Ruthenium(ii) arene complexes with chelating chloroquine analogue ligands: Synthesis, characterization and in vitro antimalarial activity

Article abstract of DOI:10.1039/c2dt12083f

Three new ruthenium complexes with bidentate chloroquine analogue ligands, [Ru(η⁶-cym)(L¹)Cl]Cl (1, cym = p-cymene, L¹ = N-(2-((pyridin-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine), [Ru(η⁶-cym)(L²

Full text of DOI:10.1039/c2dt12083f

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Dalton
Transactions
Cite this: Dalton Trans., 2012, 41, 2764
www.rsc.org/dalton
PAPER
Ruthenium(II) arene complexes with chelating chloroquine analogue ligands:
Synthesis, characterization and in vitro antimalarial activity†
a
a
b
c
c
b
Lotta Glans, Andreas Ehnbom, Carmen de Kock, Alberto Martínez, Jesús Estrada, Peter J. Smith,
d
c
a
Matti Haukka, Roberto A. Sánchez-Delgado* and Ebbe Nordlander*
Received 2nd November 2011, Accepted 5th December 2011
DOI: 10.1039/c2dt12083f
6
1
Three new ruthenium complexes with bidentate chloroquine analogue ligands, [Ru(η -cym)(L )Cl]Cl
1
6
(
1, cym = p-cymene, L = N-(2-((pyridin-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine), [Ru(η -
2
2
cym)(L )Cl]Cl (2, L = N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-4-
amine) and [Ru(η -cym)(L )Cl] (3, L = N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-
-amine) have been synthesized and characterized. In addition, the X-ray crystal structure of 2 is reported.
The antimalarial activity of complexes 1–3 and ligands L , L and L , as well as the compound N-(2-
bis((pyridin-2-yl)methyl)amino)ethyl)-7-chloroquinolin-4-amine (L ), against chloroquine sensitive and
6
3
3
4
1
2
3
4
(
chloroquine resistant Plasmodium falciparum malaria strains was evaluated. While 1 and 2 are less active
2
than the corresponding ligands, 3 exhibits high antimalarial activity. The chloroquine analogue L also
shows good activity against both the chloroquine sensitive and the chloroquine resistant strains. Heme
aggregation inhibition activity (HAIA) at an aqueous buffer/n-octanol interface (HAIR ) and
50
lipophilicity (D, as measured by water/n-octanol distribution coefficients) have been measured for all
ligands and metal complexes. A direct correlation between the D and HAIR50 properties cannot be made
because of the relative structural diversity of the complexes, but it may be noted that these properties are
3
enhanced upon complexation of the inactive ligand L to ruthenium, to give a metal complex (3) with
promising antimalarial activity.
recommended first line treatment for infection by the most lethal
of the malaria parasites, Plasmodium falciparum.
Introduction
2
Malaria has been afflicting mankind for 500 000 years, and in
the 21st century the disease remains as lethal as ever. Recent
reports by WHO estimate 234 million cases and 860 000 deaths
in 2008, numbers that are representative for recent years.
Although there is cause for optimism within some areas of the
battle against malaria - especially as the funding available for
malaria control has increased dramatically - serious obstacles
remain, e.g. the resistance to common antimalarial drugs. The
question of resistance is now more current than ever, after
Chloroquine (CQ, Fig. 1) is one of the most successful anti-
malarial drugs and was for decades the primary chemotherapy
1
5
used to treat malaria. In its erythrocytic phase, the malaria para-
2
site digests red blood cells, releasing lethal free heme. In the
acidic food vacuole of the parasite, heme is detoxified through a
biomineralization process which converts it into hemozoin, a
non-toxic crystalline dimer that forms extended chains via inter-
6
molecular hydrogen bonding. The commonly accepted mechan-
ism of action of chloroquine and related 4-aminoquinoline drugs
involves binding hematin and inhibiting its conversion into
3
,4
reports of resistance to artemisinins, drugs that are part of the
6
–11
hemozoin.
It has been shown that the 7-chloro-4-aminoqui-
noline nucleus of chloroquine is responsible for inhibition of
a
Inorganic Chemistry Research Group, Chemical Physics, Center for
Chemistry and Chemical Engineering, Lund University, Box 124,
SE-221 00 Lund, Sweden. E-mail: Ebbe.Nordlander@chemphys.lu.se;
Fax: +46 46 222 4119; Tel: +46 46 222 8118
b
Division of Pharmacology, Department of Medicine, University of
Cape Town Medical School, Observatory 7925, South Africa
c
Department of Chemistry, Brooklyn College and The Graduate Center,
The City University of New York, CUNY, 2900 Bedford Avenue,
Brooklyn, New York 11210, U.S.A
d
Department of Chemistry, University of Eastern Finland, Box 111,
FIN-80101 Joensuu, Finland
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2dt12083f
Fig. 1 Structures of chloroquine (CQ), ferroquine (FQ) and reported
ruthenium arene complexes with chloroquine as ligand.
1
2764 | Dalton Trans., 2012, 41, 2764–2773
This journal is © The Royal Society of Chemistry 2012

View Article Online
hemozoin formation, while the basic quinoline nitrogen and the
amine side chain assist accumulation of the molecule in the food
vacuole.
ability in an attempt to better account for the observed biological
properties.
12,13
Widespread resistance has rendered chloroquine useless in
most malaria-endemic countries, but it is still the recommended
treatment for Plasmodium vivax malarial cases in parts of the
Results and discussion
Synthesis and characterization
2,14
world.
Although the mechanism of resistance to chloroquine
1
4
is not yet fully understood, it seems to be related to mutations in
the Plasmodium falciparum chloroquine resistance transporter
Ligands L –L (Fig. 2) were synthesized following standard syn-
thetic protocols (cf. Experimental section). To synthesize N-(2-
((pyridin-2-yl)methylamino)ethyl)-7-chloroquinolin-4-amine
(
PfCRT) transmembrane protein, located at the membrane of the
1
3,15–18
1
digestive vacuole of the parasite.
The mutated protein is
(L ), N-(2-aminoethyl)-7-chloroquinolin-4-amine and 2-pyridine
able to recognize and expel chloroquine from the food vacuole
carboxaldehyde were stirred overnight at room temperature to
form the imine, which was in turn reduced with sodium borohy-
dride to give the desired product in good yield. However, when
this reaction was carried out with 3 equivalents of aldehyde and
sodium tris(acetoxy)borohydride as reducing agent, the disubsti-
tuted ligand N-(2-(bis((pyridin-2-yl)methyl)amino)ethyl)-7-
1
9
by mechanisms that are still subject to debate and that result in
a dramatic decrease of drug concentration at the active site.
Therefore new antimalarial drugs capable of overcoming resist-
ance are urgently needed. The ability of the PfCRT protein to
extrude chloroquine from the digestive vacuole strongly depends
on the lipophilicity of the drug as well as on its structural fea-
4
chloroquinolin-4-amine (L ) was formed. The ligand N-(2-((1-
18,20,21
tures.
In addition, there is clear evidence indicating that
methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chloroquinolin-
2
1
the formation of hemozoin crystals occurs at water/lipid
4-amine (L ) was synthesized analogously to L . In contrast, N-
22–24
interfaces.
Thus, the design of new antiplasmodial agents
(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-4-
3
aimed at CQ-resistant parasites must take into consideration not
only their heme aggregation inhibition activity (HAIA) but also
structural and physicochemical factors.
amine (L ) was isolated in the imine form, after stirring the start-
ing materials together in ethanol at room temperature. Reaction
1
2
of ligands L or L with the [(p-cymene)RuCl
] dimer in
2 2
Numerous chloroquine analogues have been synthesized
during the last decades; one of the most original, and potentially
most successful, being the organometallic derivative ferroquine
2 : 1 molar ratio in dichloromethane at room temperature gave
1
2
the complexes [RuCl(p-cymene)(L)]Cl (L = L (1); L (2)) in
3
good yields (Scheme 1). For ligand L , deprotonation by tri-
25
(Fig. 1). Ferroquine exhibits a completely restored activity
ethylamine followed by stirring with the ruthenium p-cymene
3
against resistant parasite strains and has entered phase IIb clinical
dimer gave the complex [RuCl(p-cymene)(L )] (3). All com-
2
5–28
trials in association with artesunate.
Other reported organo-
plexes were isolated as yellow or orange air-stable solids with
good solubility in polar solvents.
The structure of 2 could be confirmed by single crystal X-ray
diffraction (Fig. 3, vide infra). The NMR and mass spectra of 1
metallic and inorganic derivatives of chloroquine include ruthe-
nocene compounds and half sandwich compounds of chromium
and rhenium, as well as ruthenium, rhodium and gold coordi-
29–35
nation complexes.
Ruthenium arene half sandwich complexes have recently
received increasing attention as prospective metal-based antican-
cer drugs and a number of complexes, mostly with nitrogen and
phosphorus ligands, are being investigated for their drug candi-
36–39
date potential.
Combining the versatility of ruthenium arene
compounds with the interesting properties of metal-chloroquine
derivatives, a family of ruthenium half sandwich compounds
with chloroquine as ligand was recently reported (Fig. 1). These
compounds showed an increased antimalarial activity against
chloroquine resistant parasite strains compared to free chloro-
quine, as well as anticancer properties against several tumour
40
cell lines. A detailed study of factors relating to the antimalar-
ial mechanism of action of these complexes indicated that the
increased activity was likely to originate primarily in the major
change in lipophilicity and structure of the Ru complexes as
41
compared to chloroquine itself (see Results and Discussion).
1
4
Fig. 2 Structures of ligands L –L .
However, the coordination of chloroquine in these complexes
occurred through the quinoline nitrogen (Fig. 1), which has been
1
2
shown to be crucial for the antimalarial activity. Here we report
the synthesis and characterization of a new group of ruthenium
arene complexes containing chloroquine analogue ligands with
N,N- or Schiff base N,O-donor moieties, and an investigation of
the antimalarial activity of the ruthenium complexes as well as
the ligands. We also report some relevant physicochemical
studies of their lipophilicity and heme aggregation inhibition
and 3 strongly support that the structures of these complexes are
analogous to that of 2. The mass spectra of complexes 1–3 show
fragmentation patterns that are in agreement with bidentate
1
3
coordination of ligands L –L (see ESI†), and half sandwich
6
(η -arene)Ru complexes of ligands based on a picolylamine
motif that coordinates in a bidentate fashion to the ruthenium
42–44
atom are previously known.
Similarly, there are previous
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 2764–2773 | 2765

View Article Online
1
moiety. Hence, in the H NMR spectra of 1–3 the aromatic
protons of the p-cymene are observed as four separate doublets
and the isopropyl group as two doublets, one for each methyl
group. For 1 and 2, the methylene protons adjacent to the coordi-
nated amine show diastereotopic splitting, consistent with a
1
bidentate coordination mode. In the H NMR spectrum of 1 in
CDCl , one set of peaks was present, but upon dissolution of 1
3
in DMSO-d two sets of signals of different relative intensities
6
were observed. The less intense signal set increased with time
until equilibrium was reached, after which the distribution of
species remained stable; this is demonstrated in Fig. 4, which
shows the emergence of the second signal for the H5 proton of
the pyridine moiety over time. A similar behaviour was observed
in CD OD. The solution behaviour of 2 differed slightly from
3
1
that of 1, as the emergence of the second set of signals in the H
NMR spectrum was slower. Five days after dissolution in
DMSO-d , the relationship between the two species was 1 : 0.17
6
for 2, while for 1 a 1 : 1 relationship was reached within 24 h. It
is possible that this difference may be related to the larger ring
strain of the imidazole relative to the pyridine. In both cases,
both sets of signals differed from those of the free ligand, so
decomplexation was ruled out as a possible explanation for the
observed equilibrium. Complex 3 displayed only one set of
signals in DMSO-d , as well as in CDCl . The most likely expla-
Scheme 1 Synthesis of complexes 1–3.
6
3
nation for the observed solution behaviour of 1 and 2 is the
exchange of the coordinated chloride for a molecule of solvent
(DMSO, MeOH, vide infra). The alternative explanation that the
two observed species correspond to two diastereomers may be
ruled out since only one species is observed in CDCl3.
3 2
Fig. 3 ORTEP drawing of the molecular structure of 2·(CH ) CO,
showing the atom numbering scheme. Thermal ellipsoids are drawn at
the 50% probability level. The solvent of crystallization, the counter ion
−
(
Cl ) and all hydrogens except those of the secondary amines (N3 and
N4) have been omitted for clarity (cf. ESI†). The ruthenium centre has
6
the RRu configuration, according to the ligand priority sequence η -arene
>
Cl > Nimidazole > Namine, and the chiral aminic nitrogen shows a S
configuration. Relevant bond lengths (Å) and angles (°): Ru–N(1) 2.078
1); Ru–N(3) 2.169(1); Ru–Cl(1) 2.422(3); Ru-arene centroid 1.66;
N(1)–Ru–N(3) 76.47(5); N(1)–Ru–Cl(1) 87.07(3); N(3)–Ru–Cl(1)
5.31(3).
N
(
1
Fig. 4 H5 region of the H NMR spectrum of complex 1 at 10 min,
8
9
0 min, 3 h, 7 h and 24 h after dissolution in DMSO-d
6
.
examples of complexes analogous to 3, with the ligand coordi-
Conductivity measurements
nating to ruthenium through the imine nitrogen and the hydroxyl
45–47
oxygen.
In order to gain further knowledge of the behaviour of the new
complexes in solution and better interpret the NMR spectroscopy
results, their electrical conductance was measured. Molar con-
NMR spectroscopy
ductivity values of 279 and 293 ohm− cm mol for 1 mM
aqueous solutions of 1 and 2, respectively, are within the normal
range for three ions and indicate that these two complexes
1
2
−1
Unsymmetrical N,N- and N,O-donor ligands induce chirality at
6
the metal atom upon coordination to the (η -p-cymene)Ru frag-
ment, resulting in loss of two-fold symmetry of the p-cymene
undergo rapid hydrolysis to form [Ru(p-cymene)(ligand)(H
O]
2
2766 | Dalton Trans., 2012, 41, 2764–2773
This journal is © The Royal Society of Chemistry 2012

View Article Online
3 2
Table 1 Crystal data for 2·(CH ) CO
2
Empirical formula
fw
T/K
λ/Å
29 3 5
C H38Cl N ORu
680.06
100(2)
0.71073
Crystal system
Space group
a/Å
b/Å
c/Å
α (°)
β (°)
Triclinic
P1ˉ
11.3740(4)
12.0484(4)
12.0484(4)
99.6620(10)
106.562(2)
[
Cl] ; these conductivity values remain essentially unchanged
2
−1
2
−1
after 3 h (299 and 306 ohm cm mol for 1 and 2, respect-
ively). For complex 3, a conductivity value of 249 ohm cm
mol (or 266 ohm cm mol after 3 h) also indicates the
γ (°)
V/Å
114.2640(10)
1511.68(12)
2
1.494
0.815
−1
2-
3
−1
−1
2
−1
Z
/Mg m−
3
ρ
c
presence of three ions in solution, rather than the two expected
−1
μ(Mo-Kα)/mm
No. reflns.
3
for aquation of the complex to yield [Ru(p-cymene)(L )(H O)]
2
33271
[
Cl]; a possible explanation for this unusual value is that the
Unique reflns.
GOOF (F )
10277
1.023
0.0260
0.0277
0.0609
2
phenoxy group of the ligand is detached in solution and replaced
by a second water molecule. The H NMR spectrum of 3 in D O
shows that the ligand is no longer bidentate, as the p-cymene
peak pattern is that of a symmetric complex (i.e. two doublets
for the aromatic protons). Signals for the coordinated ligand of
1
R
R
wR
int
2
a
1
(I ≥ 2σ)
b
2
(I ≥ 2σ)
a
b
2
o
2 2
2 2 1/2
R
1
= Σ∥F | − |F
o
c
∥/Σ|F
o
|. wR
2
= [Σ[w(F
c o
− F ) ]/Σ[w(F ) ]] .
1
: 1 intensity to the p-cymene are also observed.
More interesting, the molar conductivity values of 1 mM sol-
utions of the various complexes in DMSO, the solvent in which
two sets of NMR signals were observed (and which was used in
biological experiments, see Experimental section), are 48.3
picolylamine complexes with respect to the immediate coordi-
42,43,45
nation environment of the metal.
As is expected in this
class of structures, the arene ring of the coordinated p-cymene is
essentially planar, with the distance between the ruthenium atom
and the p-cymene ring centroid being 1.66 Å and Ru–Carene
bonds ranging between 2.165(2) and 2.213(2) Å. This agrees
−1
2
−1
−1
2
−1
ohm cm mol for 1 and 52.1 ohm cm mol for 2, respect-
ively. These values are intermediate between those correspond-
4
8,49
ing to two and three ions,
which indicates partial
43,52,53
displacement of the coordinated chloride by DMSO to produce
equilibrium mixtures of [RuCl(p-cymene)(ligand)][Cl] and
well with similar complexes.
The Ru–Cl, Ru–Namine and
Ru–Nimidazole bonds (2.422(3), 2.169(1) and 2.078(1) Å, respect-
ively) are also similar to distances reported for related com-
plexes. The Ru–Namine bond is longer than the Ru–Nimidazole
[
Ru(p-cymene)(ligand)(DMSO)][Cl] (eqn (1)). Such equilibria
2
are fully consistent with the two independent sets of signals
appearing in the NMR spectra of 1 and 2. On the other hand, the
bond,
structures.
a pattern that mirrors corresponding picolylamine
−1
2
−1
42,43,45
conductivity of 3 (33.8 ohm cm mol ) corresponds to two
ions in solution, most likely resulting from full displacement of
3
the chloride to yield exclusively [Ru(p-cymene)(L )(DMSO)]Cl
(eqn (1)).
Antimalarial activity and cytotoxicity towards normal
mammalian cells
1
4
To our knowledge, the antimalarial activities of ligands L –L
X-Ray crystallography
54
have not been reported previously. Therefore, the antimalarial
1
4
The ruthenium complex 2 was characterised by X-ray crystallo-
graphy. Suitable crystals were grown from acetone at −20 °C.
The molecular structure of 2 is shown in Fig. 3 and relevant
crystallographic data are summarized in Table 1. To our knowl-
edge, this crystal structure is unique in being the first half
sandwich complex with a pendant 4-amino-7-chloroquinoline
moiety; the only previously reported structures are of metallo-
cene aminoquinoline conjugates. The complex crystallizes as a
racemate, and each molecule possesses the expected piano stool
geometry, with the two nitrogens of the imidazole and neigh-
bouring amine and the chloro ligand as the legs of the stool. The
counter ion Cl− (omitted in Fig. 3) is engaged in hydrogen
bonding with the two NH groups framing the ethylene spacer
activities of complexes 1–3 as well as of ligands L –L were
evaluated in vitro against chloroquine sensitive (D10) and chlor-
oquine resistant (Dd2) Plasmodium falciparum strains (Table 2).
4
Ligand L was included in the antimalarial study as a compari-
1
son to L , even though a corresponding metal complex had not
been synthesized. The standard drug chloroquine diphosphate
1
2
was used as reference in all assays. Ligands L and L show
3
0
good activity against both parasite strains, with the IC -values
5
0
1
for L (against D10) being just above those for chloroquine.
Ligand L exhibited medium activity against both strains (Resist-
ance Index, RI = 1.0, cf. Table 2), while L was active against
4
3
the chloroquine sensitive (CQS) strain, but inactive against the
chloroquine resistant (CQR) strain. Complexes 1 and 2 showed
medium to low activity against D10 (CQS) and 3–4 times lower
activity against Dd2 (CQR, relative to D10), while complex 3 is
very active against the CQS strain, though less active against the
(
cf. ESI†). There are very few examples of piano stool ruthenium
5
0,51
arene complexes with a bidentate imidazole ligand,
molecular structure of 2 closely resembles corresponding
but the
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 2764–2773 | 2767

View Article Online
1
4
Table 2 IC50-values of L –L and 1–3 tested in vitro for antiplasmodial activity against chloroquine sensitive (D10) and chloroquine resistant (Dd2)
strains of P. falciparum and for cytotoxicity on Chinese Hamster Ovarian (CHO) cells
a
b
Compound
D10: IC50 (μM)
Dd2: IC50 (μM)
CHO: IC50 (μM)
RI
SI
1
L
L
L
L
1
2
3
0.03 ± 0.002
0.19 ± 0.03
0.55 ± 0.30
1.3 ± 0.1
7.69 ± 0.07
4.53 ± 0.69
0.07 ± 0.007
0.025 ± 0.009 (n = 7)
—
0.22 ± 0.03
0.25 ± 0.06
>30
144 ± 50
240 ± 65
>300
>240
>160
>160
89 ± 19
—
7.3
1.3
ND
1.0
2.8
4.3
18.6
4800
1263
ND
ND
ND
ND
1271
2
3
4
1.3± 0.1
21.9 ± 1.0
19.7 ± 0.4
1.3 ± 0.1
0.143 ± 0.014 (n = 3)
—
CQ
Emetine
0.25 ± 0.03 (n = 2)
a
b
ND = not determined, n = number of data sets averaged. RI (Resistance Index) = IC50 Dd2/IC50 D10. SI (Selectivity Index) = IC50 CHO/IC50 D10.
CQR strain. The complexes and ligands were screened for cyto-
toxicity against a mammalian cell line, Chinese Hamster Ovarian
chloroquine resistance transporter, PfCRT. However, the limited
number of ligands and corresponding ruthenium arene com-
plexes prevents a clear correlation between structure and cross-
resistance.
1
2
(CHO), with emetine as reference drug. Ligands L and L
showed low cytotoxicity (IC = 144 μM and IC50 = 240 μM,
5
0
3
4
Table 2), while L and L showed no measurable cytotoxicity at
the highest tested concentration. Correspondingly, complexes 1
and 2 also showed no cytotoxicity at the highest tested concen-
tration. Complex 3 showed higher cytotoxicity than the other
two complexes (IC50 = 89 μM). In summary, all tested samples
exhibited low or no cytotoxicity.
Heme aggregation inhibition activity (HAIA) and water-octanol
distribution coefficients
As mentioned in the introduction, the design of new antiplasmo-
dial agents aimed at overcoming chloroquine resistance must
take into consideration the heme aggregation inhibition activity
(HAIA), as well as relevant physicochemical factors, such as
lipophilicity. The antimalarial potential of the Ru-chloroquine
complexes depicted in Fig. 1 against resistant strains of the Plas-
1
54
2
Ligands L
and L both show promising antimalarial
activity, but are less active than chloroquine against the CQR
2
strain. Ligand L is previously unreported and exhibits very low
toxicity against mammalian cells (Selectivity Index = 1263, cf.
41,56
Table 2). Compared to the corresponding ligands, the complexes
modium falciparum parasite has been demonstrated.
The
1
2
[
RuCl(p-cymene)(L)]Cl (L = L (1); L (2)) had significantly
incorporation of a metal centre induced important structural
modifications together with an increase in the lipophilicity of the
drug and an enhancement of the HAIR50 values (drug to hemin
ratio required to inhibit 50% of heme aggregation). This combi-
nation of features were claimed to be responsible for the high
activity observed against resistant parasites. However, complexa-
tion of the metal to chloroquine (CQ) in those cases took place
through the quinoline nitrogen, which resulted in an undesirable
decreased basicity of the new drugs, a crucial property for pH
trapping in the acidic vacuole. In the new series of complexes
reported herein, Ru(II) is coordinated to the chloroquine ana-
logues in a way that does not block the basic quinoline nitrogen,
while the enhancement of the lipophilicity compared to the free
ligand is still possible, and they were thus expected to display
interesting biological properties. It was therefore important to
gather experimental data on the above-mentioned physicochem-
ical features of the new compounds.
lower antimalarial activity. This is not surprising, since by block-
ing two of the nitrogens of the ligand, the ruthenium fragment in
1
and 2 might impede the ability of the complexes to accumulate
in the food vacuole, resulting in low activity. However, for
3
complex [RuCl(p-cymene)(L )] (3) the relationship is reversed;
the activity of the complex is notably higher than that of the
ligand. The Resistance Index for 3 is high (RI = 18), but this is
most likely a consequence of the lack of activity against the
3
CQR strain for L . The activity of 3 is considerably higher than
that of 1 and 2 (particularly against the CQS strain). Conduc-
tivity data (vide supra) suggest that the behaviour of 3 in
aqueous solution differs slightly from that of 1 and 2, which
might be related to activity.
Even though firm conclusions cannot be drawn based on
results for two parasite strains, a few points are worth noticing
regarding the cross-resistance with chloroquine of the reported
5
5
1
complexes and ligands. Ligand L is fully cross-resistant with
The results of water-octanol distribution coefficients (D) as a
measure of lipophilicity, and HAIR50 measurements are shown
in Table 3 for the ligands and metal complexes, in comparison
with chloroquine. Most of the ligands and their Ru complexes
display an increased lipophilicity at the acidic conditions of the
digestive vacuole (pH 4.9) with respect to chloroquine, which
might be a good indicator of activity against resistant parasites.
chloroquine, but exchange of the terminal pyridine group for a
2
methylimidazole as in L results in a compound that is not cross-
resistant. Most notable is the very high resistance of the Dd2
3
3
strain to L , which could suggest that L is transported out of the
food vacuole of the parasite to a higher extent than the other
3
ligands. The level of resistance to L is only partly decreased by
1
metal complexation. These results suggest that coordination to a
metal atom may limit cross-resistance with chloroquine, but does
not necessarily eliminate it. More importantly, the antimalarial
On the other hand, L shows similar lipophilicity to chloroquine,
whereas 2 is clearly more hydrophilic. Moreover, with the only
3
exception of L , all the ligands and the corresponding metal
3
activity data of L and 3 indicate that attachment of the bulky
complexes are more than twice as potent as chloroquine in their
3
41
metal group to L does not prevent interaction with the
HAIA ability at an aqueous acetate/n-octanol interface, as seen
2768 | Dalton Trans., 2012, 41, 2764–2773
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 3 Results for heme aggregation inhibition at an acetate buffer
aqueous solubility of 3 could be one of the factors involved in
the higher antimalarial activity.
(
pH
=
4.9)/n-octanol interface (HAIR50
)
and water/n-octanol
distribution coefficient (D) measurements
a
b
Compound
HAIR50
D (pH = 4.9)
D (pH = 6.6)
Conclusion
1
L
1
L
2
L
3
L
0.95
1.00
1.05
0.86
3.45
1.20
0.87
2.92
0.15 ± 0.01
0.28 ± 0.04
1.68 ± 0.09
0.007 ± 0.002
0.35 ± 0.02
0.40 ± 0.02
3.41 ± 0.62
0.15 ± 0.01
4.24 ± 0.27
8.84 ± 0.82
2.82 ± 0.16
2.06 ± 0.78
5.91 ± 0.48
1.99 ± 0.10
4.39 ± 0.54
6.61 ± 0.64
A small family of ruthenium arene half sandwich complexes
with chloroquine analogue ligands was synthesized and charac-
terized by NMR spectroscopy, mass spectrometry and, in one
case, X-ray crystallography. These complexes differ from pre-
viously reported ruthenium complexes with antimalarial activity,
as the quinoline group is pendant, leaving the quinoline nitrogen
2
3
4
c
CQ
1
2
uncoordinated. In the case of the ligands L and L , coordination
to the metal fragment did not result in increased antimalarial
activity, but these ligands themselves displayed significant
activity. However, complex 3 was considerably more active than
a
HAIR50 is the drug to hemin ratio required to inhibit 50% of heme
aggregation in comparison to a control experiment with no drug.
pH) = [compound] in n-octanol/[compound] in water at the given pH.
As reported in ref. 41
b
D
(
c
3
L and showed good activity, particularly against the chloroquine
sensitive strain of P. falciparum. The lipophilicity and heme
aggregation inhibition activity at an aqueous buffer/n-octanol
interface (HAIR ) were measured for all ligands and complexes
by the values of HAIR . Interestingly, the HAIA ability is also
5
0
50
enhanced with reference to the Ru-chloroquine complexes pre-
but a general correlation with the in vitro antimalarial activity
4
1,56
3
viously described by us (Fig. 1),
tion of L .
again with the sole excep-
could not be established. In the case of the L -3 couple, the
3
enhancement of the antiplasmodial activity upon coordination to
a metal centre was related to an increase in lipophilicity and the
HAIA ability at the buffer/n-octanol interface. The high antima-
larial activity of 3 is a promising result that warrants more
research, including further study of the structure activity relation-
Due to the high number of variables introduced in the design
of the new drug candidates tested (presence or absence of metal
moiety, charge and/or substantially different structural features)
an internal correlation among the new family of chloroquine ana-
logues and/or their metal complexes regarding the HAIA ability
and lipophilicity is not obvious. However, it may be noted that
complexation to the metal did not induce a significant modifi-
cation of the HAIA or an increase in lipophilicity at pH 4.9 of
6
ships of (η -arene)Ru complexes with bidentate chloroquine ana-
3
logue ligands. It is conceivable that structural optimization of L
could give a complex with high activity also against chloroquine
resistant strains.
1
2
the ligands in the pairs L -1, L -2, contrary to what was pre-
4
1,56
viously observed for other Ru–CQ drug candidates,
and con-
3
Experimental
trary to the results obtained for L -3. Furthermore, no clear
correlations were found between HAIR50 or lipophilicity with
the antiplasmodial activity against CQS or CQR Plasmodium
falciparum parasites. Again, this is most likely a consequence of
the complexity of the systems, i.e. the fact that the final antima-
larial activity is the result of a multifaceted balance of heme
aggregation inhibition activity, lipophilicity, structural features,
solubility and general pharmacokinetic behaviour when the drug
candidates are within biological systems. However, it is again
General
All synthetic procedures were performed under dry nitrogen
using standard Schlenk and vacuum-line techniques. Solvents
used were dried by distillation over appropriate drying reagents
and stored over molecular sieves under nitrogen. All chemicals
were purchased from Sigma Aldrich and used as received.
5
8
1
[(p-cymene)RuCl
]
and N -(7-chloroquinolin-4-yl)ethane-
2
2
3
59
worth noting the case of L and 3. The complexation of an inac-
1,2-diamine were prepared according to literature methods.
NMR spectra were recorded on a Varian Inova 500 MHz spec-
trometer using the solvent resonance as internal standard for H
tive ligand, which results in a notable increase of the HAIA
ability at the interface and a slight increase of lipophilicity at the
acidic vacuole conditions, gives a metal compound with a sig-
nificantly enhanced antimalarial activity. This indicates that, for
the series of compounds reported herein, only when the incor-
poration of a metal moiety induces important changes in the
lipophilicity and/or the HAIA ability of an inactive ligand, there
is a good correlation with the differences observed in the final
overall antiplasmodial activity of the complex.
1
13
NMR and C NMR shifts. Infrared spectra were recorded on a
Nicolet Avatar 360 FT-IR spectrometer. Electrospray ionization
(ESI) and high resolution mass spectra were recorded using a
Waters Micromass Q-Tof micro mass spectrometer or an LC-MS
Agilent 6220-TOF coupled with 1200 series HPLC. Conduc-
tivity values were obtained using 1 mM solutions of the com-
plexes in water or DMSO at various time intervals using an
Oaklon pH/Conductivity meter. Elemental analysis was per-
formed by Mikroanalytische Laboratorium Kolbe, Mülheim an
der Ruhr.
Another feature which might contribute to the increased
3
activity of 3 relative to L is aqueous solubility at physiological
pH. Coordination to an arene metal unit as a means to increasing
aqueous solubility and bioavailability of ligands with known bio-
4
2,57
logical activity has been documented in the literature.
Even
X-Ray structure determinations
though lipophilicity at vacuolar pH increases upon coordination
3
of L to the metal fragment, lipophilicity at pH 6.6 decreases. As
The crystal was immersed in cryo-oil, mounted in a Nylon loop,
and measured at a temperature of 100 K. The X-ray diffraction
3
L is virtually insoluble in water at neutral pH, the increased
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 2764–2773 | 2769

View Article Online
13
data were collected on a Bruker AXS Kappa ApexII Duo dif-
(t, 2H, J = 6.3 Hz), 2.96 (t, 2H, J = 6.3 Hz); C NMR
fractometer using Mo Kα radiation (λ = 0.71073 Å). The
APEX2 program package was used for cell refinement and
(125 MHz, CD OD) δ 152.8, 152.5, 149.7, 147.6, 136.4, 127.6,
3
60
127.1, 126.1, 124.4, 123.0, 118.8, 99.8, 47.9, 45.2, 43.5, 33.1;
−1
data reduction. The structure was solved by direct methods using
IR (KBr) vmax/cm 3260m (br), 3255m, 3063w, 2961w, 2832w,
1613m (7-chloroquinoline), 1581s (7-chloroquinoline), 1549m
(7-chloroquinoline), 1452m, 1429m, 1376m, 1289m, 1250w,
1216w, 1138m, 1106m, 1082w, 960w, 852m, 812w, 729m;
HRMS (ES+) m/z calcd. for C H N Cl 316.1329, found
61
SHELXS-97. A semi-empirical absorption correction based on
6
2
equivalent reflections (SADABS) was applied to the data.
61
Structural refinement were carried out using SHELXL-97. The
NH hydrogen atoms were located from the difference Fourier
16 19 5
map but constrained to ride on their parent atom, with Uiso
=
316.1340.
1
.5·U (parent atom). Other hydrogen atoms were positioned
eq
geometrically and were also constrained to ride on their parent
N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-chloroquinolin-
atoms, with C–H = 0.95–1.00 Å, and Uiso = 1.2–1.5 ·U (parent
eq
3
1
4
(
-amine
(L ). N -(7-chloroquinolin-4-yl)ethane-1,2-diamine
atom). The crystallographic details are summarized in Table 1.
2.026 g, 9.14 mmol) was dissolved in ethanol (90 mL). Salicy-
laldehyde (0.95 mL, 8.96 mmol) was added dropwise and the
solution was stirred at room temperature for 45 min. The solvent
was evaporated and the residue was washed with cold ethanol
Syntheses
N-(2-((pyridin-2-yl)methylamino)ethyl)-7-chloroquinolin-4-
amine
and petroleum ether. The product was obtained as a yellow solid
1
1
1
(L ). N -(7-chloroquinolin-4-yl)ethane-1,2-diamine
(
2.24 g, 77%). H NMR (500 MHz, CDCl ) δ 12.99 (br s, 1H),
3
(0.800 g, 3.6 mmol) was dissolved in methanol (30 mL). 2-pyri-
8
7
6
=
.58 (d, 1H, J = 5.3 Hz), 8.37 (s, 1H), 7.97 (d, 1H, J = 0.9 Hz),
.60 (d, 1H, J = 8.9 Hz), 7.36 (m, 2H), 7.22 (d, 1H, J = 7.5 Hz),
.98 (d, 1H, J = 8.3 Hz), 6.89 (t, 1H, J = 7.5 Hz), 6.51 (d, 1H, J
dinecarboxaldehyde (0.34 mL, 3.6 mmol) was added dropwise
and the solution was stirred for 16 h at room temperature.
Sodium borohydride (0.272 g, 7.2 mmol) was added and the
reaction mixture was stirred for an additional hour. The solvent
was removed in vacuo and the residue was dissolved in EtOAc
13
5.3 Hz), 5.20 (br s, 1H), 3.95 (m, 2H), 3.74 (m, 2H);
C
NMR (125 MHz, CDCl ) δ 167.2, 160.9, 152.0, 149.2, 149.1,
3
1
1
35.1, 132.8, 131.6, 129.0, 125.7, 120.7, 119.0, 118.5, 117.2,
17.1, 99.3, 57.7, 43.5; IR (KBr) vmax/cm− 3405s (O-H),
1
(150 mL). The organic phase was washed with brine (3 ×
5
0 mL), dried over Na SO and evaporated. The product was
2
4
2940w, 2855w, 1630m (NvC), 1613m (7-chloroquinoline),
purified by silica gel flash chromatography eluting with ethyl
acetate-methanol-triethylamine 8 : 1 : 1 (v/v). The product was
obtained as a beige solid (0.876 g, 81%). H NMR (500 MHz,
1
1
578s (7-chloroquinoline), 1541w (7-chloroquinoline), 1508w,
449w, 1425w, 1379w, 1278m, 1210w, 1143w, 895w, 761m;
1
HRMS (ES+) m/z calcd. for C H N OCl 326.1060, found
18 17 3
CDCl ) δ 8.58 (d, 1H, J = 4.4 Hz), 8.52 (d, 1H, J = 5.3 Hz),
3
326.1059.
7
.95 (d, 1H, J = 2.1 Hz), 7.81 (d, 1H, J = 8.9 Hz), 7.65 (dt, 1H,
J = 1.7 Hz, J = 7.7 Hz), 7.36 (dd, 1H, J = 2.1 Hz, J = 8.9 Hz),
N-(2-(bis((pyridin-2-yl)methyl)amino)ethyl)-7-chloroquinolin-
7
6
2
.27 (d, 1H, J = 8.0 Hz), 7.19 (dd, 1H, J = 5.1 Hz, J = 6.7 Hz),
4
1
4
-amine
(0.200 g, 0.9 mmol) was dissolved in dichloromethane (12 mL).
-pyridinecarboxaldehyde (0.17 mL, 1.8 mmol) was added drop-
(L ). N -(7-chloroquinolin-4-yl)ethane-1,2-diamine
.38 (d, 1H, J = 5.4 Hz), 6.09 (br s, 1H), 3.99 (s, 2H), 3.37 (m,
1
3
H), 3.08 (m, 2H), 2.05 (br s, 1H); C NMR (125 MHz,
2
CDCl ) δ 159.2, 151.9, 150.0, 149.3, 149.0, 136.6, 134.8, 128.5,
3
wise and the solution was stirred at room temperature for
30 min. Sodium tris(acetoxy)borohydride (0.900 g, 4.1 mmol)
was added and the reaction mixture was stirred for 17 h. A
second portion of sodium triacetoxyborohydride (0.290 g,
1
25.1, 122.4, 122.2, 121.5, 117.4, 99.1, 54.2, 47.0, 42.3; IR
−1
(KBr) vmax/cm 3221m (br), 3067w, 2962w, 2839w, 1613m (7-
chloroquinoline), 1582s (7-chloroquinoline), 1452m, 1431m,
1
8
3
380m, 1333w, 1282w, 1249w, 1204w, 1140w, 1112w, 1082w,
49w, 807w, 763m; HRMS (ES+) m/z calcd. for C H N Cl
13.12145, found 313.12215.
1
.3 mmol) was added and the stirring was continued for 3 h.
1
7 18 4
Saturated aqueous Na CO (7 mL) was added and when gas
2
3
evolution subsided, the organic phase was washed with
additional aqueous Na CO (2 × 10 mL). The organic phase was
N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)ethyl)-7-chlor-
2
3
2
1
oquinolin-4-amine (L ). N -(7-chloroquinolin-4-yl)ethane-1,2-
diamine (0.300 g, 1.35 mmol) and 1-methyl-2-imidazole carbox-
aldehyde (0.150 g, 1.35 mmol) were dissolved in methanol
dried over Na SO4 and evaporated. The crude product was
2
purified by silica gel flash chromatography eluting with
ethyl acetate-methanol-triethylamine 83 : 07:10 (v/v). The pure
1
(30 mL) and a few molecular sieves (3 Å) were added. The sol-
product was obtained as a brown oil (0.221 g, 61%). H NMR
ution was stirred for 18 h at room temperature. Sodium borohy-
dride (0.102 g, 2.7 mmol) was added and the reaction mixture
was stirred for an additional hour. Molecular sieves were filtered
off and the solvent was removed in vacuo. The residue was dis-
solved in EtOAc (50 mL). The organic phase was washed with
brine (3 × 25 mL), dried over Na SO and evaporated. The
(500 MHz, CDCl ) δ 8.57 (ddd, 2H, J = 0.8 Hz, J = 1.6 Hz, J =
3
4.9 Hz), 8.46 (d, 1H, J = 5.4 Hz), 8.32 (d, 1H, J = 9.0 Hz), 7.97
(d, 1H, J = 2.1 Hz), 7.56 (dt, 2H, J = 1.8 Hz, J = 7.7 Hz), 7.48
(br s, 1H), 7.45 (dd, 1H, J = 2.2 Hz, J = 8.9 Hz), 7.36 (d, 2H, J
= 7.8 Hz), 7.15 (ddd, 2H, J = 1.0 Hz, J = 4.9 Hz, J = 7.4 Hz),
6.25 (d, 1H, J = 5.5 Hz), 3.97 (s, 4H), 3.30 (m, 2H), 3.00 (m,
2
4
1
13
product was obtained as a white/yellow solid (0.396 g, 92%). H
2H); C NMR (125 MHz, CDCl ) δ 158.9, 151.9, 149.8, 149.2,
3
NMR (500 MHz, CD OD) δ 8.34 (d, 1H, J = 5.6 Hz), 8.09 (d,
149.0, 136.6, 134.3, 128.3, 124.9, 123.2, 122.5, 122.3, 117.7,
3
−1
1
H, J = 9.0 Hz), 7.77 (d, 1H, J = 2.1 Hz), 7.39 (dd, 1H, J = 2.2
98.7, 59.8, 51.1, 40.5; IR (KBr) vmax/cm 3199m (br), 3058m,
3006m, 2942w, 1611m (7-chloroquinoline), 1581s (7-chloroqui-
noline), 1546m (7-chloroquinoline), 1449w, 1433m, 1367w,
Hz, J = 9.0 Hz), 6.98 (d, 1H, J = 1.3 Hz), 6.86 (d, 1H, J = 1.3
Hz), 6.52 (d, 1H, J = 5.7 Hz), 3.90 (s, 2H), 3.68 (s, 3H), 3.46
2770 | Dalton Trans., 2012, 41, 2764–2773
This journal is © The Royal Society of Chemistry 2012

View Article Online
6
1
283w, 1226m, 1150m, 1076w, 850w, 759m; HRMS (ES+) m/z
(η -p-cymene)(N-(2-((2-hydroxyphenyl)methylimino)ethyl)-7-
chloroquinolin-4-amine)chlororuthenium(II), [RuCl(cymene)
L )] (3). L (0.053 g, 0.16 mmol) was dissolved in dichloro-
calcd. for C H N Cl 404.16365, found 404.16356.
2
3 23 5
3
3
(
methane (10 mL) and triethylamine (0.023 mL, 0.16 mmol) was
added. The solution was stirred at room temperature for 30 min
and [(p-cymene)RuCl ] (0.050 g, 0.08 mmol) was added. Stir-
6
(
η -p-cymene)(N-(2-((pyridin-2-yl)methylamino)ethyl)-7-
chloroquinolin-4-amine)chlororuthenium(II) chloride,
RuCl(cymene)(L )]Cl (1)
2
2
1
[
ring at ambient temperature continued overnight. The solvent
volume was reduced to approx. 2 mL and the product was preci-
pitated with diethyl ether. The precipitate was isolated via fil-
tration and washed with water (2-3 mL), diethyl ether and
petroleum ether. The product was dried under vacuum and
1
[
0
(p-cymene)RuCl2]2 (0.200 g, 0.32 mmol) and L (0.204 g,
.64 mmol) were dissolved in dichloromethane (20 mL) and
stirred at room temperature overnight. The solvent volume was
reduced to approx. 3 mL and the product was precipitated with
diethyl ether. The product was isolated via filtration and washed
with diethyl ether and petroleum ether. The product was obtained
as a fine, yellow solid (0.357 g, 91%). H NMR (500 MHz,
CDCl ) δ 9.56 (br s, 1H), 8.98 (d, 1H, J = 9.1 Hz), 8.89 (d, 1H,
J = 5.2 Hz), 8.52 (d, 1H, J = 5.4 Hz), 8.10 (br s, 1H), 7.95
d, 1H, J = 0.8 Hz), 7.83 (dt, 1H, J = 1.4 Hz, J = 7.8 Hz), 7.52
dd, 1H, J = 2.1 Hz, J = 9.0 Hz), 7.40 (t, 1H, J = 6.9 Hz), 7.30
d, 1H, J = 7.7 Hz), 6.33 (d, 1H, J = 5.4 Hz), 5.98 (d, 1H, J =
.9 Hz), 5.91 (d, 1H, J = 6.1 Hz), 5.55 (d, 1H, J = 6.0 Hz), 5.39
d, 1H, J = 6.1 Hz), 4.62 (dd, 1H, J = 6.1 Hz, J = 16.1 Hz), 3.93
dd, 1H, J = 5.4 Hz, J = 16.2 Hz), 3.62 (m, 2H), 3.56 (m, 1H),
.13 (m, 1H), 2.78 (hept, 1H, J = 6.9 Hz), 2.04 (s, 3H), 1.08
d, 3H, J = 7.0 Hz), 1.02 (d, 3H, J = 6.9 Hz); C NMR
125 MHz, CDCl ) δ 161.2, 153.6, 151.4, 150.3, 149.0, 139.2,
35.3, 127.6, 125.7, 125.1, 124.9, 121.8, 117.9, 105.7, 98.3,
7.1, 84.7, 84.3, 83.9, 82.8, 58.7, 53.7, 41.1, 30.8, 22.6, 21.4,
1
obtained as an orange solid (0.065 g, 67%). H NMR
(
500 MHz, CDCl ) δ 8.37 (d, 1H, J = 4.3 Hz), 8.02 (d, 1H, J =
3
1
1.5 Hz), 7.85 (d, 1H, J = 9.0 Hz), 7.51 (br s, 1H), 7.39 (s, 1H),
.22 (dd, 1H, J = 1.5 Hz, J = 9.1 Hz), 7.15 (m, 1H), 6.91 (d, 1H,
J = 8.5 Hz), 6.55 (d, 1H, J = 3.9 Hz), 6.48 (d, 1H, J = 7.7 Hz),
.27 (t, 1H, J = 7.3 Hz), 5.57 (d, 1H, J = 6.2 Hz), 5.52 (d, 1H,
J = 6.2 Hz), 5.46 (d, 1H, J = 5.4 Hz), 5.09 (d, 1H, J = 5.4 Hz),
.57 (m, 1H), 4.27 (m, 2H), 3.97 (m, 1H), 2.75 (hept, 1H,
J = 6.9 Hz), 2.29 (s, 3H), 1.24 (d, 3H, J = 6.9 Hz), 1.14 (d, 3H,
7
3
6
(
(
(
5
(
(
3
(
(
4
13
J = 6.9 Hz); C NMR (125 MHz, CDCl ) δ 166.0, 165.0,
3
1
1
54.8, 143.1, 139.5, 139.2, 135.6, 134.9, 127.8, 124.7, 122.1,
20.3, 118.6, 115.4, 114.7, 100.9, 98.8, 98.5, 87.7, 83.1, 81.2,
−1
1
3
80.6, 44.8, 43.8, 30.6, 22.9, 21.6, 18.9; IR (KBr) vmax/cm
3
1
1
422m (br), 3236m, 3056w, 2961w, 1615s (7-chloroquinoline),
587m (7-chloroquinoline), 1536w (7-chloroquinoline), 1467m,
449m, 1318w, 1213w, 1146w, 1091w, 1018w, 877w, 758w;
3
1
9
1
−
1
MS (ES+) m/z 596 (M+H, 100%), 560 (25, M-HCl); HRMS
8.0; IR (KBr) vmax/cm 3410m (br), 3060w, 2964w, 1610m
7-chloroquinoline), 1580s (7-chloroquinoline), 1535w (7-chlor-
oquinoline), 1451m, 1372w, 1330w, 1141w, 1088w, 765w; MS
(
ES+) m/z calcd. for C H N Cl ORu 596.0809, found
28 30 3 2
(
5
96.0822.
+
(
ES+) m/z 583 (M , 100%), 548 (30, MH - Cl); HRMS (ES+)
m/z calcd. for C H N Cl Ru 583.0969, found 583.0985;
27
31
4
2
Elemental analysis (%) calcd. for C H N Cl Ru·0.8CH Cl : C
Heme Aggregation Inhibition Activity (HAIA) at a water/
n-octanol interface
2
7
31
4
3
2
2
4
8.61, H 4.78, N 8.16, found: C 48.65, H 4.83, N 8.29.
6
(
η -p-cymene)(N-(2-((1-methyl-1H-imidazol-2-yl)methylamino)
Inhibition of the heme aggregation near the interface of aqueous
buffer/n-octanol mixtures was studied following a previously
published method. To establish a base line for the aggregation
ethyl)-7-chloroquinolin-4-amine)chlororuthenium(II)
RuCl(cymene)(L )]Cl (2). This compound was synthesized
from [(p-cymene)RuCl2]2 (0.100 g, 0.16 mmol) and L
chloride,
2
41
[
2
process, hemin was dissolved in 0.1 M NaOH solution to gener-
ate hematin and acetone was added until the acetone:water ratio
was 4 : 6; the final solution contained 15 mg hematin/ml. A
sample of this solution (200 μl) was carefully introduced close
to the interface between n-octanol (2 ml) and aqueous acetate
buffer (5 ml; pH 4.9) in a cylindrical vial with an internal diam-
eter of 2.5 cm. The mixture was incubated at 37 °C for 60 min
and at the end of the incubation period it was stirred to ensure
the transfer of all solid particles to the aqueous layer. The
product (β-hematin) was isolated by centrifugation. The pellet
was collected and washed with DMSO (4 ml), centrifuged again
for 20 min, washed with 2 ml of ethanol and finally dissolved in
25 ml of 0.1 M NaOH for spectrophotometric quantification. For
the aggregation inhibition activity measurements the appropriate
amount of each drug (23 mM in n-octanol) to yield [drug]:
[hemin] ratios in the range 1–6 was added to the acetate buffer/
n-octanol mixture; after stirring for 30 min to equilibrate the
drug between the two phases, the hematin solution was added
close to the interface and the procedure was followed as
described above. All experiments were performed in
quadruplicate.
(
0.103 g, 0.32 mmol) using the same procedure employed for
synthesis of 1 (vide supra). The product was obtained as a fine,
yellow solid (0.192 g, 95%). H NMR (500 MHz, CDCl ) δ
9
Hz), 8.34 (br s, 1H), 7.96 (d, 1H, J = 2.1 Hz), 7.50 (dd, 1H, J =
2
1
1
3
.09 (br s, 1H), 8.99 (d, 1H, J = 9.0 Hz), 8.54 (d, 1H, J = 5.4
.2 Hz, J = 9.0 Hz), 7.20 (d, 1H, J = 1.6 Hz), 6.91 (d, 1H, J =
.5 Hz), 6.36 (d, 1H, J = 5.5 Hz), 5.87 (d, 1H, J = 5.6 Hz), 5.55
(d, 1H, J = 6.0 Hz), 5.36 (d, 1H, J = 6.1 Hz), 5.28 (d, 1H, J =
5
3
3
.7 Hz), 4.27 (m, 1H), 3.91 (m, 1H), 3.65 (m, 3H), 3.57 (s, 3H),
.45 (m, 1H), 2.89 (hept, 1H, J = 6.9 Hz), 2.14 (s, 3H), 1.19 (d,
1
3
H, J = 6.9 Hz), 1.16 (d, 3H, J = 7.0 Hz); C NMR (125 MHz,
CDCl ) δ 151.2, 150.3, 149.6, 148.9, 135.5, 128.1, 127.6, 125.8,
3
1
4
3
25.1, 123.0, 117.9, 106.0, 98.1, 96.1, 83.2, 82.6, 81.0, 55.4,
6.2, 41.0, 34.8, 30.9, 22.9, 21.3, 18.1; IR (KBr) vmax/cm−
411m, 3258m, 3092w, 2964w, 2867w, 1612w (7-chloroquino-
1
line), 1579s (7-chloroquinoline), 1538w (7-chloroquinoline),
513w, 1451m, 1421, 1363, 1327, 1138w, 1088w, 869w; HRMS
ES+) m/z calcd. for C H N Cl Ru 586.1078, found 586.1077;
1
(
2
6
32
5
2
Elemental analysis (%) calcd. for C H N Cl Ru·0.6CH Cl : C
4
2
6
32
5
3
2
2
7.47, H 4.97, N 10.41, found: C 47.47, H 5.02, N 10.14.
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 2764–2773 | 2771

View Article Online
Determination of partition coefficients
dilution technique was applied to all the test samples. The
highest concentration of solvent to which the cells were exposed
had no measurable effect on the cell viability (data not shown).
The 50% inhibitory concentration (IC -values) were obtained
from full dose-response curves, using a nonlinear dose-response
curve fitting analysis via Graph Pad Prism v.4.0 software.
The distribution of all complexes between n-octanol and water
was studied by use of the stir-flask method.
6
3–65
A mixture of
50
1
0 ml n-octanol and 10 ml of water (each saturated in the other)
was stirred for 30 min at the desired temperature after adding the
right amount of the sample to be analyzed (∼ 0.7 mmol). The
pH was adjusted to the desired value by addition of either a 0.1
M solution of H PO or NaOH (10–20 μl). Once the equilibrium
Acknowledgements
3
4
was reached, the organic and aqueous phases were separated and
centrifuged. Finally, the concentration of drug in each phase was
measured spectrophotometrically in order to determine values of
D = [drug](in n-octanol)/[drug](in water). Experiments were
carried out in quadruplicate.
This research has been funded by the Swedish International
Development Cooperation Agency (SIDA) and the Swedish
Research Council (VR). Financial support from the NIH through
Grant
# 5SC1GM89558-2 to R.A. S.-D. is gratefully
acknowledged.
Determination of in vitro antiplasmodial activity
References
Two strains of Plasmodium falciparum were used in this study -
the chloroquine sensitive strain D10 and the chloroquine resistant
strain Dd2. Continuous in vitro cultures of asexual erythrocyte
stages of P. falciparum were maintained using a modified
1
2
R. Carter and K. N. Mendis, Clin. Microbiol. Rev., 2002, 15, 564–594.
WHO Malaria Report 2009, http://www.who.int/malaria/
world_malaria_report_2009
A. M. Dondorp, S. Yeung, L. White, C. Nguon, N. P. J. Day, D. Socheat
and L. von Seidlein, Nat. Rev. Microbiol., 2010, 8, 272–280.
A. M. Dondorp, F. Nosten, P. Yi, D. Das, A. P. Phyo, J. Tarning,
K. M. Lwin, F. Ariey, W. Hanpithakpong, S. J. Lee, P. Ringwald,
K. Silamut, M. Imwong, K. Chotivanich, P. Lim, T. Herdman, S. S. An,
S. Yeung, P. Singhasivanon, N. P. J. Day, N. Lindegardh, D. Socheat and
N. J. White, N. Engl. J. Med., 2009, 361, 455–467.
3
4
6
6
method of Trager and Jensen. Quantitative assessment of anti-
plasmodial activity in vitro was determined via the parasite
lactate dehydrogenase assay using a modified method described
67
by Makler. The test samples were tested in triplicate on one
−1
occasion. The test samples were prepared as a 20 mg ml stock
solution in 100% DMSO. Samples were tested as a suspension if
not completely dissolved. Stock solutions were stored at −20 °C.
Further dilutions were prepared on the day of the experiment.
Chloroquine (CQ) was used as the reference drug in all exper-
iments. A full dose-response was performed for all compounds
to determine the concentration inhibiting 50% of parasite growth
5
I. M. Hastings, P. G. Bray and S. A. Ward, Science, 2002, 45, 2001–
2002.
6 L. M. B. Ursos and P. D. Roepe, Med. Res. Rev., 2002, 22, 465–491.
7
8
K. A. de Villiers and T. J. Egan, Molecules, 2009, 14, 2868–2887.
T. J. Egan and H. M. Marques, Coord. Chem. Rev., 1999, 190-192, 493–
517.
9 A. Leed, K. DuBay, L. M. B. Ursos, D. Sears, A. C. De Dios and
P. D. Roepe, Biochemistry, 2002, 41, 10245–10255.
1
0 P. J. Rosenthal, Ed., Antimalarial Chemotherapy: mechanism of action,
resistance and new directions in drug discovery, Humana Press, New
Jersey, 2001.
(
IC –value). Test samples 1 and 2 were tested at a starting con-
50
−1
centration of 100 μg ml , which was then serially diluted 2-fold
in complete medium to give 10 concentrations; with the lowest
11 C. R. Chong and D. J. Sullivan, Biochem. Pharmacol., 2003, 66, 2201–
−1
2212.
concentration being 0.2 μg ml . The same dilution technique
12 T. J. Egan, R. Hunter, C. H. Kaschula, H. M. Marques, A. Misplon and
3
4
was used for all samples. Test samples L and L were tested at
J. Walden, J. Med. Chem., 2000, 43, 283–291.
−
1
1
a starting concentration of 10 μg ml . Test samples L and 3
13 S. R. Cheruku, S. Maiti, A. Dorn, B. Scorneaux, A. K. Bhattacharjee,
W. Y. Ellis and J. L. Vennerstrom, J. Med. Chem., 2003, 46, 3166–3169.
14 R. G. Ridley, Nature, 2002, 415, 686–693.
−
1
were tested at a starting concentration of 1000 ng ml . Test
2
−1
sample L was tested at a starting concentration of 100 ng ml
15 C. P. Sanchez, J. E. McLean, W. Stein and M. Lanzer, Biochemistry,
−
1
against the CQS strain and 1000 ng ml against the CQR strain.
2004, 43, 16365–16373.
−
1
CQ was tested at a starting concentration of 100 ng ml against
16 C. P. Sanchez, J. E. McLean, P. Rohrbach, D. A. Fidock, W. D. Stein and
−1
M. Lanzer, Biochemistry, 2005, 44, 9862–9870.
the CQS strain and 1000 ng ml against the CQR strain. The
highest concentration of solvent to which the parasites were
exposed to had no measurable effect on the parasite viability
1
1
7 P. D. Roepe, Biochemistry, 2011, 50, 163–171.
8 D. A. van Schalkwyk and T. J. Egan, Drug Res. Updates, 2006, 9, 211–
226.
(
data not shown).
19 P. G. Bray, M. Mungthin, I. M. Hastings, G. A. Biagini, D. K. Saidu,
V. Lakshmanan, D. J. Johnson, R. H. Hughes, P. A. Stocks,
P. M. O’Neill, D. A. Fidock, D. C. Warhurst and S. A. Ward, Mol. Micro-
biol., 2006, 62, 238–51.
Cytotoxicity assay
2
2
0 D. C. Warhurst, Malaria J., 2003, 2, 31–43.
1 D. Warhurst, J. Craig, I. Adagu, D. Meyer and S. Lee, Malaria J., 2003,
Cytotoxicity was assessed against a Chinese Hamster Ovarian
(
2, 26–30.
CHO) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
22 T. J. Egan, J. Inorg. Biochem., 2008, 102, 1288–1299.
6
8
diphenyltetrazoliumbromide (MTT) assay. The test samples
were tested in triplicate on one occasion. The test samples were
prepared as a 20 mg ml stock solution in 100% DMSO.
Dilutions were prepared on the day of the experiment. Emetine
was used as the reference drug in all experiments. The initial
concentration of emetine was 100 μg mL , which was serially
diluted in complete medium with 10-fold dilutions to give six
concentrations, the lowest being 0.001 μg mL . The same
23 T. J. Egan, Mol. Biochem. Parasitol., 2008, 157, 127–136.
24 A. N. Hoang, K. K. Ncokazi, K. A. de Villiers, D. W. Wright and T.
J. Egan, Dalton Trans., 2010, 39, 1235–1244.
−1
2
5 C. Biot, G. Glorian, L. A. Maciejewski, J. S. Brocard, O. Domarle,
G. Blampain, P. Millet, A. J. Georges and J. Lebibi, J. Med. Chem.,
1997, 40, 3715–3718.
−
1
26 http://clinicaltrials.gov/ct2/show/NCT00988507
2
2
7 http://clinicaltrials.gov/ct2/show/NCT00563914
8 C. Biot, N. Chavain, F. Dubar, B. Pradines, X. Trivelli, J. Brocard,
I. Forfar and D. Dive, J. Organomet. Chem., 2009, 694, 845–854.
−1
2772 | Dalton Trans., 2012, 41, 2764–2773
This journal is © The Royal Society of Chemistry 2012

View Article Online
2
3
3
3
3
3
3
3
9 P. Beagley, M. A. L. Blackie, K. Chibale, C. Clarkson, J. R. Moss and P.
J. Smith, Dalton Trans., 2002, 4426–4433.
0 P. Beagley, M. A. L. Blackie, K. Chibale, C. Clarkson, R. Meijboom,
J. R. Moss, P. J. Smith and H. Su, Dalton Trans., 2003, 3046–3051.
1 R. A. Sánchez-Delgado, M. Navarro, H. A. Pérez and J. A. Urbina, J.
Med. Chem., 1996, 39, 1095–1099.
48 W. J. Geary, Coord. Chem. Rev, 1971, 7, 81–122.
49 A. Apelblat, J. Solution Chem., 2011, 40, 1234–1257.
50 P. Govindaswamy, P. Carroll, C. Sinha and M. R. Kollipara, J. Coord.
Chem., 2007, 60, 97–107.
51 H. Mishra and R. Mukherjee, J. Organomet. Chem., 2006, 691, 3545–
3555.
52 L. K. Filak, G. Mühlgassner, M. A. Jakupec, P. Heffeter, W. Berger,
V. B. Arion and B. K. Keppler, J. Biol. Inorg. Chem., 2010, 15, 903–918.
53 S. Grgurić-Sipka, I. Ivanović, G. Rakić, N. Todorović, N. Gligorijević,
S. Radulović, V. B. Arion, B. K. Keppler and Z. L. Tesić, Eur. J. Med.
Chem., 2010, 45, 1051–1058.
54 Int. Pat., 97/18193, 1997
55 We thank a reviewer for drawing our attention to these factors relating to
the cross-resistance with chloroquine
56 A. Martínez, C. S. K. Rajapakse, B. Naoulou, Y. Kopkalli, L. Davenport
and R. A. Sánchez-Delgado, J. Biol. Inorg. Chem., 2008, 13, 703–712.
57 W. F. Schmid, R. O. John, V. B. Arion, M. A. Jakupec and B. K. Keppler,
Organometallics, 2007, 26, 6643–6652.
58 M. A. Bennett and A. K. Smith, J. Chem. Soc., Dalton Trans., 1974, 2,
233–241.
59 K. Yearick, K. Ekoue-Kovi, D. P. Iwaniuk, J. K. Natarajan, J. Alumasa,
A. C. de Dios, P. D. Roepe and C. Wolf, J. Med. Chem., 2008, 51, 1995–
1998.
2 M. Navarro, H. A. Pérez and R. A. Sánchez-Delgado, J. Med. Chem.,
1
997, 40, 1937–1939.
3 M. Navarro, F. Vásquez, R. A. Sánchez-Delgado, H. A. Pérez, V. Sinou
and J. Schrével, J. Med. Chem., 2004, 47, 5204–5209.
4 R. Arancibia, F. Dubar, B. Pradines, I. Forfar, D. Dive, A. H. Klahn and
C. Biot, Bioorg. Med. Chem., 2010, 18, 8085–8091.
5 L. Glans, D. Taylor, C. de Kock, P. J. Smith, M. Haukka, J. R. Moss and
E. Nordlander, J. Inorg. Biochem., 2011, 105, 985–90.
6 S. H. van Rijt and P. J. Sadler, Drug Discov. Today, 2009, 14, 1089–
1
097.
3
3
3
4
7 C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391–401.
8 G. Süss-Fink, Dalton Trans., 2010, 39, 1673–1688.
9 G. Gasser, I. Ott and N. Metzler-Nolte, J. Med. Chem., 2011, 54, 3–25.
0 C. S. K. Rajapakse, A. Martínez, B. Naoulou, A. A. Jarzecki, L. Suárez,
C. Deregnaucourt, V. Sinou, J. Schrével, E. Musi, G. Ambrosini, G.
K. Schwartz and R. A. Sánchez-Delgado, Inorg. Chem., 2009, 48, 1122–
1131.
4
1 A. Martínez, C. S. K. Rajapakse, D. Jalloh, C. Dautriche and
R. A. Sánchez-Delgado, J. Biol. Inorg. Chem., 2009, 14, 863–871.
2 W. F. Schmid, R. O. John, G. Mühlgassner, P. Heffeter, M. A. Jakupec,
M. Galanski, W. Berger, V. B. Arion and B. K. Keppler, J. Med. Chem.,
60 APEX2 - Software Suite for Crystallographic Programs, Bruker AXS
Inc., Madison, Wisconsin, USA, 2009
61 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2008,
64, 112–122.
4
2
007, 50, 6343–6355.
62 G. M. Sheldrick, SADABS - Bruker AXS scaling and absorption correc-
tion, Bruker AXS Inc., Madison, Wisconsin, USA, 2008
63 L. G. Danielsson and Y. H. Zhang, Trends Anal. Chem., 1996, 15, 188–
196.
64 C. Rappel, M. Galanski, A. Yasemi, L. Habala and B. K. Keppler, Elec-
trophoresis, 2005, 26, 878–884.
65 OECD, Guidelines for testing of chemicals, OECD, Paris, 1995.
66 W. Trager and J. B. Jensen, Science, 1976, 193, 673–675.
67 M. T. Makler and D. J. Hinrichs, Am. J. Trop. Med. Hyg., 1993, 48, 205–
210.
4
4
4
4
4
3 D. Carmona, M. P. Lamata, F. Viguri, R. Rodríguez, F. J. Lahoz,
I. T. Dobrinovitch and L. A. Oro, Dalton Trans., 2008, 3328–3338.
4 S. J. Dickson, M. J. Paterson, C. E. Willans, K. M. Anderson and
J. W. Steed, Chem. Eur. J., 2008, 14, 7296–7305.
5 P. Govender, A. K. Renfrew, C. M. Clavel, P. J. Dyson, B. Therrien and
G. S. Smith, Dalton Trans., 2011, 40, 1158–1167.
6 H. Brunner, R. Oeschey and B. Nuber, Dalton Trans., 1996, 1499–
1
7 H. Brunner, T. Zwack, M. Zabel, W. Beck and A. Böhm, Organometal-
lics, 2003, 22, 1741–1750.
508.
68 T. Mosmann, J. Immunol.Methods, 1983, 65, 55–63.
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 2764–2773 | 2773