.2 Hz, 3H, Bu-CH3). C NMR (101 MHz, CDCl3)
δ,173.13, 169.35, 143.18, 135.78 (NCHN), 134.92 (NCHN), 134.53, 128.56, 128.36, 127.98, 127.08,
126.14, 124.84, 122.44, 120.67, 119.87, 117.88, 50.46, 49.34, 32.20, 32.06, 23.08, 19.53, 19.39,
18.48, 18.00, 16.53, 13.48, 13.38. ESI-MS (m/z): calcd for C17H24N3: 270.19702, found: 270.19351,
[3-nBu-1-(2,6-dimethylphenyl)iminyl-C3H3N2]+.
[3-iPr-1-{C(C6H5)N(C6H5)}C3H3N2]+Cl- (L5) 1-isopropylimidazole (1.14 g, 10.35 mmol), the
1
N-phenylbenzenecarboximidoyl chloride (2.23 g, 10.34 mmol), Yield: 85% (2.88 g, 8.84 mmol). H
NMR (500 MHz, CDCl3) isomer 1: isomer 2 = 1:0.78 (molar ratio). isomer 1: δ 9.14 (s, 1H, NCHN),
7.89 (m, 2H, imidazole-H), 7.44 (m, 1H, Ar-H), 7.40 (m, 1H, Ar-H), 7.30 (t, 2H, Ar-H), 7.21 – 7.19
(d, J = 2.7 Hz, 3H, Ar-H), 6.93(d, J = 2.7 Hz, 3H, Ar-H), 4.79 – 4.68 (m, 1H, iPr-CH3), 1.62 (d, J =
6.6 Hz, 6H, N-iPr-CH3). isomer 2: δ 12.91 (s, 1H, NCHN), 7.67 (d, J = 6.0 Hz, 2H, imidazole-H),
7.58-7.52 (m, 1H, Ar-H), 7.51 – 7.48 (m, 2H, Ar-H), 7.39 – 7.36 (m, 2H, Ar-H), 7.19 – 7.14 (m, 5H,
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i
Ar-H), 3.80 – 3.68 (m, 1H, Pr-CH3), 1.73 (d, J = 6.6 Hz, 6H, N-iPr-CH3). 13C NMR (101 MHz,
CDCl3) δ 171.05, 165.90, 138.18, 134.92 (NCHN), 133.85 (NCHN), 132.06, 131.78, 129.83, 129.52,
129.27, 129.04, 128.99, 128.70, 127.40, 127.28, 125.62, 124.95, 124.44, 121.37, 120.49, 120.17,
118.22, 54.19, 52.64, 23.18, 22.88. ESI-MS(m/z): calcd for C19H20N3: 290.16572, found: 290.16464,
[3-iPr-1-{C(C6H5)N(C6H5)}C3H3N2]+.
[3-Me-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+Cl- (L6) 1-methylimidazole (0.28 g, 3.41 mmol),
the N-(2,6-diisopropylphenyl)-acetimidoyl chloride (0.79 g, 3.32 mmol), Yield: 61% (0.65 g, 2.03
mmol). This ligand is previously known.[21a]
[3-Et-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+Cl- (L7) 1-ethylimidazole (0.32 g, 3.33 mmol), the
N-(2,6-diisopropylphenyl)-acetimidoyl chloride (0.80 g, 3.36 mmol), Yield: 68% (0.75 g, 2.25
mmol). 1H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:0.68 (molar ratio). isomer 1: δ 11.97 (s,
1H, NCHN), 8.22 (s, 1H, imidazole-H), 7.57 (s, 1H, imidazole-H), 7.18 (s, 3H, Ar-H), 4.69 (q, 2H,
N-CH2Me), 2.68 – 2.59 (m, 2H, iPr-CH), 2.56 (s, 3H, imine-CH3), 1.71 (t, 3H, N-CH2CH3), 1.17 (d,
J = 6.8 Hz, 6H, iPr-CH3), 1.12 (d, J = 6.1 Hz, 6H, iPr-CH3). isomer 2: δ 9.55 (s, 1H, NCHN), 7.36 (s,
1H, imidazole-H), 7.23 (s, 1H, imidazole-H), 7.21 – 7.16 (m, 3H, Ar-H), 4.40 (q, 2H, N-CH2Me),
3.08 – 3.22 (m, 2H, iPr-CH), 2.26 (s, 3H, imine-CH3), 1.59 (t, 3H, N-CH2CH3), 1.25 – 1.19 (dd, J =
i
6.8 Hz, 12H, Pr-CH3). 13C NMR (101 MHz, CDCl3) δ 173.59, 170.05, 146.85, 146.49, 136.50
(NCHN), 134.87 (NCHN), 129.08, 128.32, 125.50, 123.92, 123.53, 123.41, 122.08, 120.21, 119.95,
117.78, 44.75, 28.75, 28.50, 24.41, 23.67, 23.21, 22.84, 15.72. ESI-MS (m/z): calcd for C19H28N3:
298.22832, found: 298.22723, [3-Et-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+.
[3-iPr-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+Cl- (L8) 1-isopropylimidazole (0.39 g, 3.54 mmol),
the N-(2,6-diisopropylphenyl)-acetimidoyl chloride (0.83 g, 3.49 mmol), Yield: 63% (0.77 g, 2.21
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mmol). 1H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:0.2 (molar ratio). isomer 1: δ 12.08 (s,
1H, NCHN), 8.23 (s, 1H, imidazole-H), 7.51 (s, 1H, imidazole-H), 7.18 (s, 3H, Ar-H), 5.41 – 5.29
(m, 1H, iPr-CH), 2.67 – 2.61 (m, 2H, iPr-CH), 2.61 (s, 3H, imine-CH3), 1.73 (d, J = 6.6 Hz, 6H,
iPr-CH3), 1.17 (d, J = 6.8 Hz, 6H, iPr-CH3), 1.12 (d, J = 6.8 Hz, 6H, iPr-CH3). isomer 2: δ 9.29 (s, 1H,
NCHN), 7.40 (s, 1H, imidazole-H), 7.24 (s, 1H, imidazole-H), 7.21 – 7.10 (s, 3H, Ar-H), 4.75 – 4.83
(m, 1H, iPr-CH), 3.22 – 3.07 (m, 2H, iPr-CH), 2.26 (s, 3H, imine-CH3), 1.61 (d, J = 6.6 Hz, 6H,
iPr-CH3), 1.25 – 1.19 (dd, J = 6.8 Hz, 12H, iPr-CH3, iPr-CH3). 13C NMR (101 MHz, CDCl3) δ 173.60,
170.15, 140.70, 137.51 (NCHN), 136.50 (NCHN), 133.69, 128.26, 125.46, 123.92, 123.50, 123.36,
120.10, 119.96, 118.47, 117.94, 54.25, 52.63, 28.72, 28.46, 24.41, 23.68, 23.21, 23.07, 22.82, 17.06.
ESI-MS
(m/z):
calcd
for
C20H30N3:
312.24397,
found:
312.24039,
[3-iPr-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+.
[3-nBu-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+Cl- (L9) 1-butylimidazole (0.42 g, 3.38 mmol), the
N-(2,6-diisopropylphenyl)-acetimidoyl chloride (0.80 g, 3.36 mmol), Yield: 49% (0.60 g, 1.66
mmol). 1H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:1.35 (molar ratio). isomer 1: δ 12.00 (s,
1H, NCHN), 8.21 (s, 1H, imidazole-H), 7.50 (s, 1H, imidazole-H), 7.17 – 7.19 (s, 3H, Ar-H), 4.61 (t,
J = 7.2 Hz, 2H, nBu-CH2), 2.68 – 2.58 (m, 2H, iPr-CH), 2.56 (s, 3H, imine-CH3), 2.08 – 1.98 (m, 2H,
nBu-CH2), 1.48 (dq, J = 15.0, 7.5 Hz, 2H, nBu-CH2), 1.16 (d, J = 6.6 Hz, 6H, iPr-CH3), 1.11 (d, J =
6.6 Hz, 6H, iPr-CH3), 1.03 – 0.99 (t, 3H, nBu-CH3). isomer 2: δ 9.48 (s, 1H, NCHN), 7.32 – 7.46 (s,
1H, imidazole-H), 7.29 (s, 1H, imidazole-H), 7.28 – 7.20 (s, 3H, Ar-H), 4.32 (t, J = 7.2 Hz, 2H,
nBu-CH2), 3.08 – 3.21 (m, 2H, iPr-CH), 2.26 (s, 3H, imine-CH3), 1.93 – 1.85 (m, 2H, nBu-CH2), 1.40
– 1.32 (dq, J = 15.0, 7.5 Hz, 2H, nBu-CH2),1.26 – 1.18 (m, 12H, iPr-CH3), 0.97 (t, 3H, nBu-CH3). 13C
NMR (101 MHz, CDCl3) δ 169.98, 146.86, 146.48, 136.51 (NCHN), 135.15 (NCHN), 131.48,
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129.08, 128.33, 125.51, 123.93, 123.53, 123.42, 120.47, 119.96, 117.68, 50.57, 49.40, 32.24, 28.76,
28.76, 28.50, 24.42, 23.67, 23.24, 22.74, 19.60, 19.42, 17.02, 13.40. ESI-MS(m/z): calcd for
C21H32N3: 326.25962, found: 326.25842, [3-nBu-1-(2,6-diisopropylphenyl)iminyl-C3H3N2]+.
[3-iPr-1-{C(C6H5)N(2,6-Me2C6H3)}C3H3N2]+Cl- (L10) 1-isopropylimidazole (1.05 g, 9.53 mmol),
the N-(2,6-dimethylphenyl)benzenecarboximidoyl chloride (2.12 g, 8.70 mmol), Yield: 72% (2.23 g,
1
6.30 mmol). H NMR (500 MHz, CDCl3), isomer 1: isomer 2 = 1:0.66 (molar ratio). isomer 1: δ
10.46 (s, 1H, NCHN), 7.94 (s, 2H, imidazole-H), 7.50 – 7.47 (m, 1H, Ar-H), 7.40 (q, J = 8.0 Hz, 4H,
Ar-H), 6.91 (dt, J = 8.9, 5.7 Hz, 3H, Ar-H), 5.54 (m, 1H, iPr-CH), 2.05 (s, 6H, o-Ar-CH3), 1.69 (d, J
= 6.6 Hz, 6H, iPr-CH3). isomer 2: δ 9.41 (s, 1H, NCHN), 8.00 (d, 2H, imidazole-H), 7.98 – 7.96 (m,
1H, Ar-H), 7.76 (d, J = 6.0 Hz, 2H, Ar-H), 7.58 – 7.47 (m, 2H, Ar-H), 7.35 (s, 2H, Ar-H), 7.29 (s,
1H, Ar-H), 4.95-4.87 (m, 1H, iPr-CH), 2.30 (s, 6H, o-Ar-CH3), 1.58 (d, J = 6.6 Hz, 6H, iPr-CH3). 13C
NMR (101 MHz, CDCl3) δ 172.50, 166.07, 148.18, 143.25, 136.31 (NCHN), 135.80 (NCHN),
134.40, 134.24, 133.77, 132.43, 131.72, 129.36, 128.83, 128.66, 128.17, 127.44, 127.28, 126.39,
124.72, 120.70, 120.12, 118.31, 54.14, 52.52, 23.20, 22.88, 18.51, 18.47. ESI-MS(m/z): calcd for
C21H24N3: 318.19702, found: 318.19586, [3-iPr-1-{C(C6H5)N(2,6-Me2C6H3)}C3H3N2]+.
Synthesis of the complexes
General method: the imidazolium salt L (0.10 mmol) was dissolved in 15.0 mL of dry
dichloromethane and the solution added to a round-bottomed flask containing stirrer. Ag2O (0.12
mmol) was then added and the reaction mixture stirred in the absence of light for 6 h. The mixture
was filtration through Celite to remove excess Ag2O. The filtrate was transferred to another
round-bottomed flask that was charged with [(η6-p-cymene)RuCl2]2 (0.05 mmol), the mixture was
stirred at ambient temperature for overnight. KPF6 (0.60 mmol) was added with stirring and further
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stir 30 min at ambient temperature. The crude product was filtered and the solvent was removed
under reduced pressure. The resulting solid was recrystallized via a method of diffusion at room
temperature.
1
[(η6-p-cymene)Ru(L1)Cl]PF6 (1) Yield: 42.4 mg (66%). H NMR (500 MHz, CDCl3) δ 7.54 (s, 1H,
imidazole-H), 7.29 (d, J = 5.2 Hz, 1H, imidazole-H), 7.24 (dd, J = 8.7, 4.0 Hz, 3H, Ar-H), 5.67 (d, J
= 5.4 Hz, 1H, p-cymene-H), 5.33 (d, J = 6.1 Hz, 1H, p-cymene-H), 4.98 (d, J = 6.2 Hz, 1H,
p-cymene-H), 4.87 (d, J = 6.1 Hz, 1H, p-cymene-H), 4.15 (s, 3H, N-CH3), 2.54 – 2.63 (m, 1H,
p-cymene-CH-iPr2), 2.38 (s, 3H, o-aniline-CH3), 2.34 (s, 3H, o-aniline-CH3), 2.24 (s, 3H,
imine-CH3), 2.07 (s, 3H, p-cymene-CH3), 1.18 (d, J = 7.0 Hz, 3H, p-cymene-CH3-iPr), 1.14 (d, J =
6.8 Hz, 3H, p-cymene-CH3-iPr). 13C NMR (101 MHz, CDCl3) δ 189.03 (NHC carbon-Ru), 163.73,
146.76, 132.40, 129.93, 129.13, 128.99, 128.20, 126.11, 118.07, 112.24, 106.90, 91.20, 86.85, 85.27,
84.98, 38.61, 31.57, 23.88, 21.65, 20.06, 19.23, 17.94, 14.97. ESI-MS (m/z): calcd for
C24H31ClN3Ru: 498.046, found: 497.903, [(η6-p-cymene)Ru(L1)Cl]+. Elemental analysis: calcd (%)
for C24H31N3ClRuPF6: C, 44.83; H, 4.86; N, 6.53; found: C, 44.99; H, 4.71; N, 6.69.
1
[(η6-p-cymene)Ru(L2)Cl]PF6 (2) Yield: 38.1 mg (58%). H NMR (500 MHz, CDCl3) δ 7.60 (d, J =
2.3 Hz, 1H, imidazole-H), 7.33 (d, J = 2.3 Hz, 1H, imidazole-H), 7.29 – 7.26 (m, 1H, Ar-H), 7.25 –
7.22 (m, 2H, Ar-H), 5.66 (d, J = 6.2 Hz, 1H, p-cymene-H), 5.31 (d, J = 6.1 Hz, 1H, p-cymene-H),
4.98 (d, J = 6.2 Hz, 1H, p-cymene-H), 4.88 (d, J = 6.1 Hz, 1H, p-cymene-H), 4.51 (q, J = 7.4 Hz, 2H,
N-CH2Me), 2.65 – 2.54 (m, 1H, p-cymene-CH-iPr2), 2.38 (s, 3H, o-aniline-CH3), 2.34 (s, 3H,
o-aniline-CH3), 2.22 (s, 3H, imine-CH3), 2.05 (s, 3H, p-cymene-CH3), 1.64 (t, J = 7.3 Hz, 3H,
N-CH2CH3), 1.16 (d, J = 7.0 Hz, 3H, p-cymene-CH3-iPr), 1.13 (d, J = 6.8 Hz, 3H,
13
p-cymene-CH3-iPr). C NMR (101 MHz, CDCl3) δ 188.00 (NHC carbon-Ru), 163.62, 146.78,
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132.41, 129.94, 129.17, 129.00, 128.22, 123.45, 118.58, 112.66, 106.25, 91.03, 87.06, 85.14, 85.10,
46.88, 31.51, 23.79, 21.71, 20.11, 19.18, 17.95, 15.46, 15.01. ESI-MS (m/z): calcd for C25H33ClN3Ru:
512.141, found: 511.907, [(η6-p-cymene)Ru(L2)Cl]+. Elemental analysis: calcd (%) for
C25H33N3ClRuPF6: C, 45.70; H, 5.06; N, 6.40; found: C, 45.86; H, 5.11; N, 6.39.
1
[(η6-p-cymene)Ru(L3)Cl]PF6 (3) Yield: 58.4 mg (87%). H NMR (500 MHz, CDCl3) δ 7.62 (d, J =
2.3 Hz, 1H, imidazole-H), 7.31 (d, J = 2.3 Hz, 1H, imidazole-H), 7.24 (dd, J = 8.6, 4.5 Hz, 3H,
Ar-H), 5.58 (d, J = 6.2 Hz, 1H, p-cymene-H), 5.30 (d, J = 6.0 Hz, 1H, p-cymene-H), 5.04 – 4.93 (m,
2H p-cymene-H and N-CH-iPr2), 4.91 (d, J = 6.0 Hz, 1H, p-cymene-H), 2.56 – 2.64 (m, 1H,
p-cymene-CH-iPr2), 2.38 (s, 3H, o-aniline-CH3), 2.33 (s, 3H, o-aniline-CH3), 2.22 (s, 3H,
imine-CH3), 2.08 (s, 3H, p-cymene-CH3), 1.79 (d, J = 6.8 Hz, 3H, N-CH3-iPr), 1.56 (d, J = 6.6 Hz,
3H, N-CH3-iPr), 1.18 (d, J = 7.0 Hz, 3H, p-cymene-CH3-iPr), 1.14 (d, J = 6.8 Hz, 3H,
p-cymene-CH3-iPr). 13C NMR (101 MHz, CDCl3) δ186.85 (NHC carbon-Ru), 163.63, 146.78,
132.45, 129.93,129.30, 129.00, 128.23, 120.25, 119.21, 112.95, 90.49, 87.24, 85.43,84.80, 54.39,
31.45, 23.82, 23.67, 22.96, 21.81, 20.12, 19.28, 17.97, 15.02. ESI-MS (m/z): calcd for
C26H35ClN3Ru: 526.156, found: 526.1267, [(η6-p-cymene)Ru(L3)Cl]+. Elemental analysis: calcd (%)
for C26H35N3ClRuPF6: C, 46.53; H, 5.26; N, 6.26; found: C, 46.39; H, 5.31; N, 6.50.
1
[(η6-p-cymene)Ru(L4)Cl]PF6 (4) Yield: 50.0 mg (73%). H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H,
imidazole-H), 7.25 (s, 4H, imidazole-H and Ar-H), 5.50 (d, J = 5.9 Hz, 1H, p-cymene-H), 5.33 (d, J
= 5.5 Hz, 1H, p-cymene-H), 4.91 (dd, J = 10.8, 6.4 Hz, 2H, p-cymene-H), 4.37 – 4.50 (m, 2H,
nBu-CH2), 2.68 – 2.59 (m, 1H, p-cymene-CH-iPr2), 2.39 (s, 3H, o-aniline-CH3), 2.34 (s, 3H,
o-aniline-CH3), 2.24 (s, 3H, imine-CH3), 2.09 (s, 3H, p-cymene-CH3), 2.04 – 1.98 (m, 2H, nBu-CH2),
1.46 – 1.53 (m, 2H, nBu-CH2), 1.18 (d, J = 6.9 Hz, 3H, p-cymene-CH3-iPr), 1.14 (d, J = 6.8 Hz, 3H,
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p-cymene-CH3-iPr), 1.04 (t, J = 7.5 Hz, 3H, nBu-CH3).13C NMR (101 MHz, CDCl3) δ 187.86 (NHC
carbon-Ru), 163.68, 146.79, 132.39, 129.91, 129.31, 129.02, 128.23, 123.96, 118.42, 113.24, 105.62,
90.79, 87.20, 85.18, 84.95, 51.59, 31.79, 31.46, 23.76, 21.59, 20.14, 19.98, 19.18, 17.99, 15.01,
13.77. ESI-MS (m/z): calcd for C27H37ClN3Ru: 540.172, found: 539.978, [(η6-p-cymene)Ru(L4)Cl]+.
Elemental analysis: calcd (%) for C27H37N3ClRuPF6: C, 47.34; H, 5.44; N, 6.13; found: C, 47.56; H,
5.31; N, 6.19.
[(η6-p-cymene)Ru(L5)Cl]PF6 (5) Yield: 39.5 mg (56%). 1H NMR (500 MHz, DMSO) δ 7.99 (d, J =
2.4 Hz, 1H, imidazole-H), 7.52 (dd, J = 13.7, 4.8 Hz, 4H, imidazole-H and Ar-H), 7.39 (s, 4H, Ar-H),
7.29 (t, J = 8.3 Hz, 3H, Ar-H), 6.01 (d, J = 6.1 Hz, 1H, p-cymene-H), 5.52 (d, J = 6.1 Hz, 1H,
p-cymene-H), 5.47 (d, J = 6.2 Hz, 1H, p-cymene-H), 5.42 (d, J = 6.1 Hz, 1H, p-cymene-H), 5.08 –
4.99 (m, 1H, N-CH-iPr2), 2.57 – 2.52 (m, 1H, p-cymene-CH-iPr2), 2.11 (s, 3H, p-cymene-CH3), 1.75
(d, J = 6.7 Hz, 3H, N-CH3-iPr), 1.45 (d, J = 6.6 Hz, 3H, N-CH3-iPr), 1.04 (d, J = 6.9 Hz, 3H,
p-cymene-CH3-iPr), 1.02 (d, J = 6.9 Hz, 3H, p-cymene-CH3-iPr). 13C NMR (101 MHz, DMSO) δ
188.86 (NHC carbon-Ru), 162.47, 150.11, 132.46, 129.75, 129.65, 129.60, 128.10, 125.81, 123.83,
123.69, 121.76, 120.83, 107.57, 92.39, 89.07, 88.19, 85.00, 54.32, 31.37, 23.73, 22.93, 22.70, 22.55,
19.14. ESI-MS (m/z): calcd for C29H33ClN3Ru: 560.141, found: 559.984, [(η6-p-cymene)Ru(L5)Cl]+.
Elemental analysis: calcd (%) for C29H33N3ClRuPF6: C, 49.40; H, 4.72; N, 5.96; found: C, 48.99; H,
4.81; N, 5.73.
1
[(η6-p-cymene)Ru(L6)Cl]PF6 (6) Yield: 56.6 mg (81%). H NMR (500 MHz, CDCl3) δ 7.54 (d, J =
1.9 Hz, 1H, imidazole-H), 7.44 (t, J = 7.7 Hz, 1H, imidazole-H), 7.39 – 7.35 (m, 1H, Ar-H), 7.35 –
7.30 (m, 2H, Ar-H), 5.97 (d, J = 6.3 Hz, 1H, p-cymene-H), 5.21 (d, J = 6.0 Hz, 1H, p-cymene-H),
5.05 (d, J = 6.2 Hz, 1H, p-cymene-H), 5.02 (d, J = 6.1 Hz, 1H, p-cymene-H), 4.16 (s, 3H, N-CH3),
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3.54 – 3.45 (m, 1H, o-aniline-CH-iPr2), 2.55 – 2.43 (m, 2H, o-aniline-CH-iPr2 and
p-cymene-CH-iPr2), 2.42 (s, 3H, p-cymene-CH3), 2.25 (s, 3H, imine-CH3), 1.45 (d, J = 6.7 Hz, 3H,
o-aniline-CH3-iPr2), 1.26 (d, J = 6.6 Hz, 3H, o-aniline-CH3-iPr2), 1.17 (d, J = 7.0 Hz, 3H,
p-cymene-CH3-iPr), 1.15 (d, J = 6.6 Hz, 3H, o-aniline-CH3-iPr2), 1.13 (d, J = 6.9 Hz, 3H,
p-cymene-CH3-iPr), 0.95 (d, J = 6.8 Hz, 3H, o-aniline-CH3-iPr2). 13C NMR (101 MHz, CDCl3) δ
188.57 (NHC carbon-Ru), 164.16, 143.86, 143.06, 140.25, 129.07, 126.37, 125.78, 124.81, 118.19,
111.27, 109.07, 90.40, 86.46, 85.96, 82.64, 38.72, 31.04, 27.84, 27.69, 25.75, 25.07, 23.99, 23.83,
22.16, 19.14, 17.29. ESI-MS (m/z): calcd for C28H39ClN3Ru: 554.188, found: 554.019,
[(η6-p-cymene)Ru(L6)Cl]+. Elemental analysis: calcd (%) for C28H39N3ClRuPF6: C, 48.10; H, 5.62;
N, 6.01; found: C, 48.32; H, 5.51; N, 6.09.
[(η6-p-cymene)Ru(L7)Cl]PF6 (7) Yield: 53.5 mg (75%). 1H NMR (500 MHz, CDCl3) δ 7.60 (s, 1H,
imidazole-H), 7.43 (t, J = 7.7 Hz, 1H, imidazole-H), 7.36 (d, J = 6.9 Hz, 2H, Ar-H), 7.34 (d, J = 7.7
Hz, 1H, Ar-H), 5.95 (d, J = 6.3 Hz, 1H, p-cymene-H), 5.20 (d, J = 6.0 Hz, 1H, p-cymene-H), 5.04 (t,
J = 5.6 Hz, 2H, p-cymene-H), 4.47 – 4.60 (q, J = 7.2 Hz, 2H, N-CH2Me), 3.45 – 3.55 (m, 1H,
o-aniline-CH-iPr2), 2.56 – 2.43 (m, 2H, o-aniline-CH-iPr2 and p-cymene-CH-iPr2), 2.42 (s, 3H,
p-cymene-CH3), 2.24 (s, 3H, imine-CH3), 1.64 (t, J = 7.3 Hz, 3H, N-CH2CH3), 1.45 (d, J = 6.7 Hz,
3H, o-aniline-CH3-iPr2), 1.25 (d, J = 6.6 Hz, 3H, o-aniline-CH3-iPr2), 1.16 (d, J = 7.0 Hz, 3H,
p-cymene-CH3-iPr), 1.14 (d, J = 6.6 Hz, 3H, o-aniline-CH3-iPr2), 1.11 (d, J = 6.8 Hz, 3H,
p-cymene-CH3-iPr), 0.94 (d, J = 6.8 Hz, 3H, o-aniline-CH3-iPr2). 13C NMR (101 MHz, CDCl3) δ
187.60 (NHC carbon-Ru), 164.10, 143.87, 143.05, 140.23, 129.09, 125.80, 124.81, 123.61, 118.73,
110.81, 109.16, 90.41, 86.67, 85.85, 82.73, 46.92, 31.03, 27.85, 27.68, 25.74, 25.07, 23.85, 22.34,
19.07, 17.39, 15.31. ESI-MS (m/z): calcd for C29H41ClN3Ru: 568.203, found: 568.008,
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[(η6-p-cymene)Ru(L7)Cl]+. Elemental analysis: calcd (%) for C29H41N3ClRuPF6: C, 48.84; H, 5.79;
N, 5.89; found: C, 48.89; H, 5.82; N, 5.95.
[(η6-p-cymene)Ru(L8)Cl]PF6 (8) Yield: 43.6 mg (60%). 1H NMR (500 MHz, CDCl3) δ 7.69 (s, 1H,
imidazole-H), 7.44 (t, J = 7.7 Hz, 1H, imidazole-H), 7.41 – 7.30 (m, 3H, Ar-H), 5.88 (d, J = 5.8 Hz,
1H, p-cymene-H), 5.19 (d, J = 6.0 Hz, 1H, p-cymene-H), 5.07 (d, J = 6.0 Hz, 1H, p-cymene-H), 5.05
(d, J = 5.9 Hz, 1H, p-cymene-H), 5.01 – 4.92 (m, 1H, N-CH-iPr2), 3.54 – 3.47 (m, 1H,
o-aniline-CH-iPr2), 2.48 – 2.53 (m, 2H, o-aniline-CH-iPr2 and p-cymene-CH-iPr2), 2.43 (s, 3H,
p-cymene-CH3), 2.25 (s, 3H, imine-CH3), 1.81 (d, J = 6.6 Hz, 3H, N-CH3-iPr), 1.56 (d, J = 6.4 Hz,
3H, o-aniline-CH3-iPr2), 1.45 (d, J = 6.5 Hz, 3H, N-CH3-iPr), 1.25 (d, J = 6.6 Hz, 3H,
o-aniline-CH3-iPr2), 1.18 (d, J = 6.6 Hz, 3H, p-cymene-CH3-iPr), 1.15 (d, J = 6.4 Hz, 3H,
o-aniline-CH3-iPr2), 1.12 (d, J = 6.8 Hz, 3H, p-cymene-CH3-iPr), 0.94 (d, J = 6.8 Hz, 3H,
o-aniline-CH3-iPr2). 13C NMR (101 MHz, CDCl3) δ 186.34 (NHC carbon-Ru), 164.09, 143.87,
143.09, 140.35, 129.09, 125.80, 124.82, 120.53, 119.31,110.90, 109.14, 89.75, 86.92, 86.46, 82.10,
54.42, 30.91, 27.83, 27.67, 25.76, 25.09, 23.96, 23.84, 22.59, 22.50, 19.16, 17.42. ESI-MS (m/z):
calcd for C30H43ClN3Ru: 582.219, found: 582.42, [(η6-p-cymene)Ru(L8)Cl]+. Elemental analysis:
calcd (%) for C30H43N3ClRuPF6: C, 49.55; H, 5.96; N, 5.78; found: C, 49.35; H, 5.81; N, 5.88.
1
[(η6-p-cymene)Ru(L9)Cl]PF6 (9) Yield: 51.1 mg (69%). H NMR (500 MHz, CDCl3) δ 7.59 (d, J =
2.1 Hz, 1H, imidazole-H), 7.43 (t, J = 7.7 Hz, 1H, imidazole-H), 7.38 – 7.32 (m, 2H, Ar-H), 7.31 (d,
J = 2.1 Hz, 1H, Ar-H), 5.83 (d, J = 6.2 Hz, 1H, p-cymene-H), 5.20 (d, J = 6.0 Hz, 1H, p-cymene-H),
5.05 (d, J = 6.1 Hz, 1H, p-cymene-H), 4.99 (d, J = 6.2 Hz, 1H, p-cymene-H), 4.39 – 4.54 (m, 2H,
nBu-CH2), 3.56 – 3.45 (m, 1H, o-aniline-CH-iPr2), 2.60 – 2.45 (m, 2H, o-aniline-CH-iPr2 and
p-cymene-CH-iPr2), 2.42 (s, 3H, p-cymene-CH3), 2.25 (s, 3H, imine-CH3), 1.99 – 2.03 (m, 2H,
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n
nBu-CH2), 1.46 – 1.50 (m, 2H, Bu-CH2), 1.45 (d, J = 6.7 Hz, 3H, o-aniline-CH3-iPr2), 1.25 (d, J =
6.6 Hz, 3H, o-aniline-CH3-iPr2), 1.17 (d, J = 7.0 Hz, 3H, p-cymene-CH3-iPr), 1.15 (d, J = 6.6 Hz, 3H,
n
o-aniline-CH3-iPr2), 1.11 (d, J = 6.8 Hz, 3H, p-cymene-CH3-iPr), 1.02 (t, J = 7.3 Hz, 3H, Bu-CH3),
0.94 (d, J = 6.8 Hz, 3H, o-aniline-CH3-iPr2). 13C NMR (101 MHz, CDCl3) δ 187.46 (NHC
carbon-Ru), 164.14, 143.87, 143.04, 140.33, 129.10, 125.82, 124.83, 124.15, 118.53, 110.43, 109.51,
90.28, 86.77, 85.79, 82.66, 51.60, 31.61, 31.00, 27.85, 27.70, 25.80, 25.10, 25.04, 23.89, 23.80,
22.29, 19.95, 19.09, 17.37, 13.78. ESI-MS (m/z): calcd for C31H45ClN3Ru: 596.235, found: 596.2108,
[(η6-p-cymene)Ru(L9)Cl]+. Elemental analysis: calcd (%) for C31H45N3ClRuPF6: C, 50.23; H, 6.12;
N, 5.67; found: C, 50.38; H, 6.21; N, 5.54. Crystals of complex 9 qualified for X-ray analysis were
obtained by slow diffusion of petroleum ether into a concentrated solution of complex 9 in ethyl
acetate.
1
[(η6-p-cymene)Ru(L10)Cl]PF6 (10) Yield: 47.7 mg (65%). H NMR (500 MHz, DMSO) δ 8.01 (d, J
= 2.4 Hz, 1H, imidazole-H), 7.59 (d, J = 2.4 Hz, 2H, imidazole-H and Ar-H), 7.55 (t, J = 7.4 Hz, 1H,
Ar-H), 7.43 (s, 2H, Ar-H), 7.24 (d, J = 7.4 Hz, 1H, Ar-H), 7.14 (t, J = 7.6 Hz, 1H, Ar-H), 6.96 (d, J =
7.5 Hz, 2H, Ar-H), 6.13 (d, J = 6.3 Hz, 1H, p-cymene-H), 5.28 (d, J = 6.0 Hz, 1H, p-cymene-H),
5.21 (d, J = 6.3 Hz, 1H, p-cymene-H), 5.00 – 5.07 (m, 1H, N-CH-iPr2), 4.99 (d, J = 5.9 Hz, 1H,
p-cymene-H), 2.57 (s, 3H, o-aniline-CH3), 2.55 – 2.51 (m, 1H, CH-iPr2), 2.21 (s, 3H, p-cymene-CH3),
1.78 (s, 3H, o-aniline-CH3), 1.73 (d, J = 6.7 Hz, 3H, N-CH3-iPr), 1.46 (d, J = 6.6 Hz, 3H, N-CH3-iPr),
1.16 (d, J = 6.9 Hz, 3H, p-cymene-CH3-iPr), 1.14 (d, J = 6.9 Hz, 3H, p-cymene-CH3-iPr). 13C NMR
(101 MHz, DMSO) δ 192.68 (NHC carbon-Ru), 168.39, 153.04, 138.11, 137.97, 134.41, 134.23,
133.76, 133.66, 132.70, 131.14, 126.87, 126.07, 115.18, 113.74, 97.93, 91.75, 90.96, 89.80, 60.14,
58.91, 36.40, 28.71, 28.07, 27.65, 27.53, 25.40, 23.92, 23.46. ESI-MS (m/z): calcd for
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