Synthesis, structures, and antimalarial activities of some silver(I), gold(I) and gold(III) complexes involving N-heterocyclic carbene ligands

Article abstract of DOI:10.1016/j.ejmech.2012.11.038

A series of mono-and dinuclear silver(I) and mononuclear gold(I) complexes containing bis(N-heterocyclic carbene) (NHC) or N-functionalized NHC ligands were synthesized and fully characterized by spectroscopic methods and, in some cases, by single crystal

Full text of DOI:10.1016/j.ejmech.2012.11.038

European Journal of Medicinal Chemistry 60 (2013) 64e75
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structures, and antimalarial activities of some silver(I), gold(I) and
gold(III) complexes involving N-heterocyclic carbene ligands
,
b
,
a
,
b
,
,
b
,
,b,c,
*
Catherine Hemmert a , Aymeric Fabié c, Aude Fabre a c, Françoise Benoit-Vical a
,
*
,
b
Heinz Gornitzka a
a CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, F-31077 Toulouse cedex 4, France
b Université de Toulouse, UPS, INP, LCC, F-31077 Toulouse, France
c Service de Parasitologie-Mycologie and Faculté de Médecine de Rangueil, Centre Hospitalier Universitaire de Toulouse, Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of mono-and dinuclear silver(I) and mononuclear gold(I) complexes containing bis(N-hetero-
cyclic carbene) (NHC) or N-functionalized NHC ligands were synthesized and fully characterized by
spectroscopic methods and, in some cases, by single crystal X-ray diffraction. The in vitro antiplasmodial
and antifungal activities of a previously described family of N-functionalized bis(imidazolium) proligands
and their corresponding silver(I), gold(I) and gold(III) complexes but also the new here described
compounds were investigated in a chloroquine-resistant strain of Plasmodium falciparum, and against
two Candida strains, respectively. For the first family, interesting antiplasmodial and antifungal activities
were found for the dinuclear silver(I) species but they also showed strong hemolytic properties.
Pharmaco-modulations leading to the second series of complexes allowed notably increase in the
antiplasmodial activity, in particular of the mononuclear gold(I) complexes with IC50 values up to
330 nM, without any hemolysis.
Received 27 September 2012
Received in revised form
22 November 2012
Accepted 26 November 2012
Available online 7 December 2012
Keywords:
N-heterocyclic carbene
Silver
Gold
Organometallic complexes
Antiplasmodial drugs
Ó 2012 Published by Elsevier Masson SAS.
1. Introduction
Many new AuI-or AuIIIeNHC(s)-catalyzed reactions were recently
devised [6]. AuI-and AuIIIeNHC compounds are generally readily
Coinage metal-NHCs (NHC ¼ N-heterocyclic carbene) are widely
studied for their intriguing structural properties and numerous
applications; in particular, a newly emerging interest in medicinal
applications of stable NHC(s) complexes has recently expanded.
Due to their easy preparation in mild conditions via the Ag2O route
and their ability as transmetalating agents, AgIeNHCs have become
the most synthesized and studied among coinage metal-NHCs [1].
Furthermore, Youngs and co-workers first demonstrated the
exceptional antimicrobial efficacy of AgIeNHCs against a broad
spectrum of both gram-positive and gram-negative bacteria as well
as fungi [2e4]. The same research group recently investigated the
anticancer activity of AgIeNHCs stable to light and water [5]. The
interest in AuI-and AuIIIeNHC complexes has surged in the past
decade because of their easy preparation via the Ag carbene
transfer route and the variety in properties and applications [1].
prepared, stable to air and moisture, and some of them, especially
dinuclear gold(I) and polynuclear gold(I)-heterometal species
display long-lived intense photoluminescence at room temperature
[1]. Biomedical applications of gold complexes based on NHCs are
beginning to unfold. In particular, given that these ligands are
extremely good
sedonors, they form strong Au-carbene bonds,
giving stable AuIeNHC complexes that are insensitive to biologi-
cally important thiol groups. AuIeNHCs have shown potential
medical applications [7], especially as anticancer [8e12], antiar-
thritis [13], and antimicrobial agents [14]. It has to be mentioned
that gold-based compounds, including some gold(I)-NHCs, show
anti-mitochondrial activity, a promising mode of action to fight
cancer. Their antitumor activity may stem from the lipophilic and
cationic properties, allowing their accumulation in mitochondria of
tumor cells with great specificity [7].
In biomedical research, one of the most important fields
concerns infectious disease. Candidemia are opportunistic fungal
infections causing alarming problems with serious infections in
immunologically compromised people [15]. Candida albicans and
Candida glabrata are the most frequent Candida isolates [15]b.
Malaria remains the most important parasitic disease affecting
humans. Recent reports estimate more than one million deaths in
* Corresponding authors. CNRS, LCC (Laboratoire de Chimie de Coordination), 205
route de Narbonne, BP 44099, F-31077 Toulouse cedex 4, France. Tel.: þ33
561553187; fax: þ33 561553003.
E-mail addresses: hemmert@lcc.toulouse.fr (C. Hemmert), Francoise.Vical@
inserm.fr (F. Benoit-Vical).
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.11.038

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
65
2010 [16]. The major antimalarial drug namely chloroquine (CQ) is
today almost completely ineffective, due to parasite resistance. In
the approach to find new compounds against infective diseases,
Navarro reviewed many gold complexes showing potential anti-
parasitic properties [17]. Gold is among the most ancient of all
metals used in medicine and its current use has allowed informa-
tion regarding toxicological and clinical administration to become
available along with valuable studies concerning its metabolism
and molecular targets. Due to the urgent need to develop new
drugs, gold compounds appear as good candidates in the design of
novel metal-based anti-malaria drugs. For example, the CQ
complex of triphenylphosphine gold(I) was found to be more active
in vitro than CQ against two CQ-resistant strains of Plasmodium
falciparum (IC50 (50% inhibitory doses) ¼ 5.1e23 nM) and also
in vivo against Plasmodium berghei [17].
In recent years, we focused our research on a series of hetero-
ditopic polydentate bis(NHC) ligands designed for the complexa-
tion of transition metals [18e21]. We developed short and
modulable synthetic routes for the preparation of functionalized
bis(NHC) precursor ligands and their AgI-, AuI-and AuIIIebis(NHC)
complexes (see Scheme 1 [22] for some examples). Such ligands
were found to allow the stabilization of bimetallic silver and gold
complexes, exhibiting sometimes particularly short metalemetal
contacts, thus favoring the occurrence of metallophilic interac-
tions [17,18]. In the case of silver and gold complexes, we studied
the influence of the non-coordinating functionalized side arms on
the luminescent properties [19,21].
their antiplasmodial activity and to remove their hemolytic
characteristics.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of N-functionalized mono and bis(imidazolium)
salts
The bis(imidazolium) salts 16 and 17 and the imidazolium salt 20
were obtained from commercially available reagents, after a qua-
ternization step of 1-methylimidazole by reaction with half an
equivalent of dibromoalkane (dibromomethane for 16 and 1,2-
dibromoethane for 17), or with one equivalent of 2-chloro-N-phe-
nylacetamide for 20 (Scheme 2) [23]. The proligand 23 was prepared
by alkylation of (1R)-2-(1H-imidazol-1-yl)-1-phenylethanol [18]
with methyliodide, followed by an ion-exchange with ammonium
hexafluorophosphate (Scheme 2). The generation of the quinoline
derivative 25 requires more drastic reaction conditions to activate
the 2-chloroquinoline (excess of 1-methylimidazole, toluene,
130 ꢀC), followed by an anionic metathesis (Scheme 3). 5-(Bromo-
methyl)-2,20-bipyridine was synthetized in three steps according
literature procedures [24,25]. 2-Acetylpyridine and iodine were
heated in pyridine to give the corresponding pyridinium salt, which
was then reacted with metacrolein in formamide in the presence of
NH4OAc to afford 5-methyl-2,20-bipyridine. The last step involves
the conversion of the methyl group to the corresponding bro-
moethyl by treatment with N-bromosuccinimide and azoisobutyr-
onitrile. The carbene precursor 28 was prepared under the same
conditions as 25, starting from 1-methylimidazole and 5-(bromo-
methyl)-2,20-bipyridine (Scheme 4).
In this work, we aim to test a first series of air and moisture
stable dinuclear silver and gold N-functionalized NHC complexes
(Scheme 1) against P. falciparum, the parasite responsible of
malaria. In addition, we report the preparation and characterization
of a second series of mono-and dinuclear silver(I) and mononuclear
gold(I) complexes, in order to examine how modifications of
the different building blocks of the ligands can influence the bio-
logical activity of the corresponding complexes. The pharmaco-
modulation of these complexes permitted a notably increase in
The most notable features in the 1H and 13C spectra of the
mono-and bis-imidazolium salts are the resonances for imidazo-
lium protons (H2) located between 9.07 and 10.48 ppm and the
corresponding imidazolium carbons (C2) in the range of 134.9e
139.0 ppm. The FAB-MS spectra of all compounds exhibit
Scheme 1. First series of dinuclear complexes.

66
C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
Scheme 2. Synthesis of proligands 16, 17, 20 and 23, silver(I) complexes 18, 19, 21 and 24 and gold(I) complex 22.
the classical peak corresponding to [M ꢁ X]þ cations (X ¼ Cl, Br
or PF6).
2.1.2. Synthesis and characterization of mono-and dinuclear
silver(I) and gold(I) bis(carbene) complexes
In the bis(imidazolium) salts 16 and 17, the two azolium
rings are held together by an aliphatic bridge and contain
methyl groups. The other imidazolium precursors are func-
tionalized by various groups, namely amide (20), alcohol (23)
and nitrogen containing heterocyles (quinoline for 25 and
bipyridine for 28).
All silver complexes were prepared by deprotonation of mono-
or bis(imidazolium) salts 16, 17, 20, 23, 25 and 28, with the mild
Scheme 3. Synthesis of proligand 25, silver(I) complex 26 and gold(I) complex 27.
Scheme 4. Synthesis of proligand 28, silver(I) complex 29 and gold(I) complex 30.

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
67
base Ag2O in a suitable dry solvent (dichloromethane or methanol)
at room temperature, except for the dinuclear complexes 18 and 19
(50 ꢀC) (Schemes 2e4). The complexation of the bis(imidazolium)
precursors 16 and 17 to silver(I) in equimolar amounts led probably
to coordination polymers of the general formula [AgI2{bis(NHC)}2]
[AgBr2]2 (formula proposed from the elemental analysis) with
interactions between the cationic silver ions and the AgBr2 anions,
which are totally insoluble; an ion exchange with silver(I) nitrate
was thus necessary to obtain 18 and 19 as soluble compounds in
protic solvents.
For complexes 21, 24, 26 and 29, a stoichiometry of one half
equivalent of Ag2O for one equivalent of ligand precursor was used.
Silver(I) complexes 24, 26 and 29 were obtained as the desired
mononuclear cationic species [AgI(NHC)2][X] (X ¼ Cl, Br or PF6).
However, under our reaction conditions (ratio silver salt/ligand
precursor), we obtained the neutral dinuclear dimeric complex 21,
involving amido-NHC ligands. Ghosh and coll. previously reported
a series of structurally diverse gold and silver complexes ranging
from mononuclear ionic (NHC-amide)2MþClꢁ type complexes to
large 12-membered macrometallacyclic (NHC-amido)2M2 species,
prepared by varying the N-substituent of the amido-functionalized
side arm from a t-butyl to a 2,6-dii-propylphenyl moiety; they
obtained neutral dinuclear complexes when starting from stoi-
chiometric quantities of Ag2O and imidazolium salts whereas
cationic mononuclear complexes were isolated with a ratio of one
half Ag2O for one proligand. In their case, the authors explain the
observed structural diversity as a direct consequence of the varia-
tion of the N-substituent of the amido-functionalized side arm,
despite a common synthetic route [26]. Thus, we can state that it is
difficult to predict the coordination patterns of Ag(I) compounds
involving NHC(s) ligands. The solid state structure depends on the
substituents of the imidazolium ring, the anion, the solvent and the
temperature used and is not directly correlated to the stoichiom-
etry of the starting materials.
Fig. 1. Molecular structure of 22 (50% probability level for the thermal ellipsoids). Only
the cationic part is shown for clarity. Selected bond lengths [A] and angles [ ]: Au C1
2.016(2), Au C13 2.018(2), C1 Au C13 178.40(5).
ꢀ
ꢀ
reaction conditions with the presence of the amide protons at
11.58 ppm in the 1H NMR spectrum. The elemental analysis of the
gold(I) complexes correspond to the general [AuL2][X] formula
(X ¼ Cl for 22, PF6 for 27 and Br for 30) and the FAB-MS spectra
exhibit the classical peak corresponding to the cationic fragment
[M ꢁ X]þ.
2.1.3. Crystal structures of 22, 26, 27 and 30
Crystals of the gold complex 22 involving the amide function-
alized carbene ligands were obtained by slow evaporation of
dichloromethane. The asymmetric unit contains one gold cation
coordinated by two of the ligands, one chlorine anion and one
molecule of water. The cationic part (see Fig. 1) shows a classical
linear coordination of the gold atom. It is remarkable, that the
amide functions of the ligands are involved in hydrogen bonds
formed either with the anions or with water molecules (see Fig. 2).
The ligands form hydrogen bonds exclusively by the NH function
and not at all by the C]O. In this network each chlorine atom is
linked to one NH and to two water molecules, whereas each water
molecule is linked to two chlorine atoms and one NH from the
ligands.
Crystals of the silver complex 26 were obtained by slow evap-
oration from an acetonitrile solution. As shown in Fig. 3 the silver
cation is coordinated in a linear manner by two carbene units.
In the case of 26 neither classical hydrogen-bonds nor short
metalemetal interactions are present in the crystalline solid state.
Short contacts between CeH groups and fluorine atoms with C.F
The formation of the silver(I) complexes was confirmed by the
absence of the 1H NMR resonance of the acidic imidazolium H2, and
for complex 21, the lack of the amide protons at 11.14 ppm. The 13C
NMR spectra exhibit a resonance signal located between 180.3 and
182.7 ppm, ascribed to the carbenic carbon atoms C2, which is
consistent with the reported values for other cationic mononuclear
and dinuclear silver(I) NHC complexes [1]. Except for compound 24,
in which the carbene resonance appears as one doublet with
a coupling constant of J107,109
¼ 180.1 Hz, all the other
AgeC
complexes show only a singlet for the C2 carbons. There is no
evidence of coordination of the metal by a nitrogen atom of the
quinoline or bipyridine moieties in complexes 26 and 29. The FAB-
MS spectra showed mass peaks corresponding to the cations of
dinuclear species [AgI2L2NO3]þ for 18 and 19, mononuclear species
[AgIL2]þ for 24, 26 and 29 and also a mononuclear fragment
[AgI(NHC-amide)2]þ for 21, the parent peak [AgI2 (NHC-
amido)2 þ Hþ]þ being not detected by FAB spectrometry. The
elemental analyses are consistent with the proposed structures
depicted in Schemes 2e4.
The three cationic mononuclear gold(I) complexes 22, 27 and 30
were prepared according a classical way, from the N-functionalized
imidazolium salts 20, 25 or 28 and one half equivalent of Au(SMe2)
Cl with sodium acetate as a mild base in hot N,N-dimethylforma-
mide (120 ꢀC). NMR spectroscopy unequivocally demonstrates the
formation of the gold(I) complexes; the 13C spectra show the
resonance for the carbene carbon atoms at 188.1, 182.9 and
183.2 ppm, for 22, 27 and 30, respectively. These values are in the
range of reported values for AuIeNHC complexes having CeAueX
(X ¼ halide) or CeAueC motifs [1]. It has to be pointed out that
in contrast to the silver(I) complex 21, the corresponding gold(I)
analog 22 is a cationic mononuclear species as expected from the
Fig. 2. Hydrogen-bond-network in the solid state of 22.

68
C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
Fig. 5. Solid state structure of 27 (50% probability level for the thermal ellipsoids). Only
the cationic part is shown for clarity. Selected bond lengths [A] and angles [ ]: Au C1
2.035(6), C1 Au C1A 179.999(1).
Fig. 3. Molecular structure of 26 (50% probability level for the thermal ellipsoids). Only
the cationic part is shown for clarity. Selected bond lengths [A] and angles [ ]: Ag C1
2.093(2), C1 Au C1A 180.0.
ꢀ
ꢀ
ꢀ
ꢀ
groups and bromine anions with C.Br distances between 2.811
and 3.030 A, reflecting the higher charge density of bromine in
comparison to PF6, can be observed. As shown in Fig. 8 also this
system is arranged in layers of cations and anions, respectively.
ꢀ
distances between 3.015 and 3.058 A can be observed. As shown in
ꢀ
Fig. 4 the system is arranged in layers of cations and anions,
respectively.
Crystals of the corresponding gold complex 27 were also ob-
tained by slow evaporation from an acetonitrile solution. As in the
case of the silver complex 26 the gold cation is coordinated in
a linear manner by two NHC ligands (see Fig. 5) and neither clas-
sical hydrogen-bonds nor short metalemetal interactions are
present in the crystalline solid state of 27. Very short contacts
between CeH groups and fluorine atoms with C.F distances
2.2. Antimalarial and antifungal activities
The results of the preliminary antiplasmodial tests of the first
series of molecules (Scheme 1) are presented in the Table 1. Fifteen
molecules, involving bis(imidazolium) salts and their corre-
sponding dinuclear silver(I), gold(I) and gold(III) complexes were
tested against the CQ-resistant Plasmodium falciparum strain
FcM29-Cameroon (IC50 of CQ ¼ 445 nM) for doses ranging from
ꢀ
between 2.992 and 3.012 A can be observed. As shown in Fig. 6 the
system is arranged in layers of cations and anions, respectively.
In the case of compound 30 slow evaporation from a chloroform
solution gave suitable crystals for an X-ray analysis. As in the other
compounds, the metal cation is linearly coordinated by two ligands.
Apart from the cationic unit shown in Fig. 7, only the bromine anion
is present in the asymmetric unit.
0.05
showed
values from 1.2 to 2.6
these molecules appeared stable since they kept their anti-
m
g/mL to 50
very promising antiplasmodial activity with IC50
M when tested immediately. Interestingly,
m
g/mL. All the [AgIebis(NHC)]2 complexes 5e8
a
m
In complexes 22, 26 and 27 a trans-arrangement of the func-
tionalized side arms related to the coordination-axis can be
observed. Only in 30 the functionalized side arms are on the same
side, an arrangement which is not supported by interactions
between the two bipyridyl groups. The perturbation of the CeAueC
angle, 174.9ꢀ instead of 180ꢀ, may be due to steric repulsion
resulting from this arrangement.
As in the case of complexes 26 and 27 neither classical
hydrogen-bonds nor short metalemetal interactions are present in
the crystalline solid state of 30. Very short contacts between CeH
plasmodial activity with similar IC50 values (1.5e2
mM) after
storage several days at 4 ꢀC. Three compounds, namely the [AuIe
bis(NHC)]2 complex 11 and two of the three [AuIIIebis(NHC)]2
complexes 14e15 showed moderate activity with IC50 values
between 9 and 15
mM. We can note the weak stability of
compound 11 which totally lost its activity after conservation of
the stock solution in DMSO at 4 ꢀC for 6 days. Finally, eight species
including the bis(imidazolium) salts (1e4) and most of the [AuIe
bis(NHC)]2 complexes (9, 10 and 12) and one gold(III) complex
(13) showed no activity with IC50 values ranging from 30 to higher
than 110 mM.
Fig. 4. Crystal packing of 26 with a view in direction of b.
Fig. 6. Crystal packing of 27 with a view in direction of b.

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
69
indicative of the role of the metallic centre in the biological
activity of the resulting complexes. For the dinuclear gold
complexes, the oxidation state of the gold cation seems to have an
effect because the higher-valent gold(III) complexes 14e15 exhibit
a moderate but better activity than their gold(I) counterparts,
which are ineffective. These results are probably due to the high
stability of the dinuclear gold(I) complexes, which could be
attributed both to the protective cage effect of the ligands and to
aurophilic interactions. Finally, the highest activity for this first
series was found for the dinuclear silver(I) complexes 5e8 but
they are also toxic. The examples of silver compounds used against
malaria are scarce. In 2003, the in vitro antimalarial activity
against a CQ-sensitive FCR-3 strain was evaluated for a series of
metalloporphyrins, among which a silver(I) protoporphyrin IX
with a moderate IC50 value of 15.5
m
M (IC50 ¼ 0.022
mM for CQ)
was described [27]. In an old patent, it was reported that silver
sulfadiazine in doses of 1.050 mg/kg/day, when administrated
orally or subcutaneously, once a day for five days cured CF-1 mice
of Plasmodium berghei with a minimal toxicity but at higher doses
death occurred within 24 h [28]. Even if the issue of silver toxicity
is still under debate, silver is known to be one of the least toxic
metals [2].
Fig. 7. Solid state structure of 30 (50% probability level for the thermal ellipsoids). Only
the cationic part is shown for clarity. Selected bond lengths [A] and angles [ ]: Au C1
2.020(4), Au C16 2.019(4), C1 Au C16 174.91(17).
ꢀ
ꢀ
According to these first results, pharmaco-modulations of our
compounds were carried out in order to increase the anti-
plasmodial activity and to decrease their cytotoxic effect by
decreasing hemolysis. First, we have prepared dinuclear [AgIe
bis(NHC)]2 complexes bearing simple methyl groups on the nitro-
gens of the rings (18, 19), in order to see if the functionalized arms
in the first series could have an influence on the antiplasmodial
activity. Furthermore, we were interested in the synthesis of
various mononuclear AgI(NHC)2 and AuI(NHC)2 which have a less
protected environment around the metal and in particular a better
delivery of the metallic cation. The results of the antiplasmodial
tests of the second series of molecules are presented in the Table 2.
Nine new silver(I) and gold(I) complexes were tested against the
same CQ-resistant strain FcM29-Cameroon for doses ranging from
The antifungal assay showed that the same 5e8 compounds
with the best antiplasmodial activity presented also the best
antifungal activities against both Candida strains with MIC ranging
from 0.8
tested here had no activity against neither Candida albicans nor
Candida glabrata (MIC > 50 g/ml corresponding to values ranging
from >27 to >107 M, according to molar weight of compounds).
mM to 2 mM whereas all the eleven other compounds
m
m
The MIC values of compounds 5e8 appeared as very interesting
since in the same range values that the antifungal control drug 5-
Fluorocytosine (MIC: 0.7e1.6 mM). Unfortunately these four best
antiplasmodial and antifungal silver(I) compounds 5e8 also
showed strong hemolytic properties on the parasite culture and
even at the weakest doses tested (0.5
mg/ml). The absence of
specific activity of the compounds 5e8 against pathogens is
representative of a toxic activity of these compounds. This is
confirmed by the high hemolysis reported on parasite cultures
with these same four compounds. These preliminary results do
not allow the extraction of structureeactivity relations based on
the key groups of the N-functionalized bis(NHC) used in this first
series of compounds. Nevertheless, some interesting experimental
facts deserve further attention and a general trend appears from
the IC50 values. First, the control tests with the proligands are
0.01
series of complexes was highly improved with IC50 values ranging
from 0.33 to 4.1
M except for the complex AuIebis(NHC) (22)
showing no activity even at 15 M. The molecules with the best
antiplasmodial activity are the two dinuclear [AgIebis(NHC)]2
complexes 18 and 19 with IC50 values of 1.2 and 1.5 M and the
two mononuclear AuIebis(NHC) 27 and 30 with IC50 values of 1.1
and 0.33 M, respectively.
mg/mL to 10 mg/mL. The antimalarial activity of this second
m
m
m
m
Except the silver(I) complex 24, which decomposes in the solid
state after six months and more rapidly in solution, all the other AgI
and AuI complexes had the same activity whatever the test in
extemporaneous conditions or after conservation of the stock
solution in DMSO at 4 ꢀC for 4e6 days. Very interestingly, this new
series of complexes had lost their hemolytic properties. No
hemolysis was observed for any compound of this new series and
even at the highest doses tested (10 mg/ml). Concerning the dinu-
clear silver(I) compounds, it seems that the aliphatic side arms
have no real influence on the activity because the IC50 values are
very close, the main difference being the hemolysis for the first
series, when compared to the second one, which cannot be
explained at this stage of the work. The neutral complex 21, in
which the silver cations are coordinated by one NHC and one
amido group and the mononuclear silver(I) complexes 24 and 26
are slightly less effective than 18 and 19. This could be attributed to
the fact that first, two NHCs stabilize the silver better than a het-
eroditopic NHC-amido ligand and second, that bis(NHC) ligands
can stabilize dinuclear species with metallophilic interactions,
thereby controlling a slower release of the Agþ in the culture
medium. Taking into account that silver is more labile than gold
Fig. 8. Crystal packing of 30 with a view in direction of a.

70
C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
Table 1
Antiplasmodial and antifungal activities screening of the first series of compounds 1e15.
Antiplasmodial activitya
Antifungal activityb
Hemolysisc
Compounds
Extempore
g/ml
Stored
g/ml
C. albicans
C. glabrata
m
m
M
m
m
M
m
M
mM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>50
>50
>50
25
1.1
1.8
1.7
1.8
>50
>50
22
>50
48
17
23
ND
>92
>107
>89
43
1.2
1.8
>50
>50
>50
30
2
2
>110
>107
>89
52
2.1
2
>92
>107
>89
>86
1.6
1.3
2
1.4
>39
>39
>34
>33
>32
>28
>27
1.6
>92
>107
>89
>86
1
0.8
1.5
1.5
>39
>39
>34
>33
>32
>28
>27
0.7
ꢁ
ꢁ
ꢁ
ꢁ
þ
þ
þ
þ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ND
e
e
1.4
1.5
2
1.7
1.5
1.8
>50
>50
>50
>50
>50
25
>39
>39
15
>33
30
>39
>39
>34
>33
>32
14
9
13
33
18
5-Fluorocytosined
Artemisinind
CQd
3.10ꢁ3 ꢂ 0.01 (10.10ꢁ3
m
M)
M)
ND
ND
230.10ꢁ3 ꢂ 0.4 (445.10ꢁ3
m
a
IC50 values against the P. falciparum strain FcM29 are reported in
m
g/ml and in mM. Data represented the IC50 values obtained in one preliminary experiment and for each
one both kinds of conservation (extemporaneously or after conservation of the stock solution in DMSO at 4 ꢀC for 4e6 days).
b
Antifungal activity on both C. albicans and C. glabrata are expressed in mM.
c
þHemolysis on P. falciparum parasite culture. ꢁNo hemolysis on P. falciparum parasite culture.
d
5-Fluorocytosine and, artemisinin and CQ were routinely tested as antifungal and antiplasmodial control drugs, respectively.
(silvereNHC complexes are often used for transmetallation reac-
tions), a good protection of the silver cation is needed to ensure the
slow release of silver ions, in order to avoid AgCl precipitation in
the culture medium. This is in strong contrast with the gold(I)
compounds, for which a drastic increase of the parasite growth
inhibition is obtained for the mononuclear complexes 27 and 30,
both containing nitrogen heterocyclic functionalized arms, namely
quinoline and bipyridine moieties. It has to be noted that CQ
contains a quinoline entity. The improved potency of 27 and 30
against the CQ-resistant strain FcM29-Cameroon confirmed our
hypothesis of a better drug-delivery in less protected gold con-
taining species. It should be mentioned that the potential of gold
derivatives as drugs against parasitic diseases has been very little
explored until very recently. Messori and coll. have demonstrated
that the anti-arthritic drug auranofin displays very pronounced
antiplasmodial effects, which are probably mediated by severe
oxidative stress originating from Plasmodium falciparum thio-
redoxin reductase (TrxR) inhibition [29]. Interestingly, several AuIe
NHC complexes tested as anticancer agents inhibit the activity of
TrxR, leading to cell death through a mitochondrial apoptotic
pathway [10]. Gold(I) complexes involving functionalized alkynes
have shown low in vitro activity against the malaria parasite strains
3D7 (CQ-sensitive, IC50 of CQ ¼ 0.01
m
M) and K1 (CQ-resistant, IC50
of CQ ¼ 0.3 M) with IC50 values between 7.2 and 23
m
mM [30]. The
same authors described a series of mono-and dinuclear gold(I)
phosphine complexes containing mono-anionic seleno-and thio-
semicarbazones ligands; the IC50 results showed that the sulfur
containing compounds exhibit activity similar to CQ against the
3D7 strain (IC50 ¼ 7.06 and 10.7 nM, IC50 of CQ ¼ 8.84 nM) [31].
Similarly, Chibale and coll. reported gold(I) and gold(III) thio-
semicarbazone complexes, which exhibit moderate in vitro activity
Table 2
Antiplasmodial activity of the second series of AgI and AuI complexes 18, 19, 21, 22,
24, 26, 27, 29 and 30 against the P. falciparum strain FcM29-Cameroon.
against two strains D10 (CQ-sensitive, IC50 of CQ ¼ 0.0173
M) with IC50 ranging from
M for the gold(I) compounds and IC50 ranging from
M for the gold(III) complexes [32]. Very recently,
mM) and
W2 (CQ-resistant, IC50 of CQ ¼ 0.095
m
Hemolysisc
1.36 to 6.92
m
Antiplasmodial activity
Extemporea
3.04 to >20
m
Compounds
Storeda
g/mlb
Cronje and coll. reported preliminary in vitro antimalarial activities
of N-heterocyclic ylideneamine gold(I) against P. falciparum; the
coordination of ancillary phosphine or NHC ligands to these
m
g/mlb
m
Mb
m
m
Mb
18
19
21
22
24
26
27
29
0.87 ꢂ 0.6
1.08 ꢂ 0.3
2.5 ꢂ 0.5
>10
1.2
1.5
4.0
>15
3.9
4.1
1.1
2.8
0.33
1.05 ꢂ 0.2
1.9 ꢂ 0.8
3.8 ꢂ 0.5
>10
1.4
2.6
6.1
>15
e
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
complexes results in better activities with an IC50 value of 5.1
for the NHC derivative [33].
mg/ml
2.7 ꢂ 0.6
2.8 ꢂ 0.9
0.9 ꢂ 0.1
1.45 ꢂ 0.8
0.25 ꢂ 0.03
e
3.1 ꢂ 0.8
1.4 ꢂ 0.6
2.2 ꢂ 0.9
0.37 ꢂ 0.2
4.6
1.8
3.3
0.49
3. Conclusion
In summary, a series of mono-and dinuclear silver(I) and
mononuclear gold(I) complexes based on NHC ligands were
prepared and characterized. The in vitro anti-malaria activity of two
different families of compounds has been assessed against the
chloroquine-resistant P. falciparum strain FcM29-Cameroon. For the
first tested family, including N-functionalized bis(imidazolium)
proligands and their corresponding silver(I), gold(I) and gold(III)
complexes, the dinuclear silver(I) compounds have shown the best
antiplasmodial and antifungal activities but also strong hemolytic
30
Artemisinind
CQd
4.10ꢁ3 ꢂ 0.2 (14.10ꢁ3
mM)
260.10ꢁ3 ꢂ 0.5 (503.10ꢁ3
mM)
a
For each compound, data were obtained both extemporaneously or after
conservation of the stock solution in DMSO at 4 ꢀC for 4e6 days.
b
IC50 values are reported in mg/ml and in mM. Data represented the mean of 4e8
independent experiments.
c
þHemolysis on P. falciparum parasite culture. ꢁNo hemolysis on P. falciparum
parasite culture.
d
Both antiplasmodial control drugs artemisinin and CQ were routinely tested.

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
71
properties. From these preliminary results, pharmaco-modulations
have been realized to obtain the second series of silver(I) and
gold(I) complexes. Their antiplasmodial activity was significantly
increased and no hemolysis was observed. It seems that the pres-
ence of nitrogen containing heterocycles in the series of mono-
122.1 (2C, C5), 58.4 (1C, C6), 36.7 (2C, C7). MS (FABþ): m/z 257
[M ꢁ Brꢁ]þ.
4.1.1.2. 3-Methyl-1-[(3-methyl-1H-imidazol-3-ium-1-yl)ethyl]-1H-
imidazol-3-ium dibromide (17). Dibromoethane (1.08 mL,
12.6 mmol) was added to an acetonitrile solution (10 mL) of 1-
methylimidazole (2 mL, 25.1 mmol) and the mixture was stirred
at 80 ꢀC for 4 h. After cooling to room temperature, the desired
white solid was filtered off and dried under vacuum (1.864 g, 42%
yield). Anal. Calcd. For C10H16N4Br2: C, 34.11; H, 4.58; N, 15.91.
Found: C, 33.99; H, 4.37; N, 15.82. 1H NMR (250 MHz, DMSO-d6)
nuclear gold(I) complexes has
a great importance on the
antiplasmodial activity, probably related to the lipophilicity, the
basicity, and structural features of these compounds. The activities
of metal NHCs as antimalarials have not been extensively explored
and this work is also among the first to investigate silver complexes
as potential antimalarials. Further studies for the design of metallo-
drugs combining gold(I)-NHC covalently bound to well-known
antimalarial drugs or analogs are ongoing in our laboratories.
d
(ppm) ¼ 9.21 (s, 2H, H2), 7.76 (s, 2H, H5), 7.71 (bs, 2H, H4), 4.75 (s,
4H, H6), 3.87 (s, 6H, H7). 13C NMR (63 MHz, D2O)
d
(ppm) ¼ 136.8
(2C, C2), 124.8 (2C, C4), 122.4 (2C, C5), 49.0 (2C, C6), 36.5 (2C, C7). MS
(FABþ): m/z 271 [M ꢁ Brꢁ]þ.
4. Experimental section
4.1.1.3. 3-Methyl-1-[(phenylcarbamoyl)methyl]-1H-imidazol-3-ium
chloride (20). 1-Methylimidazole (0.735 mL, 9.22 mmol) and 2-
chloro-N-phenylacetamide (0.782 g, 4.61 mmol) were stirred in
acetonitrile (20 mL) at 80 ꢀC for 12 h. After cooling to room
temperature, the desired white solid was filtered off and dried
under vacuum (0.889 g, 77% yield). Anal. Calcd. For C12H14N3OCl: C,
57.26; H, 5.61; N, 16.69. Found: C, 57.28; H, 5.71; N, 16.71. 1H NMR
4.1. General information
Unless otherwise stated, all reactions were performed in air.
CH2Cl2, CH3CN and DMF were dried over 4 A molecular sieves. All
ꢀ
other reagents were used as received from commercial suppliers.
All reactions involving silver compounds were performed with the
exclusion of light. 2-Chloro-N-phenylacetamide [23], 2-(1H-imi-
dazol-1-yl)-1-phenylethanol [18], 5-(bromomethyl)-2,20-bipyr-
idine [24,25] were prepared according literature procedures. 1H
(250, 300 or 400 MHz) and 13C spectra (63 or 75 MHz) were
recorded at 298 K on Bruker ARX250, Bruker DPX300 or Bruker
AV400 spectrometers in D2O, CDCl3, CD3CN, CD3OD or DMSO-d6 as
solvents. Elemental analyses were carried out by the “Service de
Microanalyse du Laboratoire de Chimie de Coordination” (Tou-
louse). Mass spectrometry analysis were performed on a Nermag
R1010 (FABþ/meta-nitrobenzylalcohol (MNBA)) spectrometer, by the
“Service de Spectrométrie de Masse de Chimie UPS-CNRS”
(Toulouse).
(250 MHz, DMSO-d6)
d
(ppm) ¼ 11.14 (bs, 1H, NH), 9.22 (s, 1H, H2),
7.88 (d, 2H, H4, H5), 7.66 (d, 2H, HAr, 3J ¼ 7.7 Hz), 7.34 (t, 2H, HAr,
3J ¼ 7.6 Hz), 7.09 (t, 1H, HAr, 3J ¼ 7.1 Hz), 5.31 (s, 2H, H6), 3.93 (s, 3H,
H8). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) ¼ 164.2 (1C, C7), 139.0
(1C, C2), 138.4 (1C, CAr), 129.3.2 (2C, CAr), 124.3 (1C, C5), 124.2 (1C,
CAr), 123.5 (1C, C4), 119.6 (2C, CAr), 51.7 (1C, C6), 36.3 (1C, C8). MS
(FABþ): m/z 216 [M ꢁ Clꢁ]þ.
4.1.1.4. 1-[(2R)-Hydroxy-2-phenylethyl]-3-methyl-1H-imidazol-3-
ium hexafluorophosphate (23). An excess of iodomethane
(0.288 mL, 4.64 mmol) was added to an acetonitrile solution
(40 mL) of (1R)-2-(1H-imidazol-1-yl)-1-phenylethanol (0.437 g,
2.32 mmol) and the mixture was stirred at 75 ꢀC overnight. After
cooling to room temperature, the desired white solid was filtered
off and dried under vacuum (0.529 g, 69% yield). The solid was
dissolved in a mixture of H2OeMeOH (20 mLe10 mL) and the
4.1.1. Preparation of imidazolium salts
The following picture describes the numbering of H (1H NMR)
and C (13C NMR). These notations are used in the following
experimental section.
4.1.1.1. 3-Methyl-1-[(3-methyl-1H-imidazol-3-ium-1-yl)methyl]-1H-
imidazol-3-ium dibromide (16). An excess of dibromomethane
(3.5 mL, 50.2 mmol) was added to an acetonitrile solution
(10 mL) of 1-methylimidazole (2 mL, 25.1 mmol) and the
mixture was stirred at 80 ꢀC for 4 h. After cooling to room
temperature, the desired white solid was filtered off and dried
under vacuum (1.443 g, 34% yield). Anal. Calcd. For C9H14N4Br2:
C, 31.98; H, 4.17; N, 16.57. Found: C, 31.96; H, 3.92; N, 16.56. 1H
subsequent addition of an aqueous solution (10 mL) of NH4PF6
(0.261 g, 1.60 mmol) afforded a white precipitate, which was
collected by filtration and dried under vacuum (0.522 g, 94% yield).
Anal. Calcd. For C12H15N2OPF6: C, 41.39; H, 4.34; N, 8.04. Found: C,
41.18; H, 4.19; N, 8.11. 1H NMR (250 MHz, DMSO-d6)
d
(ppm) ¼ 9.07
(s, 1H, H2), 7.67 (bs, 2H, H4, H5), 7.39 (m, 5H, HAr), 6.00 (bs, 1H, OH),
4.94 (m 1H, H7), 4.41 (d, 1H, H6, 2J ¼ 14.7 Hz), 4.23 (dd, 1H, H6,
2J ¼ 13.2 Hz, 3J ¼ 9.1 Hz), 3.87 (s, 3H, H8). 13C NMR (75 MHz, CDCl3)
NMR (250 MHz, DMSO-d6)
H5), 7.83 (bs, 2H, H4), 6.76 (s, 2H, H6), 3.92 (s, 6H, H7). 13C NMR
(63 MHz, DMSO-d6)
(ppm) ¼ 138.0 (2C, C2), 124.8 (2C, C4),
d
(ppm) ¼ 9.52 (s, 2H, H2), 8.07 (s, 2H,
d
(ppm) ¼ 141.7 (1C, CAr), 137.4 (1C, C2), 128.8 (3C, CAr), 128.3 (1C,
C5),126.4 (2C, CAr),123.5 (1C, C4), 71.1 (1C, C7), 56.1 (1C, C6), 36.3 (1C,
C8). MS (FABþ): m/z 155 [M ꢁ PFꢁ6 ]þ.
d

72
C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
4.1.1.5. 3-Methyl-1-(quinolin-2-yl)-1H-imidazol-3-ium hexafluorophos-
phate (25). 1-Methylimidazole (4 mL, 47.3 mmol) and 2-
chloroquinoline (2.23 g, 13.63 mmol) were stirred in toluene
(20 mL) at 130 ꢀC for 24 h. After cooling to room temperature, the
desired brown solid was filtered and dried under vacuum (1.106 g, 28%
yield). The solid was dissolved in a mixture of H2OeMeOH (5 mLe
10 mL) and the subsequent addition of an aqueous solution (10 mL)
of NH4PF6 (0.746 g, 4.574 mmol) afforded a white precipitate, which
was collected by filtration and dried under vacuum (1.30 g, 96% yield).
Anal. Calcd. For C13H12N3PF6: C, 43.96; H, 3.41; N, 11.83. Found: C,
Found: C, 33.57; H, 3.75; N, 19.06. 1H NMR (250 MHz, DMSO-d6)
(ppm) ¼ 7.40 (s, 4H, H5), 7.36 (s, 4H, H4), 4.68 (s, 8H, H6), 3.77
d
(s, 12H, H7). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) ¼ 180.3 (4C, C2),
123.5 (4C, C5), 122.9 (4C, C4), 51.4 (4C, C6), 38.8 (4C, C7). MS (FABþ):
m/z 658 [M ꢁ NOꢁ3 ]þ.
4.1.2.3. Complex 21. A Schlenk tube was charged with silver(I)
oxide (0.046 g, 0.199 mmol), 20 (0.100 g, 0.399 mmol) and dry
CH2Cl2 (25 mL). The resulting mixture was stirred under a nitrogen
atmosphere at room temperature overnight. A solid precipitated.
The solvent was evaporated under vacuum, to afford the desired
white complex (0.115 g, 89% yield). Anal. Calcd. For C24H24N6O2Ag2:
C, 44.74; H, 3.75; N, 13.05. Found: C, 44.57; H, 3.57; N, 13.48. 1H
43.91; H, 3.28; N, 11.79. 1H NMR (300 MHz, CD3CN)
d
(ppm) ¼ 9.47 (s,
1H, H2), 8.69 (d, 1H, H8, 3J ¼ 8.8 Hz), 8.28 (pseudo-t, 1H, H5,
3,4J ¼ 1.7 Hz), 8.12 (d, 2H, H10, H13
,
3J ¼ 9.2 Hz), 7.96 (t, 1H, H12
,
3J ¼ 7.0 Hz), 7.86 (d, 1H, H7, 3J ¼ 8.8 Hz), 7.80 (t,1H, H11, 3J ¼ 7.9 Hz), 7.62
NMR (300 MHz, CDCl3)
d
(ppm) ¼ 7.79 (d, 4H, HAr, 3J ¼ 7.5 Hz), 7.29
(pseudo-t, 1H, H4, 3,4J ¼ 1.7 Hz), 4.03 (s, 3H, H15). 13C NMR (63 MHz,
(d, 2H, H5, 3,4J ¼ 1.7 Hz), 7.24 (t, 4H, HAr, 3J ¼ 7.9 Hz), 7.09 (t, 2H, HAr,
CD3CN)
d
(ppm) ¼ 146.1 (1C, C6), 145.0 (1C, C14), 141.4 (1C, C8), 134.9
3J ¼ 7.4 Hz), 6.93 (d, 2H, H4, 3,4J ¼ 1.7 Hz), 5.32 (s, 4H, H6), 3.74 (s, 6H,
(1C, C2), 131.8 (1C, C12), 128.5 (1C, C13), 128.2 (1C, C10), 128.1 (2C, C9,
C11), 124.9 (1C, C7), 119.4 (1C, C5), 111.8 (1C, C4), 36.6 (1C, C15). MS
(FABþ): m/z 210 [M ꢁ PFꢁ6 ]þ.
H8). 13C NMR (75 MHz, CDCl3)
d
(ppm) ¼ 182.4 (2C, C2), 165.4 (2C,
C7), 138.4 (2C, CAr), 128.7 (4C, CAr), 124.1 (2C, C5), 123.4 (2C, C4), 121.5
(2C, CAr), 119.9 (4C, CAr), 54.7 (2C, C6), 38.7 (2C, C8). MS (FABþ): m/z
537 [Ag(LH)2]þ.
4.1.1.6. 3-Methyl-1-{[6-(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imida-
zol-3-ium bromide (28). 1-Methylimidazole (2.02 mL, 25.32 mmol)
and 5-(bromomethyl)-2,20-bipyridine (1.802 g, 7.234 mmol) were
stirred in toluene (40 mL) at 130 ꢀC for 24 h. After cooling to
room temperature, the desired beige precipitate was filtered off and
dried under vacuum (1.385 g, 58% yield). Anal. Calcd. For
C15H15N4Br.0.3H2O: C, 53.52; H, 4.67; N, 16.64. Found: C, 53.40; H,
4.1.2.4. Complex 24. A Schlenk tube was charged with silver(I)
oxide (0.100 g, 0.431 mmol), 23 (0.300 g, 0.862 mmol) and dry
MeOH (15 mL). A solution of NaOH (1 N, 5 mL) in H2O was added
and the resulting mixture was stirred under a nitrogen atmosphere
at room temperature overnight. After filtration through a pad of
celite, the solvents were evaporated to dryness, and the brown solid
obtained was dried under vacuum (0.252 g, 89% yield). Anal. Calcd.
For C24H28N4O2AgPF6: C, 43.85; H, 4.29; N, 8.52. Found: C, 43.57; H,
4.62; N, 16.61. 1H NMR (300 MHz, CDCl3)
d
(ppm) ¼ 10.76 (s, 1H, H2),
8.85 (d, 1H, H16
,
3J ¼ 1.8 Hz), 8.67 (m, 1H, H15), 8.42 (d, 1H, H12
,
3J ¼ 8.3 Hz), 8.35 (d,1H, H9, 3J ¼ 7.8 Hz), 8.15 (dd,1H, H13, 3J ¼ 8.3 Hz,
4J ¼ 2.3 Hz), 7.83 (td, 1H, H8, 3J ¼ 7.8 Hz, 4J ¼ 1.8 Hz), 7.50 (pseudo-t,
1H, H5, 3,4J ¼ 1.7 Hz), 7.37 (pseudo-t,1H, H4, 3,4J ¼ 1.7 Hz), 7.34 (m,1H,
H14), 5.85 (s, 2H, H6), 4.08 (s, 3H, H17). 13C NMR (75 MHz, CDCl3)
4.64; N, 8.35. 1H NMR (400 MHz, DMSO-d6)
d
(ppm) ¼ 7.42e7.24
(m, 14H, H4, H5, HAr), 4.94 (d, 2H, H7, 3J ¼ 4.5 Hz), 4.29 (m, 4H,
H6), 3.78 (s, 6H, H8). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) ¼ 182.4
(2C, C2, J107,109
¼ 180.1 Hz), 147.3 (2C, CAr), 127.9 (4C, CAr), 126.8
AgeC
d
(ppm) ¼ 156.7 (1C, C10), 155.0 (1C, C11), 149.5 (1C, C15), 149.2 (1C,
(4C, CAr), 126.5 (2C, CAr), 122.7 (2C, C5), 121.5 (2C, C4), 78.8 (2C, C7),
C16), 137.8 (1C, C2), 137.0 (2C, C8, C13), 129.4 (1C, C7), 124.1 (1C, C14),
122.5 (1C, C5),121.2 (1C, C4),111.8 (2C, C9, C12), 50.1 (1C, C6), 36.8 (1C,
C17). MS (FABþ): m/z 251 [M ꢁ Brꢁ]þ.
60.8 (2C, C6), 38.4 (2C, C8). MS (FABþ): m/z 511 [M ꢁ PF6ꢁ]þ.
4.1.2.5. Complex 26. A Schlenk tube was charged with silver(I)
oxide (0.098 g, 0.422 mmol), 25 (0.300 g, 0.845 mmol), dry CH2Cl2
(25 mL) and NH4PF6 (0.035 mg, 0.211 mmol). A solution of NaOH
(1 N,15 mL) in H2O was added and the resulting mixture was stirred
under a nitrogen atmosphere at room temperature for 2 h. After
filtration through a pad of celite, the organic layer was extracted,
dried over Na2SO4, filtered and evaporated to dryness, affording
a pink solid (0.279 g, 98% yield). Crystals suitable for X-ray
diffraction analysis were obtained by slow evaporation from an
acetonitrile solution of 26. Anal. Calcd. For C26H22N6AgPF6: C,
46.52; H, 3.30; N, 12.52. Found: C, 46.57; H, 3.64; N, 12.35. 1H NMR
4.1.2. Preparation of silver(I) complexes
4.1.2.1. Complex 18. A Schlenk tube was charged with silver(I)
oxide (0.206 g, 0.888 mmol), 16 (0.338 g, 0.888 mmol) and dry
MeOH (5 mL). The resulting mixture was stirred under a nitrogen
atmosphere at 50 ꢀC for 8 h. A white solid precipitated. After
filtration, the solid was suspended in a mixture of H2OeMeOH
(15 mLe15 mL) and Ag(NO3) (0.115 g, 0.339 mmol) was added.
After stirring for 2 h at room temperature, the solution was filtered
through a pad of celite and the solvents were evaporated under
vacuum, to afford the desired white complex (0.196 g, 73% yield).
Anal. Calcd. For C18H24N10Ag2O6: C, 31.23; H, 3.49; N, 20.24. Found:
C, 31.16; H, 3.62; N, 20.46. 1H NMR (300 MHz, DMSO-d6)
(400 MHz, CD3CN)
d
(ppm) ¼ 8.22 (d, 2H, H8, 3J ¼ 8.8 Hz), 7.99 (d,
2H, H5, 3J ¼ 1.7 Hz), 7.84 (d, 2H, H7, 3J ¼ 8.8 Hz), 7.71 (m, 2H, H11),
7.50 (d, 2H, H4, 3J ¼ 1.7 Hz), 7.44e7.40 (m, 6H, H10, H12, H13), 4.05 (s,
d
(ppm) ¼ 7.89 (s, 4H, H5), 7.55 (s, 4H, H4), 6.67 (bs, 4H, H6), 3.86 (s,
6H, H15). 13C NMR (63 MHz, DMSO-d6)
d
(ppm) ¼ 182.7 (2C, C2),
12H, H7). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) ¼ 181.6 (4C, C2),
149.6 (2C, C6), 145.6 (2C, C14), 140.8. (2C, C8), 131.2 (2C, C12), 128.2
(2C, C13), 127.7 (2C, C10), 127.5 (2C, C9), 127.1 (2C, C11), 125.1 (2C, C7),
120.6 (2C, C5), 113.9 (2C, C4), 39.9 (2C, C15). MS (FABþ): m/z 525
[M ꢁ PFꢁ6 ]þ, 316 [LAg]þ.
124.8 (4C, C5), 122.2 (4C, C4), 63.5 (2C, C6), 39.0 (4C, C7). MS (FABþ):
m/z 630 [M ꢁ NOꢁ3 ]þ, 567 [M ꢁ 2NOꢁ3 ꢁ Hþ]þ.
4.1.2.2. Complex 19. A Schlenk tube was charged with silver(I)
oxide (0.197 g, 0.852 mmol), 17 (0.300 g, 0.852 mmol) and dry
MeOH (5 mL). The resulting mixture was stirred under a nitrogen
atmosphere at 50 ꢀC for 8 h. A white solid precipitated. After
filtration, the solid was suspended in a mixture of H2OeMeOH
(20 mLe20 mL) and Ag(NO3) (0.119 g, 0.703 mmol) was added.
After stirring for 2 h at room temperature, the solution was filtered
through a pad of celite and the solvents were evaporated under
vacuum, to afford the desired white complex (0.248 g, 81% yield).
Anal. Calcd. For C20H28N8Ag2N2O6: C, 33.35; H, 3.92; N, 19.45.
4.1.2.6. Complex 29. A Schlenk tube was charged with silver(I)
oxide (0.071 g, 0.305 mmol), 28 (0.202 g, 0.610 mmol) and dry
CH2Cl2 (8 mL). The resulting mixture was stirred under a nitrogen
atmosphere at room temperature overnight. After filtration
through a pad of celite, the solvent was evaporated under vacuum,
to afford the desired beige complex (0.181 g, 86% yield). Anal. Calcd.
For C30H28N8AgBr: C, 52.34; H, 4.10; N, 16.28. Found: C, 52.57; H,
4.34; N, 16.35. 1H NMR (300 MHz, CDCl3)
H15), 8.62 (m, 2H, H16), 8.42 (m, 4H, H9, H12), 7.85 (m, 2H, H13), 7.74
d
(ppm) ¼ 8.70 (m, 2H,

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
73
(td, 2H, H8, 3J ¼ 7.8 Hz, 4J ¼ 1.8 Hz), 7.35 (m, 2H, H14), 7.04 (d, 2H, H5,
3J ¼ 1.7 Hz), 6.97 (d, 2H, H4, 3J ¼ 1.7 Hz), 5.41 (s, 4H, H6), 3.90 (s, 6H,
N, 11.24. 1H NMR (250 MHz, CD3CN)
d
(ppm) ¼ 8.14 (d, 2H, H8,
3J ¼ 8.7 Hz), 8.06 (d, 2H, H9, 3J ¼ 8.7 Hz), 7.95 (d, 2H, H5, 3J ¼ 2.0 Hz),
7.82 (m, 4H, H7, H12), 7.70 (td, 2H, H11, 3J ¼ 7.1 Hz, 4J ¼ 1.3 Hz), 7.56
(td, 2H, H10, 3J ¼ 6.9 Hz, 4J ¼ 1.2 Hz), 7.46 (d, 2H, H4, 3J ¼ 2.0 Hz), 4.04
(s, 6H, H15). 13C NMR (75 MHz, CD3CN-DMSO-d6/0.9e0.1)
H17). 13C NMR (75 MHz, DMSO-d6)
d
(ppm) ¼ 181.0 (2C, C2), 155.5
(2C, C10), 155.2 (2C, C11), 149.8 (2C, C15), 149.1 (2C, C16), 137.8 (2C,
C13), 137.1 (2C, C8), 133.8 (2C, C7), 124.8 (2C, C14), 123.9 (2C, C5), 122.6
(2C, C4),121.0 (2C, C12),120.9 (2C, C9), 51.8 (2C, C6), 38.7 (2C, C17). MS
(FABþ): m/z 607 [M ꢁ Brꢁ]þ, 357 [AgL]þ.
d
(ppm) ¼ 182.9 (2C, C2), 149.3 (2C, C6), 146.2 (2C, C14), 139.5 (4C,
C8), 130.9 (2C, C12), 128.3 (2C, C13), 127.9 (2C, C10), 127.7 (2C, C9),
127.4 (2C, C11), 123.9 (2C, C7), 121.0 (2C, C5), 115.7 (2C, C4), 38.7 (2C,
C15). MS (FABþ): m/z 615 [M ꢁ PFꢁ6 ].
4.1.3. Preparation of gold(I) complexes
4.1.3.1. Complex 22. Under a nitrogen atmosphere, sodium acetate
(0.079 g, 0.958 mmol) was added to a mixture of 20 (0.200 g,
0.798 mmol) and Au(SMe2)Cl (0.118 g, 0.399 mmol) in dry DMF
(4 mL) at 100 ꢀC. The mixture was then heated to 120 ꢀC and this
temperature was maintained for 2 h. After cooling to room
temperature, a white solid precipitated. After filtration, complex 22
was dried under vacuum (0.171 g, 65% yield). Crystals suitable for X-
ray diffraction analysis were obtained by slow evaporation from
a dichloromethane solution of 22. Anal. Calcd. For C24H26N6O2AuCl:
C, 43.48; H, 3.95; N, 12.68. Found: C, 43.39; H, 3.88; N, 12.61. 1H
4.1.3.3. Complex 30. Under a nitrogen atmosphere, sodium acetate
(0.089 g, 0.453 mmol) was added to a mixture of 28 (0.300 g,
0.906 mmol) and Au(SMe2)Cl (0.133 g, 0.453 mmol) in dry DMF
(6 mL) at 100 ꢀC. The mixture was then heated to 120 ꢀC and this
temperature was maintained for 2 h. After cooling to room
temperature, a white solid precipitated. After filtration, complex 30
was dried under vacuum (0.218 g, 64% yield). Crystals suitable for
X-ray diffraction analysis were obtained by slow evaporation from
a chloroform solution of 30. Anal. Calcd. For C30H28N8AuBr: C,
46.35; H, 3.63; N, 14.41. Found: C, 46.69; H, 3.23; N, 14.42. 1H NMR
NMR (300 MHz, CDCl3)
d
(ppm) ¼ 11.58 (s, 2H, NH), 7.85 (d, 4H, HAr,
3J ¼ 8.0 Hz), 7.26e7.22 (m, 6H, H5, HAr), 7.05 (t, 2H, HAr, 3J ¼ 7.94 Hz),
(300 MHz, CDCl3)
d
(ppm) ¼ 8.64 (m, 4H, H15, H16), 8.34 (d, 2H, H12
,
6.99 (d, 2H, H4, 3J ¼ 1.1 Hz), 5.47 (s, 4H, H6), 3.87 (s, 6H, H8). 13C NMR
3J ¼ 8.2 Hz), 8.30 (d, 2H, H9, 3J ¼ 8.0 Hz), 7.82e7.75 (m, 4H, H8, H13),
7.34 (d, 2H, H5, 3J ¼ 1.8 Hz), 7.32e7.28 (m, 2H, H14), 7.18 (d, 2H, H4,
3J ¼ 1.8 Hz), 5.60 (s, 4H, H6), 3.90 (s, 6H, H17). 13C NMR (75 MHz,
(75 MHz, CDCl3eCD3OD/0.4e0.6)
d
(ppm) ¼ 188.1 (2C, C2), 168.1
(2C, C7), 140.4 (2C, CAr), 131.5 (4C, CAr), 127.3 (2C, C5), 126.0 (2C, CAr),
125.1 (2C, C4), 122.5 (4C, CAr), 56.1 (2C, C6), 40.3 (2C, C8). MS (FABþ):
m/z 627 [M ꢁ Clꢁ]þ.
CD3CN-DMSO-d6)
d
(ppm) ¼ 183.2 (2C, C2), 154.7 (2C, C10), 154.0
(2C, C11), 148.8 (2C, C15), 147.6 (2C, C16), 137.4 (2C, C13), 136.1 (2C, C8),
132.7 (2C, C7), 124.2 (2C, C14), 123.4 (2C, C5), 122.1 (2C, C4), 120.7 (2C,
C12), 120.5 (2C, C9), 50.6 (2C, C6), 37.3 (2C, C17). MS (FABþ): m/z 697
[M ꢁ Brꢁ].
4.1.3.2. Complex 27. Under a nitrogen atmosphere, sodium acetate
(0.106 g, 1.29 mmol) was added to a mixture of 25 (0.204 g,
0.575 mmol) and Au(SMe2)Cl (0.169 g, 0.575 mmol) in dry DMF
(6 mL) at 100 ꢀC. The mixture was then heated to 120 ꢀC and this
temperature was maintained for 3 h. After cooling to room
temperature, the solvent was evaporated and the white solid ob-
tained was dried under vacuum (0.218 g, 85% yield). Crystals suit-
able for X-ray diffraction analysis were obtained by slow
evaporation from an acetonitrile solution of 27. Anal. Calcd. For
C26H22N6AuPF6: C, 41.07; H, 2.92; N, 11.05. Found: C, 40.80; H, 2.79;
4.1.4. Crystallographic data for 22, 26, 27 and 30
All data were collected at low temperature using oil-coated
shock-cooled crystals on a Bruker-AXS APEX II diffractometer
ꢀ
with MoK
a
radiation (
l
¼ 0.71073 A). The structures were solved by
direct methods [34] and all non-hydrogen atoms were refined
anisotropically using the least-squares method on F2 [35]. The data
are summarized in Table 3. CCDC-884301 (22), CCDC-884302 (26),
Table 3
Crystal data. Parameters for data collection and structure refinements of 22, 26, 27 and 30.
Complex
22
26
27
30
Empirical formula
Formula weight
T/K
C24H28AuClN6O3
680.94
173(2)
Triclinic
P‾1
10.3446(7)
11.4826(8)
11.6938(8)
96.711(3)
107.935(3)
96.914(3)
1294.4(2)
2
C52H44Ag2F12N12P2
1342.67
173(2)
Monoclinic
C2/c
25.400(11)
7.2786(4)
14.2569(6)
e
C26H22AuF6N6P
760.43
173(2)
Monoclinic
C2/c
25.459(4)
7.242(2)
14.154(3)
e
C30H28AuBrN8
777.48
173(2)
Triclinic
P‾1
9.9307(4)
10.7149(5)
14.8413(8)
93.436(2)
102.189(2)
111.718(2)
1417.4(2)
2
Crystal system
Space group
ꢀ
a/A
ꢀ
b/A
ꢀ
c/A
ꢀ
ꢀ
a
b
g
/
/
103.078(2)
e
102.591(5)
e
ꢀ
/
3
ꢀ
V/A
2591.7(2)
2
1.721
2546.9(7)
4
1.983
Z
rcalcd/Mg mꢁ3
1.747
5.823
1.822
6.335
m
/mmꢁ1
0.912
5.913
F(000)
668
1344
1472
756
Crystal size/mm3
0.40 ꢃ 0.30 ꢃ 0.20
5.12e30.51
52295
7865
0.0242
0.40 ꢃ 0.20 ꢃ 0.20
5.11e28.28
15221
3196
0.0261
0.40 ꢃ 0.10 ꢃ 0.10
5.16e26.37
9367
2579
0.0792
0/185
0.40 ꢃ 0.10 ꢃ 0.10
2.33e26.48
42250
5829
0.0774
q
Range for data collection/ꢀ
Reflections collected
Independent reflections
R (int)
Restraints/parameters
0/334
1.027
0/202
1.018
0/363
1.000
Goodness-of-fit on F2
0.931
R1 (I > 2
wR2 (I > 2
R1 (all data)
s
(I))
0.0156
0.0354
0.0188
0.0364
0.0270
0.0588
0.0422
0.0636
0.0324
0.0517
0.0747
0.0599
1.009
0.0320
0.0459
0.0565
0.0506
s
(I))
wR2 (all data)
ꢀꢁ3
Drmax/e A
0.821
0.302
0.783

74
C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
CCDC-884303 (27) and CCDC-884304 (30) contain the
Supplementary crystallographic data. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
This test permitted to detect the hemolytic compounds (with
concentration tested from 0.01 g/mL to 50 g/mL) that weaken red
m
m
blood cells and induce erythrocyte membrane bursting with
subsequent release of hemoglobin. Water known to involve the
lysis of red blood cells was used as positive control and RPMI alone
was used as negative control. Hemolysis was considered as positive
when the supernatant of the parasite culture was brown and the
erythrocyte pellet totally liquid.
4.2. Antiplasmodial tests
The CQ-resistant FcM29-Cameroon strain of Plasmodium falci-
parum was cultured continuously according to the modified Trager
and Jensen’s method [36]. The antiplasmodial activity was evalu-
ated by the radioactive micro-method described by Desjardins et al.
[37] with modifications [36]b. The antiplasmodial tests of mole-
cules were performed from one to eight different times in triplicate
in 96-well culture plates (TPP) on synchronous cultures at ring
stages.
Acknowledgments
This work was supported by the Centre National de la Recherche
Scientifique (CNRS) and the University Paul Sabatier Toulouse (BQR
grant).
The antiplasmodial drug controls, CQ and artemisinin, were
routinely tested on this strain and the IC50 values were ranged from
445 nM to 503 nM and from 10 nM to 14 nM, respectively. CQ was
dissolved directly in the culture medium. The compounds and
artemisinin were dissolved in dimethyl sulfoxide (DMSO, Sigma)
(stock solution: 1 mg/mL) and further diluted in the culture
medium so that the final DMSO concentration never exceeded 1%.
For each experiment, we verified that this 1% concentration of
DMSO did not affect parasite growth. Parasite growth was esti-
mated by [3H]-hypoxanthine incorporation (Perkin Elmer). The
control parasite culture (RPMI with 5% of human serum alone or
with 1% DMSO) was referred to as 100% growth.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.11.038.
References
[1] J.C.Y. Lin, R.T.W. Huang, C.S. Lee, A. Bhattcharyya, W.S. Hwang, I.J.B. Lin, Chem.
Rev. 109 (2009) 3561e3598.
[2] K.M. Hindi, M.J. Panzner, C.A. Tessier, C.L. Cannon, W.J. Youngs, Chem. Rev. 109
(2009) 3859e3884.
[3] M.J. Panzner, C.A. Tessier, W.J. Youngs, in: D. Morales-Morales, C.M. Jensen (Eds.),
The Chemistry of Pincer Compounds, first ed,, Elsevier, 2007, pp. 139e150.
[4] A. Kascatan-Nebioglu, M.J. Panzner, C.A. Tessier, C.L. Cannon, W.J. Youngs,
Coord. Chem. Rev. 251 (2007) 884e895.
[5] D.A. Medvetz, K.M. Hindi, M.J. Panzner, A.J. Ditto, Y.H. Hun, W.J. Youngs, Met.-
Based Drugs (2008) 7. 384010.
[6] M. Marion, S.P. Nolan, Chem. Soc. Rev. (2008) 1776e1782.
[7] P.J. Barnard, S.J. Berners-Price, Coord. Chem. Rev. 251 (2007) 1889e1902.
[8] M.-L. Teyssot, A.-S. Jarrousse, M. Manin, A. Chevry, S. Roche, F. Norre,
C. Beaudoin, L. Morel, D. Boyer, R. Mahiou, A. Gautier, Dalton Trans. (2009)
6894e6902.
The ranges of concentrations of the molecules tested were for
the first preliminary series from 0.05
mg/mL to 50
mg/mL and for the
second series from 0.01 g/mL to 10
m
m
g/mL. Each solution was
tested both in extemporaneous conditions and after a conservation
of the stock solution in DMSO at 4 ꢀC for 2e8 days in order to
evaluate the stability of the biological activity of the compounds
tested.
[9] S. Ray, R. Mohan, J.K. Singh, M.K. Samantaray, M.M. Shaikh, D. Panda, P. Ghosh,
J. Am. Chem. Soc. 129 (2007) 15042e15053.
4.3. Antifungal tests
[10] J.L. Hickey, R.A. Ruhayel, P.J. Barnard, M.V. Baker, S.J. Berners-Price,
A. Filipovska, J. Am. Chem. Soc. 130 (2008) 12570e12571.
[11] R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L.A. Onambele,
M. Stefanopoulou, Y. Geldmacher, W.S. Sheldrick, G. Wolber, A. Prokop,
S. Wölfl, I. Ott, J. Med. Chem. 53 (2010) 8608e8618.
[12] J. Weaver, S. Gaillard, C. Toye, S. Macpherson, S.P. Nolan, A. Riches, Chem. Eur.
J. 17 (2011) 6620e6624.
[13] D. Krishnamurthy, M.R. Karver, E. Fiorillo, V. Orrú, S.M. Stanford, N. Bottini,
M. Barrios, J. Med. Chem. 51 (2008) 4790e4795.
The C. albicans American Type Culture Collection 90028 (ATCC
90028) and Candida glabrata (#0500024220) were used to evaluate
the antifungal activity of the 15 first molecules. A micro-dilution
method adapted from the Clinical and Laboratory Standards Insti-
tute (NCCLS-M27A) [38] was used. The culture medium used was
[14] I. Özdemir, A. Denizci, H.T. Öztürk, B. Çetinkaya, Appl. Organomet. Chem. 18
(2004) 318e322.
[15] (a) R.A. Calderone, Candida and Candidiasis, ASM Press, Washington, DC,
2002, 451;
RPMI 1640 (Sigma, France) with 2 mM L-Glutamine and 0.165 M
morpholinopropanesulfonic acid (MOPS) buffer (Sigma, France).
Prior to testing, the isolate was sub-cultured on Sabouraud dextrose
agar (Bio-Mérieux, France) and was incubated at 35 ꢀC during 48 h.
Inoculates were prepared by suspending the yeast in 1.0 mL of
a sterile saline solution and adjusting to a final concentration of
(b) M.A. Pfaller, D.J. Diekema, R.N. Jones, H.S. Sader, A.C. Fluit, R.J. Hollis,
S.A. Messer, The SENTRY Participant Group, J. Clin. Microbiol. 39 (2001) 3254e
3259.
[16] C.J. Murray, L.C. Rosenfeld, S.S. Lim, K.G. Andrews, K.J. Foreman, D. Haring,
N. Fullman, M. Naghavi, R. Lozano, A.D. Lopez, Lancet 379 (2012) 413e431.
[17] (a) M. Navarro, Coord. Chem. Rev. 253 (2009) 1619e1626;
(b) M. Navarro, H. Pérez, R.A. Sánchez-Delgado, J. Med. Chem. 40 (1997)
1937e1939.
2.5 ꢃ 106 yeast cells/mL. Each well of 96-well plates received 100
mL
of this suspension and 100 mL of various concentrations of drugs
(with the maximum concentration at 50
mg/mL), and the plates
incubated for 48 h at 35 ꢀC. All antifungal assays were only per-
formed from drug solutions diluted extemporaneously. The results
were determined using a spectrophotometer (Elx 808, Vetra
Microplate Reader, Avantec) at a wavelength of 550 nm. The MIC
(Minimal Inhibitory Concentration) values were determined
graphically by plotting concentrations of tested drugs versus
percentage of yeast inhibition. For comparison, the control 5-
fluorocytosine was routinely tested as a reference in this test and
gave MIC similar to the ATCC reference values.
[18] F. Jean-Baptiste dit Dominique, H. Gornitzka, C. Hemmert, J. Organomet.
Chem. 693 (2008) 579e583.
[19] F. Jean-Baptiste dit Dominique, H. Gornitzka, A. Sournia-Saquet, C. Hemmert,
Dalton Trans. (2009) 340e352.
[20] F. Jean-Baptiste dit Dominique, H. Gornitzka, C. Hemmert, Organometallics 29
(2010) 2868e2873.
[21] J. Cure, R. Poteau, I.C. Gerber, H. Gornitzka, C. Hemmert, Organometallics 31
(2012) 619e626.
[22] The synthesis of compounds 1, 2, 9, 10 and 13 was reported in references 18
and 20 and the synthesis of compounds 3e8, 11, 12, 14 and 15 will be re-
ported elsewhere in a manuscript in preparation
[23] C.-Y. Liao, K.-T. Chan, J.-Y. Zeng, C.-H. Hu, C.-Y. Tu, H.M. Lee, Organometallics
26 (2007) 1692e1702.
[24] C. Wan Ying Chung, P.H. Toy, J. Comb. Chem. 9 (2007) 115e120.
[25] L. Labat, J.-F. Lamère, I. Sasaki, P.G. Lacroix, L. Vendier, I. Asselberghs, J. Pérez-
Moreno, K. Clays, Eur. J. Inorg. Chem. (2006) 3105e3113.
[26] M.K. Samantaray, K. Pang, M.M. Shaikh, P. Ghosh, Inorg. Chem. 47 (2008)
4153e4165.
4.4. Hemolysis assays
The evaluation of hemolysis induced by compounds was per-
formed on parasite cultures after 48 h-incubation time at 37 ꢀC.

C. Hemmert et al. / European Journal of Medicinal Chemistry 60 (2013) 64e75
75
ꢁ
[27] K. Begum, H.-S. Kim, V. Kumar, I. Stojilkovic, Y. Wataya, Parasitol. Res. 90
(2003) 221e224.
[33] J. Coetzee, S. Cronje, L. Dobrzanska, H.G. Raubenheimer, G. Jooné, M.J. Nell,
H.C. Hoppe, Dalton Trans. 40 (2011) 1471e1483.
[28] M.S. Wysor, US 4020150 A 19770426.
[29] A.R. Sanella, A. Casini, C. Gabbiani, L. Messori, A.R. Bilia, F.F. Vincieri, G. Majori,
C. Severini, FEBS Lett. 582 (2008) 844e847.
[30] E. Schuh, S.M. Valiadhi, M.A. Jakupec, B.K. Keppler, P. Chiba, F. Mohr, Dalton
Trans. (2009) 10841e10845.
[31] A. Molter, J. Rust, C.W. Lehmann, G. Deepa, P. Chiba, F. Mohr, Dalton Trans. 40
(2011) 9810e9820.
[32] (a) S.D. Khanye, G.S. Smith, C. Lategan, P.J. Smith, J. Gut, P.J. Rosenthal,
K. Chibale, J. Inorg. Biochem. 104 (2010) 1079e1083;
(b) S.D. Khanye, B. Wan, S.G. Franzblau, J. Gut, P.J. Rosenthal, G.S. Smith,
K. Chibale, J. Organomet. Chem. 696 (2011) 3392e3396.
[34] G.M. Sheldrick, Acta Crystallogr. A46 (1990) 467e473.
[35] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112e122.
[36] (a) W. Trager, J.B. Jensen, Science 193 (1976) 673e675;
(b) F. Benoit-Vical, J. Lelievre, A. Berry, C. Deymier, O. Dechy-Cabaret,
J. Cazelles, C. Loup, A. Robert, J.-F. Magnaval, B. Meunier, Antimicrob. Agents
Chemother. 51 (2007) 1463e1472.
[37] R.E. Desjardins, C.J. Canfield, J.D. Haynes, J.D. Chulay, Antimicrob. Agents
Chemother. 16 (1979) 710e718.
[38] NCCLS, NationalCommittee forClinicalLaboratory Standards, ReferenceMethod
forBrothDilutionAntifungalSusceptibility Testingof Yeasts, Approved Standard
M27-A National Committee for Clinical Laboratory Standards, Wayne, PA, 1997.