Adding diversity to ruthenium(II)-arene anticancer (RAPTA) compounds via click chemistry: The influence of hydrophobic chains

Article abstract of DOI:10.1016/j.jorganchem.2010.09.067

The application of click chemistry to develop libraries of organometallic ruthenium-arene complexes with potential anticancer properties has been investigated. A series of ruthenium-imidazole-triazole complexes, with hydrophobic tails, were prepared from a common precursor via click chemistry. The tail could be attached to the ligand prior to coordination to the ruthenium complex or following coordination, the former giving the product in superior yield. The complexes were screened for cytotoxicity in tumourigenic and non-tumourigenic cell lines, and while the compounds were only moderately cytotoxic, good selectivity for tumourigenic cells was observed.

Full text of DOI:10.1016/j.jorganchem.2010.09.067

Journal of Organometallic Chemistry 696 (2011) 772e779
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Adding diversity to ruthenium(II)earene anticancer (RAPTA) compounds via click
chemistry: The influence of hydrophobic chains
a
b
a,
*
Anna K. Renfrew , Lucienne Juillerat-Jeanneret , Paul J. Dyson
a
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
Institute of Pathology, University Hospital (CHUV), University of Lausanne, Lausanne, Switzerland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2010
Received in revised form
The application of click chemistry to develop libraries of organometallic rutheniumearene complexes
with potential anticancer properties has been investigated. A series of rutheniumeimidazoleetriazole
complexes, with hydrophobic tails, were prepared from a common precursor via click chemistry. The tail
could be attached to the ligand prior to coordination to the ruthenium complex or following coordi-
nation, the former giving the product in superior yield. The complexes were screened for cytotoxicity in
tumourigenic and non-tumourigenic cell lines, and while the compounds were only moderately cyto-
toxic, good selectivity for tumourigenic cells was observed.
29 September 2010
Accepted 29 September 2010
Available online 8 October 2010
Keywords:
Bioorganometallic chemistry
Anticancer drugs
Ó 2010 Elsevier B.V. All rights reserved.
Click chemistry
Metal-based drugs
Bioinorganic chemistry
1. Introduction
labile chloride ligands (Fig. 1). The in vitro cytotoxicities of these
compounds, termed RAPTA compounds, are generally modest, but
The field of metal-based anticancer agents, beginning with
cisplatin [1], remains an area of immense research interest. Many
classes of compounds have been studied including coordination
complexes [2] and organometallic compounds [3]. Cisplatin induces
apoptosis via DNA binding [4], whereas other metal compounds
exert their antiproliferative effect via inhibition of mitochondrial
activity [5], critical enzymes [6], angiogenisis [7], etc. Among the
various transition metal complexes that have been investigated,
ruthenium compounds have emerged as promising candidates, with
two ruthenium(III) compounds, NAMI-A and KP1019, presently
undergoing clinical evaluation (Fig. 1). Both compounds behave
quite differently to cisplatin; NAMI-A inhibits growth of metastatic
tumours [8], while KP1019 is active in colorectal tumours where
cisplatin exhibits limited activity [9]. Protein binding, especially to
transport proteins, appears to be essential for the modes of action of
certain ruthenium anticancer agents [10].
nevertheless some display highly promising antimetastatic activity
in vivo [11], although on certain tumour models activity towards
primary tumours is also observed [12]. Interestingly, this biological
profile is very similar to that observed for NAMI-A [13].
Consequently, in an attempt to synergistically combine key
structural features of RAPTA and NAMI-A compounds a series of Ru
(II)-arene compounds bearing 1, 2 or 3 imidazole ligands (Fig. 1)
were investigated [14]. In keeping with RAPTA and NAMI-A, the
organometallic imidazole compounds were found to show modest
in vitro activity, with IC50 values in the range 100e1000
mM
depending on the nature of the imidazole ligand. The tris-imidazole
complexes were the most cytotoxic with an approximate correlation
with compound lipophilicity. Like NAMI-A and RAPTA compounds
the Ru(II)-arene imidazole compounds tended to be selective
towards the human ovarian carcinoma A2780 and its cisplatin-
resistant variant A2780cisR cell lines relative to the model non-
tumoral human cells.
Our research has focused on the development of a series of
organometallic ruthenium(II) compounds containing a hydro-
While a large series of RAPTA-type compounds has been
prepared and evaluated with respect to their stability, reactivity to
biomolecules [15], and biological activity in vitro, and in several
cases in vivo [11,12], the possibility to add further diversity via click
chemistry [16] is very attractive. Click chemistry has previously
been exploited in the design of technetium radiolabelled contrast
agents [17], the synthesis of organometallic-peptide derivatives[18]
6
phobic
h
-arene moiety, a hydrophilic monodentate 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane (pta) ligand and, in most cases, two
*
Corresponding author. Tel.: þ41 (0)21 693 98 85.
E-mail address: paul.dyson@epfl.ch (P.J. Dyson).
0
022-328X/$ e see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2010.09.067

A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
773
N
2. Results and discussion
H
N
N
Click chemistry was used to prepare a series of imidazole ligands
with pendant chains attached. Two strategies were validated
commencing with 5-ethynyl-1-methyl-imidazole; first, the imid-
azole derivative was coordinated to the rutheniumearene complex
and reacted with azidoalkanes under copper(I) catalysed condi-
tions (Schemes 1 and 2), and second, 5-ethynyl-1-methyl-imid-
azole was derivatized under click conditions and then coordinated
to the ruthenium centre (Scheme 3).
Cl
Cl
Cl
Cl
O
Ru
S
Ru
Ru
N
N
H
Cl
Cl
N
Cl
P
N
N
N
N
N
R
R
NAMI-A
RAPTA-C
Fig. 1. Selected ruthenium complexes under investigation as antitumour agents.
2 2
h ] with 2
6-p-cymene)Cl
In detail, the reaction of the dimer [Ru(
equivalents of 5-ethynyl-1-methyl-imidazole in chloroform yielded
1/2
6
[
2
Ru(h -p-cymene)(5-ethynyl-1-methyl-imidazole)Cl ] as the only
Cl
RuII
Cl
Cl
RuII
product (Scheme 1). In contrast, the analogous reaction in ethanol
affords a dark brown precipitate that exhibits very broad signals in
the H NMR spectrum. Electrospray ionisation mass spectrometry
Cl
1
(
ESI-MS) of the product revealed a parent ion at m/z 608.2 tentatively
þ
assigned to [Ru(5-ethenyl-1-methyl-imidazole)
4
2
Cl ] (Scheme 1). It
would therefore appear that the alkyne groups are reduced under
the reaction conditions (although it cannot be ruled out that two of
the ethynyl-substituents are fully hydrogenated and two remain
unchanged).
N
N
CHCl3,
Δ
EtOH, Δ
N
N
Cycloaddition reactions between [Ru(
-methyl-imidazole)Cl ] and 1-azido-1H,1H,2H,2H-perfluorodecane
were attempted under various conditions using CuSO as the cata-
h
6-p-cymene)(5-ethynyl-
1
2
N
N
N
N
4
Cl
N
N
lyst with a 2-fold excess of sodium ascorbate as the reducing agent.
RuII
Cl
RuIII
t
Reactions were carried out over 24 h in water: butanol (1:1) or
Cl
N
water:methanol (1:1) as the solvent, under reflux or at room
temperature, with catalyst loadings ranging from 2 to 10%. The
desired product was not isolated under any of the conditions
employed with unreacted azidoperfluorodecane and a black para-
magnetic solid resulting from the decomposition of the complex. It
has previously been noted that click reactions involving compounds
with metal-chloride bonds are problematic [20].
Cl
N
N
N
Scheme 1. Influence of the solvent in the reaction of [Ru( 6
ethynyl-1-methyl-imidazole.
2 2
h -p-cymene)Cl ] with 5-
To prevent undesirable side reactions at the ruthenium centre,
a kinetically more inert ruthenium complex was used in which the
two chloride ligands were replaced by a chelate 1,1-cyclobutane-
dicarboxylate (A) or oxalate ligand (B) [21]. Reaction of the complexes
under the conditions shown in Scheme 2 resulted in the formation of
the desired cycloaddition products in moderate (21% for C) and good
(74% for D) yield.
and in the synthesis of platinum complexes showing promising
anticancer activity [19]. We have recently shown that RAPTA-type
compounds with perfluorinated alkyl chains are highly cytotoxic,
and consequently, in this paper we explored various approaches
based on click chemistry to add alkyl chains to RAPTA-type
compounds, and subsequently evaluated their cytotoxicity.
N C H C F
2 4 8 17
3
N
N
N
0
.2 CuSO
4
Ru
O
Ru
O
0
.4 Na Ascorbate
:1 tBuOH:H O
C H C F
2 4 8 17
O
O
O
N
O
N
1
2
N
N
O
A
O
C
2
1%
N C H C F
2 4 8 17
3
N
N
N
0
.2 CuSO
4
Ru
O
Ru
0
.4 Na Ascorbate
C H C F
2 4 8 17
O
O
N
N
1:1 tBuOH:H O
O
2
O
N
O
N
O
O
B
D
74%
Scheme 2. Click chemistry with (inert) ruthenium(II) complexes.

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A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
N C H R
3
2 4
0
.2 CuSO
4
N
N
0.4 Na Ascorbate
N
N
N
t
1
:1 BuOH:H O
2
N
N
C H R
2
4
L1 - L5
Ar
1
/2
Cl
Cl
Cl
Ru
Ru
Cl
Ar
2
N
N
N
N
N
N
N
N
CHCl , Δ
CHCl , Δ
3
3
N
N
C H R
C H R
2
4
2
4
Ar
N
Ar
N
Cl
N
N
N
N
N
Ru
Ru
N
C H R
N
C H R
2 4
Cl
2
4
N
Cl
Cl
N
N
.
2 Cl, 4 Cl, 5 Cl, 7 Cl
.
.
.
1, 6 - 8
N
N
RC H
N
2
4
NH BF , pta MeOH
MeOH
NH BF , pta
4 4
4
4
Ar
N
BF4
N
N
N
Ru
C H R
N
P
2 4
N
Cl
N
N
.
1
.
BF4 - 8 BF4
Arene = phenyl-2-ethanol
Arene = p-cymene
.
.
7
8
BF
BF
4
: R = C10
: R = C
H
21
1
2
3
4
5
6
BF
BF
BF
BF
BF
BF
4
4
4
4
4
4
: R = C
: R = C
: R = C10
6
H
H
H
13
,
.
.
.
.
.
.
4
2
H
4
C
8 17
F
8
17
21
: R = C
: R = C
: R = C
2
2
2
H
H
H
4
4
4
C
C
(OC
4
F
F
9
8
17
2
H
4
)
3
NH
2
Scheme 3. Synthesis of triazoleeimidazole ligands via copper(I)-catalyzed Huigsen cycloaddition and subsequent reactions to form the ruthenium(II)-arene complexes.
1
Reaction of 5-ethynyl-1-methyl-imidazole with azidoalkanes
All the compounds were characterised by H NMR spectroscopy
31
under ‘click’ conditions affords the expected 1,2,3-triazole products
in excellent yield (Scheme 3). This approach was used to prepare
a series of imidazole ligands with alkyl, perfluoroalkyl and poly-
ether chains. Reaction of 2 equivalents of these imidazole ligands
using 2D COSY to assign the signals (and for 1$BF
spectroscopy), ESI-MS and elemental analysis (with the exception of
5$Cle5$BF and 7e7$BF , where the highly perfluorinated chain
prevents accurate determination of the C, H and N composition). In
each case, only one regioisomer was obtained in the synthesis of
ligandsL1eL6. On coordination to the ruthenium centre, the aromatic
imidazoleandtriazoleprotons, H , H andH (see Experimental) were
shifted to higher frequency (ca. 0.2e1 ppm) with the affect most
pronounced for the bis-imidazole complexes 2$Cle7$Cl. No evidence
of chelation through the triazole group was observed; the additional
electronegative nitrogen atom of triazole groups with respect to
imidazole typically results in poor donor ligands [22]. Compounds
4 4
e8$BF by P NMR
4
4
with the appropriate ruthenium(II)-arene dimer gave complexes of
6
formula [Ru(
h
-arene)(im)Cl
2
] (complexes 1e8 in Scheme 3). In the
1
2
4
presence of 4 equivalents of the imidazole ligands, the corre-
sponding bis-imidazole complexes (type c) were obtained. RAPTA-
imidazole compounds 1$BF
either the mono or bis-imidazole complexes with a slight molar
excess of pta and NH BF . Compounds 1$BF e8$BF are soluble in
chlorinated organic solvents, 1$BF e5$BF
water and the polyether derivative 6$BF
Analogues of 3$BF and 4$BF
ligand, viz. 7$BF and 8$BF , were also prepared and indeed show
a higher solubility in water.
4 4
e8$BF can be prepared by reaction of
4
4
4
4
4
4
are sparingly soluble in
is highly water soluble.
4
1$BF
as a racemic mixture of two enantiomers.
Complexes 6$BF e8$BF (being the most water-soluble of the
series) exhibit a single peak in the P NMR spectrum in D
4 4
e8$BF have a chiralcentre at theruthenium and were obtained
4
4
with a more water soluble arene
4
4
4
4
31
2
O. The

A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
775
[11,12] are only weakly cytotoxic, and yet show antitumour activity
in vivo. It has been shown that these compounds exert other effects
on tumourigenic cells such as detachment from the primary tumour,
the migration/invasion and the re-adhesion to a new growth
substrate [11b]. Notably, these effects are more pronounced when
experiments are performed with the highly invasive MDA-MD-231
cells than with the non invasive MCF-7 or the non-tumourigenic
HBL-100 cell lines.
3. Conclusions
Several strategies were evaluated based on click chemistry to add
diversity to RAPTA-type compounds including the use of a kineti-
6
cally stable precursor, i.e. [Ru(h -cymene)(oxalato)(ethynyl-im)],
which allows diverse substituents to be ‘clicked’ directly onto the
ruthenium complex. Moreover, it was shown that the click reaction
provides access to a series of functionalised imidazole ligands, not
6
easily prepared by other methods, from which cationic [Ru(
h
-
Fig. 2. Plot of surface tension versus e log[complex] in PBS for 6$BF
4 4
5 7$BF A and
þ
8
$BF :.
4
arene)Cl(im)pta] complexes could be readily prepared. In this
specific study the effect of chain length and hydrophobicity on both
micelle formation and in vitro cytotoxic activity was evaluated for
the ruthenium derivatives. The complexes do not form micelles in
aqueous solution, but in keeping with other classes of compounds
25], there is a direct correlation between in vitro cytotoxic activity
and lipophilicity, with 7$BF and 8$BF being more cytotoxic than
the parent complex RAPTA, and with selectivity towards tumouri-
genic cell lines maintained.
spectrum is unchanged following addition of 100 mM NaCl, indi-
cating that aquation does not readily occur, and over a 72 h period
no indication of aquation or decomposition was observed.
The surface tension of the water-soluble complexes, 6$BF
was measured in PBS using a Du-Nouy ring tensiometer (Fig. 2). As
expected, 6$BF , containing a hydrophilic polyether chain, demon-
strates the lowest surface activity. No evidence of micelle formation
which would be indicated by a plateau in the plot of surface tension
4 4
e8$BF ,
[
4
4
4
(
vs. concentration) was observed for any of the complexes up to the
most concentrated solutions accessible (1e2 mM). These surface
tension data exclude the possibility of the involvement of micelles in
the in vitro anticancer studies (see below).
The antiproliferative activity of 1$BF e8$BF was evaluated
4 4
against the A2780 ovarian cancer cell line and cisplatin-resistant
strain A2780cisR, using the MTT assay which measures the meta-
4
. Experimental
6-p-cymene)Cl 6-phenyl-
2 2
] [27], [Ru(h
Azidoalkanes [26], [Ru(
-ethanol)Cl ] [28] and pta [29] were prepared according to liter-
2 2
h
2
ature procedures. 1-Iodo-1H,1H,2H,2H-perfluorohexane, 1-iodo-1H,
H,2H,2H-perfluorodecane, 5-ethynyl-1-methyl-imidazole, 11-az-
ido-3,6,9-trioxaundecane-1-amine, CuSO and sodium ascorbate
1
4
4 4
bolic activity of viable cells (Table 1). Complexes 1$BF e5$BF were
were purchased from Sigma Aldrich and used as received. For reac-
tions involving ruthenium complexes all solvents were degassed
prior to use and manipulations were carried out using Schlenk
techniques. H and P{ H} NMR spectra were recorded on a Bruker
Avance DPX 400 MHz spectrometer at room temperature. H , H
and H describe aromatic protons H-ortho, H-meta and H-para
respectively. ESI-MS of the complexes were obtained on a Thermo-
finigan LCQ Deca XP Plus quadrupole ion trap instrument set in
positive mode (solvent: methanol; flow rate: 5
5
pre-dissolved in DMSO due to their low aqueous solubility.
In general, the cytotoxicities of the complexes may be correlated
to their lipophilicity e expressed as octanol:water partition coeffi-
cients. As the length of the alkyl chain increases, the lipophilicity
increases and the greater the cytotoxicity. Surprisingly there is
negligible difference between fluorous and alkyl chains of equiva-
lent length although the fluorous chains are more hydrophobic.
Compounds 7 and 8 were also evaluated in the non-tumoral human
1
31
1
o
m
,
p
ml/mn; spray voltage:
HCEC endothelial cell line displaying IC50 values >500
mM thereby
ꢀ
kV; capillary temperature: 100 C; capillary voltage: 20 V), as
demonstrating the selectivity of the compounds towards tumouri-
genic cells.
While the cytotoxicities of 1$BF e8$BF are modest, other
4 4
ruthenium drug candidates such as NAMI-A [24] and RAPTA-C
described previously [30]. Microanalyses were performed at the
EPFL. UVevis measurements were performed on a JASCO V-550
spectrophotometer.
4.1. Synthesis of the azidoalkanes
Table 1
Cytotoxicity measured as IC50 values (
following 72 h cell exposure to 1$BF e8$BF
m
M) determined using the MTT assay
and compared to log Pow coefficients.
Azidoalkanes were prepared according to a literature procedure
[26], as described below for 1-azidohexane.
4
4
Compound
A2780/
m
M
A2780cisR/
m
M
Log Powa
N3
2
$BF b
4
1
2
3
4
5
6
7
8
4
>300
75
43
151
55
>300
144
125
>300
178
64
158
34
>300
51
142
ꢂ0.33
ꢂ0.27
ꢂ0.24
0.22
b
b
b
b
1
$BF
$BF
$BF
$BF
$BF
$BF
$BF
4
4
4
4
4
4
4
3
6
5
0.06
ꢂ1.60
ꢂ0.93
ꢂ0.66
A suspension of iodohexane (5 g, 23.6 mmol) and NaN
41 mmol) in DMF (50 mL) was stirred for 72 h at room tempera-
ture. The mixture was then poured into cold water (50 mL) and the
3
(8.75 g,
a
Partition coefficient octanol:water determined according to the procedure
1
described in Ref. [23].
b
Compounds were pre-dissolved in DMSO, max. concentration in final solutions
<0.5%.
product extracted with diethyl ether (3 ꢁ 50 mL). The combined

776
A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
1
organic phases were washed with brine (2 ꢁ 50 mL), dried with
further purification. Yield 507 mg (88%). H NMR (CD Cl ): d 7.72 (s,
2 2
0
0
0
0
2
5
3
3
sodium sulphate and the solvent removed under reduced pressure
1H, H ), 7.50 (s, 1H, H ), 7.21 (s, 1H, H ), 4.42 (t, JHH ¼ 7.3 Hz, 2H,
0 0
1 2
1
to give a clear oil which was used without further purification. H
H ), 3.91 (s, 3H, N-CH
3
), 1.94e2.00 (m, 2H, H ), 1.31e1.39 (m, 14H,
0
0
0
3
1
3
3 e9
3
10
NMR (CDCl
2
3
);
d
3.27 (t, JHH ¼ 7.1 Hz, 2H, H ), 1.60 (t, JHH ¼ 7.3 Hz,
H
), 0.93 (t, JHH ¼ 6.4 Hz, 3H, H ). ESI-MS (MeOH) m/z ¼ 290.2,
2
3e5
3
6
þ
H, H ), 1.29e1.43 (m, 6H, H ), 0.91 (t, JHH ¼ 6.6 Hz, 3H, H ).
[L3-H] (100%). Anal: Calculated for C16
H
27
N
5
([L3]) C, 66.40; H,
1
3
1
ꢀ
1
-azidooctane: H NMR (CDCl
3
):
d
3.27 (t, JHH ¼ 7.0 Hz, 2H, H ),
9.40; N, 24.20. Found C, 65.78; H, 9.91; N, 24.30. m.p 60e62 C.
3
2
3e7
1
J
.62 (t,
J
HH ¼ 7.0 Hz, 2H, H ), 1.25e1.42 (m, 10H, H ), 0.91 (t,
3
8
HH ¼ 6.8 Hz, 3H, H ).
4.2.4. 1H,1H,2H,2H-perfluorohexyl-4-(1-methyl-1H-imidazol-2-
yl)-1H-1,2,3-triazole L4
1
3.26 (t, 3JHH ¼ 6.8 Hz, 2H, H ),
1
1
-azidodecane: H NMR (CDCl
3
):
d
3
2
3e9
1
.60 (t,
J
HH ¼ 7.1 Hz, 2H, H ), 1.25e1.42 (m, 14H, H ), 0.91 (t,
The product was obtained as an oil which precipitated on
3
10
J
HH ¼ 6.6 Hz, 3H, H ).
addition of hexane to yield an off white solid that was used without
1
1
-azido-1H,1H,2H,2H-perfluorohexane:
further purification. Yield 715 mg (91%). H NMR (CDCl
3
):
d
7.80 (s,
00
00
3
2
5
3
1
H, H ), 7.54 (s, 1H, H ), 7.26 (s, 1H, H ), 4.75 (t, JHH ¼ 6.8 Hz, 2H,
0 0
1 2
N3
2
H ), 3.95 (s, 3H, N-CH
3
), 2.80e2.90 (m, 2H, H ). ESI-MS (MeOH) m/
þ
1
z ¼ 396.1, [L4-H] (100%). Anal: Calculated for C H F N ([L4]) C,
(
C F )CF
3
12 10 9 5
2
4
3
6.47; H, 2.55; N, 17.72. Found C, 36.45; H, 2.14; N, 17.46. m.p
1
3.60 (t, 3JHH ¼ 6.2 Hz, 2H, H ) 2.28e2.48 (m,
1
3
H NMR (CDCl ): d
ꢀ
103e107 C.
2
2
H, H ).
1
-azido-1H,1H,2H,2H-perfluorodecane: 1H NMR (CDCl
): d 3.62
3
4
1
.2.5. 1H,1H,2H,2H-perfluorodecyl-4-(1-methyl-1H-imidazol-2-yl)-
H-1,2,3-triazole L5
1
2
(
m, 2H, H ) 2.29e2.50 (m, 2H, H ).
The product was obtained as an off white solid, which was
4
.2. Synthesis of L1eL6
washed with hexane (20 mL) to give the pure product. Yield 114 mg
00
2
1
(
1
96%) of white powder. H NMR (CDCl
3
):
d
7.77 (s, 1H, H ), 7.55 (s,
1
,2,3-triazoles were prepared according to the following
procedure:
A suspension of 5-ethynyl-1-methyl-imidazole (0.2 mL, 2 mmol),
-azidoalkane (2 mmol), CuSO (64 mg, 0.4 mmol) and sodium
00
0
5
3
3
1
H, H ), 7.26 (s, 1H, H ), 4.76 (t, JHH ¼ 6.8 Hz, 2H, H ), 3.95 (s, 3H,
0
2
N-CH
3
), 2.84e2.98 (m, 2H, H ). ESI-MS (MeOH) m/z ¼ 596.2, [L5-
þ
ꢀ
H] (100%). m.p. 110e116 C.
1
4
t
ascorbate (158 mg, 0.8 mmol) in butanol:water (20 mL, 1:1) was
stirred at room temperature for 24 h. The reaction contents was then
poured into 5% NH OH (50 mL) and the product was extracted with
4
dichloromethane (3 ꢁ 50 mL). The combined fractions were dried
with sodium sulphate and the solvent was removed under reduced
pressure to give the product as a white solid or oil.
4
1
.2.6. 2-(2-(2-(2-(4-(1-Methyl-1H-imidazol-2-yl)-1H-1,2,3-triazol-
-yl)ethoxy)ethoxy)ethoxy)- ethanamine L6
The product was obtained as a brown oil. Purification by flash
column chromatography on silica gel with ¼ 10:1 DCM:MeOH as
the elutant affording the product as a yellow oil. Yield: 442 mg
00
1
2
5
(
68%) H NMR (CDCl
3
):
d
8.08 (s, 1H, H ), 7.54 (s, 1H, H ), 7.31 (s, 1H,
0
0
0
3
3
1
H ), 4.63(t, JHH ¼ 4.9 Hz, 2H, H ), 3.96 (s, 3H, N-CH
3
), 3.90e3.95
3
''
0
0
N
5
2
3
10
3
N
N
N
(m, 2H, H ), 3.73 (t, JHH ¼ 4.9 Hz, 2H, H ), 3.69 (t, JHH ¼ 5.9 Hz,
0 0 0 0
8 4 e7 11
2
'
4
'
2H, H ), 3.60e3.66 (m, 6H, H
), 3.56e3.60 (m, 2H, H ). ESI-MS
2
''
N
(MeOH) m/z ¼ 324.4 [L6-H]þ (100%).
6
'
1
'
3
'
5
'
3
''
N
5
N
N
N
2
'
4'
4
.2.1. 1-Hexyl-4-(1-methyl-1H-imidazol-2-yl)-1H-1,2,3-triazole L1
The product was obtained as an oil which precipitated on
N
8'
2''
10'
O
O
1'
NH2
5'
O
7'
addition of hexane (10 mL) to yield an off white solid that was used
without further purification. Yield 412 mg (93%). m.p 50e53 C H
NMR (CD
4
11'
ꢀ
1
0
0
00
2
5
3
2
Cl
J
2
):
d
7.72 (s, 1H, H ), 7.51 (s, 1H, H ), 7.21 (s, 1H, H ),
0
3
1
.42 (t,
HH ¼ 6.8 Hz, 2H, H ), 3.91 (s, 3H, N-CH
3
), 1.97
0
0
0
3
2
3 e5
(
t,
J
J
HH ¼ 6.8 Hz, 2H, H ), 1.33e1.40 (m, 6H, H
), 0.91 (t,
4.3. Synthesis of 1e6 and 8
2
The [Ru( ] complexes were prepared according
6-arene)(im)Cl
0
3
6
þ
HH ¼ 6.8 Hz, 3H, H ). ESI-MS (MeOH) m/z ¼ 234.2, [L1-H] (100%).
Anal: Calculated for C12
Found C, 61.53; H, 8.74; N, 29.76.
H
19
N
5
([L1]) C, 61.77; H, 8.21; N, 30.02.
h
6
to the procedure described for 1. [Ru(
2 2
h -p-cymene)Cl ] (155 mg,
0.25 mmol) and L1 (117 mg, 0.5 mmol) were stirred in chloroform
4
.2.2. 1-Octyl-4-(1-methyl-1H-imidazol-2-yl)-1H-1,2,3-triazole L2
The product was obtained as an oil which precipitated on
for 8 h under reflux. The solvent volume was reduced to ca. 5 mL
and the product precipitated with diethyl ether (10 mL), filtered
and washed with diethyl ether (2 ꢁ 10 mL) and pentane
addition of hexane to yield an off white solid that was used without
further purification. Yield 486 mg (93%). H NMR (CDCl
1
00
3
):
d
7.67
(2 ꢁ 10 mL). Yield: 192 mg (81%), orange powder.
00
00
2
5
3
3
1
2
5
(
2
s, 1H, H ), 7.52 (s, 1H, H ), 7.24 (s, 1H, H ), 4.41 (t, JHH ¼ 6.8 Hz,
H NMR (CD
00
3
2
Cl
2
):
d
7.94 (s, 1H, H ), 7.80 (s, 1H, H ), 7.51 (s, 1H,
0
0
1
2
3
3
H, H ), 3.95 (s, 3H, N-CH
3
), 1.96e2.01 (m, 2H, H ), 1.31e1.39 (m,
H ), 5.47 (d, JHH ¼ 5.9 Hz, 2H, H
m
), 5.28 (d, JHH ¼ 5.9 Hz, 2H, H
o
),
0
0
0
3
e7
3
8
3
1
10H, H
), 0.90 (t,
J
HH ¼ 6.0 Hz, 3H, H ). ESI-MS (MeOH) m/
4.42 (t, JHH ¼ 7.0 Hz, 2H, H ), 4.02 (s, 3H, N-CH
3
), 2.92e3.05 (m, 1H,
0
2
þ
z ¼ 261.1, [L2-H] (100%). Anal: Calculated for C14
23
H N
5
([L2]) C,
CH(CH
HH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
), 2.17 (s, 3H, arene-CH
3
), 1.92e1.99 (m, 2H, H ), 1.36 (d,
0 0
3
3 e5
6
5
4.34; H, 8.87; N, 26.80. Found C, 63.45; H, 8.24; N, 25.46. m.p
J
3
)
2
), 1.26e1.34 (m, 6H, H
), 0.89e0.93
-p-cymene)Cl (L1-
-p-cymene)
0
ꢀ
6
6
5e57 C.
(m, 3H, H ). ESI-MS (MeOH) m/z ¼ 568.8 [Ru(
h
2
þ
6
H)] (100%). Anal: Calculated for C24
H37Cl
2
N
5
Ru ([Ru(
h
4
.2.3. 1-Decyl-4-(1-methyl-1H-imidazol-2-yl)-1H-1,2,3-triazole L3
The product was obtained as an oil which precipitated on
2
Cl (L1)]) C, 50.79; H, 6.57; N,12.34. Found C, 47.76.; H, 6.46; N,11.38.
6
2: [Ru(
2 2
h -p-cymene)Cl ] (305 mg, 0.5 mmol) and L2 (261 mg,
1
addition of hexane to yield a white solid that was used without
1 mmol). Yield: 624 mg (86%), orange powder. H NMR (CDCl ):
3

A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
777
0
0
00
2
5
3
d
7.97 (s, 1H, H ), 7.70 (s, 1H, H ), 7.54 (s, 1H, H ), 5.48 (d,
(2 ꢁ 10 mL) to afford a yellow powder. Yield: 220 mg (94%). The
BF salt was obtained by addition of 1 equiv. NaBF to a dichloro-
methane solution of 2$Cl (5 mL). The NaCl precipitate was removed
by filtration and diethyl ether (5 mL) added to precipitate the
product.
3
3
J
HH ¼ 5.5 Hz, 2H, H
m
), 5.31 (d,
J
HH ¼ 5.5 Hz, 2H, H
o
), 4.40 (t,
4
4
0
3JHH ¼ 7.0 Hz, 2H, H ), 4.04 (s, 3H, N-CH
1
), 2.96e3.03 (m, 1H, CH
3
0
2
(
CH
J
3
)
2
), 2.00 (s, 3H, arene-CH
3
), 1.89e1.96 (m, 2H, H ), 1.34 (d,
0 0
3 -7
3
HH ¼ 8.8 Hz, 6H, CH(CH
3
)
2
), 1.26e1.34 (m, 10H, H
), 0.88e0.94
-p-cymene)Cl (L2-
-p-cymene)
0
00
00
2
8
6
1
2
(
m, 3H, H ). ESI-MS (MeOH) m/z ¼ 568.8 [Ru(
h
2
3
H NMR (CDCl ): d 8.50 (s, 2H, H ), 8.49 (s, 2H, H ), 8.43 (s, 2H,
þ
6
5
3
3
H)] (100%). Anal: Calculated for C24
H
37Cl
2
N
5
Ru ([Ru(
h
H ), 5.88 ( JHH ¼ 5.9 Hz, 2H, H
m
), 5.85 ( JHH ¼ 5.9 Hz, 2H, H
o
), 4.40 (t,
), 2.47e2.54 (m, 1H, CH
), 1.32 (m, 20H,
0
3
1
Cl
2
(L2)]) C, 50.79; H, 6.57; N,12.34. Found C, 47.76.; H, 6.46; N,11.38.
J
HH ¼ 7.1 Hz, 4H, H ), 4.04 (s, 6H, N-CH
3
0
6
2
3
: [Ru(
h
-p-cymene)Cl
2
]
2
(155 mg, 0.25 mmol) and L3 (145 mg,
(CH
3
)
2
), 1.93e1.99 (m, 4H, H ), 1.89 (s, 3H, arene-CH
3
0
0
1
3 7
3
3
1
mmol). Yield: 414 mg (79%), orange powder. H NMR (CDCl
3
):
7.98 (s, 1H, H ), 7.78 (s, 1H, H ), 7.53 (s, 1H, H ), 5.47 (d,
H
), 1.19 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
), 0.89 (t, JHH ¼ 6.6 Hz, 6H,
0
0
00
0
2
5
3
8
6
þ
2
d
H ). ESI-MS (MeOH) m/z ¼ 792.9 [Ru(
h -p-cymene)Cl(L2) ]
3
3
J
HH ¼ 5.6 Hz, 2H, H
m
), 5.30 (d,
J
HH ¼ 5.6 Hz, 2H, H
o
), 4.42 (t,
(100%).
0
3JHH ¼ 7.3 Hz, 2H, H ), 3.99 (s, 3H, N-CH
1
), 2.93e3.01 (m, 1H, CH
4$Cl: [Ru(
(316 mg, 0.8 mmol). Yield: 309 mg (91%), pale yellow powder. H
2 2
h ] (100 mg, 0.16 mmol) and L4
6-p-cymene)Cl
3
0
2
1
(
CH
J
3
)
2
), 2.17 (s, 3H, arene-CH
3
), 1.91e2.05 (m, 2H, H ), 1.33 (d,
0
0
00
00
3
3
3 e9
2
5
HH ¼ 7.8 Hz, 6H, CH(CH
3
)
2
), 1.21e1.33 (m, 14H, H
), 0.89e0.93
-p-cymene)Cl (L3-
3
NMR (CDCl ): d 8.81 (s, 2H, H ), 8.73 (s, 2H, H ), 8.31 (s, 2H, H ),
10
6
3
3
(
m, 3H, H ). ESI-MS (MeOH) m/z ¼ 596.4 [Ru(
h
2
h
5.85 (d, JHH ¼ 5.9 Hz, 2H, H
m
), 5.81 (d, JHH ¼ 5.9 Hz, 2H, H
o
), 4.77
), 2.82e2.97 (m, 4H,
), 1.20 (d,
0
þ
6
3
1
H)] (100%). Anal: Calculated for C26
H45Cl
2
N
5
O
2
Ru ([Ru(
-p-
(t, JHH ¼ 7.1 Hz, 4H, H ), 4.05 (s, 6H, N-CH
3
0
2
cymene)Cl
H, 7.12; N, 10.98.
: [Ru(
6-p-cymene)Cl
mmol). Yield: 560 mg (80%), yellow orange powder. H NMR
2
(L3]$2H
2
O) C, 50.23; H, 7.34; N, 10.85. Found C, 49.86.;
H ), 2.48e2.58 (m, 1H, CH(CH
3
)
2
), 1.91 (s, 3H, arene-CH
3
3
6
J
HH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
). ESI-MS (MeOH) m/z ¼ 1061.6 [Ru(
(100%). Calculated for C34 10Ru ([Ru
]BF ) C, 35.57; H, 2.99; N, 12.20. Found C,
h -
þ
4
h
2
]
2
(305 mg, 0.5 mmol) and L4 (395 mg,
p-cymene)Cl(L4)
2
]
H34BClF22N
1
6
1
(
h
-p-cymene)Cl(L4)
2
4
0
0
00
3
2
5
(
CDCl
J
3
)
d
: 7.95 (s, 1H, H ), 7.88 (s, 1H, H ), 7.63 (s, 1H, H ), 5.50 (d,
35.65; H, 2.64; N, 12.15.
3
3
6
HH ¼ 5.6 Hz, 2H, H
m
), 5.33 (d,
J
HH ¼ 5.6 Hz, 2H, H
o
), 4.80 (t,
), 2.98e3.05 (m, 1H, CH
), 1.33 (d,
5$Cl: [Ru(
2 2
h -p-cymene)Cl ] (100 mg, 0.16 mmol) and L5
0
1
1
J
HH ¼ 7.1 Hz, 2H, H ), 3.81 (s, 3H, N-CH
3
(476 mg, 0.8 mmol). Yield: 467 mg (81%), yellow powder. H NMR
0
00
00
3
2
2
5
(
CH
3
)
2
), 2.82e2.96 (m, 2H, H ), 2.24 (s, 3H, arene-CH
3
3
(CDCl ): d 8.84 (s, 2H, H ), 8.75 (s, 2H, H ), 8.28 (s, 2H, H ), 5.85 (d,
6
3
3
J
HH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
). ESI-MS (MeOH) m/z ¼ 666.1 [Ru(
h
-p-
J
J
HH ¼ 5.9 Hz, 2H, H
m
), 5.81 (d,
J
HH ¼ 5.9 Hz, 2H, H
o
), 4.77 (t,
), 2.79e3.00 (m,
), 1.20
0
þ
3
1
cymene)Cl(L4)] (100%). Anal: Calculated for C22
H24Cl
2
F
9
N
5
Ru ([Ru
HH ¼ 7.1 Hz, 4H, H ), 4.05 (s, 6H, N-CH
3
0
6
2
(h
-p-cymene)Cl
2
(L4)]) C, 37.67; H, 3.45; N, 9.98. Found C, 36.71; H,
4H, H ), 2.48e2.58 (m, 1H, CH(CH
3
)
2
), 1.92 (s, 3H, arene-CH
3
3
3
.27; N, 9.94.
(d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
). ESI-MS (MeOH) m/z ¼ 1457.7 [Ru
(100%).
-4-phenyl-2-ethanol)Cl
L3 (197 mg, 0.68 mmol). Yield: 279 mg (89%), yellow powder. H
6-p-cymene)Cl
]
(305 mg, 0.5 mmol) and L5 (595 mg,
(h -p-cymene)Cl(L5) ]
6
þ
5
: [Ru(
h
2
2
2
1
6
1
mmol). Yield: 823 mg (91%), red orange powder. H NMR (CDCl
3
):
7.93 (s, 1H, H ), 7.84 (s, 1H, H ), 7.73 (s, 1H, H ), 5.51 (d,
7$Cl: [Ru(
h
2 2
] (116 mg, 0.34 mmol) and
0
0
00
3
2
5
1
d
0
0
00
3
3
3
2
5
J
HH ¼ 5.6 Hz, 2H, H
m
), 5.36 (d,
J
HH ¼ 5.6 Hz, 2H, H
o
), 4.81 (t,
), 2.95e3.02 (m, 1H, CH
), 1.32 (d,
NMR (CDCl
3
):
d 8.39 (s, 2H, H ), 8.30 (s, 2H, H ), 8.18 (s, 2H, H ),
0
3JHH ¼ 6.8 Hz, 2H, H ), 3.71 (s, 3H, N-CH
1
5.94 ( JHH ¼ 5.4 Hz, 2H, H
3
), 5.88 ( JHH ¼ 5.4 Hz, 2H, H
3
3
m
o
), 4.42e4.47
0
2
3
5
3
(
CH
3
)
2
), 2.78e2.93 (m, 2H, H ), 1.74 (s, 3H, arene-CH
3
(m, 1H, H
J
p
), 4.40 (t, JHH ¼ 7.3 Hz, 4H, H ), 4.01 (s, 6H, H ), 3.93 (t,
6
3
3
J
HH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
). ESI-MS (MeOH) m/z ¼ 866.0 [Ru(
h
-p-
HH ¼ 5.6 Hz, 2H, CH
2
OH), 2.62 (t,
J
HH ¼ 5.4 Hz, 2H,
þ
6
7
cymene)Cl(L5)] (100%).
ArCH
J
2
), 2.92e2.98 (m, 4H, H ) 1.24e1.42 (m, 24H, H ) 0.90 (t,
6
3
8
6
6
: [Ru(
h
-p-cymene)Cl
2
]
2
(98 mg, 0.06 mmol) and L6 (40 mg,
HH ¼ 6.6 Hz, 6H, H ). ESI-MS (MeOH) m/z ¼ 853.8 [Ru(
h
-phenyl-
þ
0
.12 mmol). Yield: 120 mg (87%), slightly hydroscopic yellow solid.
ethanol)Cl(L3)] (100%). Anal: Calculated for C39
H
62Cl
2
N
10ORu ([Ru
0
0
00
3
1
2
5
6
H NMR (CDCl
3
):
d
8.26 (s, 1H, H ), 8.06 (s, 1H, H ), 7.53 (s, 1H, H ),
(h
-phenyl-ethanol)Cl(L3)
2
]Cl) C, 54.53; H, 7.28; N, 16.31. Found C,
3
3
5
.90 (d, JHH ¼ 5.9 Hz, 2H, H
m
), 5.86 (d, JHH ¼ 5.9 Hz, 2H, H
o
), 4.63 (t,
53.86; H, 7.69; N, 16.12.
0
3
1
3
J
HH ¼ 4.9 Hz, 2H, H ), 4.01 (s, 3H, N-CH
3
), 3.93 (t, JHH ¼ 4.9 Hz, 2H,
0
0
0
0
2
3
10
4 e8
H ), 3.73 (d, JHH ¼ 4.4 Hz, 2H, H ), 3.64e3.70 (m, 8H, H
), 3.62
), 1.90 (s, 3H,
). ESI-MS (MeOH) m/
-p-cymene)Cl(L6)] (100%). Anal: Calculated for
4.5. Synthesis of 1$BF
4 4
e8$BF
0
3
11
(
d, JHH ¼ 4.9 Hz, 2H, H ), 2.49e2.58 (m, 1H, CH(CH
3 2
)
arene-CH
z ¼ 621.6 [Ru(
Ru [Ru(
N, 12.42. Found C, 45.66; H, 6.63; N, 12.20.
3
), 1.19 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
The [Ru(
h
6-arene)(im)(pta)Cl]BF
4
complexes were prepared
. A solution of 1
BF (51 mg,
6
þ
h
according to the procedure described for 1$BF
(220 mg, 0.4 mmol), pta (66 mg, 0.42 mmol) and NH
4
6
C
26
H
44Cl
2
N
6
O
4
h
-p-cymene)Cl(L6)]$H
2
O) C, 46.15; H, 6.55;
4
4
0.5 mmol) in methanol was stirred at room temperature for 1 h. The
solvent was removed under reduced pressure and product extrac-
ted with dichloromethane (20 mL) and filtered through celite. The
filtrate was concentrated in vacuo to ca. 5 mL and diethyl ether
(20 mL) added to precipitate a hydroscopic, orange solid. Yield:
227 mg (76%), orange powder.
6
8
: [Ru(
2 2
h -phenyl-2-ethanol)Cl ] (116 mg, 0.34 mmol) and L5
130 mg, 0.68 mmol). Yield: 226 mg (71%), yellow powder. H NMR
1
(
(
0
0
00
3
2
5
CDCl
J
3
):
HH ¼ 6.0 Hz, 2H, H
m, 1H, H
d 8.00 (s, 1H, H ), 7.84 (s, 1H, H ), 7.68 (s, 1H, H ), 5.68 (d,
3
3
m
), 5.66 (t, JHH ¼ 5.8 Hz, 2H, H
o
), 4.81e4.87
0
1
(
CH
2
p
), 4.79 (t, JHH ¼ 7.1 Hz, 2H, H ), 4.09e4.16 (m, 2H,
0
0
3
1
2
5
2
OH), 4.00 (s, 3H, N-CH
3
), 2.93 (t,
J
HH ¼ 5.4 Hz, 2H, ArCH
2
),
3
H NMR (CDCl ): d 8.41 (s, 1H, H ), 8.31 (s, 1H, H ), 7.58 (s, 1H,
00
0
2
6
3
3
3
.82e2.91 (m, 2H, H ). ESI-MS (MeOH) m/z ¼ 840.2 [Ru(
h
-phenyl-
H ), 5.88 (d, JHH ¼ 5.6 Hz, 2H, H
m
), 5.85 (d, JHH ¼ 6.0 Hz, 1H, H
o
),
0
o
þ
3
3
ethanol)Cl (L5)] (100%).
5.73 (d, JHH ¼ 4.6 Hz, 1H, H
), 4.79 (s, 6H, pta), 4.43 (t, JHH ¼ 7.3 Hz,
0
1
2
2
2
H, H ), 4.33 (d, JHH ¼ 13.6 Hz, 3H, pta), 4.28 (d, JHH ¼ 14.2 Hz, 3H,
4
.4. Synthesis of 2$Cl, 4$Cl, 5$Cl and 7$Cl
pta), 4.13 (s, 3H, N-CH
arene-CH
3
), 2.65e2.72 (m, 1H, CH(CH
3
)
2
), 2.13 (s, 3H,
0
0
0
3
2
3 e5
3
), 1.99 (t, JHH ¼ 7.2 Hz, 2H, H ), 1.31e1.42 (m, 6H, H
),
The [Ru( 6
to the procedure described for 2$Cl [Ru(
.16 mmol) and L2 (208 mg, 0.8 mmol) were stirred in chloroform
h
-arene)(im)
Cl]Cl complexes were prepared according
1.29 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
), 1.17 (d, JHH ¼ 7.1 Hz, 6H, CH
3
2
3
2
0
6
0
3
6
31
h
-p-cymene)Cl
2
]
2
(100 mg,
(CH
3
)
2
) 0.91 (t, JHH ¼ 6.8 Hz, 3H, H ). P NMR (CDCl
3
):
d
ꢂ30.6 (s,
6
þ
0
pta). ESI-MS (MeOH) m/z ¼ 661.2 [Ru(
(100%). Anal: Calculated for C28 45BClF
) C, 44.96; H, 6.06; N, 14.98. Found C, 44.18; H, 5.97; N,
h -p-cymene)Cl(L1)pta]
6
for 8 h under reflux. The solvent volume was reduced to ca. 5 mL
and the product precipitated with diethyl ether (10 mL), filtered
and washed with diethyl ether (2 ꢁ 10 mL) and then pentane
H
4 8
N PRu ([Ru(h -p-cymene)Cl
(L1)pta]BF
4
14.31.

778
A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
2
$BF
4
: 2 (200 mg, 0.35 mmol), pta (61 mg, 0.39 mmol) and
(51 mg, 0.5 mmol). Yield: 221 mg (81%), hydroscopic, orange
p-cymene)Cl(L6)pta]BF
42.78; H, 6.65; N, 14.25.
7$BF : 7$Cl (279 mg, 0.3 mmol), pta (50 mg, 0.32 mmol) and
NH BF
3
NMR (CDCl ): d 8.26 (s, 1H, H ), 8.14 (s, 1H, H ), 7.61 (s, 1H, H ),
4 2
$2H O) C, 42.56; H, 6.47; N, 13.96. Found C,
NH
solid. H NMR (CDCl
4
BF
4
0
0
1
2
5
3
):
d
8.39 (s, 1H, H ), 8.09 (s, 1H, H ), 7.57 (s, 1H,
4
0
0
3
3
3
1
H ), 5.92 (d, JHH ¼ 5.6 Hz, 2H, H
m
), 5.87 (d, JHH ¼ 5.2 Hz, 1H, H
o
),
4
4
(40 mg, 0.41 mmol). Yield: 191 mg (82%), yellow powder. H
00 00
0
o
3
3
2
5
3
5
2
.74 (d, JHH ¼ 4.6 Hz, 1H, H
), 4.84 (s, 6H, pta), 4.41 (t, JHH ¼ 7.1 Hz,
0
1
2
2
3
H, H ), 4.36 (d, JHH ¼ 14.6 Hz, 3H, pta), 4.31 (d, JHH ¼ 14.8 Hz, 3H,
6.16e6.22 (m, 1H, H
m
), 6.13 (d,
J
HH ¼ 5.6 Hz, 1H, H
o
), 6.08 (d,
), 4.95 (t, JHH ¼ 6.1 Hz,
0
0
3
3
pta), 4.12 (s, 3H, N-CH
3
), 2.65e2.75 (m, 1H, CH(CH
3
)
2
), 2.14 (s, 3H,
J
HH ¼ 5.9 Hz, 1H, H
o
), 5.94e6.01 (m 1H, H
m
0
0
0
0
3
2
3 e7
1
2
arene-CH
3
), 1.99 (t, JHH ¼ 7.1 Hz, 2H, H ), 1.34e1.43 (m, 10H, H
),
2H, H ), 4.66e5.03 (m 1H, H
p
), 4.55 (d, JHH ¼ 12.8 Hz, 3H, pta), 4.40
), 3.89
OH), 2.59 (t, JHH ¼ 6.0 Hz, 2H, ArCH ),
), 0.87 (t,
3
3
2
1
(
.23 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
), 1.17 (d, JHH ¼ 6.8 Hz, 6H, CH
(s, 6H, pta), 4.20 (d, JHH ¼ 14.0 Hz, 3H, pta), 4.07 (s, 3H, N-CH
3
0
0
3
8
31
3
3
CH
3
)
2
) 0.89 (t, JHH ¼ 6.6 Hz, 3H, H ). P NMR (CDCl
3
):
d
ꢂ29.8 (s,
(d, JHH ¼ 5.9 Hz, 2H, CH
2
2
0
0
0
6
þ
2
3 e9
pta). ESI-MS (MeOH) m/z ¼ 689.3 [Ru(
h
-p-cymene)Cl(L2)pta]
1.88e2.08 (m, 2H, H ), 1.23e1.48 (m, 14H, H
0
6
3
10 31
(
(
6
100%). Anal: Calculated for C30
L2)pta]BF $5H O) C, 41.60; H, 6.87; N, 12.94. Found C, 41.49; H,
.81; N, 12.61.
$BF : 3 (280 mg, 0.46 mmol), pta (78.5 mg, 0.5 mmol) and
BF (102 mg, 1 mmol). Yield: 295 mg (79%), slightly hydro-
4 8
H49BClF N PRu ([Ru(
h
-p-cymene)Cl
J
HH ¼ 7.2 Hz, 3H, H ). P NMR (CDCl
3
)
d
: ꢂ29.9. ESI-MS (MeOH)
6
þ
4
2
m/z ¼ 692.0 [Ru(
h
-phenyl-2-ethanol)Cl(L3)pta] (100%). Anal:
6
Calculated for C29
H
4 8
47BClF N OPRu ([Ru(h -phenyl-2-ethanol)Cl
3
4
(L3)pta]BF ) C, 44.77; H, 6.09; N, 14.40. Found C, 44.16; H, 6.69; N,
4
NH
4
4
14.76.
00
2
1
scopic, orange powder. H NMR (CDCl
3
):
d
8.40 (s, 1H, H ), 8.11 (s,
8$BF
NH BF
powder. H NMR (acteone-d
7.69 (s,1H, H ), 6.16e6.22 (m,1H, H
6.06 (d,
4
: 8 (100 mg, 0.12 mmol), pta (20 mg, 0.13 mmol) and
00
5
3
3
1
H, H ), 7.58 (s, 1H, H ), 5.93 (d, JHH ¼ 5.8 Hz, 2H, H
m
), 5.88 (d,
4
4
(30 mg, 0.28 mmol). Yield: 118 mg (89%), pale yellow
0
00
3
3
1
2
5
J
HH ¼ 5.6 Hz, 1H, H
o
), 5.80 (d,
J
HH ¼ 5.6 Hz, 1H, H
o
), 4.82 (s,
6
):
d
8.61 (s, 1H, H ), 8.28 (s, 1H, H ),
0
00
3
1
2
3
3
6
H, pta), 4.40 (t, JHH ¼ 7.3 Hz, 2H, H ), 4.31 (d, JHH ¼ 12.8 Hz, 3H,
m
), 6.13 (d, JHH ¼ 5.6 Hz,1H, H
o
),
0
0
2
3
pta), 4.33 (d,
J
HH ¼ 13.6 Hz, 3H, pta), 4.10 (s, 3H, N-CH
3
),
J
HH ¼ 5.9 Hz, 1H, H
o
), 5.94e6.01 (m 1H, H
m
), 4.95 (t,
0
3
1
2
.63e2.74 (m, 1H, CH(CH
3
)
2
), 2.14 (s, 3H, arene-CH ), 2.01 (t,
3
J
HH ¼ 6.1 Hz, 2H, H ), 4.87e4.81 (m 1H, H
p
), 4.72 (s, 6H, pta), 4.36 (d,
0
0
0
3
2
3 e9
2
2
J
HH ¼ 6.9 Hz, 2H, H ), 1.32e1.41 (m, 14H, H
), 1.22 (d,
J
HH ¼ 15.2 Hz, 3H, pta), 4.26 (d, JHH ¼ 14.6 Hz, 3H, pta), 4.05 (s, 3H,
0
3JHH ¼ 7.0 Hz, 6H, CH(CH
)
2
), 1.17 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
0
)
N-CH
3
OH), 2.80e3.05 (m, 2H, H ),
2
3
3
3
), 3.87 (d, JHH ¼ 5.9 Hz, 2H, CH
2
0
3
10
31
3
31
0
.91 (t, JHH ¼ 6.8 Hz, 3H, H ). P NMR (CDCl
3
):
d
ꢂ30.0 (s, pta).
2.60 (t, JHH ¼ 5.9 Hz, 2H, ArCH
2
). P NMR (acteone-d
6
)
d
: ꢂ28.3.
6
þ
6
þ
ESI-MS (MeOH) m/z ¼ 717.4 [Ru(
h
-p-cymene)Cl(L3)pta] (100%).
ESI-MS (MeOH) m/z ¼ 993.2 [Ru(
h -phenyl-2-ethanol)Cl(L5)pta]
6
Anal: Calculated for C32
H53BClF
4
N
8
PRu ([Ru(
h
-p-cymene)Cl(L3)
(100%).
4
pta]BF ) C, 47.80; H, 6.64; N, 13.93. Found C, 47.06; H, 6.57; N,
13.60.
4.6. Aqueous phase studies
4
$BF
4
: 4 (200 mg, 0.28 mmol), pta (54 mg, 0.32 mmol) and
(44 mg, 0.43 mmol). Yield: 212 mg (82%), orange powder.
NH
4
BF
4
Hydrolysis was studied using 1 mM solutions of 6$BF
4
e8$BF
4
in
0
0
00
3
1
2
5
ꢀ
H NMR (CDCl
3
):
d
8.63 (s, 1H, H ), 8.08 (s, 1H, H ), 7.64 (s, 1H, H ),
D
2
O that were incubated at 37 C over 72 h. Aliquots were taken at
3
3
31
5
(
.84 (d, JHH ¼ 5.6 Hz, 2H, H
m
), 5.76 (d, JHH ¼ 4.6 Hz, 1H, H
o
), 5.70
regular intervals and analysed by P NMR spectroscopy. Surface
tension was measured on a Sigma 703-Automatic Tensiometer using
the Du-Nouy ring method [31]. The instrument was calibrated with
PBS. Compounds were serially diluted in PBS and surface tension
measurements made in triplicate. Partition coefficients were deter-
mined according to the shake flask method described in reference
23.
0
o
0
3
3
1
d, JHH ¼ 4.2 Hz, 1H, H
), 4.79 (t, JHH ¼ 7.3 Hz, 2H, H ), 4.59 (s, 6H,
2
2
pta), 4.26 (d, JHH ¼ 14.9 Hz, 3H, pta), 4.15 (d, JHH ¼ 15.8 Hz, 3H,
0
2
pta), 4.13 (s, 3H, N-CH
3
), 2.85e3.01 (m, 2H, H ), 2.59e2.69 (m, 1H,
3
CH(CH
3
)
2
), 2.11 (s, 3H, arene-CH
3
), 1.27 (d,
J
HH ¼ 6.8 Hz, 6H, CH
3
0
31
(
d
CH
3
)
2
), 1.17 (d,
J
HH ¼ 6.6 Hz, 6H, CH(CH
3
)
2
). P NMR (CDCl
3
):
6
ꢂ34.0 (s, pta). ESI-MS (MeOH) m/z ¼ 822.9 [Ru(
h
-p-cymene)Cl
þ
6
(
L4)pta] (100%). Anal: Calculated for C28
H
36BClF13
N
8
PRu ([Ru(
h
-
p-cymene)Cl(L4)pta]BF
4
) C, 36.96; H, 3.99; N, 12.31. Found C, 35.14;
4.7. Cell culture
H, 3.65; N, 12.20.
5
$BF
BF
H NMR (CDCl
4
: 5 (200 mg, 0.22 mmol), pta (40 mg, 0.25 mmol) and
Human A2780 and A2780cisR ovarian carcinoma cell lines were
obtained from the European Collection of Cell Culture (ECACC,
Porton Down, Wiltshire, UK). The cells were grown routinely in
NH
4
4
(46 mg, 0.44 mmol). Yield: 210 mg (86%), yellow powder.
0
0
00
3
1
2
5
3
): d 8.63 (s, 1H, H ), 8.07 (s, 1H, H ), 7.58 (s, 1H, H ),
3
3
5
.81 (d, JHH ¼ 5.1 Hz, 2H, H
m
), 5.76 (d, JHH ¼ 5.0 Hz, 1H, H
o
), 5.56
RPMI 1640 medium containing 10% foetal calf serum (FCS) and
0
o
0
3
3
1
ꢀ
(
d, JHH ¼ 4.2 Hz, 1H, H
), 4.78 (t, JHH ¼ 7.6 Hz, 2H, H ), 4.69 (s, 6H,
antibiotics at 37 C and 6% CO
2
until 70e80% confluence. For
M, Ebewe, obtained from the
2
2
pta), 4.28 (d, JHH ¼ 14.9 Hz, 3H, pta), 4.18 (d, JHH ¼ 14.9 Hz, 3H,
A2780cisR cells, cisplatin solution (1
m
0
2
pta), 4.13 (s, 3H, N-CH
3
), 2.80e3.05 (m, 2H, H ), 2.59e2.72 (m, 1H,
Pharmacy of the CHUV) was added every 2e3 passages to maintain
cisplatin resistance. Human HCEC (human cerebral endothelial
cells) cell line was a kind gift of D. Stanimirovic, Ottawa, Canada.
The cells were routinely grown in DMEM medium containing 4.5 g/
3
CH(CH
3
)
2
), 2.13 (s, 3H, arene-CH
3
), 1.28 (d, JHH ¼ 6.8 Hz, 6H, CH
3
0
31
(
CH ), 1.18 (d,
3
)
2
J
HH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
). P NMR (CDCl
3
):
6
þ
d
ꢂ33.1. ESI-MS (MeOH) m/z ¼ 1024 [Ru(
h -p-cymene)Cl(L5)pta]
ꢀ
(
100%).
$BF
BF
L glucose, 10% foetal calf serum (FCS) and antibiotics at 37 C and 6%
6
4
: 6 (130 mg, 0.12 mmol), pta (20 mg, 0.13 mmol) and
(40 mg, 0.41 mmol). Yield: 77 mg (74%), yellow brown
2
CO until confluence. All cell culture reagents were obtained from
Gibco, Invitrogen, Basel, Switzerland.
NH
4
4
0
0
1
2
5
3
powder. H NMR (CDCl ): d 8.65 (s, 1H, H ), 8.10 (s, 1H, H ), 7.85 (s,
00
3
3
3
1
H, H ), 5.80 (d, JHH ¼ 5.9 Hz, 2H, H
m
), 5.65 (d, JHH ¼ 5.0 Hz, 2H,
4.8. Evaluation of cytotoxicity (MTT assay)
0
3
1
H
o
), 4.68 (s, 6H, pta), 4.64 (t,
J
HH ¼ 4.6 Hz, 2H, H ), 4.08 (d,
2
2
J
HH ¼ 10.0 Hz, 3H, pta), 4.03 (d, JHH ¼ 6.6 Hz, 3H, pta), 4.00 (s, 3H,
Cytotoxicity was determined using the MTT assay (MTT ¼
0
0
10
2
3
N-CH
3
), 3.90e3.95 (m, 2H, H ), 3.73 (t, JHH ¼ 4.4 Hz, 2H, H ), 3.70
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide,
0
0
0
3
8
4 e7
(
t,
J
J
HH ¼ 5.6 Hz, 2H, H ), 3.62e3.68 (m, 6H, H
), 3.62 (d,
), 1.89 (s, 3H,
). P NMR (CDCl ):
-p-cymene)Cl
Sigma). Cells were seededin96-well plates asmonolayers with100 ml
0
3
11
HH ¼ 3.7 Hz, 2H, H ), 2.47e2.61 (m, 1H, CH(CH
3
)
2
of cell solution (approximately 20,000 cells) per well and pre-incu-
bated for 24 h in medium supplemented with 10% FCS. Compounds
were dissolved directly in the culture medium, oras a DMSO solution,
and serially diluted to the appropriate concentration (final DMSO
3
31
arene-CH
3
), 1.20 (d, JHH ¼ 6.8 Hz, 6H, CH(CH
3
)
2
3
6
d
ꢂ35.1 (s, pta). ESI-MS (MeOH) m/z ¼ 780.6 [Ru(
h
þ
6
(
L6)pta] (100%). Anal: Calculated for: C32
H
58BClF
4
N
9
O
5
PRu [Ru(
h
-

A.K. Renfrew et al. / Journal of Organometallic Chemistry 696 (2011) 772e779
779
concentration <0.5%). Cell culture media was aspirated and100
the compound solution was added to each well and the plates were
incubated foranother72 h. Subsequently,10 l perwell of MTT (5 mg/
m
l of
[14] C.A. Vock, C. Scolaro, A.D. Phillips, R. Scopelliti, G. Sava, P.J. Dyson, J. Med.
Chem. 49 (2006) 5552e5561.
[15] (a) C. Scolaro, A.B. Chaplin, C.G. Hartinger, A. Bergamo, M. Cocchietto,
m
B.K. Keppler, G. Sava, P.J. Dyson, Dalton Trans. (2007) 5065e5072;
mL solution in phosphate buffered saline) was added to the cells and
the plates were incubated for a further 2 h. The culture medium was
aspirated, and the purple formazan crystals formed by the mito-
chondrial dehydrogenase activity of metabolically active viable cells
were dissolved in DMSO. The optical density, directly proportional to
the number of surviving cells, was quantified at 540 nm using
a multiwell plate reader (iEMS Labsystems, BioConcepts, Allschwil,
Switzerland) and the fraction of surviving cells was calculated from
the absorbance of untreated control cells. Evaluation is based on
means from 2 independent experiments, each comprising 3 repli-
cates per concentration level.
(b) M. Groessl, C.G. Hartinger, P.J. Dyson, B.K. Keppler, J. Inorg. Biochem. 102
(
(
2008) 1060e1065;
c) A. Dorcier, C.G. Hartinger, R. Scopelliti, R.H. Fish, B.K. Keppler, P.J. Dyson,
J. Inorg. Biochem. 102 (2008) 1066e1076;
(d) C. Scolaro, C.G. Hartinger, C. Allardyce, B.K. Keppler, P.J. Dyson, J. Inorg.
Biochem. 102 (2008) 1743e1748;
(e) C.G. Hartinger, A. Casini, C. Duhot, Y.O. Tsybin, L. Messori, P.J. Dyson,
J. Inorg. Biochem. 102 (2008) 2136e2141;
(f) A.K. Renfrew, A.D. Phillips, A. Egger, C.G. Hartinger, S.S. Bosquain,
A.A. Nazarov, B.K. Keppler, L. Gonsalvi, M. Peruzzini, P.J. Dyson, Organome-
tallics 28 (2009) 1165e1172;
(g) M. Groessl, Y.O. Tsybin, C.G. Hartinger, B.K. Keppler, P.J. Dyson, J. Biol.
Inorg. Chem. 15 (2010) 677e688;
(
(
h) A.E. Egger, C.G. Hartinger, A.K. Renfrew, P.J. Dyson, J. Biol. Inorg. Chem. 15
2010) 919e927.
[
16] T.R. Chan, R. Hilgraf, K.B. Sharpless, V.V. Fokin, Org. Lett. 6 (2004) 2853e2855.
Acknowledgment
[17] (a) T.L. Mindt, H. Struthers, L. Brans, T. Anguelov, C. Schweinsberg, V. Maes,
D. Tourwe, R. Schibli, J. Am. Chem. Soc. 128 (2006) 15096e15097;
(
(
(
(
b) T.L. Mindt, R. Schibli, J. Org. Chem. 72 (2007) 10247e10250;
c) T.L. Mindt, C. Muller, M. Melis, M. de Jong, R. Schibli, Bioconjug. Chem. 19
2008) 1689e1695;
We thank the Swiss National Science Foundation for financial
support.
d) T.L. Mindt, C. Schweinsberg, L. Brans, A. Hagenbach, U. Abram, D. Tourwé,
E. Garcia-Garayoa, R. Schibli, Chemmedchem 4 (2009) 529e539;
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675e3677;
b) S.D. Koster, J. Dittrich, G. Gasser, N. Hüsken, I.C. Henao Castaneda, J.L. Lios,
C.O. Della Vedova, N. Metzler-Nolte, Organometallics 27 (2008) 6326e6332;
c) A. Pinto, U. Hoffmanns, M. Ott, G. Fricker, N. Metzler-Nolte, ChemBioChem
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