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Cytotoxic hydrogen bridged ruthenium quinaldamide complexes showing induced cancer cell death by apoptosis.
University of Huddersfield Repository
Lord, Rianne Michaela, Allison, Simon J., Rafferty, Karen, Ghandhi, Laura, Pask, Christopher M.
and McGowan, Patrick C.
Cytotoxic Hydrogen Bridged Ruthenium Quinaldamide Complexes Showing Induced Cancer Cell
Death by Apoptosis
Original Citation
Lord, Rianne Michaela, Allison, Simon J., Rafferty, Karen, Ghandhi, Laura, Pask, Christopher M.
and McGowan, Patrick C. (2016) Cytotoxic Hydrogen Bridged Ruthenium Quinaldamide
Complexes Showing Induced Cancer Cell Death by Apoptosis. Dalton Trans.. ISSN 1477-9226
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Cytotoxic Hydrogen Bridged Ruthenium Quinaldamide Complexes
Showing Induced Cancer Cell Death by Apoptosis
Rianne M. Lord,a* Simon J. Allison,b Karen Rafferty,a Laura Ghandhi,a Christopher M. Paska and
Patrick C. McGowana*
This report presents the first known p-cymene ruthenium quinaldamide complexes which are stablized by a hydrogenbridging atom, [[{(p-cym)RuIIX(N,N)}{H+}{(N,N)XRuII(p-cym)}][PF6] (N,N = functionalised quinaldamide and X = Cl or Br).
These complexes are formed by a reaction of [p-cymRu(-X)2]2 with a functionalised quinaldamide ligand. When filtered
over NH4PF6, and under aerobic conditions the equilibrium of NH4PF6 NH3 + HPF6 enables incorporation of HPF6 and the
stabilisation of two monomeric ruthenium complexes by a bridging H+, which are counter-balanced by a PF6 counterion. Xray crystallographic analysis is presented for six new structures with O···O distances of 2.430(3)-2.444(17) Å, which is
significant for strong hydrogen bonds. Chemosensitivity studies against HCT116, A2780 and cisplatin-resistant A2780cis
human cancer cells showed the ruthenium complexes with a bromide ancillary ligand to be more potent than those with a
chloride ligand. The 4'-fluoro compounds show a reduction in potency for both chloride and bromide complexes against all
cell lines, but an increase in selectivity towards cancer cells compared to non-cancer ARPE-19 cells, with a selectivity index
> 1. Mechanistic studies showed a clear correlation between IC50 values and induction of cell death by apoptosis.
Introduction
There are only a small number of reports on the synthesis and
isolation of transition metal hydrogen-bridging complexes.
Usually solvent molecules provide the H+ source and few
researchers suggest the possibility of the reagent NH4PF6
providing the source of H+. Peacock et al. were amongst the
first to isolate and characterise by X-ray crystallographic
analysis, cobalt, manganese and chromium hydrogen-bridged
structures, [CoIII(L·H3L)CoIII][PF6]3,1 [MnII(L·H3L)MnIV]][PF6]3,2
and [CrIII(L·H3L)CrIII][PF6]3,3 (LH3 = N,N’,N”-tris[(2S)-2hydroxypropyl]-1,4,7-triazacyclononane) respectively. Some of
these compounds have been studied using circular dichroism,
magnetic susceptibility and cyclic voltammetry, in order to
understand their spin states and oxidation states.2 Ward et al.
have also synthesised and characterised by X-ray
crystallographic analysis nickel and copper hydrogen-bridged
structures, [NiII(L·HL)]2[PF6]2,4 (L = 6-(2-hydroxyphenyl)-2,2’bipyridine) and [CuII(L2)]·(HPF6)0.5·H2O,5 (L = 6,6’-bis(2hydroxyphenyl)-2,2’-bipyridine)
respectively.
All
these
compounds have a shortened O···O bond distance, averaging
2.34 Å, indicative of strong hydrogen-bonds. In all cases, the
hydrogen atoms could not be located in the crystal structures;
however their presence is needed in order to balance the PF 6
counterions. Nothing has yet been reported on the possible
applications of such compounds, therefore we report here the
application of hydrogen-bridged ruthenium complexes as
possible anti-cancer agents.
In a search for less toxic and more potent alternatives to
cisplatin, organometallic complexes have shown promising
activity as anti-cancer agents.6-16 Ruthenium-based complexes
are some of the most promising, with reported selective
potency in vitro and in vivo.17-22 McGowan et al. have
synthesised a range of ruthenium metal complexes for their
uses as anticancer agents.23-26 The work published on
ruthenium quinaldamides showed that under inert
atmosphere conditions, the filtering over NH4PF6 yielded the
ruthenium quinaldamide monomers. 27 The use of dry
conditions avoids the hydrolysis of NH4PF6 to NH3 and HPF6.
These monomeric complexes show low IC50 values against a
range of cell lines and also form adducts with guanine
nucleotides. Herein, we present the same synthetic strategy
using aerobic conditions and show that the monomers are no
longer stable under these conditions and the HPF6 present
from the hydrolysis of NH4PF6, stabilises two ruthenium
quinaldamide species, [{(p-cym)RuIIX(N,N)}{H+}{(N,N)XRuII(pcym)}][PF6], with incorporation of HPF6. This motif has
previously been reported for our ruthenium picolinamide
complexes, in which an average O···O bond distance of 2.43 Å
was observed.17 The X-ray crystallographic data was reported,
however, chemosensitivity studies were not determined.
a. School of Chemistry, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT,
p.c.mcgowan@leeds.ac.uk
b. Department of Pharmacy, School of Applied Sciences, University of Huddersfield,
Huddersfield, HD1 3DH
† Electronic Supplementary Information (ESI) available: Experimental procedures
for compounds, cell line experimental, apoptosis studies and crystal structure
determination details. The cifs for complexes 1-6 were deposited to the CCDC with
codes 1472239-1472244.
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Herein, chemosensitivity studies have been carried out against
HCT116 (human colon carcinoma), A2780 (human ovarian
carcinoma) and A2780cis (cisplatin resistant A2780) cancer cell
lines, and against non-cancer ARPE-19 (human retinal
epithelium) cells. Studies investigated whether the complexes
might induce apoptosis (programmed cell death), a cell death
mechanism that is commonly suppressed in cancers. This was
measured in the HCT116 cancer cells by loss of mitochondrial
membrane potential which is an early marker of apoptosis.
Cell images by phase contrast microscopy at various timepoints after compound addition suggested induction of cell
death rather than growth arrest. Apoptotic analyses revealed a
clear correlation between chemosensitivity and levels of
apoptosis, whereby the most active compound induces the
highest percentage of apoptosis.
Results and discussion
Using a modification of the previously established literature
method by Bennett et al.,28, 29 the ruthenium(II) p-cymene
halide complexes were synthesised by dissolving RuIIIX3.xH2O
(X = Cl or Br) and -terpinene in ethanol, then heating to reflux
for 16 hours. The resulting dark red powder was filtered and
washed with ice cold ethanol to yield the desired starting
ruthenium p-cymene dimer. Upon addition of two equivalents
of a substituted quinaldamide in ethanol and filtering over
NH4PF4, the reaction mixture formed a pale orange precipitate
and yielded complexes 1-6 as analytically pure products
(Scheme 1). Single orange-red crystals suitable for X-ray
crystallographic analysis were obtained for complexes 1-6.
They crystallised in either a triclinic P1 (1-3, 5-6) or
monoclinic C2/c (4) space group. All of the angles around the
metal centre show the geometry expected for pseudo
octahedral compounds which is common for half-sandwich
“piano-stool” structures (Tables 1). The angles between the
metal and bidentate ligands are in the range 75.4(2)-87.5(3)°,
with the remaining three coordination sites occupied by the pcymene ligand, with the angles observed for their centroids to
the halide or bidentate ligand ranging between 126.81134.78°. Molecular structures for complexes 1-6 are shown in
Scheme 1 Synthetic route for the synthesis of ruthenium quinaldamide
complexes 1-6 via addition of a functionalised quinaldamide ligand to [pcymRuX(-X)]2.
Figure 1, with displacement ellipsoids placed at the 50%
probability level and hydrogen atoms and PF6 anions omitted
for clarity. The proton bridging between the two carbonyl
oxygens provides the +1 charge, which is counter-balanced by
the PF6 anion, and both metal centres are in their +2 oxidation
state. The two such monomer units [p-cymRuII(N,N)X] are held
together by one intermolecular hydrogen bond which links
O(1) and O(1’). The short O···O distances of 2.439(3)-2.444(17)
Å (Table 2), which are only slightly longer than double of a
typical O-H distance, are at the lower limit for a pair of
hydrogen-bonded oxygen atoms, indicative of strong hydrogen
bonds. This was also reported for nickel complexes synthesised
by Ward et al., in which they observed O···O distances of 2.372.39 Å.4, 30, 31 However, weak asymmetric O-H···O hydrogen
bonds more typically have O···O distance > 2.7 Å. 32 As shown in
the previously reported structures by Ward et al.,4 the
hydrogen-bridging complexes are further stabilized by
intermolecular interactions. The packing diagrams for
complexes 1-6 are presented in Figures S1-2, and show that
these ruthenium quinaldamide complexes have several
intramolecular and intermolecular interactions (Table S1a-f),
which could also contribute to the stability of the dimers. All
the complexes have their aromatic quinaldamide rings brought
into close proximity, with relatively short π-π stacking
interactions of 3.753-3.919 Å. X-ray crystallographic data is
also presented in the supplementary information (Table S2).
Table 1 Selected bond lengths (Å) and bond angles (°) for complexes 1-6, with s.u.s in parenthesis
Bond Length (Å)
Ru(1)-N(1)
Ru(1)-N(2)
Ru(1)-X(1)
Ru(1)-Cg(4)
Bond Angles (°)
N(1)-Ru(1)-N(2)
N(1)-Ru(1)-X(1)
N(2)-Ru(1)-X(1)
N(1)-Ru(1)-Cg(4)
N(2)-Ru(1)-Cg(4)
X(1)-Ru(1)-Cg(4)
1
2.100(2)/2.035(2)
2.1222(19)/2.113(2)
2.3854(6)/2.4093(9)
1.6735(10)/1.6954(12)
1
75.42(8)/76.99(8)
86.47(2)/87.29(6)
83.65(6)/83.26(6)
126.83(7)/129.68(8)
134.73(7)/133.88(7)
130.53(4)/127.74(5)
2
2.130(2)
2.076(2)
2.3969(9)
1.6803(12)
2
76.10(8)
86.15(6)
87.61(7)
132.47(8)
128.01(8)
128.83(5)
3
2.0800(18)
2.1368(18)
2.3962(6)
1.6814(9)
3
76.00(7)
87.76(5)
86.32(5)
128.18(6)
132.57(6)
128.45(4)
4
2.079(2)
2.136(2)
2.5438(5)
1.7006(14)
4
76.32(9)
86.94(6)
84.45(6)
128.78(7)
134.15(7)
128.12(5)
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5
2.130(3)
2.085(3)
2.5328(5)
1.6930(18)
5
76.13(11)
85.89(8)
87.50(8)
132.41(10)
128.37(10)
128.79(6)
6
2.076(3)
2.123(3)
2.5402(5)
1.6953(17)
6
76.52(11)
87.03(8)
84.03(8)
128.46(11)
133.79(10)
128.73(7)
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1
2
3
4
5
6
Figure 1 Molecular structures for compounds 1-6. Hydrogen atoms and PF6 anions are omitted for clarity and displacement ellipsoids are shown only for heteroatoms,
at the 50% probability level.
Table 2 Hydrogen bonding donor-acceptor distances (Å) for complexes 1-6
Compound
1
2
3
4
5
6
O(1)···O(1’) (Å)
2.425(3)
2.420(4)
2.442(3)
2.439(3)
2.444(17)
2.448(15)
Chemosensitivity Studies
Chemosensitivity studies were undertaken using the MTT
assay and IC50 values were determined against HCT116 (human
colon carcinoma), A2780 (human ovarian carcinoma) and
A2780cis (cisplatin resistant A2780 cells) cell lines, exposed to
each of compounds 1-6 or cisplatin (Table 3). Against all three
cancer cell lines the Ru-Br complexes were consistently more
active than the Ru-Cl analogues. The 4-fluoro compounds 2
and 5 are the least active when compared to the other fluoro
compounds, however, changing from Ru-Cl (compound 2, 39.2
± 0.8 M) to Ru-Br (compound 5, 8.7 ± 0.4 M) there is a > 4fold increase in cytotoxicity. The results show that the 2’,4’difluoro compounds 3 and 6 are the most active, and when
comparing Ru-Cl (compound 3, 5.9 ± 0.2 M) and Ru-Br
(compound 6, 3.9 ± 0.3 M) there is a 1.5-fold increase in
cytotoxicity. They is an increase in cytotoxicity when compared
Figure 2 The resistance factor for the compounds as indicated. This is defined as
the IC50 in A2780cis divided by IC50 in A2780 cells. An RF of 1 indicates equal
potency against both cell lines. An RF > 1 indicates that the A2780cis is more
resistant than A2780. An RF < 1 indicates that the A2780cis is more sensitive
than the A2780 cells.
to the 2'-fluoro compounds 1 and 4, but to a similar degree in
both cancer and non-cancer cells. Most of the compounds
were more active against A2780 cells than A2780cis cells, but
the level of resistance is much less than for that of cisplatin
(Figure 2 and Table S3, SI). Compound 2 is the least potent but
showed similar activity towards A2780 and A2780cis cancer
cells, and was more active against all three cancer cell lines
when compared to non-cancer ARPE-19 cells. The results show
that potency is dependent on position of the fluoro.
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Table 3 IC50 values (M) for cisplatin and compounds 1-6 against HCT116, A2780, A2780cis and ARPE-19 cell lines
Compound
Cisplatin
1
2
3
4
5
6
HCT116
N.D.
6.7 ± 0.5
39.2 ± 0.8
5.9 ± 0.2
4.02 ± 0.11
8.7 ± 0.4
3.9 ± 0.3
IC50 values (M) ± Standard Deviation
A2780
A2780cis
1.00 ± 0.16
10.6 ± 0.9
4.8 ± 0.6
11.7 ± 0.6
17.56 ± 1
22 ± 1
4.0 ± 0.5
7.4 ± 0.5
3.2 ± 0.4
5.46 ± 0.17
9±3
14 ± 2
3.0 ± 0.4
6.5 ± 0.9
ARPE-19
6 ± 126
4.3 ± 0.3
> 50
3.7 ± 0.8
3.06 ± 0.09
10.4 ± 3.3
2.9 ± 0.2
substituent and increasing the number of electron
withdrawing substituents increased the potency by > 6-fold.
Selectivity for Cancer Cells
One of the major limitations of existing anti-cancer drugs is
their poor selectivity towards cancer cells, restricting the
drugs’ dosage. As well as causing harmful side effects for the
patient, this dose-limiting toxicity impacts upon treatment
effectiveness. . Comparing the response of tumour cell lines to
non-cancer ARPE-19 cells provides a preliminary indication
oselectivity. Whilst compounds 1, 3, 4 and 6 show no
selectivity towards cancer cells (ratio of IC50 values in ARPE-19
cells to cancer cells ≤ 1), compounds 2 and 5 showed evidence
of selectivity to certain cancer cells (Figure 3 and. Table S4, SI).
Compound 2 in particular demonstrated selectivity against all
the cancer cell lines tested with selectivity ranging from 2.85
to 1.27 fold increased chemosensitivity towards cancer cells
compared to ARPE-19 non-cancer cells (HCT116: 1.27; A2780:
2.85; A2780cis: 2.29; Figure 3 and Table S3, SI). However,
compound 2 is the least active compound against all cancer
cell lines tested.
Figure 3 Show the selectivity index defined as the IC 50 in ARPE-19 divided by IC50
relevant cancerous cells. An SR = 1 indicates equitoxic potency against tumour
and normal cells. An SR > 1 indicates preferential selectivity for tumour cells
compared to normal cells. An RF < 1 indicates poor selectivity (greater
cytotoxicity towards ARPE cells compared to normal cells)
Induction of Cancer Cell Death by Apoptosis
IC50 values determined by chemosensitivity studies using the
MTT assay indicates the concentration of drug required for a
50% reduction in cell number. This provides invaluable
information about the activity of the drug against the cell line
but does not distinguish between effects on cell proliferation
and effects on cell survival. The observed activity of these
compounds towards the cell lines could be caused by induction
of cell growth arrest or the compounds may cause cell death.
Cell images under phase contrast microscopy at various timepoints after compound addition suggested induction of cell
death as suggested by an increase in the proportion of nonadhered cells rather than growth arrest. Using flow cytometry
and staining for loss of mitochondrial membrane potential the
percentage of apoptotic cells were quantified following
incubation of HCT116 cells with 0–60 µM of compounds 1-6
for 72 hours (Figure 4 and Table S4, SI).
The 2’,4’-difluoro compounds 3 and 6, which were the most
active compounds in the MTT chemosensitivity studies, also
induced significant levels of apoptotic cell death against
HCT116 cancer cell lines (Figure 4 and Table S5, SI) in a doseresponsive manner. A 72 hour exposure of HCT116 cells to 20
Figure 4 % of apoptosis for control and compounds 1-6 against HCT116 cells at
concentrations ranging from 0-60 M.
M of compound 3 resulted in ~76% of cells in early stages of
apoptosis and compound 6 which was the most active against
the MTT assay showed significant apoptosis, with 84%
apoptotic cells. In contrast, compounds 2 and 5 were the least
active compounds against the MTT assay and show the least
amount of apoptotic cells. Compound 2 is the least active of
this series of compounds, and a higher concentration of 60 M
of the compound had to be used to induce apoptosis, resulting
in only 33% apoptotic cells. Whereas the more active
compound 5 induces apoptosis at a concentration of 20 M
and gave 35% apoptotic cells. These observations indicate a
clear correlation between IC50 value and levels of apoptosis
induced.
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Conclusions
using DENZO and SCALEPACK programs.35 Structure solution by
direct methods was achieved through the use of SHELXS
programs,36 and the structural model refined by full matrix
least squares on F2 using SHELX97 Unless otherwise stated,
hydrogen atoms were placed using idealised geometric
positions (with free rotation for methyl groups), allowed to
move in a “riding model” along with the atoms to which they
were attached, and refined isotropically. Molecular graphics
were plotted using OLEX237 and Mercury.38 Editing of CIFs and
construction of tables of bond lengths and angles were
achieved using WC39 and PLATON.30
We report the successful synthesis of ruthenium hydrogenbridged complexes from the reaction of [p-cymRuX2]2 (X = Cl or
Br) with a functionalised quinaldamide ligand. The reaction
conditions differ from our previously synthesised ruthenium
quinaldamide complexes, as here we utilise aerobic conditions
and show the hydrolysis of the NH4PF6 reagent yields the
stabilisation of these unusual H+ bridged complexes, counterbalanced by a PF6 anion. These compounds have been tested
against HCT116, A2780 and A2780cis cancer cells, and results
show the 2’,4’-difluoro compounds 3 (X = Cl) and 6 (X = Br) are
the most potent against all cell lines. The di-substituted
compounds are more potent than the mono-substituted,
showing the number and position of the fluoro group is
important to the potency. Across all cell lines, the 4-fluoro
compounds 2 and 5 are the least active, with >6-fold decrease
in potency observed against HCT116 cancer cells. The most
significant results when comparing the different ancillary
ligands are that the chloride compounds 1-3 are general less
active than the bromide complexes 4-6, with up >4-fold
increase in IC50 values observed against HCT116 cells.
Induction of cell death by apoptosis was investigated and this
showed a clear correlation between IC50 values and levels of
apoptosis induced. However, the results also indicate the
importance to consider selectivity and ability to overcome
drug resistance as well as potency with compound appearing
the most promising by these important criteria.
Experimental
Materials
All chemicals were supplied by Sigma-Aldrich Chemical Co.,
Acros Organics, Strem Chemical Co. and BOC gases.
Functionalised quinaldamide ligands were prepared by
adaptations of literature methods.33 Deuterated NMR solvents
were supplied by Sigma-Aldrich Chemical Co. or Acros
Organics.
Analysis
All NMR spectra were recorded on a Bruker DPX 300 or a
Bruker DPX 500 spectrometer. Microanalyses were recorded at
the University of Leeds Microanalytical Service. Mass Spectra
were recorded on a Micromass ZMD spectrometer with
electrospray ionisation and photoiodide array analyser at the
University of Leeds Mass Spectrometry Service.
Chemosensitvity Studies
In vitro chemosensitivity tests were performed at the
University of Huddersfield, against HCT116 (human colon
carcinoma), A2780 (human ovarian carcinoma) and A2780cis
(cisplatin resistant A2780 cells) cancer cell lines, and against
ARPE-19 (human retinal epithelial non-cancer) cells. ARPE-19
cells were obtained from the American Type Culture
Collection. Cancer cell lines were routinely maintained as
monolayer cultures in appropriate medium (RPMI 1640
supplemented with 10% foetal calf serum, sodium pyruvate (1
mM) and L-glutamine (2 mM) ARPE-19 cells were cultured in
DMEM-F12 medium containing 10% foetal calf serum. For
chemosensitivity studies, cells were incubated in 96-well plates
at a concentration of 2 × 103 cells per well and the plates were
incubated for 24 hours at 37 °C in an atmosphere of 5% CO2
prior to drug exposure. Compounds or cisplatin were each
dissolved in dimethylsulfoxide to provide stock solutions that
were diluted to provide a range of final concentrations. Drug
solutions were added to cells (the final DMSO concentrations
was less than 0.1% (v/v) in all cases) and incubated for 5 days
at 37°C in an atmosphere of 5% CO2. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (20 L, 5 mg mL−1)
was added to each well and incubated for 4 hours at 37°C in an
atmosphere of 5% CO2. All solutions were then removed by
pipetting and 150 μL of dimethylsulfoxide added to each well
in order to dissolve the purple formazan crystals absorbance of
each well at 540 nm measured by spectrophotometer. Lanes
containing medium only and 100% cells were used as blanks
for the spectrophotometer and 100% cell survival respectively.
Cell survival was determined as the true absorbance of treated
cells divided by the true absorbance of controls and expressed
as a percentage. The IC50 values were determined from dose
response curves of % survival against drug concentration. Each
experiment was repeated three times and a mean value
obtained and stated as IC50 (μM) ± SD.
X-ray Crystallography
A suitable single crystal was selected and immersed in an inert
oil. The crystal was then mounted on a glass capillary or nylon
loop and attached to a goniometer head on Nonius KappaCCD
area detector diffractometer using graphite monochromated
Mo-K radiation ( = 0.71073 Å) and a Bruker X8 Apex
diffractometer.. The crystal was cooled and data measured at
148-150K by an Oxford Cryostream low temperature device. 34
The full data sets were recorded and the images processed
Induction of Cancer Cell Death by Apoptosis
HCT116 cells were incubated in T-25 flasks and diluted to
concentrations of 2.5 x 104 cells/flask (0.5 x 104 cells/ mL) using
complete RMPI 1640 medium. These were incubated for 24
hours at 37°C in an atmosphere of 5.0% CO2. Complexes were
dissolved in dimethylsulfoxide and then further diluted with
RMPI 1640 to obtained concentrations ranging from 0-60 M.
The cells were then incubated with the varying concentrations
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of complex for 72 hours, media/drug solutions were removed
and flasks were washed with PBS (5 mL), adding all collected
supernatants to a centrifuge tube. Trypsin (1 mL/flask) was
added to each flask and then incubated for 5 minutes until a
single cell suspension was obtained. The trypsin was then
neutralised with medium (5 mL) and the whole contents of the
flask transferred to the same centrifuge tube. The tube was
centrifuged at 1000 rcf for 3-5 minutes, the supernatant
removed and the pellet re-suspended in PBS (1 mL). The 1 mL
suspension was transferred to an Eppendorf tube and
centrifuged at 1500 rpm for 5 minutes. The supernatant was
removed and the pellet stained with JC-1 in order to stain for
loss of mitochondrial membrane potential and apoptosis. This
was performed as per the manufacturer’s protocol
(Chemometec) and cell samples were analysed using an
NC3000 flow cytometer (Chemometec).
Characterisation
Compound 1. Yield: 72 mg, 0.06 mmol, 86 %. ES-MS (+)
(MeOH):
m/z
501.1
[RuC26H26N2OF]+,
581.02
+
[RuC26H25N2OFBr] . Anal. Calc.: C 49.8, H 4.2, N 4.7%. Anal.
Found: C 49.5, H 4.0, N 4.3%. 1H NMR: (CD3OD, 500 MHz, 298
K) δ 8.92 (d, 1H, 3J(1H-1H) = 8.8 Hz, quin), 8.60 (d, 1H, 3J(1H-1H)
= 8.4 Hz, quin), 8.15-8.04 (m, 3H, quin), 7.89 (td, 2H, 3J(1H-1H) =
7.9 Hz, 4J(1H-19F) = 0.8 Hz, ar), 7.33-7.30 (m, 2H, ar), 7.23-7.20
(m, 1H, ar), 5.72 (d, 1H, 3J(1H-1H) = 6.1 Hz, p-cym), 5.58 (d, 1H,
3J(1H-1H) = 5.9 Hz, p-cym), 5.41 (d, 1H, 3J(1H-1H) = 6.1 Hz, pcym), 4.81 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym), 2.30-2.27 (m, 1H,
CH(CH3)2), 2.14 (s, 3H, CH3), 0.93 (d, 3H, 3J(1H-1H) = 6.9 Hz,
CH(CH3)2), 0.80 (d, 3H, 3J(1H-1H) = 6.9 Hz). 13C{1H} NMR:
(CD3OD, 125 MHz, 298 K) δ 168.6 (Q, C-O), 158.2 (d, Q, C-F,
1J(13C-19F) = 267.9 Hz), 150.0 (Q), 141.2 (CH, quin), 140.0 (Q),
132.6 (CH, quin), 131.7 (CH, quin), 131.6 (CH, quin), 130.1 (CH,
quin), 129.3 (Q), 128.8 (d, CH, 3J(13C-19F) = 7.5 Hz, ar), 128.2
(CH, ar) 125.6 (CH, ar), 122.5 (CH, quin), 116.9 (d, CH, 2J(13C-19F)
= 21.4 Hz, ar), 105.4 (Q), 101.7 (Q), 101.4 (Q), 86.7 (CH, p-cym),
86.4 (CH, p-cym), 85.5 (CH, p-cym), 85.3 (CH, p-cym), 32.4
(CH(CH3)2), 22.2 (CH(CH3)2), 22.0 (CH(CH3)2), 19.6 (CH3).
Compound 2. Yield: 39 mg, 0.03 mmol, 36%. ES-MS (+)
(MeOH): m/z 501.1 [RuC26H24N2OF]+. Anal. Calc.: C 50.3, H 4.9,
N 4.4%. Anal. Found: C 49.2, H 4.7, N 4.7%. 1H NMR: (CD3OD,
500 MHz, 298 K) δ 8.94 (d, 1H, 3J(1H-1H) = 8.9 Hz, quin), 8.61 (d,
1H, 3J(1H-1H) = 8.4 Hz, quin), 8.14-8.11 (m, 2H, quin), 8.05 (td,
1H, 3J(1H-1H) = 6.9 Hz, 4J(1H-1H) = 1.6 Hz, quin), 7.87-7.79 (m,
3H, quin+ar), 7.16 (td, 2H, 3J(1H-1H) = 7.9 Hz, 3J(1H-1H) = 5.1
Hz), 5.71 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym), 5.53 (d, 1H, 3J(1H1H) = 5.9 Hz, p-cym), 5.41 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym),
4.75 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym), 2.31-2.22 (m, 1H,
CH(CH3)2), 2.22 (s, 3H, CH3), 0.98 (d, 3H, 3J(1H-1H) = 6.9 Hz,
CH(CH3)2), 0.85 (d, 3H, 3J(1H-1H) = 6.9 Hz, CH(CH3)2). 13C{1H}
NMR: (CD3OD, 125 MHz, 298 K) δ 169.1 (Q, C-O), 162.0 (d, Q,
C-F, 1J(13C-19F) = 241.3 Hz), 158.3 (Q), 150.4 (Q), 149.3 (Q),
141.6 (CH, quin), 133.0 (CH, quin), 132.0 (CH, quin), 131.6 (CH,
quin), 130.5 (Q), 130.1 (CH, quin), 129.4 (d, 2 x CH, 3J(13C-19F) =
7.5 Hz, ar), 123.0 (CH, quin), 116.5 (d, 2 x CH, 2J(13C-19F) = 22.6
Hz, ar), 105.1 (Q), 102.7 (Q), 87.2 (CH, p-cym), 87.0 (CH, pcym), 86.3 (CH, p-cym), 85.8 (CH, p-cym), 32.8 (CH(CH3)2), 22.6
(CH(CH3)2), 22.5 (CH(CH3)2), 20.1 (CH3).
Compound 3. Yield: 65 mg, 0.05 mmol, 76%. ES-MS (+)
(MeOH): m/z 519.1 [RuC26H24N2OF2]+. Anal. Calc.: C 48.4, H
4.0, N 4.3%. Anal. Found: C 48.9, H 3.8, N 4.4%. 1H NMR:
(CD3OD, 500 MHz, 298 K) δ 8.92 (d, 1H, 3J(1H-1H) = 8.8 Hz,
quin), 8.61 (d, 1H, 3J(1H-1H) = 8.4 Hz, quin), 8.14 (dd, 1H, 3J(1H1H) = 8.2 Hz, 4J(1H-1H) = 1.5 Hz, quin), 8.10 (d, 1H, 3J(1H-1H) =
8.4 Hz, quin), 8.05 (td, 1H, 3J(1H-1H) = 7.2 Hz, 4J(1H-1H) = 1.5 Hz,
quin), 7.94-7.84 (m, 2H, quin+ar), 7.15 (td, 1H, 3J(1H-1H) = 8.9
Hz, 3J(1H-19F) = 2.6 Hz, ar), 6.99 (td, 1H, 3J(1H-1H) = 8.0 Hz, 4J(1H1H) = 1.4 Hz, ar), 5.72 (d, 1H, 3J(1H-1H) = 6.1 Hz, p-cym), 5.58 (d,
1H, 3J(1H-1H) = 5.9 Hz, p-cym), 5.43 (d, 1H, 3J(1H-1H) = 6.1 Hz, pcym), 4.80 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym), 2.30-2.25 (m, 1H,
CH(CH3)2), 2.22 (s, 3H, CH3), 0.93 (d, 3H, 3J(1H-1H) = 6.9 Hz,
CH(CH3)2), 0.81 (d, 3H, 3J(1H-1H) = 6.9 Hz, CH(CH3)2). 13C{1H}
NMR: (CD3OD, 125 MHz, 298 K) δ 169.0 (Q, C-O), 161.9 (d, 2 x
Q, C-F, 1J(13C-19F) = 233.9 Hz) 157.1 (Q), 150.0 (Q), 141.3 (CH,
quin), 132.6 (2 x CH, quin), 131.8 (CH, quin), 131.2 (CH, quin),
130.1 (d, CH, 3J(13C-19F) = 18.9 Hz, ar), 122.5 (CH, quin), 112.3
(d, CH, 2J(13C-19F) = 25.2 Hz, ar), 105.0 (d, CH, 2J(13C-19F) = 61.6
Hz, ar), 104.3 (Q), 101.7 (Q), 88.7 (CH, p-cym), 86.0 (CH, pcym), 85.6 (CH, p-cym), 85.2 (CH, p-cym), 32.5 (CH(CH3)2), 22.2
(CH(CH3)2), 22.0 (CH(CH3)2), 19.6 (CH3).
Compound 4. Yield: 82 mg, 0.06 mmol, 83%. ES-MS (+)
(MeOH): m/z 501.091 [RuC26H25N2OF]+. Anal. Calc.: C 47.8, H
3.8, N 4.3%. Anal. Found: C 47.3, H 3.9, N 4.6%. 1H NMR:
(CD3OD, 500 MHz, 298 K) δ 8.92 (d, 1H, 3J(1H-1H) = 8.8 Hz,
quin), 8.60 (d, 1H, 3J(1H-1H) = 8.4 Hz, quin), 8.15-8.04 (m, 3H,
quin), 7.89 (td, 2H, 3J(1H-1H) = 7.9 Hz, 5J(1H-1H) = 0.8 Hz,
quin+ar), 7.33-7.30 (m, 2H, ar), 7.23-7.20 (m, 1H, ar), 5.74 (d,
1H, 3J(1H-1H) = 6.0 Hz, p-cym), 5.60 (d, 1H, 3J(1H-1H) = 6.0 Hz, pcym), 5.49 (d, 1H, 3J(1H-1H) = 6.3 Hz, p-cym), 4.83 (d, 1H, 3J(1H1H) = 6.3 Hz, p-cym), 2.33-2.24 (m, 1H, CH(CH ) ), 2.06 (s, 3H,
3 2
CH3), 0.97 (d, 3H, 3J(1H-1H) = 6.9 Hz, CH(CH3)2), 0.82 (d, 3H,
3J(1H-1H) = 6.9 Hz, CH(CH ) ). 13C{1H} NMR: (CD OD, 125 MHz,
3 2
3
298 K) δ 168.7 (Q, C-O), 158.4 (d, Q, C-F, 1J(13C-19F) = 249.0 Hz),
152.2 (Q), 149.7 (Q), 141.5 (CH, quin), 140.0 (Q), 132.9 (CH,
quin), 131.8 (CH, quin), 130.6 (CH, quin), 130.1 (CH, quin),
128.8 (d, CH, 3J(13C-19F) = 7.5 Hz, ar), 128.2 (CH, ar), 125.7 (CH,
ar), 122.4 (CH, quin), 116.9 (d, CH, 2J(13C-19F) = 21.3 Hz, ar),
104.4 (Q), 102.7 (Q), 102.0 (Q), 86.8 (CH, p-cym), 86.0 (CH, pcym), 85.7 (CH, p-cym), 85.4 (CH, p-cym), 32.3 (CH(CH3)2) 22.2
(CH(CH3)2), 22.0 (CH(CH3)2), 18.9 (CH3).
Compound 5. Yield: 77 mg, 0.06 mmol, 69%. ES-MS (+)
(MeOH): m/z 501.1 [RuC26H25N2OF]+. Anal. Calc.: C 47.5, H 3.8,
N 4.3%. Anal. Found: C 47.2, H 3.7, N 5.2%. 1H NMR: (CD3OD,
500 MHz, 298 K) δ 8.93 (d, 1H, 3J(1H-1H) = 8.8 Hz, quin), 8.62 (d,
1H, 3J(1H-1H) = 8.3 Hz, quin), 8.13 (t, 2H, 3J(1H-1H) = 6.9 Hz,
quin), 8.07 (td, 1H, 3J(1H-1H) = 6.9 Hz, 4J(1H-1H) = 1.4 Hz, quin),
7.86 (td, 1H, 3J(1H-1H) = 6.9 Hz, 4J(1H-1H) = 1.4 Hz, quin), 7.777.74 (m, 2H, ar), 7.17 (t, 2H, 3J(1H-1H) = 6.8 Hz, ar), 5.71 (d, 1H,
3J(1H-1H) = 6.1 Hz, p-cym), 5.52 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-
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cym), 5.45 (d, 1H, 3J(1H-1H) = 6.1 Hz, p-cym), 4.78 (d, 1H, 3J(1H1H) = 5.9 Hz, p-cym), 2.25-2.22 (m, 1H, CH(CH ) ), 2.13 (s, 3H,
3 2
CH3), 0.98 (d, 3H, 3J(1H-1H) = 6.9 Hz, CH(CH3)2), 0.84 (d, 3H,
3J(1H-1H) = 6.9 Hz, CH(CH ) ). 13C{1H} NMR (CD OD, 125 MHz,
3 2
3
298 K) δ 168.8 (Q, C-O), 161.4 (d, Q, C-F, 1J(13C-19F) = 238.9 Hz),
158.2 (Q), 149.7 (Q), 148.8 (Q) 141.4 (CH, quin), 132.8 (CH,
quin), 131.7 (CH, quin), 131.2 (CH, quin), 130.5 (Q), 130.1 (CH,
quin), 128.8 (d, 2 x CH, 3J(13C-19F) = 7.5 Hz, ar), 122.5 (CH, quin),
116.1 (d, 2 x CH, 2J(13C-19F) = 22.7 Hz, ar), 103.6 (Q), 102.9 (Q),
87.0 (CH, p-cym), 86.7 (CH, p-cym), 85.5 (CH, p-cym), 85.4 (CH,
p-cym), 32.3 (CH(CH3)2), 22.2 (CH(CH3)2), 22.0 (CH(CH3)2), 18.9
(CH3).
Compound 6. Yield: 84 mg, 0.06 mmol, 79%. ES-MS (+)
(MeOH): m/z 519.1 [RuC26H24N2OF2]+. Anal. Calc.: C 45.3, H
4.6, N 3.9%. Anal. Found: C 45.0, H 4.8, N 4.4%. 1H NMR:
(CD3OD, 500 MHz, 298 K) δ 8.92 (d, 1H, 3J(1H-1H) = 8.8 Hz,
quin), 8.61 (d, 1H, 3J(1H-1H) = 8.4 Hz, quin), 8.14 (dd, 1H, 3J(1H1H) = 8.2 Hz, 4J(1H-1H) = 1.5 Hz, quin), 8.10 (d, 1H, 3J(1H-1H) =
8.4 Hz), 8.05 (td, 1H, 3J(1H-1H) = 7.2 Hz, 4J(1H-13C) = 1.5 Hz,
quin), 7.97-7.84 (m, 2H, quin+ar). 7.15 (td, 1H, 3J(1H-1H) = 8.9
Hz, 3J(1H-19F) = 2.6 Hz, ar), 6.99 (td, 1H, 3J(1H-1H) = 8.0 Hz, 4J(1H13F) = 1.4 Hz, ar), 5.72 (d, 1H, 3J(1H-1H) = 6.1 Hz, p-cym), 5.58 (d,
1H, 3J(1H-1H) = 5.9 Hz, p-cym), 5.43 (d, 1H, 3J(1H-1H) = 6.1 Hz, pcym), 4.80 (d, 1H, 3J(1H-1H) = 5.9 Hz, p-cym), 2.30-2.25 (m, 1H,
CH(CH3)2, p-cym), 2.07 (s, 3H, CH3), 0.93 (d, 3H, 3J(1H-1H) = 6.9
Hz, CH(CH3)2), 0.81 (d, 3H, 3J(1H-1H) = 6.9 Hz, CH(CH3)2); 13C{1H}
NMR: (CD3OD, 125 MHz, 298 K) δ 169.0 (Q, C-O), 162.8 (d, Q,
C-F, 1J(13C-19F) = 244.0 Hz), 160.9 (d, Q, C-F, 1J(13C-19F) = 244.0
Hz), 157.1 (Q), 150.0 (Q), 141.3 (CH, quin), 134.7 (Q), 132.6 (2 x
CH, quin), 131.8 (CH, quin), 131.2 (CH, quin), 130.0 (d, CH,
3J(13C-19F) = 18.9 Hz, ar), 128.0 (Q), 122.5 (CH, quin), 112.3 (d,
CH, 2J(13C-19F) = 25.2 Hz, ar), 105.0 (d, CH, 2J(13C-19F) = 61.6 Hz,
ar), 101.7 (Q), 101.4 (Q), 88.7 (CH, p-cym), 86.0 (CH, p-cym),
85.6 (CH, p-cym), 85.2 (CH, p-cym), 32.2 (CH(CH3)2), 22.2
(CH(CH3)2), 22.0 (CH(CH3)2), 19.6 (CH3).
Acknowledgements
We wish to thank the EPSRC for funding, the technical staff at
the University of Leeds and Mr Colin Kilner and Dr. James
Mannion for help with X-ray crystallography.
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