Tetrasubstituted imidazolium salts as potent antiparasitic agents against African and American trypanosomiases

Article abstract of DOI:10.3390/molecules23010177

Imidazolium salts are privileged compounds in organic chemistry, and have valuable biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can display antiparasitic activity against T. cruzi. After developing a faci

Full text of DOI:10.3390/molecules23010177

molecules
Communication
Tetrasubstituted Imidazolium Salts as Potent
Antiparasitic Agents against African and
American Trypanosomiases
Ouldouz Ghashghaei 1, Nicola Kielland 1, Marc Revés 1, Martin C. Taylor 2, John M. Kelly 2,
Ornella Di Pietro 3, Diego Muñoz-Torrero 3 ID , Belén Pérez 4 and Rodolfo Lavilla 1,
*
ID
1
Laboratory of Organic Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona,
Barcelona Science Park, Baldiri Reixac 10-12, 08028 Barcelona, Spain; ouldouz.ghashghaei@gmail.com (O.G.);
nicola.kielland@gmail.com (N.K.); revesmarc@gmail.com (M.R.)
Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine,
London WC1E 7HT, UK; Martin.Taylor@lshtm.ac.uk (M.C.T.); john.kelly@lshtm.ac.uk (J.M.K.)
Laboratory of Pharmaceutical Chemistry (CSIC Associated Unit), Faculty of Pharmacy and Food Sciences,
and Institute of Biomedicine (IBUB), University of Barcelona, 08028 Barcelona, Spain;
od265@cam.ac.uk (O.D.P.); dmunoztorrero@ub.edu (D.M.-T.)
2
3
4
Department of Pharmacology, Therapeutics and Toxicology, Institute of Neurosciences,
Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain; belen.perez@uab.cat
Correspondence: rlavilla@ub.edu; Tel.: +34-934037106
*
Received: 29 November 2017; Accepted: 13 January 2018; Published: 16 January 2018
Abstract: Imidazolium salts are privileged compounds in organic chemistry, and have valuable
biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can
display antiparasitic activity against T. cruzi. After developing a facile methodology for the synthesis
of tetrasubstituted imidazolium salts from propargylamines and isocyanides, we screened a small
library of these adducts against the causative agents of African and American trypanosomiases. These
compounds display nanomolar activity against T. brucei and low (or sub) micromolar activity against
T. cruzi, with excellent selectivity indexes and favorable molecular properties, thereby emerging as
promising hits for the treatment of Chagas disease and sleeping sickness.
Keywords: imidazolium salts; isocyanides; antiparasitic agents; Trypanosoma brucei; Trypanosoma cruzi
1. Introduction
Chagas disease and sleeping sickness are caused by infection with the protozoan parasites
Trypanosoma cruzi and Trypanosoma brucei sp., respectively, and have been classified as Neglected
Tropical Diseases. Sleeping sickness threatens millions of people in 36 countries in sub-Saharan Africa.
Chagas disease is estimated to affect around 8 million people in endemic areas of 21 Latin American
countries. The population at risk of these parasitic diseases normally has limited access to medical
assistance, as they live in rural areas of developing countries. Moreover, due to immigration, war,
and poverty, the control of infection can be complicated. Indeed, in recent years, an increasing number
of Chagas disease cases have been reported in the US, European countries, and Canada [1,2].
Both infections are transmitted by insect vectors. In the case of Chagas disease, transmission
can also occur by the congenital route, food contamination, blood transfer, and organ transplantation.
Pathology in Chagas disease is mainly associated with the chronic stage, which occurs years after
the initial infection, with cardiomyopathy being a common outcome. In African trypanosomiasis,
second-stage disease—which occurs once parasites breach the blood brain barrier (BBB)—is the critical
phase, being fatal unless treated. Due to serious side effects and other limitations of the currently
available drugs, there is an urgent need to develop efficient medications against these diseases [3–7].
Molecules 2018, 23, 177; doi:10.3390/molecules23010177
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Imidazolium ions are valuable heterocyclic scaffolds due to their broad applications, especially in
organocatalysis, and in the synthesis of liquid crystals. Recently, the biological activities of imidazolium
and benzimidazolium ions has been reviewed, assessing their toxicity and particularly their wide
range of uses in medicinal chemistry [
8]. In 2014, Robello et al. [9] reported that simple symmetric
imidazolium salts
A
(Figure 1) with bulky aromatic moieties on the N1 and N3 positions are active
against T. cruzi. The putative biological target of these compounds has been postulated as being linked
with the parasite mitochondrion. The major differences between these organelles in parasites and
mammals could explain the selective toxicity of the imidazolium salts toward trypanosomes [
In this respect, we recently reported the potent antitrypanosomal activity of a family of azine-fused
aminoimidazolium salts (Figure 1) arising from a trimethylsilyl-catalyzed interaction of azines with
8,9].
B
isocyanides in a multicomponent manner [10]. Analogously, these compounds also presented two
diversity points at the N atoms, and the most active hits displayed aliphatic residues at these positions.
Having developed a modular methodology for the facile synthesis of complex imidazolium
salts [11], the goal of the present work was to study the antiparasitic activity of more complex
unsymmetrical tetrasubstituted imidazolium salts
1 (Figure 1) against both African and American
trypanosomiases. Relevantly, the target compounds feature a novel substitution pattern affecting not
only the N atoms, but also at C atoms at positions 4 and 5.
Figure 1. Structures of imidazolium salts active against Trypanosoma species: disubstituted imidazolium
salts
A [9], amino isoquinoline-imidazolium salts B [10], and the novel tetrasubstituted imidazolium salts 1.
2. Results and Discussion
2.1. Chemistry
Compounds 1a
aldehydes, terminal alkynes, and isocyanides [11]. First, the amine, aldehyde, and terminal alkyne
substrates condense through a multicomponent A3 reaction to yield propargyl amines in high
–e can be easily accessed through the novel two-step condensation of amines,
2
yields [12]. In the second step, the formed propargylamine and isocyanide interact through an
acid-catalyzed insertion-cyclization and isomerization process to give the desired imidazolium salts
1a–e in good-to-moderate yields (Scheme 1) [11]. Interestingly, this two-step sequence can be performed
in tandem. A similar process was reported by Zhu, featuring a dual catalytic system of AgOTf and
Yb(OTf)3 [13]. The nature, scope, and commercial availability of the starting materials, the mild
reaction conditions, and the modular access to imidazolium salts
1 (Table 1), with four diversity
points, makes this synthetic approach ideal for medicinal chemistry, especially for hit finding through
screening. In this respect, is relevant to comment that restrictions on the involved chemistry and
the practical access to the alkyne inputs guided the initial screen on the substituent range at this
initial stage. The A3 reaction with primary amines works better with anilines and aromatic aldehydes,
and for these reason, R1 and R2 are substituted phenyl rings. On the other hand, there is a limited
choice of commercially available alkynes; the most suitable/cheap are arylacetylenes (R3 being also
phenyl or p-Me-phenyl), whereas there is a wider scope on R4 as many isocyanides (aliphatic, aromatic,
functionalized) conveniently react in the second step of the sequence [11].

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Scheme 1. Synthetic access to the imidazolium salts 1. ACN: Acetonitrile.
Table 1. Structural description of imidazolium salts 1.
Compd.
R1
R2
R3
R4
1a
1b
1c
1d
1e
p-Me-Ph
p-MeO-Ph
p-MeO-Ph
p-Me-Ph
p-Me-Ph
Ph
Ph
Ph
p-Me-Ph
tert-Bu
p-MeO-Ph
tert-Bu
2-naphthyl
-COOH
p-Me-Ph
p-Cl-Ph
p-Cl-Ph
Ph
Ph
Ph
2.2. Biological Activity against T. brucei and T. cruzi
Next, we evaluated the trypanocidal activity of salts
the epimastigote form of T. cruzi (Materials and Methods) (Table 2). Compounds 1a–d
1
against bloodstream forms of T. brucei and
displayed
a potent bioactivity profile at low or sub-micromolar (T. cruzi) or nanomolar levels (T. brucei).
For comparison, the EC50 value for nifurtimox—a drug used to treat T. brucei and T. cruzi infections—is
approximately 2 µM for both parasites [14]. In contrast to compounds 1a–d, salt 1e was significantly less
active, possibly due to its zwitterion character in physiological conditions, which could compromise
its ability to cross biological membranes. Compounds 1b and 1d exhibited the highest potency against
both parasites, being clearly more active against T. brucei. Interestingly, both compounds feature
aromatic moieties on N1 and N3. The cytotoxicity of these compounds was evaluated using L6 rat
myoblast cells (Materials and Methods). They displayed high selectivity indexes, particularly with
T. brucei, suggesting good potential for use as antitrypanosomal drugs.
Table 2. Biological activity of compounds 1 vs. bloodstream form T. brucei and T. cruzi epimastigotes.
1a
1b
1c
1d
1e
1
0.18 ± 0. 01
0.28 ± 0.07
33.90 ± 3.30
188
1.85 ± 0.29
3.41 ± 0.14
18
0.04 ± 0.00
0.05 ± 0.00
4.60 ± 0.38
118
0.44 ± 0.01
1.29 ± 0.05
10
0.18 ± 0.01
0.20 ± 0.01
9.69 ± 0.58
54
1.72 ± 0.39
2.98 ± 0.19
5.6
0.07 ± 0.00
0.09 ± 0.00
4.09 ± 0.28
56
0.54 ± 0.08
1.00 ± 0.04
7.6
5.26 ± 0.01
EC50 (T. brucei, µM)
2
6.79 ± 0.09
EC90 (T. brucei, µM)
EC50 (L6 cells, µM)
>130
>24
>20
>20
-
SI 3 (T. brucei)
EC50 (T. cruzi, µM)
EC90 (T. cruzi, µM)
SI (T. cruzi)
1
EC50: effective concentration to reduce parasite growth by 50%; 2 EC90: effective concentration to reduce parasite
growth by 90%; 3 SI: Selectivity index; ratio of mammalian cell and parasite EC50 values.
2.3. Molecular Properties of Compounds 1
A number of molecular properties are usually considered at early stages of the drug discovery
process for the prediction of some important pharmacokinetic attributes. High oral bioavailability
is a desirable feature for most drug candidates. According to the widely known Lipinski’s rules,
good oral bioavailability can be expected for compounds with molecular weight below 500 daltons,
calculated log P (as a measure of lipophilicity) below 5, and no more than five hydrogen bond donors

Molecules 2018, 23, 177
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and ten hydrogen bond acceptors [15]. Additionally, other molecular properties have been reported to
influence oral bioavailability, and hence are also commonly taken into account in early-phase drug
discovery. Thus, low molecular flexibility—measured by the number of rotatable bonds, with a cutoff
value of 10, and low polar surface area, measured as the sum of surface contributions of polar atoms
or as the sum of tabulated surface contributions of polar fragments (topological polar surface area or
TPSA) [16], with a cutoff value of 140 Å2—are also delimiters of good oral bioavailability [17].
Brain penetration is also a desirable pharmacokinetic attribute for antitrypanosomal agents,
purported to be effective in the late stage of sleeping sickness, when the T. brucei parasites invade
the central nervous system (CNS). Brain permeation is usually determined experimentally through
the well-known in vitro parallel artificial membrane permeability assay for blood-brain barrier
(PAMPA-BBB) [18]. Of note, researchers from Pfizer recently disclosed an in silico method for the
evaluation of the likeliness of a given compound to act in the CNS [19], which is based on the calculation
of the so-called CNS multiparameter optimization (CNS MPO) algorithm [20,21]. This algorithm can
be rapidly calculated from several of the above-mentioned molecular properties—namely, molecular
weight, calculated log P, hydrogen bond donors, and TPSA, together with calculated log D and pKa of
the most basic center [20]. Compounds with CNS MPO desirability scores over 4 (in a scale from 0
to 6) tend to display favorable pharmacokinetic and safety attributes for CNS drugs.
To get insight into the potential oral bioavailability and brain permeability of the target
compounds, we experimentally determined their PAMPA-BBB permeabilities through the method of
Di et al. [18] and calculated the molecular properties that influence oral bioavailability and the CNS
MPO scores, using the Molinspiration calculation software [22], the ChemAxon Marvin Suite 17.28
(for clog D (at pH 7.4) and pKa values), and a Pfizer’s Excel active table, which is available online [20
]
(Table 3). With regard to the experimentally determined PAMPA-BBB permeabilities (Pe), according
to the limits established by Di et al. [18], compound 1e is the only one with low BBB permeation
(Pe < 1.89), probably as a consequence of its zwitterionic character at the pH of the assay, which would
preclude its permeation by passive diffusion, whereas the Pe values of the rest of compounds are in the
range of uncertain permeability (5.16 > Pe 10−6 cm s−1 > 1.89). However, Pe values of compounds 1a
and 1b are interestingly close to the threshold for high BBB permeability (Pe > 5.16). Interestingly, CNS
MPO scores close to the threshold for a full alignment of pharmacokinetic properties were found for
most of our compounds (see Table 3 and Supplementary Information, SI), which further supports their
usefulness in late-stage sleeping sickness. Moreover, we found that these compounds are compliant
with Lipinski’s rules, and have TPSA values and a number of rotatable bonds (nrotb) that are favorable
for a good oral bioavailability (Table 3).
Table 3. Molecular properties of compounds 1.
1a
1b
1c
1d
1e
PAMPA-BBB (Pe)
CNS MPO
miLogP
MW
4.2
3.3
3.30
381.54
0
4.9
3.5
3.93
461.58
0
2.6
3.7
4.04
446.01
0
2.4
1.4
5.67
486.04
0
1.6
5.8
−1.35
382.46
0
nOHNH
nON
2
4
3
2
4
nviolations
TPSA
0
8.82
5
0
0
1
8.82
5
0
27.29
7
18.05
6
48.95
6
nrotb
CNS MPO: CNS multiparameter optimization; MW: molecular weight; nOHNH: number of hydrogen bond donors;
nON: number of hydrogen bond acceptors; nrotb: number of rotatable bonds; nviolations: number of violations
of Lipinski’s rules; PAMPA-BBB (Pe): parallel artificial membrane permeability assay for blood-brain barrier
permeability; miLogP: LogP calculated using Molinspiration; TPSA: topological polar surface area.

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3. Materials and Methods
3.1. Compound Preparation General Methods
N-(aryl)propargylamines
2 were prepared from the corresponding amines, aldehydes,
and terminal alkynes by a modification of Li and Wei’s protocol [12]: In a Schlenk tube, 2.0 mmol
(1.00 equiv.) of the aldehyde and 2.2 mmol (1.1 equiv.) of the amine were dissolved in 5 mL of
◦
THF under N2 atmosphere. The tube was then sealed and the mixture was heated at 60 C until
complete consumption of the aldehyde ( 2 h). Next, CuBr (30 mol %), RuCl3 (3 mol %), and the alkyne
≈
(2.4 mmol, 1.2 equiv.) were added. The mixture was stirred at room temperature (
≈
30 min), and then
◦
heated at 50 C until complete consumption of the imine (
≈
24 h). Afterwards, the solution was cooled,
20 mL). The combined organic phases were
20 mL), dried (anh. MgSO4), and concentrated under vacuum. Purification by
flash chromatography on silica gel (hexanes/AcOEt) afforded the corresponding propargylamines.
Tetrasubstituted imidazolium salts were prepared from the corresponding propargylamines
and isocyanides through the recent procedure reported by the group [11]: Propargylamine (0.5 mmol,
1 equiv.) was dissolved in THF (1.5 mL) and ACN (1.5 mL) under N2 atmosphere. Next, the isocyanide
(0.5 mmol, 1 equiv.) was added. A 4 M HCl solution in dioxane (125 L, 1 equiv.) was added and the
mixture stirred at room temperature until total consumption of the propargylamine ( 4 h). Next the
reaction was quenched with saturated Na2CO3 aqueous solution (20 mL) and extracted with AcOEt
3 × 10 mL). The combined organic phases were dried (anh. MgSO4) and concentrated under vacuum
to afford the corresponding imidazolium salts . Analytically pure samples of the products were
poured into water (40 mL), and extracted with DCM (3
washed with water (2
×
×
1
2
µ
≈
(
1
obtained by flash chromatography on silica gel (hexanes/EtOH 9:1 to 7:3 v/v).
For detailed protocols and compound characterization, see Reference [11] and the Supporting
Information.
3.2. Biological Activity Determination
3.2.1. T. brucei Culturing and Evaluation of Trypanocidal Activity
Bloodstream form T. brucei (strain 221) were grown as described previously [23]. Compound
activity was determined by culturing parasites in the presence of different concentrations so that the
levels which inhibited growth by 50% (EC50)−a1nd 90% (EC90) could be calculated. Logarithmically
growing T. brucei were diluted to 2.5
×
104 mL and aliquoted into 96-well plates. Then, compounds
◦
were added at a range of concentrations and the plates incubated at 37 C. Each compound was tested
in triplicate. Resazurin was added after 48 h and the plates incubated for a further 16 h. The plates
were then read in a Spectramax plate reader. Results were analyzed using GraphPad Prism.
3.2.2. T. cruzi Culturing and Evaluation of Trypanocidal Activity
T. cruzi epimastigotes (CL Brener stain) were cultured at 28 ◦C [24]. Activity was determined
using 96-well plate assays. Cultures were diluted to 2.5
of compound concentrations, and incubated for 4 days. Resazurin was added and the plates incubated
for a further 3 days, then read in a Spectramax plate reader as outlined above.
×
105 mL−1, aliquoted in triplicate at a range
3.2.3. Cytotoxic Activity against Rat Skeletal Myoblast L6 Cells
Cytotoxicity was assessed using microtiter plates following a previously described protocol [25].
Briefly, L6 cells were seeded at 1
concentrations. The plates were incubated for 6 days at 37 ◦C and then 20
each well. After a further 8 h incubation, fluorescence was determined using a Spectramax plate reader.
×
104 mL−1 in 200
µ
L of growth medium at different compound
µL resazurin was added to

Molecules 2018, 23, 177
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3.3. Cheminformatics Calculations
The Molinspiration package [22] was used to calculate the molecular properties (miLog P,
topological polar surface area (TPSA), molecular weight (MW), number of hydrogen bond acceptors
(nON), number of hydrogen bond donors (nOHNH, number of rotatable bonds (nrotb)), and number
of violations of Lipinski’s rules (nviolations). The CNS MPO scores were calculated according to
Reference [20]. For the full data, see the Supplementary Materials.
3.4. Determination of Brain Permeability: PAMPA-BBB Assay
The in vitro permeability (Pe) of the tested compounds and fourteen reference drugs through
a lipid extract of porcine brain membrane was assessed by using a parallel artificial membrane
permeation assay. Commercial drugs and the target compounds were tested using a mixture of
phosphate-buffered saline (PBS): EtOH 70:30. The assay was validated by comparing the experimental
permeability with the reported values of the reference drugs. A lineal correlation between experimental
and reported permeability values of the fourteen commercial drugs was established (experimental
Pe = 1.6374
×
reported Pe
−
1.3839; R2 = 0.9203). From this equation and the limits established
by Di et al. [18] for BBB permeation, three ranges of permeability were considered: compounds
of high BBB permeation (CNS+): Pe (10−6 cm s−1) > 5.16; compounds of low BBB permeation
(CNS
−
): Pe (10−6 cm s−1) < 1.89; and compounds of uncertain BBB permeation (CNS
): 5.16 >
±
Pe (10−6 cm s−1) > 1.89.
4. Conclusions
In conclusion, we have demonstrated that complex tetrasubstituted imidazolium salts are active
antiparasitic agents against both African and American trypanosomiases. The remarkably high
potency, good selectivity indexes, and favorable physicochemical/pharmacokinetic properties of
these compounds make them promising hits for future studies. These properties can be further
optimized by exploiting the several diversity points of the scaffold and its multicomponent origin.
Moreover, the synthetic approach described here will facilitate the generation of new compounds and
the assessment of structure-activity relationship SAR due to its modular nature.
Supplementary Materials: The following file is available online: a pdf file containing the experimental procedures
for the synthesis of the compounds, their chemical characterization, additional data on the calculations, PAMPA
and biological activity determinations.
Acknowledgments: We acknowledge support from DGICYT–Spain (CTQ-2015-67870P, SAF2014-57094-R),
and the Generalitat de Catalunya (2014 SGR 52, 137 and 1189). J.M.K. acknowledges support from the British
Heart Foundation.
Author Contributions: O.G. and R.L. conceived and designed the experiments; O.G., N.K. and M.R. performed
the chemical experiments; M.C.T. and J.M.K. did the biological testing; O.D.P., D.M.-T. and B.P. calculated the
physicochemical profiles and the PAMPA scores. All authors analyzed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–e are not available from the authors.
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