In vitro screening of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives as antiprotozoal agents and docking studies on Trypanosoma cruzi CYP51

Article abstract of DOI:10.1016/j.ejmech.2016.02.028

Sterol 14α-demethylase (CYP51) is a key enzyme involved in the survival and virulence of many parasite protozoa, such as Trypanosoma and Leishmania species, thus representing a valuable drug target for the treatment of Kinetoplastid diseases. A set of azole-based compounds selected from an in-house compound library was in vitro screened against different human protozoan parasites. Several compounds showed selective activity against Trypanosoma cruzi, with compound 7 being the most active (IC₅₀ = 40 nM). Given the structural similarity between the compounds here reported and known CYP51 inhibitors, a molecular docking study was performed to assess their binding with protozoal target and to rationalize the biological activity data.

Full text of DOI:10.1016/j.ejmech.2016.02.028

Accepted Manuscript
In vitro screening of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives as antiprotozoal
agents and docking studies on T. cruzi CYP51
Daniela De Vita, Dr., Francesca Moraca, Claudio Zamperini, Fabiana Pandolfi,
Roberto Di Santo, An Matheeussen, Louis Maes, Silvano Tortorella, Luigi Scipione
PII:
S0223-5234(16)30108-8
10.1016/j.ejmech.2016.02.028
EJMECH 8379
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2015
Revised Date: 18 January 2016
Accepted Date: 10 February 2016
Please cite this article as: D. De Vita, F. Moraca, C. Zamperini, F. Pandolfi, R. Di Santo, A.
Matheeussen, L. Maes, S. Tortorella, L. Scipione, In vitro screening of 2-(1H-imidazol-1-yl)-1-
phenylethanol derivatives as antiprotozoal agents and docking studies on T. cruzi CYP51, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.028.
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ACCEPTED MANUSCRIPT
In vitro screening of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives as antiprotozoal agents
and docking studies on T. cruzi CYP51
Daniela De Vita,a* Francesca Moraca,c Claudio Zamperini,c Fabiana Pandolfi,a Roberto Di Santo,a,b
An Matheeussen,d Louis Maes,d Silvano Tortorellaa and Luigi Scipionea
a Department of “Chimica e Tecnologie del Farmaco”, Sapienza University of Rome, Piazzale Aldo
Moro, 5, 00185 Rome, Italy
b
“Istituto Pasteur-Fondazione Cenci Bolognetti”, Department of “Chimica e Tecnologie del
Farmaco”, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
c
Department of “Biotecnologie, Chimica e Farmacia”, “University of Siena”, Via A. Moro 2,
53100 Siena, Italy
d
Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Faculty of Pharmaceutical,
Biomedical and Veterinary Sciences, Antwerp University, 2610 Antwerp, Belgium
* Corresponding author: Dr. Daniela De Vita, Department of “Chimica e Tecnologie del Farmaco”,
Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy.
Email: daniela.devita@uniroma1.it

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Graphical abstract
F
EtO S
2
N
N
O
N
H
O
N
N
T. cruzi IC50 = 40 nM

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Abstract
Sterol 14α-demethylase (CYP51) is a key enzyme involved in the survival and virulence of many
parasite protozoa, such as Trypanosoma and Leishmania species, thus representing a valuable drug
target for the treatment of Kinetoplastid diseases. A set of azole-based compounds selected from an
in-house compound library was in vitro screened against different human protozoan parasites.
Several compounds showed selective activity against T. cruzi, with compound 7 being the most
active (IC50 = 40nM). Given the structural similarity between the compounds here reported and
known CYP51 inhibitors, a molecular docking study was performed to assess their binding with
protozoal target and to rationalize the biological activity data.
Highlights
ꢀ A number of azoles with selective activity against T. cruzi were identified.
ꢀ The most active compound possesses 40 nM IC50.
ꢀ The biological activity of pure enantiomers was evaluated.
ꢀ Molecular docking on T. cruzi CYP51 was assessed to rationalize biological data.
Keywords
Trypanosoma cruzi; Azoles; CYP51.

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1. Introduction
Trypanosoma cruzi (T. cruzi) is an intracellular protozoan parasite responsible for Chagas disease
(CD), considered one of the neglected diseases [1]. It is transmitted to the mammalian host
primarily by infected faeces of blood-sucking triatomine bugs, also known as “kissing bugs”. Other
modes of transmission include infected blood transfusion, vertical transmission or infected organ
transplantation [2]. CD occurs mainly in Latin America, however, the number of infections in the
United States of America, Canada, many European and some Western Pacific countries continues to
increase because of population mobility [3]. To date, CD treatment includes the nitro-derivatives
benznidazole and nifurtimox [4]. Despite their significant activity in congenital and adult acute T.
cruzi infections, their use presents major drawbacks, including long duration of therapy, poor
efficacy in the chronic phase, different efficacy related to geographical area and several adverse
reactions [5]. These facts motivate to continue the research and development of new more safe and
effective drugs [6,7].
Sterol 14α-demethylase (CYP51) represents a main target to develop new drugs for the treatment of
CD. This enzyme catalyzes the removal of 14α-methyl group of sterols, an essential step in sterol
biosynthesis leading to the formation of cholesterol in vertebrates, ergosterol in fungi, and a variety
of 24-alkylated ergosterol derivatives in plants and protozoa [8]. Unlike mammals that can
accumulate cholesterol from diet, blocking of ergosterol production in fungi and protozoa is lethal:
it affects cytokinesis, stops cell growth, and eventually leads to collapse of the cellular membrane
[9]. Due to the similarity in sterol composition, several antifungal agents have been tested against T.
cruzi. Among these, posaconazole and the ravuconazole prodrug E1224 have recently terminated
Phase-2 clinical trials for treatment of chronic Chagas disease [10]. These studies have led to
disappointing conclusions about their effectiveness in chronic CD, mailnly due to poor
pharmacokinetic properties. Moreover, the chemical synthesis of posaconazole and ravuconazole is
coumbersome and very expensive. Nevertheless, research for inhibitors specifically designed for the

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CYP51 of T. cruzi (CYP51Tc), less expensive and endowed with optimal pharmacokinetic
properties, is still promising [11].
In previous studies [12, 13], we identified promising imidazole derivatives with a high and selective
in vitro activity against intracellular amastigotes of T. cruzi (Tulahuen C2C4). The most potent
among them (I - III) showed IC50-values in the low nM range and are reported in Chart 1. These
compounds were found to inhibit the T. cruzi sterol 14α-demethylase and, as demonstrated by the
co-crystals, they fit into the deepest segment of the CYP51 cavity and disrupt the heme support
from the protein moiety (compound II) or block the entrance into the CYP51 substrate access
channel (compound III).
Chart 1: lead compounds identified in our previous studies
Here, we report the in vitro antiprotozoal activity evaluation of eight compounds selected from our
in-house imidazole library; they are structurally correlated with already described I - III and some
of them possess antifungal activity [14,15].
All selected molecules possess the three principal pharmacophoric features of CYP51 inhibitors: i)
an iron chelating nitrogen containing heterocycle; ii) an hydrophobic group quite close to it; iii) a
second hydrophobic group (Chart 1). Furthermore, we tried to rationalize the biological data by

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means of a molecular docking study based on known crystal structures of T. cruzi CYP51
(CYP51Tc).
Compounds 1 and 2 (logP 5.963 and 5.662 respectively) were chosen to verify how the presence of
a halogen atom (chlorine or fluorine) on the first hydrophobic group influences the antiparasitic
activity in comparison to compound I (logP 5.566). Compound 3 (logP 7.236) was chosen in order
to verify the effect of more bulky and hydrophobic group in the same position. In addition, we have
chosen compounds 4-8 to evaluate how side chains with different size and polarity in the second
hydrophobic group affect the activity. In particular, compounds 5 (logP 4.405) and 6 (logP 4.138)
possess a more polar piperazine moiety instead of the aromatic ring closer to carbonyl linker (5 and
6 vs I); compound 4 (logP 3.775) with respect to 5 and 6 contains a more polar furoylpiperazine
moiety, present in some compounds with antitrypanosomal activity [16,17]. Finally, compounds 7
(logP 4.096) and 8 (logP 5.011) have been selected for their similitude with compound III (logP
8.155) but with more polar groups in the terminal portion of the side chain
2. Results and discussion
2.1 Chemistry
Compounds 1, 2, 4-8 were previously synthesized and evaluated for their antifungal activities
[14,15]. The new compound 3 was prepared as previously reported for compounds 1 and 2 [15]. In
brief, 1-(biphenyl-4-yl)-2-(1H-imidazol-1-yl)ethanol was activated as alcoholate using NaH in dry
CH3CN and then the biphenyl-4-carbonyl chloride was added (Scheme 1).

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Scheme 1: Reagents and conditions for 1-8. a: NaH, CH3CN, r.t., 2 h. b: biphenyl-4-carbonyl chloride. c: dry CH3CN,
triphosgene, r.t., overnight; d: TEA, opportune amine, r.t., overnight.
The synthesis of 4-8 was accomplished by the activation of the –OH group of 2-(1H-imidazol-1-yl)-
1-phenylethanol with triphosgene in dry CH3CN to obtain the desired chloroformate; then TEA and
the selected amines were added to the chloroformate stirring overnight (scheme 1) [14].
Commercially available amines were used to synthesize compounds 4-6; otherwise the side chains
of 7 and 8 were prepared as described in the Scheme 2. The synthesis of the amines 11 and 12
started by condensation of 1-fluoro-4-nitrobenzene with the appropriate commercial piperazine
refluxed for 2h in CH3CN. The obtained nitro compounds (9 and 10) were reduced to the
corresponding amines (11 and 12) with 50 psi of H2 for 4h in a Parr apparatus, using Pd/C (5%) as
catalyst. All the selected compounds contain a chiral carbon atom and were obtained as racemic
mixtures.

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NO2
NH2
H
N
a
b
N
N
N
N
R
N
R
R
11: R = -C3H8O2S
12
(9-10)
: R = -C6H6O2
Scheme 2: Reagents and conditions. a: CH3CN, reflux, 2h; b: H2, Pd/C, 50 psi, r.t., 4h.
In order to evaluate if the pure enantiomers possess different biological activity and if this can be
related to the absolute configuration, we synthesized some of the selected compounds in the
enantiomerically pure form. The pure enantiomers of esters 1 and 2 and carbamates 5, 6 and 8 were
obtained as described for racemates from enantiomerically pure R or S 1-(4-fluorophenyl)-2-(1H-
imidazol-1-yl)ethanol and R or S 1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethanol, as previously
reported [12].
2.2 Antiparasitic activity
All compounds were in vitro tested vs amastigote stage of T. cruzi, Tulahuen CL2, β-galactosidase
strain (nifurtimox-sensitive). In addition the screening was extended to include the activity of
compounds 1-8 towards the other human protozoan parasites Leishmania infantum (MHOM/MA-
BE/67 amastigotes), T. brucei (Squib-427 strain, suramin-sensitive) and Plasmodium falciparum
(Chloroquine-resistant K1-strain). The in vitro screening was carried out according to the
previously described procedures [18]. The results are reported in Table 1. All the racemic azole
derivatives show an anti-T. cruzi activity with IC50-values ranging from 0.04 to 5.84 µM. In
particular, 1, 2, 5-8 were more active than benznidazole used as reference drug. The most active
compound 7 showed IC50 values of 40 nM and it proved to be approximately fifty times more active
than benznidazole and eight times less active than azole reference compound, posaconazole (Table
1).

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Table 1. In vitro antiparasitic activity and cytotoxicity of compounds 1-8.
Tca
0.48
0.20
0.56
0.71
1.80
0.49
2.72
5.84
0.87
1.00
1.32
1.68
1.23
5.44
0.04
0.79
1.72
0.13
1.95
-
Lic
12.7
>64.0
32.0
32.2
>64.0
34.6
8.0
Tbd
32.4
32.4
30.1
32.2
>64.0
33.7
29.1
>64.0
8.0
32.2
>64
27.2
32.2
>64
>64.0
32.9
>64
>64
-
Pfe
1.9
2.0
2.1
0.6
2.3
2.0
7.7
10.7
2.4
2.2
1.5
2.6
1.2
1.3
17.2
4.0
4.0
5.2
-
MRC-5f
7.2
7.8
8.2
7.8
>64
8.0
8.8
>64.0
8.2
8.7
8.5
15.5
8.7
8.2
>64
26.4
26.5
37.5
-
SIg
15.0
39.0
14.6
10.9
>35.6
16.3
3.2
>10.9
9.4
8.7
6.4
9.2
Cmp
1
(R)-1
(S)-1
2
(R)-2
(S)-2
3
LogPh
5.963
5.662
7.236
3.775
4.405
>64.0
8.0
4
5
(R)-5
(S)-5
6
(R)-6
(S)-6
7
12.7
12.7
27.2
20.3
>64
38.0
28.9
>64
>64
-
4.138
7.0
1.5
>1600
33.4
15.4
288.5
-
4.096
5.011
8
(R)-8
(S)-8
BNZ
MIL
SUR
CHL
TAM
POS
10.4
-
-
-
-
-
-
-
-
-
-
-
-
-
0.02
-
-
-
-
-
0.14
-
10.9
0.005 b
Data represent IC50-values given in ꢀM. a: T.cruzi Tulahuen CL2 amastigote stage; b: Ref 13; c: L. infantum
MHOM/MA/67/ITMAP263; d: T. brucei rhodesiense Squib-427 strain, suramin-sensitive, trypomastigote stage; e: P.
falciparum K1, Chloroquine-resistant, erythrocytic stages; f: Human fetal lung fibroblast (MRC-5) cell line toxicity; g:
selectivity index calculated as IC50MRC-5/IC50Tc; h: LogP were calculated by QikProp ver. 4.2 [18]; BNZ = benznidazole,
MIL = miltefosine; Sur = suramin; CHL = chloroquine; TAM = tamoxifen; POS = posaconazole.
The antiparasitic activity and cytotoxicity data show that the imidazole derivatives 1-8 are highly
selective towards T. cruzi. In particular, compound 7 possess the highest activity (T. cruzi IC50 0.04
µM) and the lowest cytotoxicity (MRC-5 IC50 > 64 µM and a SI >1600), making it an excellent
candidate for a new series of anti-Chagas imidazole derivatives.
Compounds 1, 3 and 5 presented an antileishamial activity similar to miltefosine but they showed a
cytotoxicity on MRC-5 cell line of the same magnitude, thus indicating the lack of selectivity
towards L. infantum.
Furthermore, compound 2 showed antiplasmodial activity (IC50 0.6 µM), approximately twice than
the reference drug chloroquine, and good selectivity against P. falciparum, with an low cytotoxicity
vs MRC-5 cell (MRC-5 IC50 7.8 µM).

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None of studied azole compounds showed a higher or comparable activity than the reference drug
suramine towards T. brucei, which generally demonstrates lower sensitivity vs this class of
compounds.
The high selectivity of studied compounds toward T. cruzi over T. brucei and L. infantum could be
probably due also to the difference in the CYP51 active site volume and surface area smaller in T.
brucei and L. infantum than in T. cruzi that could favour the access of drugs in the active site. [12]
The analysis of the anti-Chagas activity data of the pure enantiomers highlights two significant
aspects: i) in all cases, we found a small difference in activity in a pair of enantiomers reaching
maximum one order of magnitude; ii) the most active enantiomers do not have the same absolute
configuration, for compound 1, 5 and 6 we found that the most active was the R-enantiomer, on the
contrary, for compounds 2 and 8, S resulted the most active enantiomer. These results are in good
agreement with our previously reported data for similar compounds [13], which showed that both
enantiomers of are able to bind and inhibit the CYP51Tc.
2.3 Molecular docking
To rationalize the effect of different side chains on the CYP51Tc as the target protein, due to the
high structural similarity to previously reported CYP51 inhibitors [13], a docking study was
performed. Recently, CYP51Tc was solved with the compounds LFT (IIa), LFS (IIb) and LFD
(III), (PDB ID: 4CK9, 2.74 Å resolution; 4CKA, 2.70 Å resolution; 4CK8, 2.62 Å resolution) [13].
The coordinates of CYP51Tc co-crystallized with III (PDB ID: 4CK8) were downloaded from the
Protein Data Bank (PDB) because of the highest resolution, and used for the subsequent docking
studies. The enzyme structure was pretreated by means of the Protein Preparation Wizard of
Maestro9.9 suite [20], deleting crystallization water molecules, adding hydrogen atoms, filling in
missing side chains, and assigning the correct ionization state of the co-crystallized ligands at
physiological pH (7 ± 2.0) using Epik2.9 [21], and the correct amino acids protonation state at the
same pH using Prime [22]. The azole derivatives were designed by means of Maestro9.9 and

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pretreated with LigPrep3.1 [23]. Their ionization state was checked at physiological pH (7 ± 2.0)
using Epik2.9. Since all compounds have a chiral center, the molecular modeling study was
performed considering both –R and –S enantiomers. Docking studies were performed in the active
site of the pretreated CYP51Tc by means of Glide6.4 [24], using the Standard Precision (SP) scoring
function. Docking protocol was validated reproducing the co-crystal binding pose of the compound
III (Supportive information). The protein-ligand interaction energy (PLIE) was estimated for the
selected docking poses by means of SZYBKI 1.8.0.1 [25] using the Poisson-Boltzmann solvation
model.
The only difference between compounds 1 (IC50 = 0.48 µM) and 2 (IC50 = 0.71 µM) and the already
tested 2-(1H-imidazol-1-yl)-1-phenylethyl biphenyl-4-carboxylate (I, IC50 = 0.014 µM vs T. cruzi
amastigote stage cells) [12] is the substitution of the hydrogen atom by a chlorine in compound 1
and by a fluorine in compound 2. Both the halogens are oriented towards a little hydrophobic cavity
formed by A287, F210, Y116 and L127. The lone pair oxygen of Y116 faces the halogens atoms in
both compounds’ poses. Hence, the decrease in the activity of compounds 1 and 2 with respect to
compound I can be ascribed to a little electron repulsion effect between F and Cl and the Y116 lone
pair oxygen.
The further decrease of T. cruzi inhibition exerted by compound 3 (IC50 = 2.72 µM) could be
ascribed to a worse coordination at the heme iron, compared to that of the other more active
compounds (Figure 1), since the coordination angle between the lone pair N of the azole ring and
the iron should be perpendicular [13]. In addition the protein-ligand interaction energy (PLIE) is not
so favored: the R-enantiomer shows a PLIE = -5.17 kcal/mol and the S enantiomer a PLIE = 0.31
kcal/mol. Since the activity is referring to the racemic mixture, we can consider an average of -2.43
kcal/mol.

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Figure 1: compound (R)-3 docking binding pose (orange stick). The hydrophobic cavity surrounding the heme
prosthetic group is shown in gray stick and the HC2 in purple stick. Cys422 coordinating the heme iron is represented in
light green stick and the heme prosthetic group in yellow stick. Coordination at the heme iron is represented as black
dotted lines. Atoms are colored according to their atom types and non-polar hydrogen atoms are omitted.
Compound 4 (IC50=5.84 µM) shows both a Cl inserted in the little hydrophobic cavity as described
for compounds 1 and 2, and also a furanyl moiety with lone pair oxygen oriented towards the lone
pair S of M360 in the (R)- enantiomer and towards the backbone oxygen of Val102 in the (S)-
enantiomer, that can contribute to the decrease of its activity due to a repulsive effect (Figure 2).
Indeed, the PLIE for its S enantiomer is in a positive range (+3.21 kcal/mol) and for the R
enantiomer is reasonable (-9.91 kcal/mol), but the IC50 is referring to the racemic mixture, hence we
can consider an average of -3.35 kcal/mol. Compounds 5 (IC50 = 0.87 µM) and 6 (IC50 = 1.68 µM)
are characterized by a NO2 group that decreases their overall hydrophobicity and hence the
permeability through parasite’s cell membrane, being a reasonable explanation of the decrease of
activity, even if the docking study indicates a good coordination at the heme iron and seem to be
well stabilized in the active site of the enzyme by hydrophobic interactions into the hydrophobic
cavity surrounding the prosthetic group, as also shown by the favored PLIE values (Figure 2, Table
2). In addition, in compound 6, the para-Cl substituent at the phenyl ring is replaced by a para-F
substituent that can further contribute to the decrease of the overall hydrophobicity of the
compound.

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Figure 2: docking binding pose of compound (S)-4 (left, cyan stick) and (S)-5 (right, pale violet stick). Color code is
according to Figure 1.
The docking pose of the most active compounds 7 and 8 (Figure 3) shows that their side chain
explores a cavity surrounding the prosthetic group, formed by Ile45, Phe48, Gly49, Ile72, Pro210,
Ala211, Val213, Phe214 (for convenience thereafter referred to HC 2), showing a similar binding
mode to that of the co-crystallized III. This evidence together with their higher hydrophobicity and
the very low PLIE value could explain their high activity.
Figure 3: docking binding pose of the most active compounds (S)-7 (left, green stick) and (R)-8 (right, pale cyan stick).
Color code is according to Figure 1.

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Table 2. Protein-Ligand interaction energy values (PLIE) of the different enantiomers binding poses in CYP41 4CK8
coordinates
Compound
(R)-1
(S)-1
PLIE (kcal/mol)
-9.34
-1.87
(R)-2
(S)-2
-10.30
-9.53
(R)-3
(S)-3
-5.17
0.31
(R)-4
(S)-4
(R)-5
(S)-5
-9.91
+3.21
-6.35
-3.96
(R)-6
(S)-6
-4.19
-4.63
(R)-7
(S)-7
(R)-8
(S)-8
-14.90
-22.19
-11.10
-25.86
2.4 SAR analysis on anti-T. cruzi activity
Due to the high structural similarity of the studied compounds with known CYP51Tc inhibitors and
on the basis of our recent publication on X-Ray structures of CYP51Tc co-crystallized with IIa, IIb
and III, we judge this enzyme as the target of imidazole derivatives 1-8. For these reasons, we
carried out a docking study and a PLIE estimation to explore their possible binding orientation
within the catalytic site of CYP51Tc. The results of the modeling study, combined with the
hydrophobicity of the side chain, let us to propose a possible explanation of the different biological
activity of the studied compounds. From the docking study, it is clear that the coordination at the
heme iron is fundamental for the discrimination between active, less active and inactive
compounds. Only small hydrophobic groups (chlorine or fluorine) are allowed in the 4 position of
the phenyl ring of 1-(phenyl)-2-(1H-imidazol-1-yl)-ethanol derivatives. The replacement of the
chlorine with a fluorine atom (1 vs 2 and 5 vs 6) causes only small differences in the
antitrypanosomal activity. In general, chloro- derivatives were found to be slightly more active than
fluoro- analogues, probably due higher hydrophobicity. The presence of a more bulky phenyl group
was detrimental for the biological activity (3 vs 1 and 2) because of the worst influence on the

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heme-iron coordination of the imidazole ring. Otherwise, the presence of a second aromatic ring in
the lateral side chain appears to be fundamental as shown by comparing compound 4, the worse of
the series, with compounds 5 and 6.
The increase in length of the side chain in compounds 7 and 8 compared to 5 and 6 leads to an
increase of anti-T. cruzi activity. Moreover, the side chains of 7 and 8 are more rigid in the initial
part due to the presence of the aromatic ring, and this can orient the chain deeper into the
hydrophobic cavity above the heme group.
2.5 Stability studies
Because the synthesized compounds contain ester or carbamate functions, potentially susceptible to
hydrolysis, we evaluated the chemical stability of the most active compounds 7 and 8 in acid
medium. The possible hydrolysis reaction of 7 and 8 was followed by 1H NMR during 24 hrs at 27
°C in the test tube, dissolving 10-4 mol of each compound in 0.6 mL of DCl/D2O (pH 2). The
appearance of the methine and methylene proton signals exclusively related to the alcohol produced
by hydrolysis were chosen as a prove of the reaction. For both the compounds 7 and 8, no free
alcohol signals were observed in the experimental conditions thus demonstrating their stability to
hydrolysis under the described conditions.
Compounds 7 and 8 were also evaluated for their metabolic stability by treatment with liver
microsomal protein. Each compound (50 ꢀM in pH 7.4 phosphate buffer + DMSO 2%) was
incubated at 37°C for 60 min with human liver microsomal protein in the presence of a NADPH-
generating system. The reaction was stopped and the parent drug and metabolites were determined
by LC-UV-MS (see supportive information). Compounds 7 and 8 show high metabolic stability,
e.g. 99% of parent drug was not metabolized.

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3. CONCLUSIONS
In conclusion, eight imidazole derivatives were evaluated in vitro against different parasitic
protozoa as racemates and pure enantiomers. The selected compounds showed selective and high
activity towards T. cruzi amastigotes, with the most active of them, 7, having an IC50 of 40 nM and
a low cytotoxicity (MRC-5 IC50 > 64 µM). Interesting activity data were also observed against L.
infantum for the compounds 1, 3 and 5 and against P. falciparum for the compound 2 and may be
subject to further investigations.
Furthermore, compound 7 showed a good chemical and metabolic stability, that combined with the
simplicity and low cost of its chemical synthesis, make it a good lead for further structural
optimization for the anti-Chagas activity.
Acknowledgements
This work was financially supported by Miur, Italy.
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