Ruthenium(II) and osmium(II) 1,2,3-triazolylidene organometallics: A preliminary investigation into the biological activity of 'click' carbene complexes

Article abstract of DOI:10.1039/c3dt52584h

Taking advantage of the facile and versatile synthetic properties of 'click' 1,2,3-triazolylidene N-heterocyclic carbenes (tzNHC's), a range of new organometallic Ru(II) and Os(II) arene complexes containing functionalised tzNHC ligands, [M(η⁶-

Full text of DOI:10.1039/c3dt52584h

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Ruthenium(II) and osmium(II) 1,2,3-triazolylidene
organometallics: a preliminary investigation into
the biological activity of ‘click’ carbene complexes†
Cite this: Dalton Trans., 2014, 43,
1443
Kelly J. Kilpin,*a Stéphanie Crot,a Tina Riedel,a Jonathan A. Kitchenb and
Paul J. Dyson*a
Taking advantage of the facile and versatile synthetic properties of ‘click’ 1,2,3-triazolylidene N-hetero-
cyclic carbenes (tzNHC’s), a range of new organometallic Ru(II) and Os(II) arene complexes containing
functionalised tzNHC ligands, [M(η6-p-cymene)(tzNHC)Cl2] [M = Ru(II), Os(II)], have been synthesised and
fully characterised, including the X-ray crystal structure of one of the Os(II) complexes. The tzNHC ligands
remain coordinated to the metal centres under relevant physiological conditions, and following binding
to the model protein, ubiquitin. The in vitro cytotoxicity of the compounds towards human ovarian
cancer cells is dependent on the substituent on the tzNHC ligand but is generally <50 µM and in some
cases <1 µM, whilst still retaining a high degree of selectivity towards cancer cells over healthy cells
(1.85 µM in A2780 ovarian cancer cells versus 435 µM in human embryonic kidney cells in one case).
Received 18th September 2013,
Accepted 30th October 2013
DOI: 10.1039/c3dt52584h
www.rsc.org/dalton
Introduction
The introduction and concurrent success of cisplatin and
other platinum-based drugs in the clinic has undoubtedly
transformed cancer chemotherapy.1 However, the problems
associated with platinum agents, in particular their lack of
selectivity towards cancer cells, has resulted in the search for
alternative metal-based agents which overcome this limit-
ation.2 In this regard, ruthenium(III) coordination compounds,
notably NAMI-A and KP1019 (Fig. 1), are under clinical
development.3,4 As the proposed mechanism of action of such
compounds is thought to involve reduction in vivo, organome-
tallic Ru(II) compounds have also started to attract attention.5,6
Within this field, significant efforts have centered around the
development of the RAPTA-based (Ru Arene PTA, pta = 1,3,5-
triaza-7-phosphatricyclo[3.3.1.1]decane) compounds7 and
ethylene diamine [Ru-arene-en]+ compounds8 (Fig. 1).
Although complexes containing ligands other than phosphines
and amines have been investigated for their biological appli-
cations,9,10 one class of ligands which seems to have been
largely neglected are N-heterocyclic carbenes (NHC’s), which is
Fig. 1 Structures of selected Ru(III) and Ru(II) complexes developed as
anti-tumour agents.
somewhat surprising given the large research efforts and
success of Ru-NHC complexes in catalytic applications.11
The potential of metal-based NHC complexes as anti-cancer
agents has been reviewed only recently, with the bulk of
the research centered on either Group 10 or 11 metal
complexes.12–14 However, Ru(II) NHC complexes have been
touched upon, and show promising results in terms of enzyme
inhibition and in vitro anti-proliferative effects.15,16
aInstitut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de
Lausanne (EPFL), CH-1015 Lausanne, Switzerland
bChemistry, Faculty of Natural and Environmental Sciences, University of
Southampton, Highfield, Southampton, SO17 1BJ, United Kingdom
†Electronic supplementary information (ESI) available: Synthetic procedures
and characterisation data, selected NMR spectra, mass spectra. CCDC 960089.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3dt52584h
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Furthermore, studies on zebrafish embryos have demonstrated
that a selection of the complexes are essentially non-toxic,17
a
somewhat significant observation considering the high toxicity
of the clinically used platinum drugs.
Although relatively new,18 one class of topical NHC ligands,
which to the best of our knowledge remain unexplored in bio-
logical applications, are the 1,2,3-triazolylidene N-heterocyclic
carbenes.19,20 We are particularly interested in this class of
NHC as the facile, modular synthetic route via the CuAAC
(copper catalysed azide alkyne cycloaddition) ‘click’ reaction
offers the ability to readily introduce a number of functional
groups onto the parent triazole21,22 and ultimately the metal
complex23 e.g. for use in cellular targeting or enzyme inhi-
bition.24 Furthermore, the resulting strong metal–ligand bond
of the organometallic complex25 may prove advantageous in a
highly complex biological environment.
Herein, we report the synthesis, characterisation and, to the
best of our knowledge, the first investigation into the biologi-
cal activity of ruthenium(II) and osmium(II) arene compounds
containing 1,2,3-triazolylidene NHC ligands.
Results and discussion
Synthesis and characterisation
Ruthenium(II) and osmium(II) piano stool complexes contain-
ing functionalised tzNHC ligands were synthesised (Scheme 1)
using an adaption of the procedure reported by Albrecht
et al.18
In brief, the facile one-pot CuAAC methodology described
by Crowley and co-workers was used to synthesise the 1,4-di-
substituted-1,2,3-triazoles 2a–e, eliminating the need to isolate
the potentially explosive azide intermediates.26,27 Conversely,
Scheme 1 Route used to prepare Ru(II) and Os(II) 1,2,3-triazolylidene
NHC complexes. 1a/2a/3a/4a/5a: R = ethyl; 1b/2b/3b/4b/5b: R = n-hexyl;
1c/2c/3c/4c/5c: R = n-dodecyl; 1d/2d/3d/4d/5d: R = benzyl; 1e/2e/3e/
4e/5e:
R = 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl; 2f/3f/4f/5f:
R = 2,6-diisopropylphenyl.
1
2f was synthesised directly from 1-azido-2,6-diisopropyl is no longer present in the H NMR spectra of the metal com-
benzene. Regioselective methylation of the triazoles 2a–f at the plexes 4a–e and 5a–e, and the alpha protons on the N1 triazo-
N3 position was carried out using [Me3O][BF4] to afford 3a–f.28 lylidene substituent show a slight downfield shift (ca. 0.2 ppm)
Subsequent reaction of the triazolium salts 3a–f with Ag2O, fol- in the 1H NMR spectra relative to the triazolium salt precur-
lowed by addition of either [Ru(η6-p-cymene)Cl2]2 or [Os(η6-p- sors 3a–e. The 13C{1H} chemical shift of the carbene centre
cymene)Cl2]2, afforded the mononuclear piano stool com- (C-1) differs slightly for the ruthenium (ca. 160 ppm) and
plexes in good yields.18 Using this method we were able to osmium (ca. 148 ppm) complexes.
introduce the simple alkyl (ethyl 4a and 5a, hexyl 4b and 5b,
The main ion observed in the ESI mass spectra of the com-
dodecyl 4c and 5c), benzyl (4d and 5d), and diisopropylphenyl plexes arises from fragmentation via the loss of a chloride
(4f and 5f) substituents onto the tzNHC metal complex. Fur- ligand to give [M − Cl]+ type species.
thermore, as a proof of principle that the methodology can be
The molecular structure of 5d was unequivocally verified by
extended to incorporate biologically relevant functional groups single crystal X-ray crystallography on crystals grown at 4 °C by
such as glucosyl-based moieties, we also synthesised com- the slow diffusion of diethylether into a dichloromethane solu-
pounds containing a 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl tion of 5d. The molecular structure of 5d is shown in Fig. 2
group as a substituent on the tzNHC (4e and 5e). All the metal and selected bond parameters are given in the caption.
compounds were isolated as yellow to orange solids which
As expected, the complex adopts a piano-stool motif, com-
were soluble in a range of solvents (MeOH, CH2Cl2, MeCN, prising the p-cymene ‘seat’, with the two chloride ligands and
acetone, DMSO), and in addition, the presence of the carbo- the tzNHC ligand representing the three ‘legs’. The Os–tzNHC
hydrate substituent on 4e and 5e imparted good water solubi- bond length [Os(1)–C(1)] is 2.068(10) Å, with the benzyl N(1)
lity to the complexes.
substituent orientated in such a manner to minimize inter-
Characterisation data corroborate the expected structures of actions with the p-cymene ring. The Os⋯p-cymene centroid
the metal carbene complexes. Namely, the pre-carbene proton distance is [1.675(5) Å], with no appreciable tilt or offsetting of
of the triazolium salts 3a–f (H-1, ca. 8.6–8.8 ppm, CDCl3, ESI†) the p-cymene ring observed.
1444 | Dalton Trans., 2014, 43, 1443–1448
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the osmium(II) complex 5e also appears to undergo immediate
hydrolysis in aqueous phosphate buffered solution.
Biological activity
The biological activity of the compounds was initially explored
by determining their cytotoxicity against two human tumour
(A2780 and A2780R) and non-tumour (HEK) cell lines
(Table 1).
The new complexes show promising activity profiles,
notably, a number of compounds display low micromolar
activity against the tumour cell lines (<1 µM), which contrasts
with other bifunctional ruthenium arene compounds such as
RAPTA-C.33 This difference may be related to the relatively fast
hydrolysis process, at least in part, as relationships between
the rate of hydrolysis (activation) and cytotoxicity have been
demonstrated.34 Importantly, in all cases the tzNHC complexes
show selectivity towards the tumour cell lines (A2780 and
A2780R) over the non-tumourigenic HEK cell line – this is
exemplified by 4a which has a ca. 200-fold selectivity for the
A2780 cell line over the HEK cell line. In addition, such a
marked in vitro selectivity profile in this assay shows improve-
ments over the selectivity characteristics of cisplatin. It is also
evident that increasing the lipophilicity of the complex
(brought about by modifying the tzNHC alkyl sidechain) influ-
ences the in vitro activity of the complexes,35 with the activity
trends being 4a < 4b < 4c and 5a < 5b < 5c, presumably due to
increased uptake into cells.36 The relationship between the
metal ion employed, i.e. Ru(II) vs. Os(II), and the cytotoxicity for
the reported tzNHC complexes follows no obvious trends, and
prediction of the level of biological activity upon switching the
metal centre from Ru(II) to Os(II) is not straightforward. For
example, whilst the in vitro cytotoxicity of RAPTA-C and
OsAPTA-C ([Os(η6-p-cymene)Cl2(PTA)] is of a similar order of
magnitude,37 when the PTA ligand is replaced by a carbo-
hydrate-based phosphine in a similar series of compounds, the
Ru(II) complexes are more active than the Os(II) analogues.38
As RAPTA complexes are believed to interact with proteins in
preference to DNA,39 we also probed the interaction of 4b with
the model protein ubiquitin (Ub) using HR-ESI mass
Fig. 2 Molecular structure of 5d·CH2Cl2 with selected atom labelling.
Hydrogen atoms and the CH2Cl2 solvate have been omitted for clarity.
Thermal ellipsoids are drawn at 30% probability. Selected bond lengths (Å)
and angles (°): Os(1)–C(1) 2.068(10), Os(1)–Cl(1) 2.420(3), Os(1)–Cl(2) 2.445(3);
C(1)–Os(1)–Cl(1) 85.7(3), C(1)–Os(1)–Cl(2) 90.9(3), Cl(1)–Os(1)–Cl(2) 82.53(11).
Behaviour in solution
The chloride ligand(s) of ruthenium arene compounds such as
those of the RAPTA and [Ru-arene-en]+ series (Fig. 1) undergo
hydrolysis in aqueous solution, giving aquated species, which
are proposed to be the active form of the compounds inside
cells.29,30 The rate of this hydrolysis process has been
suggested to influence the biological activity of ruthenium
drugs therefore, it is useful to evaluate the behaviour of
new compounds under pseudo-physiological conditions.31,32
As such, the solution characteristics of the new ruthenium(II)
and osmium(II) tzNHC complexes were probed by a combi-
1
nation of UV-Vis spectroscopy and H NMR spectroscopy, and
compared to the RAPTA series of compounds, in particular
RAPTA-C, ([Ru(η6-p-cymene)Cl2(PTA)], due to the structural
similarity. In aqueous phosphate buffered solution (10 mM,
pH 7.4), the UV-Vis spectra of 4e was stable and unchanged
from the initial solution over a period of 7 days. However, the
UV-Vis trace differed when the spectrum was acquired in
saline phosphate buffered solution (10 mM, pH 7.4, [Cl−]
150 mM), indicating that under the former conditions hydro-
lysis of one, or both, of the chloride ligand(s) had already
taken place. Unlike RAPTA-C, which reaches an equilibrium
after ca. 20 min,29 hydrolysis of 4e must be very rapid. More-
over, as with RAPTA-C, the process is suppressed under rela-
tively high physiological chloride concentrations. Furthermore,
Table 1 Cytotoxicity (IC50, μM) of selected compounds at 72 h
A2780a
A2780Rb
HEKc
4a
5a
4b
5b
4c
5c
4d
5d
4e
5e
4f
5f
1.85 0.29
27.8 12.3
0.31 0.08
4.97 0.03
0.12 0.02
0.12 0.02
4.82 0.12
1.41 0.06
41.0 8.2
19.9 2.3
0.21 0.05
0.13 0.03
4.3 0.5
25.6 5.5
112 13
435 25
168 11
29.0 1.8
16.5 1.4
1.80 0.20
2.59 0.34
64.0 6.1
31.7 5.0
>200
1.77 0.04
14.2 3.3
0.23 0.02
0.33 0.06
37.8 7.8
2.36 0.46
>200
1
although the H NMR spectrum of 4e (D2O) is rather compli-
cated due to the presence of a number of different species (ten-
tatively assigned as hydrolysis products with the loss of either
one or both chloride ligands), there was no evidence to
suggest the loss of either the p-cymene or tzNHC ligands. The
electrospray mass spectra of an aqueous solution of 4e also
provides evidence which points towards the loss of one or both
of the chloride ligands, with the ions corresponding to both
105
5
>200
1.10 0.12
0.21 0.01
18.2 1.0
1.85 0.12
2.89 0.03
15.3 0.5
Cisplatin
a Human ovarian carcinoma cells. b Human ovarian carcinoma cells –
[4e–Cl]+ and [4e–2Cl]2+ species present (see ESI†). As with 4e, acquired resistance to cisplatin. c Human embryonic kidney cells.
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keeping with previously studied Ru(II) arene complexes, the
tzNHC examples also undergo hydrolysis in aqueous solution,
but in contrast, do not react with model proteins such as Ub
as readily. Different groups can be easily introduced onto the
tzNHC ligand via the initial CuAAC click reaction. Thus, a
number of different functionalities, e.g. targeting groups,
enzyme inhibitors, optically active fluorophores/lumophores,
may be introduced, allowing diversity to be included in the
complexes, an important aspect with respect to drug discovery.
Experimental
General
[Ru(η6-p-cymene)Cl2]2,41 and [Os(η6-p-cymene)Cl2]2,42 were pre-
pared using literature procedures, all other reagents and sol-
vents were obtained from commercial sources and used
without further purification. The general synthetic protocol for
the synthesis of the metal tzNHC complexes 4a–f and 5a–f is
outlined below, full synthetic details and characterisation data
for all compounds in this study (including 1,4-disubstituted-1-
2-3-triazoles 2a–f and the corresponding triazolium salts 3a–f)
are included in the ESI.†
Fig. 3 ESI mass spectra of (a) Ub; (b) Ub + 4b, (1 : 5 ratio) 1 day incu-
bation and (c) Ub + 4b, (1 : 5 ratio) 5 day incubation, with deconvoluted
mass spectra shown as insets.
General synthetic procedure for Ru(II) and Os(II) tzNHC
complexes 4a–f and 5a–f
The triazolium salt (1 equiv.), Ag2O (0.5 equiv.) and NMe4Cl
(1 equiv.) were stirred in CH2Cl2–MeCN (1 : 1, 10 mL) in a foil-
covered flask for 18 h to give a cloudy white solution. The
solvent was removed and the residue redissolved in CH2Cl2.
[M(η6-p-cymene)Cl2]2 (M = Ru or Os) (0.5 equiv.) was added
and the solution stirred for a further 90 min. The solution was
then filtered through a short column of celite to remove the
salt by-products, concentrated under reduced pressure and
precipitated with either Et2O or hexane at −4 °C. The solid was
isolated by filtration, washed with either Et2O or hexane and
dried in vacuo.
spectrometry. Complex 4b was incubated with Ub (5 : 1) at 37 °C
and the mass spectra were recorded after 1 and 5 days (Fig. 3).
After 1 day, peaks corresponding to a 1 : 1 Ub-complex
adduct is observed, assigned as Ub + [4b–2Cl], with the tzNHC
fragment still bound to the metal centre. After 5 days, partial
loss of the tzNHC ligand was observed, and although 4b was
present in excess, the mass spectrum still indicated the pres-
ence of unmodified Ub. This reactivity is quite distinct to that
of RAPTA-C (see ESI†), which reacts far more readily with Ub,
with loss of the pta ligand observed after 1 day, and prolonged
incubation leading to the complete disappearance of free Ub.
The osmium(II) tzNHC complex responded differently again,
and was not found to coordinate to Ub under the employed
conditions (see ESI†). The low reactivity of the tzNHC com-
pounds towards Ub, and presumably other relevant proteins,
may prove advantageous, as with some drugs (in particular cis-
platin), interactions with proteins are believed to deactivate
the drug and/or lead to adverse side effects.40
X-Ray crystallography
Single crystal X-ray diffraction data for 5d·CH2Cl2 were col-
lected using graphite monochromated Cu Kα radiation (λ =
1.54180 Å) on an Agilent Technologies SuperNova dual system
in combination with an Atlas CCD detector. Data reduction
was carried out using Crysalis PRO.43 The structure was solved
by direct methods (SHELXS-97) and refined against all F2 data
(SHELXL-97 and Shelxle).44,45 Non-hydrogen atoms were
refined with anisotropic displacement parameters. Hydrogen
atoms were positioned geometrically and refined using a
riding model with d(CHaro) = 0.93 Å, Uiso = 1.2Ueq.(C) for
aromatic, d(CH) = 0.98 Å, Uiso = 1.2Ueq.(C) for CH, 0.97 Å,
Uiso = 1.2Ueq.(C) for CH2 and 0.96 Å, Uiso = 1.2Ueq.(C) for
CH3 (Table 2).
Conclusions
For the first time, we have demonstrated that the relatively new
tzNHC ligands offer considerable potential in medicinal in-
organic/organometallic chemistry. The ruthenium(II) and
osmium(II) η6-arene complexes with these ligands show an
excellent activity profile against a range of cell lines, and in
Cell culture conditions and cytotoxicity assay
some instances, such as compound 4a, show excellent selecti- Human A2780 and A2780cisR ovarian carcinoma and HEK
vity towards tumour cell lines over non tumour lines. In (human embryonic kidney) cells were obtained from the
1446 | Dalton Trans., 2014, 43, 1443–1448
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ratio of 5 : 1 at 37 °C. Immediately prior to analysis, an aliquot
of the incubation solution was diluted with water–acetonitrile–
trifluoroacetic acid (50 : 50 : 0.1) to yield a final protein concen-
tration of 0.5 μM, and introduced by direct infusion into
the ESI source of the mass spectrometer at a flow rate of
5 μL min−1 (source voltage, +4.5 kV; capillary temperature,
180 °C; sheath gas flow, 4 L min−1). Data were processed with
DataAnalysis 4.0 SP5 software (Bruker Daltonics).
Table 2 Crystal data and structure refinement parameters for
5d·CH2Cl2
CCDC code
960089
C27H31Cl4N3Os
729.55
Empirical formula
Formula weight
Temperature
298(2) K
Wavelength
Crystal system
Space group
1.54180 Å
Monoclinic
P21/n
Unit cell dimensions
a = 6.38330(10) Å, b = 28.5636(6) Å,
c = 15.9512(3) Å; β = 98.391(2)°
2877.25(9) Å3
4
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Index ranges
1.684 Mg m−3
11.946 mm−1
1432
Acknowledgements
0.33 × 0.17 × 0.10 mm3
3.09 to 67.48°
−7 ≤ h ≤ 5, −20 ≤ k ≤ 34,
−16 ≤ l ≤ 19
10 726
This work was supported by the New Zealand Ministry of
Business, Innovation and Employment (formally Foundation
of Research, Science and Technology Postdoctoral Fellowship,
EPFL1001, to KJK) and the EPFL. We thank Dr R. Scopelliti for
the collection of the X-ray data set.
Reflections collected
Independent reflections
Completeness to theta =
67.48°
5178 [R(int) = 0.0360]
99.8%
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Semi-empirical from equivalents
0.3919 and 0.1083
Full-matrix least-squares on F2
5178/0/317
Notes and references
1.192
R1 = 0.0641, wR2 = 0.1693
R1 = 0.0713, wR2 = 0.1732
2.700 and −2.486 e Å−3
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