Synthesis and antiproliferative activity of benzimidazole-based, trinuclear neutral cyclometallated and cationic, N^N-chelated ruthenium(ii) complexes

Article abstract of DOI:10.1039/c9dt03902c

A series of 2-phenyl and 2-pyridyl tris-benzimidazole ligands was reacted with the [Ru(p-cymene)Cl₂]₂ dimer to yield the corresponding neutral cyclometallated and cationic organoruthenium(ii) complexes. All of the synthesized compoun

Full text of DOI:10.1039/c9dt03902c

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Synthesis and antiproliferative activity of
benzimidazole-based, trinuclear neutral
cyclometallated and cationic, N N-chelated
Cite this: DOI: 10.1039/c9dt03902c
^
ruthenium(II) complexes†
a
a
b
c
Athi Welsh,
Laa-iqa Rylands, Vladimir B. Arion,
Sharon Prince and
a
Gregory S. Smith
*
2 2
A series of 2-phenyl and 2-pyridyl tris-benzimidazole ligands was reacted with the [Ru(p-cymene)Cl ]
dimer to yield the corresponding neutral cyclometallated and cationic organoruthenium(II) complexes. All of
the synthesized compounds were characterized using an array of spectroscopic and analytical techniques,
1
13
including H, C nuclear magnetic resonance (NMR), infrared spectroscopy and mass spectrometry. The tri-
nuclear compounds were screened for their cytotoxicy against the MCF-7 breast cancer cell line and the
triple negative MDA-MB-231 breast cancer cell line at concentrations of 10 and 20 µM. Overall, the
6 3 6 3
2-pyridyl ligands 10 and 11, and their corresponding trinuclear complexes [16][PF ] and [17][PF ] , show
the most promising activity as these compounds significantly reduce the percentage cell survival of MCF-7
and MDA-MB-231 breast cancer cell lines at the aforementioned fixed concentrations. It was observed that
10 and [16][PF ] show potency greater than that of cisplatin against the MCF-7 breast cancer cell line, and
Received 2nd October 2019,
Accepted 13th December 2019
6
3
[
6 3
17][PF ] shows potency comparable to that of cisplatin against the MCF-7 cell line. Additionally, the syn-
DOI: 10.1039/c9dt03902c
thesized compounds were observed to have relatively low cytotoxicty towards MCF-12A breast epithelial
cells and relatively higher selectivity towards MCF-7 breast cancer cells compared to cisplatin.
rsc.li/dalton
benzimidazole derivatives show an array of biological activity,
including antiviral, antihistaminic, antiparasitic, antimalarial
Cancer is a non-communicable disease that causes abnormal and anticancer activity. Due to their vast medicinal value,
cell growth and division, and remains one of the major causes the research of benzimidazole-containing drugs is an active
Introduction
5
,6
1
of death globally, affecting the lives of approximately 600 000 and attractive field in chemistry. Based on the success of clini-
2
,3
people. The defining characteristic of cancer is the abrupt cally approved benzimidazole derivatives, such as omeprazole,
evolution of abnormal cells which overgrow, and may spread lansoprazole and albendazole, research focussing on the med-
via metastasis to other organs using either the lymphatic icinal use of benzimidazoles as chemotherapeutic agents
4
7
system or bloodstream. Despite improvements in chemo- remains promising, with several recent publications reporting
therapy treatments, the development of resistance remains the benzimidazole derivatives with various substitution patterns,
7
,8
main cause of chemotherapy failure.
possessing potent anticancer activities.
The fortuitous discovery of cis-dichloridodiammineplatinum(II),
The prevalence of the benzimidazole core in biologically
active molecules has stimulated systematic investigations of commonly known as cisplatin, by Rosenberg in 1967 paved
this class of heterocyclic compounds. It is well established that the way for the development of organometallic complexes
as an alternative class of chemotherapeutic agents. Today,
platinum-based drugs, cisplatin, carboplatin and oxaliplatin,
9
constitute as much as 70% of cancer treatment regimens.
a
Department of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town,
However, their use is limited by their severe side effects and
South Africa. E-mail: Gregory.Smith@uct.ac.za
b
Institute of Inorganic Chemistry of the University of Vienna, Währinger Strasse 42,
the development of resistance by numerous cancers. Efforts to
target resistance has resulted in a shift towards the develop-
ment of other platinum-group metals (PGMs) as potential anti-
1
090 Vienna, Austria
c
Department of Human Biology, University of Cape Town, Faculty of Health Science,
Observatory 7925, South Africa
10
cancer metallodrugs.
†
Electronic supplementary information (ESI) available. CCDC 1940327 and
940326. For ESI and crystallographic data in CIF or other electronic format see
By far, ruthenium-based drugs have been very successful
1
DOI: 10.1039/c9dt03902c
in this category as they offer more potential as cytostatic and
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1
1
cytotoxic agents with novel modes of action. Indeed, ruthe- scaffold, which were investigated for their potential anticancer
nium complexes have gained significant prominence as poten- activity against two breast cancer cell lines, the MCF-7 and
tial anticancer agents, as they display diverse and non-conven- MDA-MB-231 cell lines. The focus on breast cancer was a
1
2
tional mechanisms of action. These ruthenium complexes result of recent GLOBOCAN statistics which estimate that in
offer a range of oxidation states which are accessible under women, breast cancer is the most commonly diagnosed cancer
physiological conditions, which is unique among PGMs. This (11.6% of the total new cases) and the leading cause of cancer-
feature offers a unique advantage in rational drug design as related mortality (6.6% of total cancer deaths). In both sexes
the anticancer activity of most metal-based compounds is combined, the only cancer that has a slightly higher number
1
3–16
dependent on the metal’s oxidation state.
To date, the of new diagnoses and cancer related mortalities is lung cancer,
3
7
most successful ruthenium complexes are the New Anti- closely followed by breast cancer.
tumour Metastatic Inhibitor-A (NAMI-A) and IT-139 which
9
,17
have entered clinical trials.
NAMI-A has minimal effects on
1
8
primary tumour growth but has anti-metastatic properties.
Indeed, it can reduce metastatic mass and prevent the formation
of secondary tumours. On the other hand, IT-139 is a cytotoxic
Results and discussion
Synthesis of 2,5-disubstituted tris-benzimidazole ligands (7–12)
agent that is effective against advanced solid tumours, showing The synthesis of the 2,5-disubstituted tris-benzimidazoles
promising activity against non-small lung cancer, neuroendo- (7–12) involved three steps: (i) a nucleophilic aromatic substi-
18
N
crine and sarcoma tumours. The success of ruthenium com- tution reaction (S Ar) of tris(2-aminoethyl)amine with either
plexes and their alternate mechanisms of action suggest that they ortho-substituted fluoro- or chloro-nitrobenzenes (ii) the
may facilitate the discovery of new combination chemotherapy reduction of the nitro-functionalities to primary amines,
agents. There have been several reports of half-sandwich ruthe- and lastly (iii) the condensation–cyclisation with either benz-
nium(II)-arene complexes which have several intracellular targets. aldehyde or 2-pyridinecarboxaldehyde to afford the desired
19
These targets include DNA interaction and binding, inhibition tris-benzimidazole ligand (Scheme 1).
of enzymes which are overexpressed in human cancers including The pure ligands were isolated as coloured solids in
20
21
22
the PARP-1, CDK1, and cathepsin B enzymes, which are moderate to good yields (33–71%) and are soluble in several
known to play vital roles in the proliferation and progression of polar organic solvents including ethanol, acetone and
23,24
1
cancers, and lastly, induction of apoptosis.
dimethylsulfoxide. Comparison of the H NMR spectra of
The success of mononuclear chemotherapeutic agents also the 2-phenyl (7–9) and 2-pyridyl tris-benzimidazole ligands
pre-empted the investigation of multinuclear PGM complexes (10–12) and their corresponding tris-1,2-benzenediamines
1
as potential drug candidates. This interest was initiated by the (4–6) revealed several notable features. Firstly, the H NMR
observation that compounds bearing more than one metal spectra of the benzimidazole ligands did not possess any sig-
center have improved biological activities relative to their nificant changes in the aliphatic region, as two aliphatic
2
5–27
mononuclear counterparts.
The improved cytotoxicity of signals are observed, corresponding to the methylene protons
multinuclear macromolecules may also be attributed to the on the tris-core. However, in the aromatic region, additional aro-
2
5,28
enhanced permeability and retention (EPR) effect.
This is matic signals are observed, and these additional signals collec-
a phenomenon in which solid tumours have defective and tively integrate for either fifteen (for the 2-phenyl tris-benzimid-
permeable blood vessels, to ensure a sufficient supply of azole ligands 7–9) or twelve protons (for the 2-pyridyl tris-benz-
nutrients and oxygen to tumour tissues for rapid growth. imidazole ligands 10–12). These additional aromatic signals
Macromolecules, such as polynuclear complexes, exploit this correspond to the protons of either the 2-phenyl or 2-pyridyl
2
9
EPR effect and selectively accumulate in tumour cells. The functionalities on the 2-position of the benzimidazole motifs
most successful trinuclear candidate is BBR3464, which com- and are indicative of successful synthesis of the benzimidazole
prises three platinum(II) centres bridged by a polyamine entity. In the IR spectra of the 2-phenyl benzimidazoles (7–9),
scaffold. The polyamine scaffold, and multinuclearity of the one absorption band between 1695–1625 cm− was observed
complex results in a slightly different mechanism when inter- and this absorption band corresponds to the imine (CvN)
acting with DNA, as the complex spans a long distance along bond stretch. The IR spectra of the 2-pyridyl tris-benzimidazole
1
1
0
DNA strands. The complex is currently in phase II clinical ligands (10–12) revealed two absorption bands, one in the range
trials for the treatment of melanoma, lung, ovarian and gastric 1592–1733 cm− and another in the range 1489–1587 cm , that
1
−1
3
0
tumours. However, the study of transition metals, such as correspond to imine (CvN) bond stretches of the benzimidazole
ruthenium, for the preparation of polynuclear complexes motif and the CvN bond stretch in 2-pyridyl functionality,
3
1
opens another manifold of diversity.
The incorporation of multivalent pharmacophores and the
respectively.
The molecular structures for compounds 7 and 8 were con-
combination of these ligands with PGMs, to form trinuclear firmed in the solid state by single crystal X-ray diffraction, with
complexes, is a prolific area of research which we have pre- molecular structures depicted in Fig. 1 and 2, respectively.
3
2–36
viously explored as a strategy in anticancer drug design.
Single crystals of 7 and 8 were obtained by slow diffusion of a
This study explored triruthenium(II) complexes based on benzi- concentrated solution of either 7 or 8 in dichloromethane
midazole motifs anchored on a tris-2-(ethylamino)amine layered with ethyl acetate, at room temperature. Further crystal-
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Scheme 1 Reagents and conditions: (i) DMF/RT/24 h; (ii) NH
4
Cl/Zn/MeOH/RT/1 h; (iii) benzaldehyde/EtOH/TFA/80 °C/24 h or 2-pyridinecarboxal-
dehyde/EtOH/TFA/RT/24 h.
Fig. 2 The ORTEP drawing of the molecular structure of compound 8,
hydrogen atoms have been omitted for clarity. Thermal ellipsoids are at
the 50% probability level.
Fig. 1 The ORTEP drawing of the molecular structure of compound 7,
hydrogen atoms have been omitted for clarity. Thermal ellipsoids are at
the 50% probability level.
P ˉ1 , respectively. In 8, the F atoms of two of the three trifluoro-
methyl groups (C9A and C9C) are disordered over two sites,
with refined site occupancy factors of 0.615(15) for F1A, F2A
lographic data and refinement parameters for the two ligands and F3A, 0.315(15) for F4A, F5A and F6A, 0.778(9) for F1C, F2C
are summarised in Table 1. The compounds 7 and 8 crystal- and F3C, 0.222(9) for F4C, F5C and F6C. The imine (CvN)
lised in the trigonal space group P ˉ3 c1 and triclinic space group bond lengths in the benzimidazole cores of 7 and 8 range
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Table 1 Crystallographic data for compounds 7 and 8
appropriate 2-phenyl tris-benzimidazole ligand (7–9) was
reacted with the [Ru(p-cymene)Cl2]2 dimer in the presence
of sodium acetate (Scheme 2). The compounds 13–15
were isolated in excellent yields (84–88%) as green (13) or
dark yellow powders (14 and 15) that are air- and moisture-
stable, and are soluble in dimethylsulfoxide, acetonitrile or
acetone.
Compound 7
Compound 8
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
C
45
H
39
N
7
48 36 9 7
C H F N
881.85
173(2)
Triclinic
Pˉ1
677.83
173(2)
Trigonal
P ˉ3 c1
15.9799(10)
15.9799(10)
15.7925(9)
90
90
120
3492.4(5)
4
1.289
0.078
1432.0
0.44 × 0.26 × 0.25
MoKα (λ = 0.71073)
2.942 to 56.514
1
12.9590(8)
In the H NMR spectra of 13–15, the appearance of four
13.5286(10)
14.2464(10)
70.930(2)
64.023(2)
81.799(2)
2122.1(3)
2
1.380
0.111
908.6
(
for 13) or five (for 14 and 15) signals between 5.0 and
6.0 ppm which collectively correspond to the aromatic
protons of the p-cymene ring, confirm successful cyclometal-
lation. The appearance of two multiplets in the range
0.5–1.0 ppm is due to the methyl protons of the iso-propyl
functionality on the p-cymene ring. These signals suggest
that the methyl protons are in electronically distinct environ-
ments and thus further corroborating successful cyclometal-
lation. The difference in electronic environments may be
attributed to the stereogenic metal centers induced upon
cyclometallation. High-resolution mass spectrometry analysis
of 14 revealed an ion peak at m/z = 1157.3227 corresponding
γ (°)
Volume (Å )
3
Z
−
3
ρcalc (g cm
)
−
1
μ (mm
F(000)
)
3
Crystal size (mm )
Radiation
0.18 × 0.18 × 0.12
MoKα (λ = 0.71073)
3.18 to 56.62
2Θ range for data
collection (°)
+
+
to the fragment [M + Na − C26
H
25ClF
3
N
2
Ru] . Additionally,
upon analysis of the high-resolution mass spectrum of 15, a
from 1.317 Å to 1.320 Å and are comparable to imine bond
lengths observed in the literature for analogous 2-phenyl
benzimidazole compounds.
peak at m/z = 1494.3296 was observed which corresponds to
+
3
8,39
the [M − Cl] ion. In the IR spectra of these complexes it is
generally observed that the absorption band corresponding to
the imine (CvN) bond was at a lower wavenumber, in the
range of 1580–1596 cm , relative to the corresponding
Synthesis of neutral cyclometallated trinuclear C^N-Ru(II)-p-
cymene complexes (13–15)
−1
2
-phenyl tris-benzimidazole ligands 7–9, with imine (CvN)
−
1
Generally, the trinuclear cyclometallated complexes were absorption bands between 1625 and 1695 cm . This is due
synthesized via a C–H activation reaction, in which the to the extensive pi-backbonding between the low-spin d
6
Scheme 2 Reagents and conditions: For 13–15: (i) MeCN or DCM : EtOH (1 : 1)/NaOAc/RT/24 h; for [16][PF
4 h; (ii) DCM : EtOH (1 : 1)/NH PF /RT/1 h.
6 3 6 3
] –[18][PF ] : (i) DCM : EtOH (1 : 1)/RT/
2
4
6
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ruthenium center and the benzimidazole motif. This syner- remain intact in DMSO and do not interact with DMSO
gistic strengthening of the N–Ru(II) bond and weakening of molecules.
the imine CvN bond results in the observed shift in the
imine (CvN) bond stretch in the complexes to a lower
wavenumber.
Synthesis of a monomeric 2-pyridylbenzimidazole ligand (19)
and its corresponding cationic N^N-Ru(II)-p-cymene metal
complex ([20][PF6])
Prior to the synthesis of the mononuclear 2-pyridylbenzimida-
Synthesis of cationic N^N-Ru(II)-p-cymene metal complexes
zole ligand (19) and the corresponding Ru(II) complex ([20][PF ]),
6
(
[16][PF ] –[18][PF ] )
6 3 6 3
results from the in vitro anticancer evaluation (vide infra) of the
The synthesis of the cationic ruthenium complexes was tris-benzimidazole ligands and complexes provided the criteria
6
achieved in two steps: (i) firstly, the ruthenium [Ru(η -p- for the selection of the most active tris-benzimidazole ligand
i
Pr C
6
H
4
Me)Cl
2
]
2
dimer was reacted with the appropriate and corresponding complex (10 and [16][PF
6
]
3
, respectively).
2
-pyridyl tris-benzimidazole ligand (10–12), followed by (ii) an The synthesis of the monomeric compound 19 was carried out
anion exchange reaction with ammonium hexafluoro- using the same three-step procedure as described previously
phosphate (Scheme 2). The complexes [16][PF –[18][PF for the synthesis of the 2,5-disubstituted tris-benzimidazole
6
]
3
6 3
]
1
were isolated in moderate to good yields (51–75%) as yellow ligands. Analysis of the obtained H NMR spectra for the
hexafluorophosphate salts that are air- and moisture-stable, N-ethyl-2-nitroaniline and N -ethylbenzene-1,2-diamine syn-
1
and are soluble in dimethylsulfoxide, dichloromethane or thons, and the target 2-pyridylbenzimidazole ligand (19) are
acetone.
Comparison
16][PF −[18][PF
consistent with the proposed structures of these compounds.
1
of
the
H
NMR
spectra
of Additionally, the chemical shifts and signal multiplicities
1
[
6
]
3
6
]
3
to those of the corresponding ligands observed in each of the obtained H NMR spectra were all com-
4
0–42
1
0–12, reveals a signal attributed to the proton bonded to the parable to those reported in literature.
carbon adjacent to the pyridyl nitrogen at a significantly
higher chemical shift in the ruthenium complexes [16][PF
18][PF
the metal complexes). This is due to the bonding of the chloromethane or acetone. Comparison of the H NMR spectra
pyridyl nitrogen to the ruthenium center resulting in pi-back- of [20][PF ] to that of 19, shows the first indicator of successful
The mononuclear cationic complex [20][PF ] was isolated
6
6
]
3
–
in 81% yield as the yellow hexafluorophosphate salt that is air-
[
6 3
] (from 8.25–8.50 ppm in the ligands, to ∼9.6 ppm in and moisture-stable, and is soluble in dimethylsulfoxide, di-
1
6
bonding, which consequentially results in the reduction of complex formation, as the splitting of the signal corres-
electron density on the carbon adjacent to the pyridyl nitro- ponding to the methylene protons of the ethyl functionality
gen. Additionally, the aromatic protons of the p-cymene ring from a distinct quartet, in 19, to an intricate multiplet, in
were observed as either three ([16][PF
6 3 6
] ) or four signals [20][PF ]. This splitting is due to the chiral Ru(II) center, result-
(
for [17][PF6]3 and [18][PF ] ) in the range 6.00–6.50 ppm, ing in the diastereotopicity of these methylene protons.
6 3
corroborating the coordination of the ruthenium center Additionally, three signals for the aromatic protons of the
to both the nitrogens of the benzimidazole motif and the p-cymene ancillary ligand are observed between 6.0 and
2
-pyridyl functionalities. These signals are indicative of 6.5 ppm. Furthermore, analysis of the high-resolution mass
the stereogenicity introduced through chelation of the Ru(II) spectrum of [20][PF ] revealed a peak at m/z = 494.0948 corres-
6
+
6
center to the benzimidazole nitrogen and the 2-pyridyl nitro- ponding to the [M − PF ] ion.
gen. Generally, the IR spectra revealed that the absorption
bands corresponding to the imine (CvN) bond of the benzi-
In vitro anticancer evaluation
midazole cores and the pyridyl CvN bond are at lower wave- The antiproliferative activity of the synthesized 2-phenyl
numbers in the complexes [16][PF ] –[18][PF ] (in the ranges and 2-pyridyl tris-benzimidazole ligands and their corres-
6
3
6 3
1
−1
1
572–1674 cm− and 1464–1490 cm
for the imine and ponding triruthenium(II) complexes was evaluated in vitro
pyridyl CvN bonds respectively) relative to their corres- against the MCF-7 breast cancer cell line at an initial dose of
ponding 2-pyridyl tris-benzimidazole ligands 10–12 (with 10 µM. The initial concentration was chosen based on the
−
1
absorption bands in the ranges 1620–1733 cm
1
and National Cancer Institute (NCI), which conducts a pre-screen
586–1590 cm− for the imine and pyridyl CvN bonds, to assess a new drug candidate at 10 µM. Due to the com-
1
43
respectively). Again, this lowering of absorption bands is pounds showing very mild to no activity at 10 µM, the concen-
indicative of pi-backbonding between the imine and pyridyl tration was subsequently doubled to 20 µM. The cytotoxicity
nitrogens to the ruthenium center, resulting in weakening data obtained for the tested compounds are summarized in
of both the imine and pyridyl CvN bonds. The stability of Fig. S1† (for the 2-phenyl ligands and corresponding cyclome-
the cationic ruthenium(II) complexes ([16][PF
6
]
3
–[18][PF
6
]
3
)
tallated complexes) and Fig. S2† (for the 2-pyridyl ligands and
was investigated using UV/Vis spectroscopy at 37 °C to simu- corresponding cationic complexes). Cisplatin was included as
44
late the chemical environment prior to cell viability studies. a positive control at the reported IC50 value of 35 µM.
In the obtained UV/Vis spectra of the complexes (Fig. S35– Generally, the 2-phenyl ligands and their corresponding
S37†), no significant changes were noted after incubating the metal complexes show very mild to no activity at 20 µM as the
compounds at 37 °C for 24 and 48 h. Thus, the compounds 2-phenyl ligand series and corresponding cyclometallated
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complexes are observed to reduce cell viability to only 78–86% trends. Firstly, the cytotoxic activity of the 5-unsubstituted
Fig. S1†). However, the 2-pyridyl ligands and corresponding 2-pyridyl ligand and complex (10 and [16][PF ] , respectively) is
(
6
3
complexes display slightly improved cytotoxicity (Fig. S2†) rela- superior to that of cisplatin at both the tested concentrations.
tive to their corresponding 2-phenyl counterparts. A clear indi- Furthermore, the 5-trifluoromethyl complex ([17][PF ) was
6 3
]
cation of this is when comparing the cytotoxic activity of the noted to show activity comparable to that of cisplatin at 10 and
unsubstituted 2-phenyl (7, with a percentage cell survival of 20 µM.
7
8%) and 2-pyridyl (10, with a percentage cell survival of 43%)
As a consequence of the enhanced activity of the 2-pyridyl
ligands, and their respective cyclometallated (13, with a per- tris-benzimidazole ligands and related complexes in the
centage cell survival of 86%) and cationic complexes MCF-7 breast cancer cell line, these compounds were further
(
6 3
[16][PF ] , with a percentage cell survival of 71%). The same evaluated on the MDA-MB-231 triple-negative breast adeno-
trend was observed upon the treatment of the MCF-7 cells with carcinoma cell line. These cells represent a more aggressive,
4
5
2
0 µM of the compounds. Selected compounds were observed highly invasive and metastatic breast cancer subtype.
to display an enhanced reduction in cell viability at 20 µM rela- Cisplatin was also included as a positive control at the
4
6
tive to the 10 µM treatment. The in vitro activity of the 2-phenyl reported IC50 value of 23 µM. However, when the cytotoxic
ligands and their corresponding cyclometallated Ru(II) com- data obtained for the MDA-MB-231 cell line (Fig. S3†) were
plexes (Fig. S1†) shows that the unsubstituted 2-phenyl tris- compared to that obtained for the MCF-7 cell line, the tested
benzimidazole ligand (7) has approximately 16 and 8% greater compounds were found to display less activity in the
inhibition of cell viability compared to the corresponding tri- MDA-MB-231 cell line at 20 µM with 11 and 23% more cell via-
nuclear cyclometallated Ru(II) complex (13) at 10 and 20 µM, bility rates than that observed in the MCF-7 cell line. An excep-
respectively. However, in general, the cyclometallated com- tion was [18][PF6]3 which was 5% more potent in the
plexes (14 and 15) were observed to have slightly enhanced bio- MDA-MB-231 cell line. Comparison of the cytotoxic activity of
logical activity compared to their corresponding ligands at the most active ligands and complexes (10, 11, [16][PF
6 3
] and
both the tested concentrations. Analysis of the in vitro cytotoxic [17][PF ] ) to that of cisplatin at 20 µM in the MDA-MB-231
6
3
data obtained for the 2-pyridyl tris-benzimidazole ligands breast cancer cells reveals that all the tested compounds are
(
(
10–12) and their corresponding cationic N^N-Ru(II) complexes less active relative to cisplatin against the MDA-MB-231 breast
Fig. S2†), revealed the same trend as observed in the neutral cancer cell line (Fig. 4).
cyclometallated series. The unsubstituted 2-pyridyl ligand (10)
Based on the pre-screen data summarized in Fig. 3 and 4,
displayed more potent inhibition of cell survival compared to the 2-pyridyl ligands 10 and 11, and their corresponding tri-
its corresponding cationic complex ([16][PF ] ). On the con- nuclear cationic complexes [16][PF6]3 and [17][PF ] , respect-
6
3
6 3
trary, the trifluoromethyl ([17][PF
6
]
3
)
and the methyl ively, were selected for multidose experiments, as these com-
(
[18][PF ) complexes displayed the expected enhanced cyto- pounds showed the most promising activity at the fixed con-
6 3
]
toxicity by 10% and 16–20%, respectively, relative to their centrations of 10 and 20 µM. Briefly, the MCF-7 and the
respective ligands (11 and 12) at both tested concentrations. A MDA-MB-231 breast cancer cells were treated with a range of
comparison of the cytotoxic activity of the most active 2-pyridyl concentrations (5 to 35 µM) of the selected compounds for
trinucleating ligands (10 and 11) and their respective complexes 48 h and cell viability was measured using the MTT assay.
(
6 3 6 3
[16][PF ] and [17][PF ] ) to that of cisplatin at 10 and 20 µM Based on the data obtained, the IC50 values of the selected
in the MCF-7 breast cancer cell line (Fig. 3) reveals interesting compounds were determined and the results are summarized
in Table 2. Cisplatin was included as a positive control at the
Fig. 3 The percentage cell viability as measured by MTT assays in
MCF-7 breast cancer cells exposed to either vehicle (0.1% DMSO) or the
most active 2-pyridyl trimeric ligands (10 and 11) and their corres-
Fig. 4 The percentage cell viability as measured by MTT assays in
MDA-MB-231 breast cells exposed to either vehicle (0.1% DMSO) or the
2-pyridyl ligands (10 and 11) and their respective N^N-Ru(II) chelated
ponding trinuclear N^N-Ru(II) cationic complexes ([16][PF
17][PF ) or cisplatin at 10 and 20 µM concentrations for 48 hours.
6
]
3
and
complexes ([16][PF
20 µM) was included as the positive control.
6 3 6 3
] and [17][PF ] ) at 20 µM for 48 hours. Cisplatin (at
[
6 3
]
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Table 2 In vitro anticancer activity of the selected 2-pyridyl tris-benzimidazole ligands (10 and 11), their corresponding trinuclear cationic com-
plexes ([16][PF
6
]
3
and [17][PF
6
]
3
, respectively) and the mononuclear counterpart of the most active ligand and complex (19 and [20][PF
6
],
respectively)
IC50 (µM) ± SD
c
Compound
5-Substituent
MCF-7
MDA-MB-231
33.53 ± 1.03
MCF-12A
Selectivity index
b
b
1
0
H
H
28.65 ± 1.08
33.87 ± 1.16
38.72 ± 1.10
82.42 ± 2.29
1.35
2.43
—
3.49
—
a
[
16][PF
6
]
6
]
6
]
3
3
N/A
N/A
N/A
a
a
a
1
1
CF
CF
H
3
N/A
N/A
a
b
[
17][PF
3
35.06 ± 1.07
122.20 ± 2.62
—
—
a
1
9
N/A
—
—
a
[
20][PF
H
N/A
—
Cisplatin
—
35 (ref. 44)
23 (ref. 46)
36.32 ± 1.09
1.04
a
b
c
N/A: not active at tested concentrations. Extrapolated by GraphPad Prism V5.01. (IC50 MCF-12A/IC50 MCF-7).
IC50 values reported in the literature of 35 µM (ref. 44) and [17][PF ] , to gain insight on the selectivity of the compounds
6 3
2
3 µM (ref. 46) against the MCF-7 and MDA-MB-231 cell lines, towards breast cancer cells. The compounds were tested
respectively. At these reported IC50 values, as expected, cispla- against the MCF-12A human breast epithelial cell line, where
tin reduced the percentage cell survival to 50% in both the cisplatin was used as a positive control. The results attained
MCF-7 and MDA-MB-231 cell lines (Fig. S4 and S5†).
indicate that all of the tested compounds were less cytotoxic
The 5-unsubstituted 2-pyridyl tris-benzimidazole ligand 10 relative to cisplatin (Table 2). The compounds 10, 11, and com-
and its corresponding metal complex [16][PF ] are more active plexes [16][PF ] and [17][PF ] did not inhibit MCF-12A cell
6
3
6 3
6 3
compared to cisplatin in the MCF-7 breast cancer cell line survival below 50% (Fig. S6 and S7†). Consequently, the IC50
IC50 = 28.65 and 33.87 µM for 10 and [16][PF , respectively, values for the synthesized compounds 10, 11, [16][PF and
relative to IC50 = 35 µM for cisplatin). The enhanced activity of [17][PF ] have been extrapolated by GraphPad Prism from the
(
6
]
3
6 3
]
6 3
1
6 3 6 3
0 and [16][PF ] relative to cisplatin in the MCF-7 cell line was data summarized in Fig. S6 and S7.† The complex [17][PF ]
also observed in the pre-screen at 10 and 20 µM (Fig. 3). The has the highest selectivity, evidenced by the high IC50 value
IC50 value for the 5-trifluoromethyl substituted 2-pyridyl tris- (IC50 = 122.20 μM) and the relatively high selectivity index (SI)
benzimidazole ligand (11) could not be determined as the value of 3.49.
compound did not reduce MCF-7 cell survival below 50% at
any of the tested concentrations (Fig. S4†). The metal complex to that of cisplatin (IC50 = 36.53, SI = 1.04), it is evident that
17][PF showed comparable activity relative to cisplatin (IC50 [17][PF has activity comparable to that of cisplatin against
35.06 µM for [17][PF and IC50 = 35 µM for cisplatin), the MCF-7 cancer cell line. However, [17][PF is three times
6 3
By comparing the data attained for the complex [17][PF ]
[
6
]
3
6 3
]
=
6
]
3
6 3
]
showing an enhancement of anticancer activity due to more selective towards MCF-7 breast cancer cells relative to
the presence of the ruthenium(II) centers thus highlighting cisplatin. Although mild cytotoxic activity is often observed
the synergistic action of the metal centers and the organic in organoruthenium(II) complexes, their novel mechanisms of
4
7,48
ligand. The comparable cytotoxic activity of [17][PF ] to that action as antimetastatic and antiangiogenic agents
present
6
3
of cisplatin in the MCF-7 breast cancer cell line was also a new manifold in the design of novel anticancer agents.
reflected in the pre-screen evaluation of this compound at Therefore, it may be worthwhile investigating the possible
1
0 µM (Fig. 3).
When tested against the more aggressive triple-negative greater understanding of how these compounds exert their
MDA-MB-231 cell line, the only compound that showed signifi- anticancer effects.
mechanisms of action of these complexes in a bid to gain a
cant activity, albeit moderate activity, was the 2-pyridyl tris-
The mononuclear counterpart of the most active tris-benzi-
benzimidazole 10 (IC50 = 33.53 µM). Unfortunately, the IC50 midazole ligand and the corresponding complex, 10 and
values of all the other selected compounds, [16][PF
6 3 6 3
] , 11 and [16][PF ] , respectively, were also evaluated for their anticancer
[
17][PF ] , could not be determined as these compounds did activity against the MCF-7 breast cancer cell line. The mono-
6
3
not inhibit MDA-MB-231 cell survival to 50% or below at any of meric ligand 19 and the corresponding mononuclear cationic
the tested concentrations (Fig. S5†). Overall, the 5-unsubsti- complex [20][PF ] did not inhibit MCF-7 breast cancer cell sur-
tuted ligand 10 and its corresponding cationic trimetallic vival to 50% or below at any of the tested concentrations
complex [16][PF showed the most promising activity, with (Fig. S8†). As a result, the IC50 values of these compounds were
6
6 3
]
either activity greater than that of clinically used cisplatin (in not determined. In addition, a comparison of the results
the MCF-7 cell line) or mild activity in the MDA-MB-231 cell attained to those reported in the literature for structurally
line.
similar mononuclear and dinuclear benzimidazole-based
Cytotoxicity studies were conducted using the ligands 10 ruthenium(II) arene complexes reveals that the trinuclear com-
and 11 and their respective metal complexes, [16][PF6]3 and plexes investigated in this study show relatively enhanced
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2
2,49,50
activity.
Thus, this highlights the merit for further MCF-7 breast cancer survival to 50% or below at the tested
investigations of trinuclear systems as potential drug candi- concentrations. This further highlights the merit in investi-
dates and with a view to understanding their mechanisms of gating multimeric benzimidazole-based ligands, and also
action relative to their mononuclear and dinuclear the advantage of having more than one Ru(II) center. These
counterparts.
promising results contribute to the growing interest in multi-
nuclear organoruthenium(II) compounds as potential anti-
cancer agents, as these compounds are known to show antime-
tastatic activity and other novel mechanisms of action. Further
investigations into the possible mechanisms of action of these
Conclusions
A series of novel 5-substituted 2-phenyl (7–9) and 2-pyridyl promising compounds in this study are ongoing.
10–12) tris-benzimidazole ligands were successfully syn-
(
thesized and fully characterized. Using the synthesized tris-
benzimidazole ligands 7–12, a series of corresponding cyclo- Experimental
metallated (C^N)-Ru(II)-p-cymene (13–15) and cationic (N^N)-
Ru(II)-p-cymene ([16][PF ] –[18][PF ] ) trinuclear complexes
6 3 6 3
Materials
All reactions were carried out in an inert argon atmosphere,
unless stated otherwise. All reagents were purchased from
commercial sources (Sigma-Aldrich, Combi-blocks) and used
were prepared and fully characterised. The synthesized ligands
and complexes were all investigated for their antiproliferative
activity at fixed concentrations of 10 and 20 µM against the
MCF-7 and MDA-MB-231 breast cancer cell lines. The expected
general enhancement of the anticancer activity of the com-
pounds when tested at 20 µM compared to 10 µM was
observed in MCF-7 cells. Additionally, in general, the 2-pyridyl
ligands (10–12) showed slightly enhanced anticancer activity,
relative to their respective 2-phenyl counterparts (7–9). The
same trend was observed in the trimetallic complexes, where
the cationic complexes ([16][PF ] –[18][PF ] ) were observed to
without further purification. The [Ru(p-cymene)Cl
2 2
] dimeric
5
1
precursor was prepared following a literature method. The
1
2
2
ligand
precursors
N -(2-nitrophenyl)-N ,N -bis(2-((2-nitro-
1
phenyl)amino)ethyl)ethane-1,2-diamine and N -(2-(bis(2-((2-
5
2
aminophenyl)amino)ethyl)amino)ethyl)benzene-1,2-diamine
were synthesized following adapted literature methods.
Equipment and instrumentation
6
3
6 3
show significantly enhanced anticancer activity relative to the Reactions were monitored by TLC using aluminium-backed
corresponding cyclometallated neutral analogues (13–15). Merck silica-gel F254 plates, and compounds were visualised
From these pre-screens, 10 and 11, and their corresponding under UV-lamp. All column chromatography was carried out
trimetallic cationic complexes [16][PF
6
]
3
and [17][PF
6
]
3
,
using Fluka Silica Gel 60, 40–63 microns. Nuclear Magnetic
respectively, were selected for a further screening at various Resonance spectra were recorded on either a Bruker X400 MHz
1
13
concentrations of 5–35 µM in order to determine their IC50 spectrometer ( H at 399.95 MHz and C at 100.65 MHz) or a
1
13
values against the MCF-7 and MDA-MB-231 breast cancer cell Varian Mercury XR300 MHz ( H at 299.95 MHz,
C at
lines. From the multidose screens, 10 and the corresponding 75.46 MHz) with tetramethylsilane (TMS) as the internal stan-
complex [16][PF6]3 were observed to have cytotoxic activity dard for chemical shifts. Chemical shifts and J-coupling values
greater than that of cisplatin against the MCF-7 cell line. were reported in ppm and Hz, respectively. Infrared spec-
The complex [17][PF
comparable to cisplatin against the MCF-7 cell line. However, spectrometer using Attenuated Total Reflectance (ATR) with
6 3
]
was observed to have cytotoxicity troscopy was conducted on a Perkin-Elmer Spectrum 100 FT-IR
−
1
against the more aggressive MDA-MB-231 breast cancer cell bond vibrations measured in reciprocal centimetres (cm ).
line, the ligand 11, its corresponding metal complex [17][PF
Mass spectrometry (MS) determinations were carried out using
6 3
]
and the complex [16][PF6]3 were all inactive at the tested Electron Impact (EI) on JEOL GCmatell instrument or
concentrations. On the contrary, 10 was observed to possess Electrospray Ionisation (ESI) on a Waters API Quattro Micro
mild activity against the MDA-MB-231 cell line. In light of triple quadrupole mass spectrometer with data recorded using
their promising activity, selected compounds were evaluated the positive mode.
A Büchi Melting Point Apparatus
against the MCF-12A human breast epithelial cell line to B-540 machine was used to obtain the uncorrected melting
determine selectivity. Generally, the screened compounds points of each compound. Determination of C/H/N was done
were observed to be less cytotoxic and more selective using a 2400 CHN elemental analyzer by Perkin Elmer.
6 3
relative to cisplatin. Interestingly, the complex [17][PF ] was
observed to have activity comparable to cisplatin against the
MCF-7 breast cancer cell line and to be approximately three
The general procedure for the synthesis of tris-nitrobenzene
precursors (1–3)
times more selective for MCF-7 breast cancer cells. Lastly, the Tris(2-aminoethyl)amine (1 eq.) was dissolved in DMF (5 mL)
mononuclear complex of the most active tris-ligand and at room temperature, under argon atmosphere. Thereafter, the
complex, 10 and [16][PF ] , respectively, were synthesized and appropriate nitrobenzene (3 eq.) was added to the reaction
6
3
evaluated for their anticancer activity against the MCF-7 breast vessel and the reaction was allowed to stir at room temperature
cancer cell line. Both the monomeric ligand and the corres- for 24 h under argon, reaction progress was monitored by
ponding cationic complex, 19 and [20][PF ], did not inhibit TLC analysis. Upon completion, the reaction mixture was
6
Dalton Trans.
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diluted with a saturated brine solution (30 mL) and the argon. After 1 hour, TLC analysis showed a complete conver-
extracted with two aliquots of ethyl acetate (2 × 30 mL). The sion of the limiting reagent to a new product. The reaction
organic extracts were combined and dried over anhydrous mixture was filtered through Celite® and rinsed with copious
sodium sulfate, and excess solvent was removed in vacuo. The methanol. The filtrate was subsequently collected, and the
resultant crude product was purified using column chromato- excess solvent was reduced by rotary evaporation. The resultant
graphy to afford the desired tris-nitrobenzene product.
residue was re-dissolved in ethyl acetate (30 mL) and washed
1
2
2
N -(2-Nitrophenyl)-N ,N -bis(2-((2-nitrophenyl)amino)ethyl) with two aliquots of a saturated sodium bicarbonate solution
5
2
ethane-1,2-diamine (1). Tris(2-aminoethyl)amine (0.500 mL, (2 × 30 mL), a 1 M solution of sodium hydroxide (30 mL) and
3
1
.34 mmol) was reacted with 1-fluoro-2-nitrobenzene (1.06 mL, deionized water (30 mL). The organic extract was collected,
0.1 mmol) at room temperature for 24 h, under argon. The dried over anhydrous sodium sulfate and excess solvent was
desired compound (1) was isolated as a bright yellow solid reduced in vacuo and the resultant crude was purified using
1.11 g, 2.18 mmol). R (4 : 6 ethyl acetate : petroleum ether): column chromatography (100% ethyl acetate).
(
0
(
f
1
1
.50. Yield: 48.3%. H NMR (300 MHz, DMSO) δ (ppm): 8.21
N -(2-(Bis(2-((2-aminophenyl)amino)ethyl)amino)ethyl)benzene-
3
3
4
52
1
2
2
t, J
= 5.1 Hz, 3H, NH
̲
= 8.6 Hz, J
= 1.6 1,2-diamine (4). N -(2-Nitrophenyl)-N ,N -bis(2-((2-nitrophe-
HH
HH
3
HH
2
4
Hz, 3H, ArH
̲
), 7.46 (ddd, JHH = 8.5 Hz, JHH = 7.0 Hz, JHH
=
nyl)amino)ethyl)ethane-1,2-diamine (0.810 g, 1.59 mmol) was
), 6.64 (ddd, reacted with ammonium chloride (2.52 g, 47.0 mmol) and zinc
= 1.1 Hz, 3H, ArH ̲) , 3.45 (q, J = powder (6.16 g, 94.2 mmol) at room temperature for 24 h.
HH
1
.4 Hz, 3H, ArH ̲) , 6.98 (d, JHH = 8.0 Hz, 3H, ArH̲
2
3
JHH = 8.3 Hz, J
.1 Hz, 6H, CH
= 6.9, J
HH
13
1
6
̲
2 2
), 2.90 (t, JHH = 6.3 Hz, 6H, CH̲ ). C{ H}- The desired product (4) was isolated as a dark brown solid
1
NMR (101 MHz, DMSO) δ (ppm): 145.50, 136.87, 131.53, (0.778 g, 1.86 mmol). R
1
f
(ethyl acetate): 0.45. Yield: 97.0%. H
3
26.64, 115.55, 114.85, 52.93, 41.19. MP (°C): 103.8–105.3.
N -(2-Nitro-4-(trifluoromethyl)phenyl)-N ,N -bis(2-((2-nitro-
-(trifluoromethyl)phenyl)amino)ethyl)ethane-1,2-diamine (2). NH
NMR (300 MHz, DMSO) δ (ppm): 6.57 (dd, JHH = 7.2 Hz,
HH = 1.5 Hz, 3H, ArH
1
2
2
4
J
̲
̲
3
4
4
̲
and NH ), 3.13 (dd,
̲
2
3
Tris(2-aminoethyl)amine (0.500 mL, 3.34 mmol) and 4-chloro- CH̲ ), 2.79 (t, J = 6.6 Hz, 6H, CH̲ ). C{ H}-NMR (101 MHz,
2 HH 2
3
-nitrobenzotrifluoride (1.49 ml, 10.0 mmol) were dissolved DMSO) δ (ppm): 136.77, 135.75, 118.42, 117.48, 114.98,
in DMF and allowed to stir at room temperature for 24 h, 110.67, 53.61, 42.18. FT-IR (ATR) ν (cm− ): 3366 and 3299
1
under argon. The pure compound (2) was isolated as a bright (1° amine). MP (°C): 121.7–123.8.
1
yellow solid (0.841 g, 1.18 mmol). R
f
(2 : 1 petroleum ether :
N -(2-(Bis(2-((2-amino-4-(trifluoromethyl)phenyl)amino)ethyl)
1
ethyl acetate): 0.56. Yield: 35.3%. H NMR (300 MHz, DMSO) amino)ethyl)-4-(trifluoromethyl)benzene-1,2-diamine
(5).
3
3
1
2
2
δ (ppm): 8.45 (t, J = 5.0 Hz, 3H, NH
̲) , 8.15 (d, J = 1.6 Hz, N -(2-Nitro-4-(trifluoromethyl)phenyl)-N ,N -bis(2-((2-nitro-4-(tri-
HH
3
HH
4
3
J
H, ArH ̲) , 7.66 (dd, JHH = 9.1, JHH = 2.1 Hz, 3H, ArH̲
HH = 9.1 Hz, 3H, ArH̲
3
3
4
3
13
1
6
H, CH̲ ), 2.92 (t, JHH = 5.9 Hz, 6H, CH̲ ). C{ H}-NMR zinc powder (3.32 g, 50.8 mmol) were reacted in anhydrous
2 2
101 MHz, DMSO) δ (ppm): 146.49, 131.53, 129.96, 124.97, methanol at room temperature for 1 h, under argon. The
(
1
23.68, 115.60, 114.70, 52.17, 40.80. MP (°C): 170.6–172.1.
product (5) was isolated as a beige solid (0.660 g, 0.706 mmol).
1
2
2
N -(4-Methyl-2-nitrophenyl)-N ,N -bis(2-((4-methyl-2-nitro-
phenyl)amino)ethyl)ethane-1,2-diamine (3). Tris(2-amino-
ethyl)amine (0.500 mL, 3.34 mmol) and 4-fluoro-2-nitrotolu- 3H, ArH
ene (1.55 g, 10.0 mmol) were dissolved in DMF and allowed Hz, 3H, ArH
to stir at room temperature for 24 h, under argon. The pure 3.19 (dd, JHH = 11.9 Hz, JHH = 6.0 Hz, 6H, CH
compound (3) was isolated as a dark orange solid (0.887 g, = 6.6 Hz, 6H, CH
R (1 : 1 ethyl acetate : petroleum ether): 0.97. Yield: 83.8%.
f
1
3
H NMR (300 MHz, DMSO) δ (ppm): 6.83 (d, JHH = 2.0 Hz,
3
3
̲) , 6.74 (d, JHH = 8.3 Hz, 3H, ArH̲
3
̲
̲
̲
2
3
HH
4
3
̲
2
), 2.78 (t, JHH
1
3
1
̲
2
). C{ H}-NMR (101 MHz, DMSO) δ (ppm):
4
1
.62 mmol). R (4 : 1 petroleum ether : ethyl acetate): 0.51. 139.03, 135.08, 124.07, 116.46 (q, JC–F = 31.0 Hz), 114.70,
f
1
3
−1
Yield: 39.2%. H NMR (300 MHz, DMSO) δ (ppm): 8.08 (t, JHH 109.69, 108.28, 52.63, 41.35. FT-IR (ATR) ν (cm ): 3342 and
3
=
5.0 Hz, 3H, NH
̲
), 7.79 (d, JHH = 1.0 Hz, 3H, ArH
̲
3
4
3
1
JHH = 8.8 Hz, J
= 1.9 Hz, 3H, ArH
), 3.41 (dd, JHH = 11.5 Hz, JHH = 5.9 Hz, 6H, CH
̲
N -(2-(Bis(2-((2-amino-4-methylphenyl)amino)ethyl)amino)
HH
HH
3
4
1
3
2
H, ArH
̲
̲
), ethyl)-4-methylbenzene-1,2-diamine (6). N -(4-Methyl-2-nitro-
3
1
2
2
2 3
.87 (t, JHH = 6.2 Hz, 6H, CH̲ ), 2.20 (s, 9H, CH̲ ). C{ H}- phenyl)-N ,N -bis(2-((4-methyl-2-nitrophenyl)amino)ethyl)ethane-
NMR (101 MHz, DMSO) δ (ppm): 143.33, 137.76, 130.53, 1,2-diamine (1.11 g, 2.01 mmol) was reacted with ammonium
1
25.07, 124.02, 114.39, 52.47, 40.7, 19.36. MP (°C): chloride (3.17 g, 59.3 mmol) and zinc powder (6.76 g, 103 mmol)
1
25.6–127.3.
in anhydrous methanol, under argon for 1 h. The desired
product (6) was isolated as a brown solid (0.668 g, 1.45 mmol).
General synthesis of tris-1,2-benzenediamines (4–6)
1
R
f
(ethyl acetate): 0.14. Yield: 75.1%. H NMR (300 MHz,
3
The appropriate tris-nitrobenzene (1 eq.) was dissolved in dry DMSO) δ (ppm): 6.40 (d, JHH = 1.1 Hz, 3H, ArH
methanol (10 mL), under argon and allowed to stir for H-e, ArH
minutes. Ammonium chloride (30 eq.) and zinc (60 eq.) were 3.07 (d, JHH = 4.8 Hz, 6H, CH̲
added to the reaction vessel, under argon. The reaction vessel CH
̲
3
̲
̲
), 4.25 (t, J
̲
2
HH
3
3
5
2
1
3
1
̲
2 3
), 2.08 (s, 9H, CH̲ ). C{ H}-NMR (101 MHz, DMSO)
was the allowed to stir at room temperature for 1 h, under δ (ppm): 135.96, 134.42, 125.99, 118.60, 115.90, 111.12, 53.70,
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2.47, 20.91. FT-IR (ATR) ν (cm− ): 3318 and 3373 (1° amine). (imine CvN). MP (°C): 197.6–199.8. Purity 97% by LC
1
4
MP (°C): 128.3–130.2.
(tR 2.703 min).
Tris(2-(2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl)amine
General synthetic procedure for the tris-benzimidazole ligands
(
10). Pale brown solid (10) (0.0300g, 0.0441 mmol). R
ethyl acetate : petroleum ether): 0.22. Yield: 34.8%. H NMR
f
(7 : 3
(7–12)
1
3
The appropriate tris-1,2-benzenediamine (4–6) (1 eq.) was dis- (300 MHz, DMSO) δ (ppm): 8.30 (m, 6H, ArH
̲
=
4
solved in dry ethanol (6 mL), and allowed to stir under argon 7.8 Hz,
J
HH = 1.5 Hz, 3H, ArH
̲
̲
),
2), 2.97
). C{ H}-NMR (101 MHz, DMSO) δ
3
for 5 minutes. Benzaldehyde or 2-pyridinecarboxaldehyde (3.6 7.41–7.23 (m, 12H, ArH
̲
̲
HH
3
13
1
eq.) was added to the reaction vessel, under argon. Thereafter, (t, JHH = 6.9 Hz, 6H, CH
̲
2
magnesium sulfate (18 eq.) and trifluoroacetic acid (0.3 eq.) (ppm): 149.81, 149.37, 148.39, 142.04, 137.26, 136.22, 124.17,
were added to the reaction vessel, under argon. The reaction 124.02, 123.13, 122.32, 119.53, 110.42, 53.64, 43.38. FT-IR
mixtures with benzaldehyde were refluxed at 80 °C for (ATR) ν (cm− ): 1733 (imine CvN), 1586 (pyridyl CvN). MP
1
2
4
h
and the reactions with 2-pyridinecarboxaldehyde (°C): 183.5–184.9. Purity 99% by LC (t
were stirred at room temperature for 24 h, open to air. TLC Tris(2-(2-(pyridin-2-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-
analysis confirmed the successful conversion of starting 1-yl)ethyl)amine (11). Cream white solid (11) (0.0203 g,
materials to a new product. The reaction mixture was sub- 0.0229 mmol). R (2 : 1 petroleum ether : ethyl acetate): 0.13.
sequently filtered and the filtrate collected. The excess solvent Yield: 40.3%. H NMR (300 MHz, DMSO) δ (ppm): 8.35 (d, J =
R
2.816 min).
f
1
3
was removed by rotary evaporation, and the resultant crude 4.2 Hz, 3H, ArH ̲) , 8.29 (d, JHH = 7.9 Hz, 3H, ArH̲
3
was re-dissolved in ethyl acetate (30 mL) and washed with a ArH
saturated sodium bicarbonate solution (30 mL) and a satu- ArH), 7.42–7.33 (m, 3H, ArH
rated brine solution (30 mL). The organic extract was collected 3.03 (t,
̲
̲
3
̲
2̲ ),
HH
1
HH = 6.3 Hz, 6H, CH
and dried over anhydrous sodium sulfate, and the excess DMSO) δ (ppm): 152.29, 149.76, 149.06, 142.03, 139.03,
solvent was removed in vacuo. The resultant residue was puri- 137.96, 125.12, 125.06, 124.10, 120.03, 117.48, 112.14, 60.06,
53.86, 44.28. FT-IR (ATR) ν (cm− ): 1620 (imine CvN), 1587
1
fied using column chromatography.
Tris(2-(2-phenyl-1H-benzo[d]imidazol-1-yl)ethyl)amine (7). (pyridyl CvN). MP (°C): 201.3–202.0. Purity 94% by LC
Pale yellow solid (7) (0.0740g, 0.109 mmol). Rf (7 : 3 ethyl (tR 3.038 min).
1
acetate : petroleum ether): 0.21. Yield: 51.1%.
H
NMR
Tris(2-(5-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl)
(7 : 3
(
(
7
400 MHz, DMSO) δ (ppm): 7.68–7.62 (m, 3H, ArH
m, 6H, ArH), 7.48–7.40 (m, 9H, ArH
.20–7.15 (m, 3H, ArH
̲
f
1
̲
̲
̲
3
3
̲
̲
2
), 2.41 (400 MHz, CDCl
3
) δ (ppm): 8.41 (d, JHH = 8.0 Hz, 3H, ArH
̲
),
=
3
13
1
3
3
4
(t, JHH = 7.1 Hz, 6H, CH
̲
). C{ H}-NMR (101 MHz, DMSO) 8.30 (d, JHH = 4.5 Hz, 3H, ArH
δ (ppm): 153.59, 143.0, 135.71, 130.81, 130.08, 129.43, 129.13, 1.8 Hz, 3H, ArH
̲
̲
̲
̲
),
1
̲
2
̲
2
), 2.51 (s, 9H,
−
1
13
1
ν (cm ): 1695 (imine CvN). MP (°C): 193.6–196.2. Purity 98% CH
̲
3
3
) δ (ppm): 150.50, 149.65,
by LC (t 2.823 min).
R
−
1
Tris(2-(2-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl) 119.91, 109.4, 54.66, 44.38, 21.60. FT-IR (ATR) ν (cm ): 1726
ethyl)amine (8). Cream solid (8) (0.0952 g, 0.108 mmol). R
f
(imine CvN), 1590 (pyridyl imine CvN). MP (°C):170.1–173.5.
1
(
1 : 1 ethyl acetate : petroleum ether): 0.18. Yield: 70.3%.
NMR (300 MHz, DMSO) δ (ppm): 8.01 (s, 3H, ArH), 7.61–7.54
), 7.52 (dd, JHH = 8.7 Hz, JHH = 1.4 Hz, 3H, ArH
H
Purity 99% by LC (t 2.969 min).
R
̲
Cyclometallated Ru(II)-p-cymene metal complex (13). Tris(2-
3
4
(
7
(
(
m, 6H, ArH
.48–7.39 (m, 12H, ArH
t, J = 6.6 Hz, 6H, CH
ppm): 155.87, 142.41, 138.00, 130.51, 130.11, 129.49, 129.21, nium(II) dimer (0.0474 g, 0.0774 mmol), sodium acetate
̲
̲
(0.0353g,
3
̲) , 3.88 (t, J = 6.6 Hz, 6H, CH̲
HH
1
3
1
̲
2
4
1
5
(
26.78, 123.55 (q, JC–F = 35.9 Hz), 119.47, 117.06, 112.01, (0.00853 g, 0.103 mmol) was added and the mixture was
−
1
2.43, 42.88. FT-IR (ATR) ν (cm ): 1626 (imine CvN). MP allowed to stir at room temperature for 24 h, under argon.
2.880 min). After TLC analysis confirmed the conversion of the limiting
Tris(2-(5-methyl-2-phenyl-1H-benzo[d]imidazol-1-yl)ethyl)amine reagent to a product spot, the reaction mixture was filtered
9). White solid (9) (0.0723 g, 0.100 mmol). R (1 : 1 petroleum through Celite®. The filtrate was collected, and excess solvent
°C): 204.4–205.1. Purity 94% by LC (t
R
(
f
1
ether : ethyl acetate): 0.12. Yield: 46.4%. H NMR (300 MHz, was reduced to ca. 1 mL, and this crude mixture was subjected
CDCl ) δ (ppm): 7.58 (s, 3H, ArH), 7.55–7.48 (m, 6H, ArH), to trituration in chloroform for 24 h. A dark green crude
was isolated by suction filtration, and this was re-dissolved in
), 3.69 (t, JHH DCM (1 mL) and precipitated in pentane. The desired product
̲
̲
3
3
4
7
1
=
6
1
1
.44–7.35 (m, 9H, H-m, ArH ̲) , 7.07 (dd, JHH = 8.2 Hz, JHH =
.1 Hz, 3H, ArH
3
3
̲) , 6.78 (d, JHH = 8.2 Hz, 3H, ArH̲
3
7.0 Hz, 6H, CH
̲
), 2.50 (s, 9H, CH
̲
), 2.37 (t, J
= 7.0 Hz, (13) was isolated as a light green powder (0.0704 g,
2
3
HH
1
3
1
1
H, CH
̲
2 3
). C{ H}-NMR (101 MHz, CDCl ) δ (ppm): 153.41, 0.0473 mmol) by suction filtration. Yield: 84.9%. H NMR
3
43.07, 133.17, 132.53, 130.37, 129.92, 129.07, 128.83, 124.55, (300 MHz, DMSO) δ (ppm): 8.27 (d, JHH = 7.3 Hz, 3H, ArH
̲) ,
19.94, 109.07, 53.43, 42.78, 21.56. FT-IR (ATR) ν (cm− ): 1625 7.97 (d, J = 8.0 Hz, 3H, ArH
1
3
̲) , 7.60–7.12 (m, 12H, ArH ̲) , 7.06
HH
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3
3
(
t, JHH = 7.4 Hz, 3H, ArH
̲
̲
=
removed and the resultant crude was re-dissolved in ca. 1 mL
4
1
5
3
3.7 Hz, J
= 6.9 Hz, 3H, Arp-cye), 5.75–5.63 (m, 3H, Arp-cye), DCM and hexane (12 mL) was subsequently added to the solu-
HH
3
.37 (dt, JHH = 11.2, 5.7 Hz, 3H, Arp-cye), 5.19 (t, JHH = 5.8 Hz, tion. This resulted in the precipitation of a dark yellow-brown-
H, Arp-cye), 4.42 (s, 6H, CH ), 2.92 (bs, 6H, CH ), 1.63 (s, 9H, ish precipitate (15) which was isolated via suction filtration
), 0.70–0.54 (m, 9H, CH(CH ), 0.53–0.36 (m, 9H, (0.0638 g, 0.0417 mmol). Yield: 88.3%. H NMR (300 MHz,
1
CH
CH(CH
̲
3
p-cye
1
3
1
3
3
2
p-cye). C{ H}-NMR (101 MHz, DMSO) δ (ppm): DMSO) δ (ppm): 8.26 (t,
J
HH = 6.7 Hz, 3H, ArH
̲) , 7.72 (d,
3
1
8
(
41.34, 124.11, 123.63, 122.54, 117.91, 111.05, 89.75, 89.88,
JHH = 4.4 Hz, 3H, ArH
̲
̲
3
2.56, 81.06, 53.93, 43.59, 30.69, 22.42, 21.70, 18.84. FT-IR (m, 3H, Arp-cye), 5.68 (t, J
= 6.0 Hz, 3H, Arp-cye), 5.47–5.27
HH
ATR) ν (cm− ): 1580 (imine CvN). MP (°C): 275.1 (decomp.). (m, 3H, Arp-cye), 5.23–5.12 (m, 3H, Arp-cye), 4.54–4.23 (m, 6H,
1
MS (HR-ESI, m/z): Calculated: 1488.2580, Found: 1488.2526 CH
̲
2
), 2.84–2.74 (m, 6H, CH
2
), 2.65–2.55 (m, 6H, Ar–CH ), 2.42
3), 1.95–1.62 (m, 12H, CH̲ p-cye,
3
+
3
(
100%, [M + H] ).
Trifluoromethyl substituted Ru(II) cyclometallated complex CH
14). Tris(2-(2-phenyl-5-(trifluoromethyl)-1H-benzo[d]imidazol- (m, 9H, CH(CH
-yl)ethyl)amine (0.0210 g, 0.0227 mmol) was dissolved in δ (ppm): 157.46, 146.16, 145.41, 141.62, 141.32, 135.00, 134.15,
(d, JHH = 3.9 Hz, 3H, CH
̲
3
̲
3
)
2
̲
3
)
2
p-cye), 0.51–0.35
1
3
1
(
3 2
̲ ) p-cye). C{ H}-NMR (151 MHz, DMSO)
1
anhydrous acetonitrile (10 mL), under argon. The dichloro(p- 134.05, 133.00, 129.25, 128.10, 126.53, 124.91, 123.93, 122.46,
cymene)ruthenium(II) dimer (0.0222 g, 0.0363 mmol) and 89.88, 88.88, 82.93, 80.84, 33.43, 30.78, 24.43, 22.38, 21.79,
sodium acetate (0.0112 g, 0.136 mmol) were then added to 21.01, 18.94. MS (HR-ESI, m/z): Calculated: 1494.0329,
+
the reaction vessel, under argon. The reaction mixture was Found: 1494.3296 (70% [M − Cl] ). Elemental analysis for
O (1583.184 g mol− ): Found (%) C, 59.36%;
1
then allowed to stir at room temperature for 24 h, after which
C H84Cl N Ru ·3H
78 3 7 3 2
the complete reaction of the limiting reagent was observed H, 5.82%; N, 5.85%; Calculated (%) C, 59.18%; H, 5.73%;
upon TLC analysis. The reaction mixture was then filtered N, 6.19%.
through Celite® and the filtrate was collected. Excess solvent
was removed by rotary evaporation and the resulting crude was
re-dissolved in DCM (ca. 1 mL). Hexane was subsequently
added to the vessel, resulting in a dark yellow precipitate The ruthenium dimer [Ru(η -p-Pr C
General synthetic procedure for the cationic N,N-Ru(II)-p-
cymene metal complexes ([16][PF ] –[18][PF ] )
6
3
6 3
6
i
6
H
4
2 2
Me)Cl ] (1.5 eq.) was
(
0
14) which was isolated by suction filtration (0.0479 g, added to a stirring solution of the appropriate 2-pyridyl tris-
1
.0283 mmol). Yield: 83.3%. H NMR (300 MHz, CDCl
3
)
benzimidazole ligand (1 eq.) in
), DCM : ethanol, under argon. The reaction mixture was allowed
to stir at room temperature for 24 h, under argon. After
.7 Hz, 3H, Arp-cye), 5.66 (t, JHH = 5.4 Hz, 3H, Arp-cye), 5.36–5.16 TLC analysis confirmed the complete reaction of the limiting
a
1 : 1 solution of
3
δ (ppm): 8.30 (d, JHH = 7.5 Hz, 3H, ArH
7
5
̲
̲
3
.59–7.23 (m, 15H, ArH
̲
=
3
3
(m, 3H, Arp-cye), 5.02 (t, JHH = 5.3 Hz, 3H, Arp-cye), 3.67 (bs, 6H, reagent, the contents of the reaction flask were filtered
3
CH
̲
), 2.33–2.08 (m, 9H, CH
̲
, CH
̲
), 1.98 (dd, J
=
through Celite® and the filtrate was collected. NH PF (4 eq.)
4 6
2
2
3 2 p-cye
HH
4
3
5
.4 Hz, JHH = 2.4 Hz, 9H, CH̲3 p-cye), 0.80 (dd, JHH = 13.8 Hz, was added to the filtrate and was allowed to stir for at room
4
3
J
HH = 8.4 Hz, 9H, CH
̲
3
)2 p-cye), 0.64 (dd, JHH = 12.6 Hz, temperature for 1 h, under argon. The DCM was removed
4
1
̲
3 2
3
1
1
8
2
31.85, 130.41, 126.40, 123.88, 121.63, 116.83–114.65 (m), isolated by suction filtration, washed with cold ethanol and
13.66, 103.81–103.05 (m), 101.64–100.21 (m), 91.49, 90.30, dried.
3.33, 81.98, 53.39, 45.73 (d, J = 37.0 Hz), 44.24, 32.30, 31.09,
3.86, 23.17, 20.37, 15.50. FT-IR (ATR) ν (cm ): 1581 (imine (2-(2-phenyl-1H-benzo[d]imidazol-1-yl)ethyl)amine (0.0354 g,
Cationic N,N-Ru(II)-p-cymene metal complex ([16][PF
6 3
] ). Tris
−
1
CvN). MS (HR-ESI, m/z): Calculated: 1156.9920, Found: 0.0520 mmol) was reacted with dichloro(p-cymene)ruthenium(II)
+
1
157.3227 (40% [M + Na − C26
lysis for C78 75Cl Ru ·3H
%) C, 53.95%; H, 4.31%; N, 6.12%; Calculated (%) C, 53.69%; and stirred for 1 h. The desired product ([16][PF ] ) was iso-
H
25ClF
3 2
N Ru] ). Elemental ana- dimer (0.0478 g, 0.0780 mmol) at room temperature for
−
1
H
3
F
9
N
7
3
2
O (1745.098 g mol ): Found 24 h. NH
4 6
PF (0.0487 g, 0.299 mmol) was added to crude
(
6
3
H, 4.68%; N, 5.62%.
Methyl substituted Ru(II) cyclometallated complex (15). 0.0265 mmol). Yield: 51.1%. H NMR (300 MHz, DMSO)
Tris(2-(5-methyl-2-phenyl-1H-benzo[d]imidazol-1-yl)ethyl)amine δ (ppm): 9.69–9.55 (m, 3H, ArH), 8.14–7.92 (m, 9H, ArH),
), 6.31 (d, JHH = 6.0 Hz, 3H, Arp-cye),
lated by vacuum filtration as a dark yellow solid (0.0435 g,
1
̲
̲
3
(
0.0291 g, 0.0405 mmol) was dissolved in anhydrous aceto- 7.83–7.40 (m, 12H, ArH̲
3
3
nitrile (8 mL) under argon. To this transparent 6.27 (d, JHH = 6.0 Hz, 3H, Arp-cye), 6.13 (d, JHH = 5.1 Hz, 3H,
solution, dichloro(p-cymene)ruthenium(II) dimer (0.0384 g, Arp-cye), 6.05 (d, J JHH = 5.8 Hz, 3H, Arp-cye), 4.60–4.23 (m, 6H,
3
0
.0627 mmol), sodium acetate (0.0350 g, 0.427 mmol) was CH
2
), 3.11–2.80 (m, 6H, CH
3 p-cye), 0.91–0.73 (m, 9H, CH(CH
C{ H}-NMR (101 MHz, DMSO) δ (ppm): 157.98, 148.39,
̲
2 3 2
), 2.42–2.24 (m, 3H, CH ̲( CH )
added and the mixture was allowed to stir at room temperature p-cye), 2.13 (s, 9H, CH
for 24 h, under argon. After TLC analysis (in ethyl acetate)
̲
3
̲ )2 p-cye).
1
3
1
confirmed the complete conversion of the limiting reagent to 145.46, 140.23, 127.64, 126.90, 126.18, 124.69, 119.46, 119.31,
a product spot, the reaction mixture was filtered through 119.19, 112.94, 84.53, 83.16, 80.72, 79.64, 52.32, 44.36, 30.96,
3
1
1
Celite®. The filtrate was collected and excess solvent was 22.25, 22.13, 19.09. P{ H}-NMR (162 MHz, DMSO) δ (ppm):
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). FT-IR (ATR) ν (cm− ): 1599 ruthenium(II) dimer (0.107 g, 0.176 mmol) at room tempera-
1
−
144.13 (sep, J = 711.2 Hz, PF
6
(
imine CvN), 1481 (pyridyl CvN). MP (°C): 225.2 (decomp.). ture for 24 h. NH PF (0.0494 g, 0.301 mmol) was added
4
6
MS (HR-ESI, m/z): Calculated: 1782.9679, Found: 1783.0917 to crude and stirred for 1 h. The desired product ([20][PF
6
])
+
(
40%, [M − PF
6
] ).
was isolated by vacuum filtration as a yellow solid (0.0899 g,
1
Trifluoromethyl substituted cationic N,N-Ru(II)-p-cymene 0.141 mmol). Yield: 81.3%. H NMR (300 MHz, DMSO)
6
metal complex ([17][PF
6
]
3
). The ruthenium dimer [Ru(η -p- δ(ppm): 9.72 (d, J = 5.1 Hz, 1H, ArH
(0.0636 g, 0.104 mmol) was reacted with tris ArH), 8.36 (t, J = 7.4 Hz, 1H, ArH), 8.13 (dd, J = 6.9, 1.8 Hz, 1H,
2-(2-(pyridin-2-yl)-5-(trifluoromethyl)-1H-benzo[d]imidazol-1- ArH), 8.09–8.03 (m, 1H, ArH), 7.90–7.82 (m, 1H, ArH),
yl)ethyl)amine (0.0529 g, 0.0735 mmol) at room temperature. 7.75–7.62 (m, 2H, ArH), 6.38 (d, J = 6.1 Hz, 1H, Arp-cye), 6.33 (d,
NH PF (0.0356 g, 0.434 mmol) was added to the filtrate and J = 6.2 Hz, 1H, Arp-cye), 6.21 (d, J = 6.1 Hz, 1H, Arp-cye), 6.11 (d,
stirred for 1 h. The desired product ([17][PF ] ) was isolated J = 6.1 Hz, 1H, Arp-cye), 5.04–4.76 (m, 2H, C CH ), 2.48–2.38
̲) , 8.52 (d, J = 8.1 Hz, 1H,
i
Pr C
(
6
H
4
Me)Cl
2
]
2
̲
̲
̲
̲
̲
̲
4
6
̲H ̲
2
6
3
3
as a bright yellow solid (0.0523 g, 0.0293 mmol). Yield: 69.3%. (m, 1H, C
̲
̲
3
)
2
p-cye), 2.24 (s, 3H, CH
), 0.92–0.85 (m, 6H, CH(C
C{ H}-NMR (151 MHz, DMSO) (ppm): 157.91, 148.30, 145.64,
), 6.37–6.26 (m, 6H, Arp-cye), 6.13 (dd, JHH = 13.1 Hz, 140.86, 140.45, 135.78, 127.70, 126.81, 126.02, 125.15, 119.10,
HH = 6.5 Hz, 3H, Arp-cye), 6.07–5.99 (m, 3H, Arp-cye), 4.68–4.32 112.88, 104.79, 103.27, 86.90, 84.54, 83.16, 80.57, 40.93, 30.97,
2), 3.12–2.89 (m, 6H, CH2), 2.47–2.40 (m, 3H, 22.13, 22.10, 19.10, 15.19. MS (HR-ESI, m/z): Calculated:
2 p-cye, 2.13–2.08 (m, 9H, CH3 p-cye), 0.91–0.83 (m, 18H, 494.0937, Found: 494.0948 (100% [M − PF
̲
3
p-cye), 1.43 (t, J =
1
H NMR (600 MHz, DMSO) δ (ppm): 9.68–9.60 (m, 3H, ArH
.17–7.99 (m, 9H, ArH
̲
2
C
̲
̲
3
3 2
̲H ̲ )
p-cye).
1
3
1
8
̲) , 7.94–7.83 (m, 6H, ArH ̲) , 7.82–7.70 (m,
3
3
H, ArH
̲
4
J
(m, 6H, CH
̲
̲
+
CH
CH(CH
1
1
5
̲( CH
3
)
)
̲
6
] ). FT-IR (ATR)
1
3
1
−1
̲
3
2
p-cye). C{ H}-NMR (151 MHz, DMSO) δ (ppm): ν (cm ): 1592 (imine CvN), 1493 (pyridyl CvN). MP (°C):
58.13, 150.88, 144.84, 140.91, 139.88, 138.47, 128.22, 125.76, 186.3 (decomp.).
23.38, 116.14, 115.05, 104.51, 87.20, 84.96, 83.41, 81.69,
3
1
1
X-ray structure analysis
2.48, 45.20, 31.46, 22.31, 22.16, 19.34.
P{ H}-NMR
(
162 MHz, DMSO) δ (ppm): −144.10 (sep, J = 711.1 Hz, PF ). Single crystals of 7 and 8 were obtained by slow diffusion of a
6
1
9
F NMR (377 MHz, DMSO) δ (ppm): −59.32 (s, J = 7.4 Hz), concentrated solution of 7 or 8 in dichloromethane layered
70.34 (dd, J = 711.0, 13.7 Hz). FT-IR (ATR) ν (cm− ): 1572 with ethyl acetate, at room temperature over two days. Single-
1
−
(imine CvN), 1490 (pyridyl CvN). MP (°C): 231.2 (decomp.). crystal X-ray diffraction data were collected on a Bruker KAPPA
MS (HR-ESI, m/z): Calculated: 1986.4225, Found: 1986.0101 APEX II DUO diffractometer using graphite-monochromated
+
(
20%, [M − PF
6
] ).
MoKα radiation (λ = 0.71073 Å). Data collection was carried
Methyl substituted cationic N,N-Ru(II)-p-cymene metal out at 173(2) K. Temperature was controlled by an Oxford
6
complex ([18][PF
6
]
3
). The ruthenium dimer [Ru(η -p- Cryostream cooling system (Oxford Cryostat). Cell refinement
i
Pr C H Me)Cl ] (0.0382 g, 0.0624 mmol)was reacted with tris and data reduction were performed using the program
2-(5-methyl-2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)ethyl) SAINT. The data were scaled, and absorption correction per-
amine (0.0292 g, 0.0404 mmol) in a at room temperature. formed using SADABS. The structures were solved by direct
PF (0.0487 g, 0.299 mmol) was added to crude and stirred methods using SHELXS-97 and refined by full-matrix least-
4 6
for 1 h. The desired product ([18][PF ] ) was isolated as a squares methods based on F using SHELXL-2014 and using
bright yellow solid 0.0597 g, 0.0303 mmol. Yield: 75.0%. the graphics interface program X-Seed. The programs X-Seed
H NMR (400 MHz, DMSO) δ (ppm): 9.67–9.53 (m, 3H, H-o), and POV-Ray
.10–7.87 (m, 6H, H-n and H-d), 7.71 (ddd, JHH = 28.5 Hz, images. All non-hydrogen atoms were refined anisotropically.
HH = 16.6 Hz, JHH = 10.1 Hz, 6H, ArH ̲) , 7.54–7.27 (m, 6H, All hydrogen atoms were placed in idealised positions and
), 6.33 (t, JHH = 7.3 Hz, 3H, Arp-cye), 6.25 (d, JHH = 6.0 Hz, refined in riding models with Uiso assigned 1.2 or 1.5 times
6 4 2 2
5
4
(
5
5
5
3
NH
2
55
6
3
5
6
1
56
were used to prepare molecular graphic
2
8
J
ArH
3
6
3
4
3
3
̲
3
4
H, Ar
), 6.15 (dd, J = 13.2 Hz, J = 6.5 Hz, 3H, Arp-cye), Ueq of their parent atoms and the bond distances were con-
p-cye HH HH
3
.04 (d, JHH = 5.9 Hz, 3H, Arp-cye), 4.53–4.17 (m, 6H, CH
̲
2
), 2.95 strained in the range from 0.95 Å to 0.99 Å. CCDC 1940327 (7),
), 2.62 (s, 3H, 1940326 (8).†
̲( CH ) ), 2.13
3
(
d, JHH = 22.9 Hz, 6H, CH
CH ), 2.57 (s, 3H, CH3), 2.39–2.26 (m, 3H, CH
t, JHH = 7.1 Hz, 9H, CH3 p-cye), 0.85–0.75 (m, 18H, CH(CH
p-cye). C{ H}-NMR (101 MHz, DMSO) δ (ppm): 157.85, 148.01, The human breast adenocarcinoma cell lines, MCF-7 (oestro-
2 3
̲ ), 2.65 (s, 3H, CH̲
̲
̲
3
3 2 p-cye
3
Cell culture
(
̲
3 2
̲ )
1
3
1
1
1
4
45.52, 140.59, 140.23, 136.25, 134.35, 128.58, 127.44, 124.31, gen-receptor positive, ER+) was maintained in Roswell Park
18.31, 112.51, 87.07, 84.72, 82.96, 80.27, 55.35, 53.07, 52.53, Memorial Institute (RPMI) 1640 medium (Sigma Aldrich, USA)
3
1
1
4.24, 30.97, 22.31, 22.10, 21.64, 19.13.
P{ H}-NMR and the MDA-MB-231 (triple negative, TNBC) was maintained
(
162 MHz, DMSO) δ (ppm): −144.16 (sep, J = 711.2 Hz, PF ). in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma
6
−
1
FT-IR (ATR) ν (cm ): 1674 (imine CvN), 1464 (pyridyl CvN). Aldrich, USA). All culture medium was supplemented with
MP (°C): 236.8 (decomp.). MS (HR-ESI, m/z): Calculated: 10% heat-inactivated foetal bovine serum (FBS), 100 U mL
1
−
1
3
+
−1
823.0329, Found: 1823.1600 (80%, [M − PF + 2H] ).
penicillin and 100 μg mL streptomycin. The non-tumori-
6
Mononuclear
20][PF ].
0.0784 g, 0.352 mmol) was reacted with dichloro(p-cymene) plemented with 10% foetal bovine serum (FBS), 100 U ml
cationic
N,N-Ru(II)-p-cymene
complex genic human breast epithelial MCF-12A cells were maintained
[
(
6
1-Ethyl-2-(pyridin-2-yl)-1H-benzo[d]imidazole in complete media consisting of DMEM/Ham’s F12 sup-
−
1
Dalton Trans.
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Paper
−
1
penicillin, 0.1 μg ml cholera toxin (Sigma, St Louis, MO,
3 WHO, WHO: Cancer, http://www.who.int/mediacentre/fact-
sheets/fs297/en/, (accessed 28 June 2018).
4 A. Schroeder, D. A. Heller, M. M. Winslow, J. E. Dahlman,
G. W. Pratt, R. Langer, T. Jacks and D. G. Anderson, Nat.
Rev. Cancer, 2011, 12, 39–50.
5 M. Gaba and C. Mohan, Development of drugs based on imid-
azole and benzimidazole bioactive heterocycles: Recent
advances and future directions, Springer US, 25th edn, 2016.
−
1
USA), 0.5 μg ml hydrocortisone (Calbiochem, Billerica, MA),
1
1
0 μg ml− insulin (Novorapid; Novo Nordisk, Copenhagen,
−
1
Denmark), 20 ng ml epidermal growth factor (Gibco, Life
Technologies, Carlsbad, CA), and 5% horse serum (Highveld
Biological, Lyndhurst, South Africa). Cells were maintained at
3
2
7 °C in a 95% air and 5% CO humidified incubator and
medium was replaced every 2 to 3 days.
6
7
8
9
L. Rylands, A. Welsh, K. Maepa, T. Stringer, D. Taylor,
K. Chibale and G. S. Smith, Eur. J. Med. Chem., 2019, 161,
Cytotoxicity studies
11–21.
For the cytotoxicity assays, MCF-7, MDA-MB-231 breast cancer
cells and non-tumorigenic MCF-12A epithelial cells were
seeded in a 96-well plate at a density of 4500 cells per well,
L. T. Wu, Z. Jiang, J. J. Shen, H. Yi, Y. C. Zhan, M. Q. Sha,
Z. Wang, S. T. Xue and Z. R. Li, Eur. J. Med. Chem., 2016,
114, 328–336.
3
000 cells per well and 6000 cells per well, respectively. The
J. E. Cheong, M. Zaffagni, I. Chung, Y. Xu, Y. Wang,
F. E. Jernigan, B. R. Zetter and L. Sun, Eur. J. Med. Chem.,
cells were incubated for either 24 (for the MDA-MB-231 cell
line) or 48 h (for the MCF-7 and MCF-12A cell lines) to allow
adhesion. The cells were then treated with either the vehicle
2
018, 144, 372–385.
C. S. Allardyce and P. J. Dyson, Dalton Trans., 2016, 45,
201–3209.
(0.1% DMSO) or 10 μM or 20 μM of the test compounds
3
for 48 h. The impact of the test compounds on cell viabi-
lity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as described in lit-
1
1
0 J. Reedijk, Eur. J. Inorg. Chem., 2009, 1303–1312.
1 W. Han and P. J. Dyson, Eur. J. Inorg. Chem., 2006, 20,
4003–4018.
5
7
erature. The absorbance at 600 nm was determined for each
well using a spectrophotometer (GloMax® Explorer Multimode
Microplate Reader GM3500, Promega) and normalised to the
absorbance of the RPMI medium (for the MCF-7 cell line). To
determine the IC50 (concentration required to inhibit 50% via-
bility) of selected compounds, the cells were treated with a
range of concentrations (5–35 μM). These experiments were
performed twice in quadruplicate, and the mean cell viability
determined using GraphPad Prism V.5.01 software.
12 E. Alessio, Eur. J. Inorg. Chem., 2017, 1549–1560.
13 P. J. Dyson and G. Sava, Dalton Trans., 2006, 16, 1929–1933.
14 C. S. Allardyce, A. Dorcier, C. Scolaro and P. J. Dyson, Appl.
Organomet. Chem., 2005, 19, 1–10.
1
1
5 I. Kostova, Curr. Med. Chem., 2006, 13, 1085–1107.
6 M. Galanski, V. B. Arion, M. A. Jakupec and B. K. Keppler,
Curr. Pharm. Des., 2003, 9, 2078–2089.
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7 G. K. Gransbury, P. Kappen, C. J. Glover, J. N. Hughes,
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Metallomics, 2016, 8, 762–773.
1
8 D. S. Thompson, G. J. Weiss, S. F. Jones, H. A. Burris,
R. K. Ramanathan, J. R. Infante, J. C. Bendell, A. Ogden
and D. D. Von Hoff, J. Clin. Oncol., 2012, 30, 3033–3033.
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Conflicts of interest
The authors declare no competing financial interest.
1
2
470.
0 R. Carter, A. Westhorpe, M. J. Romero, A. Habtemariam,
C. R. Gallevo, Y. Bark and N. Menezes, Sci. Rep., 2016, 6, 1–12.
Acknowledgements
Financial support from the University of Cape Town and the 21 Z. Wang, H. Qian, S. Yiu, J. Sun and G. Zhu, J. Inorg.
National Research Foundation (NRF, UID: 114305; 111707) is
Biochem., 2014, 131, 47–55.
gratefully acknowledged. We are also thankful for several short 22 M. Martínez-Alonso, N. Busto, F. A. Jalón, B. R. Manzano,
term exchange visits between the two laboratories in South
J. M. Leal, A. M. Rodríguez, B. García and G. Espino, Inorg.
Africa and Austria and financial support through the South
Chem., 2014, 53, 11274–11288.
Africa/Austria Joint Scientific and Technological Cooperation 23 M. Maroto-Díaz, B. T. Elie, P. Gómez-Sal, J. Pérez-Serrano,
(UID: 106165 and OeAD travel grant ZA01/2017) is gratefully
R. Gómez, M. Contel and F. J. De Mata, Dalton Trans., 2016,
acknowledged.
45, 7049–7066.
2
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4 P. R. Florindo, D. M. Pereira, P. M. Borralho,
M. P. Rodrigues, M. F. M. Piedade and A. C. Fernandes,
J. Med. Chem., 2015, 58, 4339–4347.
5 N. Mohan, S. Muthumari and R. Ramesh, J. Organomet.
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